Nuclear receptors: Key regulators of somatic cell functions in the ovulatory process

Molecular Aspects of Medicine xxx (xxxx) xxx

Contents lists available at ScienceDirect

Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Nuclear receptors: Key regulators of somatic cell functions in the ovulatory process Camilla H.K. Hughes, Bruce D. Murphy * Centre de Recherche en Reproduction et Fertilit´e, Universit´e de Montr´eal, St-Hyacinthe, Qc, J2S 2M2, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords: Nuclear receptor Follicle Ovulation Steroidogenesis

The development of the ovarian follicle to its culmination by ovulation is an essential element of fertility. The final stages of ovarian follicular growth are characterized by granulosa cell proliferation and differentiation, and steroid synthesis under the influence of follicle-stimulating hormone (FSH). The result is a population of gran­ ulosa cells poised to respond to the ovulatory surge of luteinizing hormone (LH). Members of the nuclear receptor superfamily of transcription factors play indispensable roles in the regulation of these events. The key regulators of the final stages of follicular growth that precede ovulation from this family include the estrogen receptor beta (ESR2) and the androgen receptor (AR), with additional roles for others, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). Following the LH surge, the mural and cumulus granulosa cells undergo rapid changes that result in expansion of the cumulus layer, and a shift in ovarian steroid hormone biosynthesis from estradiol to progesterone production. The nuclear receptor best associated with these events is LRH-1. Inadequate cumulus expansion is also observed in the absence of AR and ESR2, but not the progesterone re­ ceptor (PGR). The terminal stages of ovulation are regulated by PGR, which increases the abundance of the proteases that are directly responsible for rupture. It further regulates the prostaglandins and cytokines associ­ ated with the inflammatory-like characteristics of ovulation. LRH-1 regulates PGR, and is also a key regulator of steroidogenesis, cellular proliferation, and cellular migration, and cytoskeletal remodeling. In summary, nuclear receptors are among the panoply of transcriptional regulators with roles in ovulation, and several are necessary for normal ovarian function.

1. Introduction Worldwide, more than 10% of women experience problems becoming pregnant or maintaining pregnancy to term (Vander Borght and Wyns, 2018). Anovulation, or failure of the ovulatory process, contributes substantially to the problem, explaining some 25% of infertility (Wang et al., 2017). Ovulation is the culmination of a complex and well-coordinated sequence of events. It is initiated by a surge of LH from the pituitary gland, and engenders rapid physiological changes in cells, as well as extensive tissue remodeling. An improved understanding of the transcriptional regulation of the events leading to and regulating ovulation is expected to contribute to the development of technology to address anovulation and has the potential to provide new targets for contraceptive development. A period of antral follicular growth precedes ovulation. During this time, the antral follicle, or follicles, secrete increasing concentrations of estradiol. Upon reaching a threshold concentration, this estradiol signals

the readiness of a follicle to ovulate by provoking release of GnRH from the surge center of the hypothalamus, which in turn causes the release of LH from the pituitary (Berga and Naftolin, 2012; Dufour et al., 2020; Plant, 2015). This LH surge triggers a complex cascade of events and induces a variety of changes in ovarian follicular cell chromatin struc­ ture and transcription factor expression, thereby dramatically altering the transcriptional landscape (Bianco et al., 2019). Following the LH surge, hyperemia of all but the apex of the ovulatory follicle ensues, the microenvironment of the follicle becomes hypoxic, the cumulus oophorus expands, and the follicle ruptures, allowing release of the cumulus oocyte complex (Duffy et al., 2019; Robker et al., 2018). The LH surge also triggers a major shift in steroid hormone synthesis, in which the estradiol production that characterized follicular devel­ opment declines and the dominant hormone becomes progesterone. An important effect of the increase in progesterone is to trigger key ovulatory events, including rupture of the periovulatory follicle (Lydon et al., 1995). Accompanying these events is the initiation of the immune

* Corresponding author. E-mail address: [email protected] (B.D. Murphy). https://doi.org/10.1016/j.mam.2020.100937 Received 22 September 2020; Received in revised form 23 November 2020; Accepted 26 November 2020 0098-2997/© 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Camilla H.K. Hughes, Bruce D. Murphy, Molecular Aspects of Medicine, https://doi.org/10.1016/j.mam.2020.100937

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

cell infiltration and angiogenesis that are the hallmarks of the process of formation of the corpus luteum. Simultaneously, the oocyte is released to the infundibulum of the oviduct. Ovulation, therefore, is the result of multiple well-integrated processes. The LH surge causes rapid modifi­ cations that ready the oocyte for fertilization, result in follicular rupture to release the oocyte, and facilitate the consequent formation of the corpus luteum. Many factors coordinate the ovulatory process, most markedly a complex network of transcription factors. Among these are the nuclear receptors, of which 48 have been identified in humans and 50 in all mammals. Twenty-four of the nuclear receptors are orphan nuclear receptors, meaning that they lack a known ligand (Meinsohn et al., 2019). Nuclear receptors were first identified as mediators of hormone ac­ tion by researchers investigating how estrogen, corticoids, and thyroid hormone elicit their physiological effects (Mangelsdorf et al., 1995). Molecular cloning provided insight into their structure and the simi­ larities they shared. It is now known that all of the steroid hormones have cognate receptors in the nuclear receptor family, as do vitamins A (retinoic acid) and D. Twelve among the known nuclear receptors, were initially classified as orphan nuclear receptors, but when their ligands were subsequently identified, these receptors were reclassified, and are known as “adopted” orphan nuclear receptors. Classifications of all nuclear receptors are shown in Table 1. In the early years of the field of transcription factor molecular biology, identification of specific targets of transcription factors required arduous promotor activity experiments. The development of chromatin immunoprecipitation technology, combined with next gen­ eration sequencing can generate data on all genes regulated by any given transcription factor, and has led to substantial advances in this field. In addition, nontargeted approaches such as FAIREseq and ATACseq have allowed detailed investigation of global open chromatin in various physiological contexts (Bianco et al., 2015; Buenrostro et al., 2013, 2015; Simon et al., 2012). This technology has recently been used to investigate the ovulatory period (Bianco et al., 2019; Dinh et al., 2019). As we look to the future of nuclear receptor study, these methods will provide a more profound understanding of the complex and inter­ connected networks of gene regulation during the ovulatory period.

Table 1 Summary of reproductive phenotypes of female mice lacking various nuclear receptors. Receptor NR0 family DAX1 (NR0B1)

SHP (NR0B2)

NR1 family THRA (NR1A1)

2. Nuclear receptors with key roles in the regulation of ovulatory processes Of the 50 known mammalian nuclear receptors, only three (CAR, TLX, PXR) are completely undetectable in the reproductive tract (Bookout et al., 2006, Table 1). The reproductive phenotypes of mice in which individual nuclear receptors have been deleted or depleted are summarized in Table 1. Remarkably, among murine models that have been generated, only deletion of PGR, LRH-1, SF-1, ESR2, and ESRRB result in a phenotype of complete infertility clearly attributable to an ovarian defect (Table 1). Among these, the defects observed in mice lacking SF-1 or ESRRB seem to relate to ovarian development or follicle formation, while defects observed in the absence of LRH-1 or PGR clearly relate directly to ovulation. Phenotypes of mice lacking ESR2 have been variable, and range from subfertile to fully infertile, in part due to ovulatory defects (Table 1). In addition, mice lacking PPARG, REVERBA, LXRA, LXRB, TR4, AR, GCNF, ESR1, or haploinsufficient for COUP-TFII are subfertile, due at least in part to ovarian defects (Table 1). There is additional evidence for roles for other nuclear re­ ceptors. While these mouse models provide substantial evidence to suggest which nuclear receptors are important in ovulation, the caveat is that models of ovarian depletion have not been generated for several nuclear receptors that could be of importance in the ovulatory process, most notably the NR4 family and the corticoid receptors, The focus of this review, therefore, is on the receptors where investigation has been conducted, with the addition of a few others with documented impor­ tance in the ovary.

Classificationa

Phenotype

Expression in ovary

Orphan nuclear receptor

CMV-cre driver: females are normal and fertile (Yu et al., 1998)

Orphan nuclear receptor

Germline deletion: viable and fertile (Wang et al., 2002)

Primarily granulosa cells (Sato et al., 2003), also detectable in theca ( Murayama et al., 2008) Detectable in ovary ( Bookout et al., 2006) and in granulosa cells ( Takae et al., 2019)

Not orphan

Germline deletion of isoform 1: Viable and fertile (Wikstr¨ om et al., 1998) Germline deletion of isoform 1 and 2: Die at approx. 5 weeks old ( Fraichard et al., 1997) Germline deletion: Fertile (Forrest et al., 1996)

THRB (NR1A2)

Not orphan

RARA (NR1B1)

Not orphan

RARB (NR1B2)

Not orphan

RARG (NR1B3)

Not orphan

PPARA (NR1C1)

“Adopted” orphan

PPARD NR1C2

“Adopted” orphan

Germline deletion: Normally fertile (Peters et al., 2000)

PPARG (NR1C3)

“Adopted” orphan

Germline deletion: Death due to placental disfunction (Barak et al., 1999) Depletion following the

Germline deletion of RARA: Most individuals die by 2 months old ( Lufkin et al., 1993) Germline deletion of RARA1 isoform: fertile and viable (Lufkin et al., 1993) Triple knockout of RARA, RARB, RARB (SF-1-cre to deplete all three receptors from DPC11 onward): normally fertile (Minkina et al., 2017) Germline deletion: Fertile (Luo et al., 1995) Triple knockout of RARA, RARB, RARB using SF-1 promotor to deplete all three receptors from DPC11 onward: normally fertile (Minkina et al., 2017) Germline deletion: fertile (Lohnes et al., 1993) Triple knockout of RARA, RARB, RARB using SF-1 promotor to deplete all three receptors from DPC11 onward: normally fertile (Minkina et al., 2017) Germline deletion: Normally fertile (Lee et al., 1995b)

Granulosa cells, cumulus, oocytes ( Aghajanova et al., 2009; Zhang et al., 1997)

Granulosa cells, cumulus, oocytes ( Aghajanova et al., 2009; Zhang et al., 1997) Cumulus (Mohan et al., 2003), pan-RAR antibody shows RAR in granulosa and to lesser extent in theca (Zhuang et al., 1994)

Lowly abundant but detectable in ovary in some studies ( Bookout et al., 2006; Minkina et al., 2017; Zhuang et al., 1994)

Granulosa cells ( Kawai et al., 2016), 2016), cumulus ( Mohan et al., 2003), pan-RAR antibody shows RAR in granulosa and to lesser extent in theca (Zhuang et al., 1994) Theca and stroma; granulosa to a lesser extent (Komar et al., 2001) Theca and stroma; granulosa to a lesser extent (Komar et al., 2001) Granulosa cells ( Komar et al., 2001)

(continued on next page)

2

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Table 1 (continued ) Receptor

REVERBA (NR1D1)

REVERBB (NR1D2)

Classificationa

“Adopted” orphan

“Adopted” orphan

Table 1 (continued ) Phenotype ovulatory signal (PPARG f/f, PGR-cre): Greatly reduced ovulation rate, impaired follicular rupture (Kim et al., 2009a) Depletion from granulosa cells (MMTV-cre, also depletes expression in mammary epithelium, oocytes, megacaryocytes, B- and T-lymphocytes, and salivary gland): Reduced fertility, smaller litter size (Cui et al., 2002) Germline deletion: females are subfertile, with only 30% of matings leading to a pregnancy, relative to 75% in WT, and litter size is reduced ( Preitner et al., 2002) Germline deletion: Normal and fertile (Cho et al., 2012)

RORA (NR1F1)

Orphan nuclear receptor

Germline deletion: Die by 3–4 wks old (Dussault et al., 1998)

RORB (NR1F2)

Orphan nuclear receptor Orphan nuclear receptor

Germline deletion: Fertile (Andr´e et al., 1998) Germline deletion: Viable and fertile (Sun et al., 2000)

“Adopted” orphan

Germline deletion: Either single knockout is subfertile, with smaller and less frequent litters, impaired ability of oocytes to resume meiosis, but normal follicle number. Greater impairment in these functions in LXRA/LXRB double knockout ( Steffensen et al., 2006). Superstimulation results in symptoms of ovarian hyperstimulation syndrome (Mouzat et al., 2009). Germline deletion: Normal and fertile (Sinal et al., 2000) Germline deletion: Subfertile to infertile, no antral follicles ( Yoshizawa et al., 1997), impaired estradiol production (Kinuta et al., 2000), defects due to calcium deficiency and rescued by calcium supplementation ( Johnson and Deluca, 2001) Germline deletion: Fertile (Xie et al., 2000)

RORC (NR1F3) LXRA (NR1H3) and LXRB (NR1H2)

FXR (NR1H4)

“Adopted” orphan

VDR (NR1I1)

Not orphan

PXR (SXR or NR1I2)

“Adopted” orphan

Expression in ovary

Receptor

Classificationa

Phenotype

Expression in ovary

CAR (NR1I3)

Orphan nuclear receptor

Germline deletion: Fertile (Wei et al., 2000)

No ovarian expression (Bookout et al., 2006)

Orphan nuclear receptor

Germline deletion: Lethal during embryonic development (Chen et al., 1994b) Germline deletion: Heterozygotes are normally fertile when bred together but produce twice as many male pups as female pups, presumably due to a male reproductive defect. Fertility of homozygotes was not tested (Gerdin et al., 2006) Germline deletion: Lethal during embryonic development (Kastner et al., 1994)

Oocytes, granulosa cells, and cumulus ( Khan et al., 2016)

NR2 family HNF4A (NR2A1) HNF4G (NR2A2)

Orphan nuclear receptor

RXRA (NR2B1)

“Adopted” orphan

RXRB (NR2B2)

“Adopted” orphan

Germline deletion: Some individuals die during development, but the females that survive are fertile (Kastner et al., 1996)

RXRG (NR2B3)

“Adopted” orphan

TR2 (NR2C1)

Orphan nuclear receptor

TR4 (NR2C2)

Orphan nuclear receptor

TLX (NR2E1)

Orphan nuclear receptor

PNR (NR2E3)

Orphan nuclear receptor Orphan nuclear receptor Orphan nuclear receptor

Germline deletion: Fertile (RXRB/RXRG double knockout are also fertile) (Krezel et al., 1996) Germline deletion: Moderately subfertile in breeding pairs in which both males and females lack TR2, but not clear to which sex the decrease is attributable (Olivares et al., 2017). Germline deletion: Defective maternal behavior or lactation, reduced fertility (Collins et al., 2004); potential role in folliculogenesis ( Chen et al., 2008). Germline deletion: Impaired mating behavior and maternal behavior, attributed to neural defects ( Monaghan et al., 1997). Germline deletion: Fertile (Akhmedov et al., 2000). Germline deletion: Perinatal death (Qiu et al., 1997). Germline deletion: Embryonic death (Pereira et al., 1999). Haploinsufficiency: Subfertile, with increased age at puberty, disrupted estrous cycles, normal response to superovulation, but reduced luteal progesterone production (Takamoto et al., 2005).

Detectable in granulosa cells ( Chen et al., 2012)

Detectable in the ovary (Bookout et al., 2006; Liu et al., 2014) Detectable in the oocyte and cumulus (Brązert et al., 2020; Celichowski et al., 2018; Molinari et al., 2016) Low to absent in ovary (Bookout et al., 2006) Moderately abundant in the ovary (Bookout et al., 2006) Granulosa cells ( Drouineaud et al., 2007)

Granulosa cells ( Takae et al., 2019) Granulosa cells and oocytes (Xu et al., 2018)

COUP-TFI (NR2F1) COUP-TFII (NR2F2)

No ovarian expression (Bookout et al., 2006)

Detectable in the ovary (Bookout et al., 2006)

Granulosa cells ( Tatone et al., 2016; Xing et al., 2002), cumulus (Mohan et al., 2003) Granulosa cells ( Schweigert and Siegling, 2001; Tatone et al., 2016; Xing et al., 2002), cumulus (Mohan et al., 2003) Granulosa cells ( Tatone et al., 2016; Xing et al., 2002) Expressed in the ovary (Bookout et al., 2006)

Oocytes and granulosa cells of secondary and tertiary follicles ( Chen et al., 2008) No ovarian expression (Bookout et al., 2006)

Low to absent in ovary (Bookout et al., 2006) Granulosa cells ( Xing et al., 2002) Theca cells, absent from granulosa cells (Sato et al., 2003; Takamoto et al., 2005)

(continued on next page)

3

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Table 1 (continued ) Receptor

EAR2 (NR2F6) NR3 family ESR1 (NR3A1)

Table 1 (continued )

Classificationa

Phenotype

Orphan nuclear receptor

Ovarian depletion from the primordial follicle forward (Amhr2cre): No ovarian phenotype, likely due to lack of expression in granulosa cells, infertile due to uterine defect that leads to placental insufficiency (Petit et al., 2007). Germline deletion: Viable and fertile ( Warnecke et al., 2005)

Not orphan

ESR2 (NR3A2)

Not orphan

ESRRA (NR3B1)

Orphan nuclear receptor

ESRRB (NR3B2)

Orphan nuclear receptor

ESRRG (NR3B3)

Orphan nuclear receptor

GR (NR3C1)

Not orphan

Germline deletion: Infertile, but not due to ovarian function. Hyperemic ovaries, no CL in the absence of gonadotropin treatment ( Dupont et al., 2000; Lubahn et al., 1993), ovulate in response to gonadotrophin treatment, at reduced rate (Rosenfeld et al., 2000). Germline deletion: Subfertile to infertile, antral follicles present, impaired LH response and cumulus expansion ( Couse et al., 2005; Krege et al., 1998; Rumi et al., 2017), impaired granulosa cell proliferation and follicle growth (Dupont et al., 2000), precocious oocyte meiotic resumption (Liu et al., 2017) Germline deletion: Viable and fertile (Luo et al., 2003) Germline deletion: Germline deletion results in death on embryonic day 8–10 because of placental abnormalities ( Luo et al., 1997). Rescue of ERRB− /− with aggregated with four-cell stage tetraploid embryos results in viable offspring, with a reduction in number of primordial germ cells, adult ovaries had oocytes, but females were infertile (Mitsunaga et al., 2004) Germline deletion: Die during the first week of life, because of heart abnormalities (Alaynick et al., Germline deletion: Perinatal death (Cole et al., 1995) Germline deletion of melanocortin receptor 2, resulting in no corticosterone and minimal aldosterone:

Expression in ovary

Receptor

Classificationa

MR (NR3C2)

Not orphan

PGR (NR3C3)

Not orphan

AR (NR3C4)

Not orphan

Granulosa cells ( Zhang and Dufau, 2001) Primarily theca and interstitial cells ( Couse et al., 2000; Sar and Welsch, 1999); lowly abundant in granulosa cells (Liu et al., 2017)

Primarily granulosa cells (Couse et al., 1997; Sar and Welsch, 1999); ( D’Haeseleer et al., 2005)

Moderately expressed in the adult ovary ( Bookout et al., 2006) Primordial germ cells of developing ovary (Mitsunaga et al., 2004); moderately expressed in adult ovary (Bookout et al., 2006)

Lowly to moderately expressed in adult ovary (Bookout et al., 2006)

NR4 family NGFIB (NR4A1 or NUR77) NURR1 (NR4A2)

Granulosa cells ( Schreiber et al., 1982)

Phenotype fertile, disrupted cyclicity, reduced ovulation rate (Chida et al., 2011; Matsuwaki et al., 2010) Germline deletion: death 1–2 weeks after birth (Berger et al., 1998) Germline deletion of the ACTH receptor (melanocortin receptor 2), resulting in no corticosterone and minimal aldosterone: fertile, disrupted cyclicity, reduced ovulation rate (Chida et al., 2011; Matsuwaki et al., 2010) Germline deletion: Normal follicular growth, but no ovulation, no rupture, trapped oocyte, luteinization occurs, cumulus expansion is normal, oocytes are fertilizable (Lydon et al., 1995, 1996; Robker et al., 2000) Germline deletion of the PR-A isoform: very similar phenotype as the above (Mulac-Jericevic et al., 2000) Germline deletion of the PR-B isoform: ovulate normally in response to superstimulation protocols ( Mulac-Jericevic et al., 2003) Ovarian depletion from granulosa cells (ESR2-cre): Phenocopy of germline deletion (Park et al., 2020) Ovarian depletion from primordial follicle forward (AMH-cre; AMHR2-cre): fertile, but litters are smaller and ovulation rate is reduced, with defect in antral follicle formation and impaired cumulus expansion (Sen and Hammes, 2010; Walters et al., 2012) Other models (ACTB-cre; transgenic cytomegalovirus (CMV)-Cre): have a similar subfertile phenotype (Hu et al., 2004; Walters et al., 2007, 2009)

Expression in ovary

More abundant in the granulosa than the theca cells ( Gomez-Sanchez et al., 2009)

Granulosa cells of preovulatory follicle following the ovulatory signal ( Park and Mayo, 1991; Robker et al., 2000)

Granulosa cells of preantral and antral follicles, in greater abundance in the cumulus than in the mural granulosa in periovulatory follicles (Hillier et al., 1997; Yazawa et al., 2013)

Orphan nuclear receptor

Germline deletion: Fertile (Cheng et al., 1997; Lee et al., 1995a)

Granulosa and theca cells (Park et al., 2003)

Orphan nuclear receptor

Germline deletion: Lethal shortly after birth ( Zetterstr¨ om et al., 1997)

Granulosa cellspecific (Park et al., 2003) (continued on next page)

4

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

et al., 1987) preceded the discovery of ESR2 (Kuiper et al., 1996); (Mosselman et al., 1996) by nearly a decade. Although these two re­ ceptors share high sequence homology, particularly in their DNA bind­ ing (95%) and the ligand binding domains (55%) (Kuiper et al., 1996), and both bind estrogens with high affinity (Kuiper et al., 1997), they are distinct in their patterns of expression in mammalian cells and tissues (Kuiper et al., 1997; Drummond and Fuller, 2010). Both ESRs are abundant in the ovary. ESR2 is mainly localized to granulosa cells (Drummond et al., 1999; Sar and Welsch, 1999; Saunders et al., 2000). ESR1 is expressed primarily in the thecal and interstitial cells (Sar and Welsch, 1999). There is, nonetheless, some evidence for ESR2 expres­ sion in the thecal layer (Saunders et al., 2000). Some reports have also suggested that there could be low expression of ESR1 in the mural and cumulus granulosa cells of antral follicles and in the granulosa cells of secondary follicles (Drummond et al., 1999; Liu et al., 2017). Loss of either ESR1 (Lubahn et al., 1993; Dupont et al., 2000) or ESR2 (Krege et al., 1998; Dupont et al., 2000) has profound effects on reproductive function, with ESR2 seeming to have more important ovarian functions than ESR1. Perhaps the steroid hormone receptor that has been most studied in regard to its role in ovulation is the progesterone receptor (PGR). There are two isoforms of PGR, PGR-A and PGR-B. They are transcribed from the same gene via two different promotors, acting at different tran­ scription start sites (O’Malley, 2005). Lydon et al. (1995) reported extensive female reproductive defects associated with germline deletion of the gene coding for both isoforms of this receptor (Lydon et al., 1995). As expected, based on the phenotype of PGR loss, PGR is scarcely expressed or undetectable in granulosa cells prior to the LH surge, but is induced dramatically by the ovulatory signal, after which it rapidly declines in expression (Hild-Petito et al., 1988; Park and Mayo, 1991). This decline is somewhat more gradual in humans as compared to other species (Choi et al., 2017b). The isoform that is of primary importance in the ovary is PGR-A; as mice lacking this isoform have a phenotype very similar to those with germline deletion (Mulac-Jericevic et al., 2000). Mice lacking the PGR-B isoform, but not the PGR-A isoform respond normally to superovulation protocols (Mulac-Jericevic et al., 2003). Androgens are key regulators in the male reproductive system, yet mounting evidence suggests a direct role for these steroid hormones in the ovary as well (Walters et al., 2008). Although some early in­ vestigations of androgen action in the ovary were confounded by aromatization of androgens into estrogens in granulosa cells (Walters et al., 2008), subsequent studies using murine models of tissue-specific AR loss demonstrated the importance of this receptor in the ovary (Table 1). The steroid hormone receptors that have been least studied in the context of reproduction are the corticoid receptors. The glucocorticoid receptor (GR) binds cortisol, corticosterone, and other glucocorticoid ligands, while the mineralocorticoid receptor (MR) is a receptor with affinity for both glucocorticoids and mineralocorticoids. These receptors are present in granulosa cells (Schreiber et al., 1982; Gomez-Sanchez et al., 2009). The regulatory role of corticosteroids in ovulation has not yet been clarified (Duffy et al., 2019).

Table 1 (continued ) Receptor

Classificationa

Phenotype

Expression in ovary

NOR1 (NR4A3)

Orphan nuclear receptor

Germline deletion: Lethal prior to birth ( Deyoung et al., 2003; Ponnio et al., 2002)

Granulosa cellspecific (Park et al., 2003)

Orphan nuclear receptor

Germline deletion: Lethal shortly after birth due to adrenocortical deficiency (Luo et al., 1994) Ovarian depletion from primordial follicle forward (SF-1 f/f, Amhr2-cre): Infertile, disrupted cyclicity, very few antral follicles and ovulations, even in response to superstimulation ( Jeyasuria et al., 2004; Pelusi et al., 2008) Ovarian depletion from the antral follicle forward (SF-1 f/f, Cyp19a1-cre or Cyp19-cre and Cyp17-cre): Normally fertile (Meinsohn et al., 2019) Germline deletion: Lethal on embryonic day 6–7 (Labelle-Dumais et al., 2006; Par´e et al., 2004) Ovarian depletion from primordial follicle forward (LRH-1 f/f, Amhr2-cre): Dominant follicles form, no ovulation, no cumulus expansion, infertile ( Bertolin et al., 2017; Duggavathi et al., 2008) Ovarian depletion from the antral follicle forward (SF-1 f/f, Cyp19a1-cre): Impaired cumulus expansion, no ovulation, infertile ( Bertolin et al., 2014, 2017)

Granulosa cells, theca cells, stroma ( Falender et al., 2003; Mendelson et al., 2005)

Germline deletion: embryonic mortality before embryonic day 10 (Chung et al., 2001) Oocyte specific depletion: prolonged estrous cycles and deficient follicular steroidogenesis (Lan et al., 2003)

Oocyte specific ( Chen et al., 1994a)

NR5 family SF-1 (NR5A1)

LRH-1 (NR5A2)

NR6 family GCNF (NR6A1)

a

Orphan nuclear receptor

Orphan nuclear receptor

Granulosa cells ( Falender et al., 2003; Mendelson et al., 2005)

2.2. Vitamin A and D receptors

Based on Meinsohn et al. (2019).

Vitamin D and the biologically active form of vitamin A, retinoic acid, both act as endogenous ligands of nuclear receptors. As expected, these nuclear receptors are important regulators of metabolic processes. In addition, there is some evidence that vitamins A and D and the nu­ clear receptors to which their active forms bind, the RARs and VDR, respectively, may be regulators of follicular growth (Table 1). Retinoic acid can bind to any one of three retinoic acid receptors, RAR-alpha, RAR-beta, and RAR-gamma (Gutierrez-Mazariegos et al., 2014), whereas there is but a single vitamin D receptor.

2.1. Steroid hormone receptors The six steroid hormone receptors: estrogen receptors 1 and 2, androgen receptor, progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor all have documented roles in regulation of reproduction (Table 1). The sex steroid hormone receptors in particular have long been established as key regulators of many reproductive processes. The estrogens elicit genomic effects through two different estrogen receptors, ERα or ESR1 and ERβ or ESR2. The discovery of ESR1 (Koike 5

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

2.3. Orphan nuclear receptors

(Table 1). The PPAR and LXR families are similar in that they both for heter­ odimers with the retinoid X receptors (RXRs) to induce transcription. The RXRs are also expressed in granulosa cells (Tatone et al., 2016), suggesting that PPARs and LXRs can mediate downstream actions in the ovary. Although these RXRs may play an important role in reproductive function through their interaction with PPARs, LXRs, or RARs, it seems that they are not required for normal reproductive function. Loss of RXRA results in embryonic death (Kastner et al., 1994), while RXRB and RXRG knockout animals are fertile (Kastner et al., 1996; Krezel et al., 1996). The RXRs appear not to be redundant, as even the combined loss of RXRB and RXRG does not result in any phenotype of reproductive impairment (Krezel et al., 1996), suggesting that RXRA alone is adequate to support the function of those nuclear receptors with which it forms a heterodimer in the reproductive system, and to support any function regulated by RXRs alone. Among other receptors that may be regulators of ovarian function, based upon mouse model studies, are GCNF and REVERBA. GCNF is oocyte-specific (Chen et al., 1994a). Mice lacking the circadian nuclear receptor REVERBA are subfertile, with a reduction in both fertile mat­ ings and litter size (Preitner et al., 2002). This difference in fertility seems to be due, at least in part to regulation of steroidogenesis by REVERBA (Chen et al., 2012). However, there i little further research is available on this nuclear receptor and none to implicate it directly in ovulation.

The receptors of the NR0 family, DAX and SHP, are unique in that they both lack DNA binding domains (Gigu`ere, 1999) and DAX addi­ tionally lacks a ligand binding pocket (Sablin et al., 2008). Therefore, both of these orphan receptors elicit their downstream effects by inter­ acting with other receptors and cofactors. DAX and SHP are negative regulators of LRH-1 and SF-1 activity, roles that have been described in detail in previous reviews (Bertolin et al., 2010; Meinsohn et al., 2019). The receptors COUP-TFI and COUP-TFII are key regulators of embryogenesis, as demonstrated by the finding that loss of either of these receptors results in pre- or perinatal death (Qui et al., 1997; Per­ eira et al., 1999). The COUP-TFs can interact with many other nuclear receptors, including PPARs, RARs, and RXRs (Pereira et al., 2000). COUPTFII was recently demonstrated to have a key role in female sexual differentiation (Zhao et al., 2017). Both COUP-TFs are expressed in the ovary, with COUP-TFI seeming to play a more important role in the granulosa cells and COUP-TFII being more important in the theca (Xing et al., 2002; Sato et al., 2003; Takamoto et al., 2005). ESRRA and ESRRB are orphan nuclear receptors that have significant homology with ESRs, bind to response elements similar to those bound by ESRs, and have been demonstrated to interact with classical ESRs in some contexts (Tanida et al., 2015). Although the most well-established roles for this receptor are in embryogenesis and energy metabolism (Festuccia et al., 2018), there is evidence for a role for this orphan nu­ clear receptor in the regulation of adult ovarian function (Table 1). The NR4A family of nuclear receptors comprises the receptors NGFIB, NURR1, and NOR1. NGFIB, the most abundant among the NR4A family members in the ovary, is expressed by both the granulosa and theca cells (Havelock et al., 2005; Li et al., 2006; Park et al., 2003). NURR1 and NOR1 are less abundant and are specific to the granulosa cells (Park et al., 2003). LRH-1 and SF-1 are orphan nuclear receptors of the NR5A family that bind to the same genomic motif, but regulate diverse reproductive and endocrine functions (Meinsohn et al., 2019). While LRH-1 is expressed specifically and abundantly in the granulosa cells of all follicles, SF-1 expression is more widespread in the ovary. The latter nuclear recep­ tor is present in the granulosa, theca, and stroma compartments of the ovary (Mendelson et al., 2005). Both LRH-1 and SF-1 have well-documented roles in regulation of reproductive processes and mice lacking LRH-1 are anovulatory, while mice lacking SF-1 have dramati­ cally reduced follicular populations (Table 1). Among orphan nuclear receptors, the receptors of the NR5A family, may be the most extensively studied for their role in reproductive function. Despite its name, testicular receptor 4 is also expressed in oocytes and granulosa cells (Collins et al., 2008) and regulates female repro­ ductive function. This receptor was initially classified as an orphan nuclear receptor, but several polyunsaturated fatty acids can act as li­ gands for TR4 (Lin et al., 2017). Although studies of this receptor in the context of the female reproductive tract have been very limited, it ap­ pears to be a potential regulator of folliculogenesis (Table 1). The liver X receptors (LXRs) were initially identified as orphan nu­ clear receptors. Subsequently, oxysterols were identified as their endogenous ligands, making these “adopted” orphan receptors (Hiebl et al., 2018). LXRA and LXRB are not isoforms of the same gene; indeed, they are encoded on separate chromosomes. LXRA is expressed specif­ ically in a few metabolically important tissues, while LXRB is widely expressed across cell types (Repa and Mangelsdorf, 2000). Both LXRs are expressed in the ovary, where they are detecable in both the granulosa cells and oocytes, but more abundant in the latter (Betowski and Semczuk, 2010; Drouineaud et al., 2007). Although the PPARs are also categorized as “adopted” orphan re­ ceptors, as a wide variety of ligands that have been identified, including fatty acids and phospholipids, and eicosanoids (Velez et al., 2013). These receptors, PPARA, PPARD, and PPARG, are important metabolic sensors, but the latter also seems to have key functions in the ovary

3. Nuclear receptors regulate antral follicle growth, estradiol synthesis, and responsiveness to gonadotropins The final period of follicular growth that occurs under the influence of FSH programs the follicle to ovulate. During this period, granulosa and theca cells act together to produce estradiol at high concentrations, and the granulosa cells proliferate. The primary causes of infertility in several nuclear receptor knockout models seem to be an inability of follicles to reach the antral stage or to progress to the preovulatory state once they have acquired an antrum. Therefore, follicular growth is pertinent to the ovulatory process. 3.1. Estrogen receptors Although phenotypes of ESR knockout mice have been variable, it has become clear through many years of investigation, that estradiol signaling under the influence of FSH is essential to follicular growth and differentiation (Couse et al., 2005; Deroo et al., 2009; Rodriguez et al., 2010). Investigations of the role of ESR1 and 2 in the ovary began in the 1990s, with studies of both ovarian development and function. Deletion of both estradiol receptors results in formation of seminiferous tubule-like structures in adult ovaries (Couse et al., 1999b; Dupont et al., 2000), demonstrating the importance of estradiol signaling in postnatal ovarian development. In contrast, loss of either estradiol receptor alone did not disrupt ovarian development (Dupont et al., 2000; Krege et al., 1998; Lubahn et al., 1993), suggesting their redundant functions. Germline deletion of ESR1 alone resulted in mice that were completely infertile, with small, hyperemic ovaries and hemorrhagic follicles (Lubahn et al., 1993). Nevertheless, these ESR1 − /− mice can ovulate in response to gonadotropin treatment, albeit at a reduced rate (Rosenfeld et al., 2000), and also ovulate normally in an ex vivo follicle culture system (Emmen et al., 2005). Moreover, mice lacking ESR1 have elevated serum LH, and the phenotype of multiple hemorrhagic follicles can be rescued by treatment with a GnRH antagonist (Couse et al., 1999a, 2003, 2004). This indicates that the primary defect driving infertility in the ESR1 knockout model is inappropriate gonadotropin signaling, rather than an ovarian defect. The reproductive phenotypes reported for mice with ESR2 germline deletion have been variable, ranging from subfertility to infertility (Antal et al., 2008; Antonson et al., 2020; Dupont et al., 2000; Krege 6

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

et al., 1998; Maneix et al., 2015; Rumi et al., 2017). The first model generated displayed impaired fertility, with a decreased rate of ovula­ tion that was evident even in response to superstimulation (Krege et al., 1998). Estradiol is a key regulator of the highly proliferative state of granulosa cells in preovulatory follicles (Robker and Richards, 1998). Therefore, it is not surprising that one effect of loss of ESR2 is impaired follicular growth (Couse et al., 2000; Dupont et al., 2000). This effect was exacerbated by the additional deletion of one allele of the ESR1 gene (Dupont et al., 2000). In addition to impaired growth, murine follicles lacking ESR2 pro­ duced less estradiol and had reduced rates of ovulation in an ex vivo follicle culture system, while follicles lacking ESR1 did not differ from wildtype follicles (Emmen et al., 2005). Also, ESR2 agonists delivered in vivo to wildtype animals dramatically increased follicle growth, while ESR1 agonists had little effect (Hegele-Hartung et al., 2004). This sug­ gests that, relative to ESR1, ESR2 is of primary importance in follicular growth. ESR1 and 2 are also regulators of follicular growth in cattle, and both increase in abundance after selection of the dominant follicle from the pool of growing follicles (Rovani et al., 2014). Conversely, there is evidence that estradiol is of lesser importance to primate follicular growth (Chaffin and Vandevoort, 2013), suggesting species-specific differences in these processes. Loss of ESR2 also impairs the granulosa cell differentiation that is a normal consequence of FSH stimulation. This has deleterious down­ stream effects, as estrogen action on these cells is a requirement for appropriate responsiveness to the LH surge (Couse et al., 2005). The phenotype of ESR2 deletion was marked by reduced steroidogenic ca­ pacity of granulosa cells and reduced aromatase and LH receptor expression. In the preovulatory follicle, the LHCGR is more abundantly expressed on the outer layer of granulosa cells, those adjacent to the basement membrane, as compared to inner layers in the follicle (Baena et al., 2020; Bortolussi et al., 1977; Peng et al., 1991; Richards et al., 1976; Zeleznik et al., 1974). FSH stimulation is necessary to the acqui­ sition of this pattern of receptor expression (Richards et al., 1976), suggesting a potential role for ESR2 in the regulation of this spatially distinct LHCGR expression in the follicle. Interestingly, the deficiency in gonadotropin-responsiveness in mice lacking ESR2 is not only a result of a lack of receptor expression. Treatment of follicles that lack ESR2 with the protein kinase-A activator forskolin, rather than LH, bypassed the LHCGR deficiency, but only partially rescued this phenotype (Deroo et al., 2009; Rodriguez et al., 2010). This implicates inadequate downstream stimulation of cAMP as another driver of the impaired ability of ESR2 − /− follicles to respond to LH. Recent studies in both mice and rats used an innovative approach in generation of two ESR2 knockout lines to investigate the possibility of estrogen responsive element-independent actions of ESR2. In one line, the ability of ESR2 to bind to its cognate DNA response element was compromised, while the other had a complete ESR2 null mutation (Maneix et al., 2015; Rumi et al., 2017). Both of these lines were completely infertile and neither could ovulate, even when super­ stimulated with the LH analog, human chorionic gonadotropin (hCG). No CL formed, despite the presence of numerous antral follicles. As in earlier mouse models (Couse et al., 2005), there were disruptions in many well-established LH-response genes, including PGR, PTGS2, and ADAMTS1 (Rumi et al., 2017). Subsequent whole transcriptome profiling experiments revealed that these ESR2 knockout rats also dis­ played disruption of gene networks consistent with the phenotype of impaired gonadotropin response and follicular maturation (Khristi et al., 2018).

cell loss of AR results in reduction in ovulation rate, litter size, antral follicle number, and follicular growth, accompanied by an increased population of unhealthy and atretic follicles (Cheng et al., 2013; Sen and Hammes, 2010; Walters et al., 2007, 2009). Androgens regulate follic­ ular growth through upregulation of FSHR (Fujibe et al., 2019; Laird et al., 2017; Xue et al., 2012), thereby regulating pathways that are activated in response to FSH, including an androgen-mediated increase in steroidogenesis, enhancement of ability of follicles to respond to FSH and IGF1, increases in the cyclin CCND2, and a concurrent suppression of the BMP and TGFB signaling pathways (Hasegawa et al., 2017; Laird et al., 2017; Xue et al., 2012). Both inadequate and excess androgen signaling can antagonize follicular growth, indicating that there is an optimal ovarian androgen concentration (Lebbe et al., 2017; Lim et al., 2017). AR also modulates follicular development by decreasing the ability of the orphan nuclear receptor NGFIB to drive excess follicular growth (Xue et al., 2012). Some years ago, it was shown that the TGFB family growth factor, GDF9, is an oocyte signaling element required for follicular development (Wu and Matzuk, 2002). One way in which ex­ erts this effect is by induction of an increase in androgen production by the follicle which then acts on the androgen receptor in the follicle to promote follicular growth (Orisaka et al., 2009). In addition to its direct role and its place downstream of other factors regulating follicular growth, AR is important for the transition from a preantral to antral follicle, thereby participating in the regulation of the formation of the follicular antrum. The evidence for this postulate comes from the observation that, in mice lacking AR in granulosa cells, there is an increase in preantral and atretic follicle numbers, with a concurrent reduction in the number of antral follicles (Sen and Hammes, 2010). Further, testosterone and DHT treatments result in stimulation of antrum formation in cultured ovarian follicles (Laird et al., 2017) and increase growth of antral follicles in vitro (Murray et al., 1998). A role for androgens is further supported by the finding that AR is primarily expressed in granulosa cells of healthy preantral and antral follicles, and declines as follicles reach the preovulatory state (Hillier et al., 1997; Jeppesen et al., 2012). Indeed, smaller (1–3 mm) bovine follicles responded to androgen treatment to a much greater extent than slightly larger (3–5 mm) antral follicles (Hickey et al., 2004). In summary, AR signaling appears to augment follicular growth and antrum formation, but is not obligatory for ovulation. Female mice lacking AR are fertile and respond to superstimulation protocols in a manner equivalent to their wild-type counterparts, indicating that ovarian function is retained in the absence of androgen signaling (Sen and Hammes, 2010). This suggests that, in the murine ovary, redundant measures are present to compensate for loss of androgen signaling. 3.3. Orphan nuclear receptors In mice lacking SF-1 in granulosa cells of all follicles, including pri­ mordial follicles, folliculogenesis is severely impaired, with dramati­ cally fewer follicles at each stage of development (Pelusi et al., 2008). In this model, granulosa cell proliferation was also blunted. It is not clear if the consequent infertility is due to failure of follicles to reach dominance and to ovulate, but in support of this postulate is the observation that very few antral follicles were present (Pelusi et al., 2008). The compromised capacity for proliferation by granulosa cells may be related to the essential role of SF-1 in steroidogenesis, including syn­ thesis of estrogens. As noted above, these hormones are key drivers of granulosa cell proliferation (Ruiz-Cort´es et al., 2005). SF-1 also induces FSHR, an action that can be repressed by the orphan nuclear receptors COUP-TFI and COUP-TFII, providing another mechanism for its regu­ lation of follicular growth (Xing et al., 2002). Preliminary findings from our laboratory indicate that mice with SF-1 depletion from the antral follicle onward are ovulatory and normally fertile (Meinsohn et al., 2019), suggesting that SF-1 is necessary for preantral follicular growth, but not for antral follicular growth and ovulation. In contrast to the phenotype observed in the absence of SF-1, mice

3.2. Androgen receptors AR is not essential for ovulation itself, given findings from the mul­ tiple models of AR depletion in which mice ovulate (Table 1). None­ theless, evidence from these models indicates that ovarian or granulosa 7

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

lacking LRH-1 have the capacity for follicular growth. Mice with LRH-1 depleted from all follicles form antral follicles apparently normally (Duggavathi et al., 2008). These animals are uniformly anovulatory for multiple reasons (Duggavathi et al., 2008; Meinsohn et al., 2019). Un­ like SF-1, LRH-1 acts directly as a mitogen in granulosa cells, and its induction of proliferation is independent of its essential role in ste­ roidogenesis (Meinsohn et al., 2018). It induces the expression of key proliferation-related genes in the ovary (Meinsohn et al., 2018). Its depletion affects follicular growth by reducing granulosa cell prolifer­ ation in the late stages of antral development. (Meinsohn et al., 2018). The discrepancies in function of LRH-1 and SF-1 indicate that these two nuclear receptors play distinct and non-redundant roles in the regulation of ovarian function. Given that they interact with the same DNA sequence, the diversity of their biological actions has been attributed to the cofactors that they recruit (Meinsohn et al., 2019). Another orphan nuclear receptor that may regulate folliculogenesis is the receptor TR4. Although there are few studies of this receptor in the context of reproduction, murine models have demonstrated that female mice lacking TR4 are subfertile and exhibit impaired maternal behavior (Collins et al., 2004). Further investigation of this model revealed that litters born were both smaller and less frequent than in wildtype coun­ terparts (Chen et al., 2008). Moreover, superovulation protocols generated fewer preovulatory follicles and few ovulations in these mice (Chen et al., 2008). This deficient folliculogenesis was attributed to increased granulosa cell apoptosis and decreased LH receptor expres­ sion, leading to reduced expression of steroidogenic genes (Chen et al., 2008; Zhang and Dufau, 2001).

corrected in part by supplementing estradiol, indicating that one component of the reproductive defect in these mice is impaired ste­ roidogenesis (Kinuta et al., 2000). A subsequent study of calcium sup­ plementation in mice with germline deletion of VDR demonstrated that a primary driver of reproductive failure was calcium deficiency. Indeed, calcium supplementation restored fertility of knockout mice to the equivalent of their wildtype counterparts (Johnson and Deluca, 2001). Nonetheless, transcriptional regulation of VDR target genes in the ovary cannot be excluded; there is evidence for VDR-mediated regulation of PTGS2 and PGE2 synthesis, key events in the ovulatory process in human luteinized granulosa cells (Wang et al., 2020).

3.4. Vitamin A and D receptors

4. Nuclear receptors are essential for expansion the cumulus oo¨phorus

The expression of retinoic acid receptors in the ovary is, at least in part, gonadotropin regulated, as FSH induces an increase in RARG protein in granulosa cells (Kawai et al., 2016) and increases granulosa cell uptake of retinol, the form of vitamin A that is predominant in the serum (Liu et al., 2018). Retinoic acid is also involved in upregulation of granulosa cell proliferation via a mechanism involving suppression of retinoic acid metabolism by the ovarian protein activin (Demczuk et al., 2016; Kipp et al., 2011). Furthermore, retinoic acid signaling pathways regulate gonadotropin responsiveness. In vitro treatment with low concentrations of retinoic acid acts synergistically with FSH to increase LHCGR, cAMP, and progesterone production, and induced demethyla­ tion of the LHCGR promotor (Bagavandoss and Midgley, 1988; Kawai et al., 2016, 2018). In contrast, saturating doses of retinoic acid reduce the expression of both LHCGR (Minegishi et al., 2000a) and FSHR (Minegishi et al., 1996, 2000b), resulting in an upstream perturbance of gonadotropin-dependent pathways. Despite evidence for regulation of follicular growth by retinoic acid, mice with germline deletion of the RARA1 isoform of RARA (Lufkin et al., 1993), RARB (Luo et al., 1995), or RARG (Lohnes et al., 1993) are normally fertile. Fertility of RARA knockout mice, which lack all iso­ forms of RARA, could not be tested, because these mice only survive for two months after birth (Lohnes et al., 1993). Moreover, depletion of all three RARs from all ovarian cells from approximately day 11 of fetal development (with SF-1 cre) was without effect on litter size, and adult ovaries appeared normal in terms of number and type of follicles (Minkina et al., 2017). Ovarian function, including LH-responsiveness and granulosa cell proliferation, was not studied in detail in the char­ acterization of this triple knockout model (Minkina et al., 2017). More research is needed to determine whether triple loss of RARs alters spe­ cific ovarian mechanisms. Early studies of mice lacking the vitamin D receptor (VDR) suggested that this receptor may also be an important regulator of follicular growth; VDR knockout mice had few antral follicles (Yoshizawa et al., 1997) and both transcript abundance and activity of aromatase were reduced, resulting in decreased estradiol synthesis relative to wild type counterparts (Kinuta et al., 2000). This defect in follicular growth was

As a follicle forms an antrum and subsequently prepares to ovulate, the granulosa cells differentiate into at least two separate lineages. The oocyte drives lineage specification of multiple layers of granulosa cells that surround it, and they differentiate into the cumulus oophorus, under the influence of BMP15 and GDF9 (Eppig et al., 1997; Peng et al., 2013; Su et al., 2007; Sugiura et al., 2007). the expansion of the cumulus follows the LH surge and is essential for oocyte maturation and natural fertilization. Cumulus expansion remodeling of the complex extracel­ lular matrix surrounding the oocyte, which is composed primarily of the glycosaminoglycan hyaluronan (Chen et al., 1993; Eppig, 1979). This ¨p et al., part of the matrix is synthesized by cumulus cell HAS2 (Fülo 1997; Sugiura et al., 2009), and is covalently bound to the serine pro­ tease inhibitor, IαI (Hess et al., 1999). The stabilizers of the hyaluronan matrix include TNFAIP6 (Fulop et al., 2003), VCAN (Russell et al., 2003), and PTX3 (Salustri et al., 2004; Varani et al., 2002), with the former specifically stabilizing hyaluronan-IαI linkages (Fulop et al., 2003). Differentiated cumulus granulosa cells do not express the LH receptor (Eppig et al., 1997). Therefore, the principal stimulatory signals for cumulus expansion after the LH surge are derived from the mural granulosa cells. These include three EGF-like ligands, AREG, EREG, and BTC (Ashkenazi et al., 2005; Park et al., 2004) and prostaglandin E, produced by the rate-limiting prostaglandin synthetic enzyme PTGS2 (Davis et al., 1999; Hizaki et al., 1999; Lim et al., 1997). There are several nuclear receptors that play roles in cumulus expansion, including LRH-1, AR, and ESR2.

3.5. Summary of the role of nuclear receptors in follicle development In summary, follicular growth, antrum formation, and differentiation under the influence of FSH are required for the formation of a peri­ ovulatory follicle (Fig. 1). The early parts of this process are regulated by the androgen receptor while the final stages are regulated in part by estradiol, primarily through ESR2. Estradiol is a regulator of granulosa cell proliferation and granulosa cell differentiation under the influence of FSH. LRH-1 is not absolutely required for antral follicle formation, but regulates granulosa cell proliferation, whereas loss of SF-1 may alter proliferation, but certainly disrupts development of follicles, resulting in numerically reduced follicle populations at each stage of follicular growth. Thus, an array of nuclear receptors, acting in concert with other transcription factors, regulate changes that complete follicle develop­ ment and ready the follicle for ovulation.

4.1. LRH-1 Mice with LRH-1 depleted from all granulosa cells and mice with LRH-1 depleted from only mural granulosa cells of antral follicles fail to undergo cumulus expansion (Bertolin et al., 2017; Duggavathi et al., 2008). In this model of depletion from the antral follicle only, LRH-1 is depleted by CYP19A1-cre in the mural granulosa, but not the cumulus cell population (Bertolin et al., 2017). The consequent compromise in cumulus expansion indicates that LRH-1 expression in the mural gran­ ulosa is required for cumulus expansion, suggesting LRH-1 regulation of 8

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Fig. 1. Nuclear receptors regulate the final stages of folliculogenesis. A summary of key functions regulated by nuclear receptors during the final period of follicular growth. Steroid hormone receptors are colored green and receptors of the NR5A family are colored blue, while others are in black. Functions are arranged temporally. Arrows indicate a positive relationship of a receptor with a function. SF-1: ste­ roidogenic factor 1; AR: androgen receptor; ESR: es­ trogen receptor; LRH-1: liver receptor homolog 1; RAR: retinoic acid receptor; FSH: follicle stimulating hormone; LH: luteinizing hormone. (For interpreta­ tion of the references to color in this figure legend, the reader is referred to the Web version of this article.)

EGF-like ligands. Indeed, the expression of the drivers of cumulus expansion AREG, EREG, and BTC was reduced after the LH surge in both models relative to control (Bertolin et al., 2017). In addition, the essential cumulus components TNFAIP6, and PTX3 were reduced in the absence of LRH-1, as were the hyaluronan receptor, CD44, and C1QBP, a hyaluronan interacting protein (Bertolin et al., 2017; Duggavathi et al., 2008). Subsequent chromatin immunoprecipitation experiments confirmed direct regulation of Tnfaip6 by LRH-1 (Bianco et al., 2019). The cumuli derived from the mice with LRH-1 depleted from the mural granulosa cells only could be induced to undergo marginal expansion with superstimulation protocols, while no such expansion occurred in the model in which LRH-1 was depleted from both mural and cumulus granulosa cells (Bertolin et al., 2017; Duggavathi et al., 2008). Impor­ tantly, oocytes from both models had no impairment in ability to become fertilized and progress normally to 2-cell and blastocyst stages, indicating that insufficient cumulus expansion did not affect oocyte competence (Bertolin et al., 2017). Cumulus cell cytoskeletal rearrangement is also required for sepa­ ration of the cumulus-oocyte complex from its connection to the mural granulosa during cumulus expansion (Kitasaka et al., 2018). Cumulus granulosa cells undergo an increase in migratory and adhesive capacity following the ovulatory signal that is unique to this cell population (Akison et al., 2012). LRH-1 regulates extensive cytoskeletal changes after the LH surge and a lack of LRH-1 results in impaired migratory capacity of granulosa cells (Bianco et al., 2019). LRH-1 may regulate cumulus expansion not only through regulation of EGF-related mecha­ nisms (Bertolin et al., 2017), but also by regulation of the cellular migration and extracellular matrix remodeling of the granulosa popu­ lation (Bianco et al., 2019).

had less dense cumuli than in controls (Hu et al., 2004). These cumuli were also more labile to enzymatic digestion (Hu et al., 2004) and had impaired expansion (Walters et al., 2012). These defects were attributed to drastically reduced HAS2 and TNFAIP6 following the LH surge (Hu et al., 2004) and thus, likely flawed formation of the cumulus extra­ cellular matrix. Inhibition of AR signaling at the time of ovulation with the AR antagonist flutamide also curtailed AREG and PTGS2 expression, whereas treatment with nonaromatizable androgens induced expression of both these genes (Yazawa et al., 2013). Action of AR on the cumulus may also alter the developmental ca­ pacity of oocytes. Granulosa cell-specific AR knockout was induced with AMH-cre, which depletes AR from granulosa cells of all follicles, including preantral and antral follicles. In this model, oocyte viability is decreased, and fewer oocytes show the capacity for fertilization and development to two-cell embryos (Walters et al., 2012). This may be due to effects of androgen signaling on oocyte maturation, as immature bovine cumulus-oocyte complexes that were matured with either aro­ matizable (testosterone) or nonaromatizable (DHT) androgens had increased oocyte cleavage rates following in vitro fertilization (Silva and Knight, 2000). DHT also increased the proportion of oocytes that reached the 8-cell stage, an effect blocked by treatment with the AR antagonist flutamide (Silva and Knight, 2000). Further confirmation came from studies showing that testosterone promotes the formation of CX37-mediated gap junctions in cumulus-oocyte complexes (Zhang et al., 2016), providing a possible mechanism of AR-mediated regulation of oocyte survival. When mice with granulosa-specific knockout of AR were compared to mice with oocyte-specific AR knockout, those with the oocyte-specific deletion exhibited normal fertility, while those with granulosa cell-specific deletion had fertility defects (Sen and Hammes, 2010). This demonstrates that the effects of AR in the ovary are likely on granulosa cells, and not the oocyte, although gap junction formation and signaling were not investigated in this study and therefore, an indirect effect on the oocyte cannot be excluded.

4.2. AR As follicles mature, AR becomes more abundant in the cumulus relative to the mural granulosa cells (Yazawa et al., 2013). This receptor remains plentiful in the compacted cumulus, but declines four-fold during cumulus expansion (Jeppesen et al., 2012). AR is essential to cumulus function, demonstrated by the findings that AR knockout mice

4.3. ESR2 Germline depletion of ESR2 also results in a phenotype of impaired 9

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

cumulus expansion, accompanied by decreased expression of the ratelimiting prostaglandin synthetic gene, PTGS2 (Couse et al., 2005). This may be due to the inadequate LH-responsiveness in this model, as discussed previously. Cumulus expansion in immature cumulus-oocyte complexes isolated from preantral follicles could be provoked by treat­ ment with 17β-estradiol (Sugiura et al., 2010). Similarly, complexes isolated from antral follicles and cultured in the absence of estradiol lose their ability to expand completely (Sugiura et al., 2010). Together, these findings suggest that estradiol signaling is important for maintaining the expansion capacity of the cumulus. Finally, cumulus ESR signaling also maintains the oocyte in meiotic arrest prior to the LH surge. The downregulation of ESR2 by the LH surge then allows a decrease in two critical genes, the natriuretic peptide precursor and the natriuretic peptide receptor-1 (Liu et al., 2017). This decline in expression is one among many important events that result in the resumption of meiosis of the oocyte.

expansion seems to be the regulation of the differences in granulosa cell LHCGR expression observed between the mural and cumulus cells. The promotor of this gene remains hypermethylated in cumulus cells throughout the ovulatory process and RA synthesis by granulosa cells was inhibited by coculture with denuded oocytes (Kawai et al., 2018). Treatment of granulosa cells with retinoic acid induces demethylation of the LHCGR promotor in the mural granulosa population (Kawai et al., 2018). This suggests that the oocyte may regulate retinoic acid synthesis in the cumulus to maintain the hypermethylated LHCGR, but not in the mural granulosa cells. 4.6. Summary of the role of nuclear receptors in expansion of the cumulus oo¨phorus The key nuclear receptors that regulate expansion of the cumulus include LRH-1, AR, and ESR2. Both estradiol and retinoic acid signaling seem to exert their effect on cumulus expansion primarily through regulation of the abundance of LHGCR. AR is expressed more abun­ dantly in the cumulus than in the mural granulosa cells during the periovulatory period and is a regulator of HAS2, which promotes syn­ thesis of the principle component of the cumulus matrix, hyaluronan. LRH-1 regulates mural granulosa production of EGF-like ligands that induce cumulus expansion and key stabilizers of the cumulus itself, including PTX3, TNFIAP6, and C1QBP. These regulatory mechanisms are summarized in Fig. 2.

4.4. PGR Mice lacking PGR have phenotypically normal cumulus expansion (Lydon et al., 1995, 1996; Park et al., 2020; Robker et al., 2000). This notwithstanding, there is evidence for PGR-mediated regulation of some key cumulus expansion genes, including the mural granulosa-derived EGF-like factors AREG, EREG, and BTC (Shimada et al., 2006). As discussed previously, a key regulator of cumulus expansion is the increase in prostaglandin synthesis that follows the ovulatory increase in PTGS2 (Davis et al., 1999; Hizaki et al., 1999; Lim et al., 1997). PTGS2 is regulated by PGR in a species-specific manner. In human (Choi et al., 2017b; Tsai et al., 2008) and bovine (Bridges et al., 2006) granulosa cells, inhibition of PGR with the antagonist RU486 (also known as mifepristone) reduced both PGE2 synthesis and PTGS2 abundance in response to hCG. Moreover, in human granulosa cells, chromatin immunoprecipitation demonstrated direct binding of PGR to the PTGS2 promotor (Choi et al., 2017b). PGR also regulates the transcription factor FOS and other factors essential to prostaglandin synthesis and transport in human granulosa cells (Choi et al., 2017b, 2018). In contrast, PGR does not appear to bind to the promotor of the mouse PTGS2 gene in the mouse (Dinh et al., 2019; Park et al., 2020), consis­ tent with the finding of Robker et al. (2000), who reported no difference in PTGS2 abundance between PGR knockout and control granulosa cells 4 h after hCG treatment. As the process of ovulation proceeds in mice, both PGE2 and PTGS2 are in greater abundance in the absence of PGR (Park et al., 2020). This mechanism will be discussed in more detail in the section below on the inflammatory response during ovulation. Overall, these data suggest that there are differences in pathways for ovulatory induction and perhaps termination of prostaglandin synthesis between rodents and humans.

5. Nuclear receptors regulate the shift from estradiol to progesterone synthesis during the ovulatory process The ovulatory follicle is highly steroidogenic, producing primarily estradiol prior to the LH surge. Granulosa cells lack CYP17A1, the ste­ roidogenic enzyme that catalyzes the conversion of progestins to an­ drogens. Therefore, theca-derived androgens provide the precursor for granulosa cell estradiol production prior to the LH surge, with granulosa cell progestins contributing to thecal androgen biosynthesis (Fortune and Armstrong, 1977, 1978; Liu and Hsueh, 1986; Makris and Ryan, 1975; Ryan et al., 1968; Short, 1962). The ovulatory signal is accom­ panied by shifts in the steroid hormone profile in both the follicular fluid and plasma, with estradiol transiently increasing and then declining, while progesterone increases stably (Chaffin et al., 1999; Weick et al., 1973). This shift to progesterone synthesis, together with the rapid in­ duction of PGR, is key regulator of the progesterone-induced changes that occur in the periovulatory follicle. Therefore, it is essential that steroidogenic pathways deviate from the primarily estrogenic ste­ roidogenesis that occurs prior to ovulation to the production of increasing concentrations of progesterone as ovulation and luteinization progress.

4.5. RARs

5.1. NR4A family orphan nuclear receptors

The retinoic acid signaling pathway also plays a part in the regula­ tion of differentiation of the cumulus cells. These cells respond via one or more of several retinoic acid receptors present on cumulus cells: RARA, RARG, RXRA, and RXRB (Mohan et al., 2003). The consequence of vitamin A deficiency was an impaired ability of the granulosa cells to respond to LH and induce PTGS2 or AREG, both key cumulus expansion regulators (Kawai et al., 2016). Although a minor diminution (approximately 20%) in ovulation rate occurred in this study, the most dramatic effect was the reduction in the develop­ mental competence of oocytes (Kawai et al., 2016). This may be explained by the finding that retinoic acid promotes the formation of essential gap junctions in the cumulus-oocyte complex (Best et al., 2015; Read and Dyce, 2019). Treatment with retinoic acid rescued some of these defects including LHCGR abundance and ovulation rate (Kawai et al., 2016). Another role of retinoic acid signaling at the time of cumulus

Among the regulators of the changing steroidogenic profile of ovulatory granulosa cells are the orphan nuclear receptors in the NR4A family, NGFIB, NURR1, and NOR1. The roles of the NR4A family in ovulation are consistent with their characterization as immediate-early genes. They are very rapidly induced by LH, hCG, or forskolin (Carletti and Christenson, 2009; Havelock et al., 2005; Park et al., 2001; Wu et al., 2005), after which they return to basal abundance. This mecha­ nism is mediated through the LH signaling pathway, via the interme­ diate signaling molecule PKC-zeta (Park et al., 2007). The rapid induction of NGFIB and NURR1 is important for the ovarian shift from estradiol to progesterone synthesis in response to the ovulatory signal, via suppression of CYP19A1 (Wu et al., 2005), a mechanism which may involve inhibition by miRNA (Wu et al., 2015). NGFIB may also mediate an increase in steroidogenesis as luteinization progresses, given that NGFIB is a regulator of HSD3B2 (Havelock et al., 2005). Despite findings implicating NR4A receptors in the suppression of 10

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Fig. 2. Nuclear receptors regulate cumulus expansion. A summary of key functions regulated by nuclear receptors during cumulus expansion. Steroid hormone receptors are colored green and receptors of the NR5A family are colored blue, while others are in black. Functions are arranged temporally. Solid ar­ rows indicate a positive relationship of a receptor with a function. The dashed arrow indicates changes in LRH-1 function, but no change in the abundance of this receptor. Specifically, AR regulates AREG, TNFIAP6, HAS2, PTGS2 and LRH-1 regulates AREG, EREG, BTC, PTX3, CD44, C1QBP, and PTGS2. SF-1: steroidogenic factor 1; AR: androgen receptor; ESR: estrogen receptor; LRH-1: liver receptor homolog 1; RAR: retinoic acid receptor; LH: luteinizing hormone; AREG: Amphiregulin; BTC: Betacellulin; CD44: CD44 molecule (hyaluronan receptor); C1QBP: Comple­ ment C1q binding protein; EGF: Epidermal growth factor; EREG: Epiregulin; HAS2: Hyaluronan synthase 2; PTGS2: Prostaglandin-endoperoxide synthase 2; PTX3: Pentraxin 3; TNFAIP6: Tumor necrosis factor alpha induced protein 6. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

estradiol production during the periovulatory period, it is not clear, based upon existing murine knockout models, if nuclear receptors in this family are required for reproductive function. Ovary-specific models of NR4A transcription factor depletion appear not to exist. Germline deletion of NGFIB does not disrupt reproductive function (Cheng et al., 1997; Lee et al., 1995a) while global knockout of NURR1 or NOR1 re­ sults in death prior to or immediately after birth (Deyoung et al., 2003; ¨m et al., 1997). The three NR4A receptors Ponnio et al., 2002; Zetterstro have nearly identical DNA-binding domains, with more than 90% shared sequence identity (Li et al., 2006), and all three bind to and elicit transcription through NGFI-B-responsive elements (NBRE) and, as homodimers, through the Nur-responsive element, NurRE (Zhao and Bruemmer, 2010). Therefore, it is possible that, in the model of germline deletion of NGFIB, NURR1 and NOR1 may compensate for the loss of NGFIB in the granulosa cells, as these receptors seem to have somewhat redundant functions in other tissues (Cheng et al., 1997; Fernandez et al., 2000). Further study, including ovarian depletion models, is required to delineate the specific functions of these orphan receptors in the ovary.

estradiol production, and that this is not corrected by the LH surge (Duggavathi et al., 2008). Moreover, these mice completely fail to make the shift from estradiol to progesterone production that normally follows the ovulatory signal (Duggavathi et al., 2008). Likewise, PGR-Cre-induced conditional deletion of LRH-1 is manifest during the ovulatory process and does not prevent ovulation, but results in a vastly under functional CL (Zhang et al., 2013). The effects of LRH-1 on ste­ roidogenesis in granulosa cells are most likely due its well-known direct regulation of multiple steroidogenic factors (Bianco et al., 2019; Kim et al., 2004; Peng et al., 2003; Zhang et al., 2013). Indeed, the identi­ fication of open chromatin by FAIRE and the LRH-1 ChIPseq by Bianco et al. (2019) demonstrated substantial changes in chromatin accessi­ bility of the Star gene following the ovulatory signal, both around the transcriptional start site and in distal intergenic and intragenic domains (Bianco et al., 2019). This provides strong evidence to indicate that the LH surge enhances the ability of LRH-1 to drive steroidogenesis. This direct regulation of Star and other genes associated with the regulation of steroidogenesis is believed to be the most significant mechanism for the LRH-1-mediated increase in progesterone (Dugga­ vathi et al., 2008). In support of this view are the findings showing that transient transfection of granulosa cells with LRH-1, together with FSH treatment, upregulated abundance of CYP11A1, HSD3B, and proges­ terone, but not estradiol, in undifferentiated granulosa cells collected from preovulatory follicles (Saxena et al., 2004). LRH-1 may also mediate changes in steroidogenesis through indirect mechanisms. In the study of Bianco et al. (2019), the significant LRH-1 regulated functions were related to cellular migration and cytoskeletal rearrangement, as predicted by analysis using the tool Metascape and confirmed via in vitro investigation of cellular migration (Bianco et al., 2019). The cytoskeleton is a regulator of cholesterol storage and trafficking, and

5.2. LRH-1 and SF-1 LRH-1 is also a key regulator of steroidogenesis in the ovary, both prior to and following the ovulatory signal. While the germline deletion of LRH-1 is lethal during early embryogenesis (Labelle-Dumais et al., 2006; Par´e et al., 2004), mice lacking a single copy survive and breed (Labelle-Dumais et al., 2007). In these haploinsufficient mice, ovulation occurs but progesterone synthesis is compromised. Mice lacking LRH-1 in granulosa cells have higher plasma concentrations of estradiol before and after LH, indicating that these mice have inappropriate excess 11

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

thus can regulate steroidogenesis, including in periovulatory granulosa cells (Flynn et al., 2016; Sewer and Li, 2008; Shen et al., 2012). Therefore, LRH-1-regulated changes in cytoskeletal rearrangement (Bianco et al., 2019) provide a further mechanism by which this essential nuclear receptor influences steroid synthesis during the peri­ ovulatory period and luteinization. In investigation of the LRH-1 cistrome, HOMER (Hypergeometric Optimization of Motif EnRichment) analysis was used to identify motifs enriched in open chromatin in both prior to and after the LH surge (Bianco et al., 2019). The LRH-1/SF-1 motif was enriched in both groups, as expected. Other classical nuclear receptor motifs identified as enriched in this analysis were ESRRB motifs both before and after LH and ESRRA and GR before LH, suggesting potential coregulation of key LRH-1 targets by these receptors. Mice lacking ESRRB die early in gestation due to a defect in placental development (Luo et al., 1997). When this placental defect is rescued by aggregating embryos with tetraploid embryonic cells, ESRRB − /− mice are viable, but have reduced number of primordial germ cells. The few females that were allowed to reach adulthood in this study had oocytes present on their ovaries, but were infertile (Mitsunaga et al., 2004). Although more research is needed to delineate the role of the ESRR family in granulosa cells, overall, these data suggest that, these receptors may be involved in LRH-1-mediated regulation of gene expression, and may also have a role in the shift in steroidogenesis during the periovulatory period. The second member of the NR5A family, SF-1, also governs compo­ nents of the steroidogenic process. It has been shown that the ovulatory signal induces association of SF-1 with the Star promotor, suggesting an important role for this nuclear receptor during the ovulatory period (Hiroi et al., 2004). As luteinization progresses, SF-1 abundance, ste­ roidogenic gene expression, and progesterone production increase in granulosa cells, while DAX1, a negative coregulator of SF-1, decreases, indicating that a decrease in DAX1 may be a permissive event that al­ lows for the increase in steroidogenesis that follows ovulation (Shimizu et al., 2009). The decline in DAX1 during luteinization may be cell type-specific, as luteinization induced an increase in DAX1 and in COUP-TFII in theca cells (Murayama et al., 2008). Female mice lacking either DAX1 or SHP are viable and fertile (Wang et al., 2002; Yu et al., 1998), indicating that any role for these receptors as individual factors in reproduction is not obligatory. Thus, the role of DAX1 and SHP during the ovulatory period, and their relative importance to regulation of LRH-1 and SF-1 remains to be determined.

progesterone. The PPARG agonist was without effect when delivered following the ovulatory signal (Funahashi et al., 2017). This mechanism is seemingly important for luteal differentiation of granulosa cells, as in another study with PPARG agonist, this time with macaque granulosa cells in vitro, there was increased abundance of cholesterol efflux-related genes and decreased abundance of key steroidogenic en­ zymes (Puttabyatappa et al., 2010). Further evidence for regulation of cholesterol homeostasis by PPARG comes from a study of obese rats, in which treatment with a PPARG agonist prior to mating mitigated obesity-induced oocyte incompetence and altered expression of ovarian somatic cell genes, including CD36, SCARB1, and FABP4 (Minge et al., 2008). Another “adopted” orphan nuclear receptor, LXR, is known to alter steroidogenic and cholesterol efflux pathways in a similar way (Drouineaud et al., 2007; Puttabya­ tappa et al., 2010) and third, FXR, about which very little is known in the ovary, seems to be a potential regulator of estradiol synthesis, through CYP19A1 (Takae et al., 2019). A reasonable conclusion from these studies is that a decrease in PPARG and LXR mediated signaling favors steroidogenesis and inhibits cholesterol efflux, thereby promoting per­ iovulatory differentiation of the granulosa cell population. The other members of this family, PPARA and PPARD appear to be less important to ovulation and regulation of steroidogenesis, although they have certainly been less studied. In murine ovaries, PPARA and PPARD are expressed primarily in the stroma and theca compartments and do not change in response to the exogenous gonadotropins equine chorionic gonadotropin (eCG) or hCG (Komar et al., 2001). In contrast, in human granulosa cells, there was a moderate increase in PPARD and a concurrent moderate decrease in PPARA in response to the ovulatory signal. Both PPARA and PPARD germline knockout mice are normally fertile (Lee et al., 1995b; Peters et al., 2000). Given the implication of PPARA in the regulation of metabolism, its role, if any, may be related to the interaction of body condition with ovarian steroidogenesis. In this context, it was recently shown that the negative effect of the adipokine, adiponectin, on aromatase expression in human granulosa cells from obese women could be rescued by treatment with a PPARA antagonist (Tao et al., 2019). There appears to be little further evidence available on the role of this nuclear receptor in ovarian steroidogenesis in the context of ovulation.

5.3. The PPAR and LXR receptors

In summary, the ovulatory shift from estradiol to progesterone biosynthesis seems to be mediated primarily by orphan nuclear re­ ceptors in the NR4A and NR5A families. The immediate early genes NGFIB, NURR1, and NOR1 are rapidly induced by LH and may mediate an early repression of follicular estradiol. In contrast, neither SF-1 (Hiroi et al., 2004) nor LRH-1 (Falender et al., 2003; Hinshelwood et al., 2005) are induced by the ovulatory signal, yet both undergo changes in their ability to regulate genes associated with steroidogenesis, presumably through regulation of chromatin accessibility or abundance or activity of cofactors or corepressors.

5.4. Summary of the role of nuclear receptors in the regulation of steroidogenesis during the periovulatory period

The nuclear receptor PPARG is involved in steroidogenesis and lipid metabolism in the ovary, as in other tissues. Reports on PPARG regu­ lation by LH dependent pathways are directly conflicting, with some authors reporting that hCG induces increases in PPARG abundance (Kim et al., 2008; Tatone et al., 2016) while others reporting that this treat­ ment decreases PPARG expression (Banerjee and Komar, 2006; Funa­ hashi et al., 2017; Komar et al., 2001; Puttabyatappa et al., 2010). Komar et al. (2001) report inconsistency in the observed decreases in PPARG expression following the ovulatory signal, with some follicles losing PPARG and others retaining expression (Komar et al., 2001). Whatever the cause, this variability could explain some of the discrep­ ancies in reports of PPARG dynamics following the LH signal. Indeed, PPARG seems to have temporally restricted effects upon ovarian func­ tion during ovulation, as some inhibition of PPARG expression is important for the ovulatory cascade to proceed normally. Evidence for this comes from a study of rats, where significant decline in PPARG occurs within 4 h of hCG treatment (Funahashi et al., 2017). These in­ vestigators showed that intrabursal injection with a PPARG agonist in tandem with the ovulatory signal, which presumably maintains the PPARG signal, resulted in a reduced ovulation rate. There was concomitant reduction in the expression of factors essential to ovulation including STAR, PTGS2, and PGE2, as well as in the synthesis of

6. Nuclear receptors promote cell survival during the periovulatory period Granulosa cells of antral follicles are highly susceptible to atresia, whereas granulosa cells of periovulatory follicles appear to be resistant to apoptotic death (Svensson et al., 2000). There is evidence that two steroid hormone receptors, AR and PGR, are key factors in maintaining cell survival as the follicle approaches ovulation. 6.1. AR In periovulatory follicles lacking AR, there is increased apoptosis, and an associated decrease in abundance of the cell cycle inhibitor p21 12

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

(Hu et al., 2004). In this model, the transient expression of PGR is compromised, indicating that the follicle failed to appropriately respond to LH. This finding is consistent with the apparent role of AR in follicular growth discussed previously; loss of AR results in increased follicular death and fewer antral follicles, indicating loss of follicles during the transition from secondary to the antral state. In support of this concept, loss of AR in granulosa cells concurred with their becoming atretic during early pregnancy (Szoltys et al., 2005).

2020). This discrepancy may be resolved by consideration of the differing temporal aspects of the two studies. The report of Park et al. (2020) is derived from ovaries taken from 6 h after the ovulatory signal, prior to ovulation (Park et al., 2020), while Akison et al. (2018) report decreased neutrophil infiltration at 16 h after the LH surge, some 2–4 h after ovulation has actually occurred. Park et al. (2020) also suggest an important role for PGR in the termination of ovulatory inflammation, with PGR signaling modestly inducing PTGS2, as described previously. There appears to be a feedback regulation of this phenomenon, in that inhibition of progesterone signaling resulted in excess expression of PTGS2 and inappropriate persistence of ovarian inflammation. PGR directly regulates the NF-κB inhibitor NFKBIA, and induction of NFKBIA promotes termination of PTGS2 expression toward the end of the ovulatory period (Park et al., 2020). Therefore, the consequence of PGR deletion is greater ovulatory inflammation with each ovulation, with the longer-term ramification of an increase in oxidative damage in ovaries of aged mice lacking PGR.

6.2. PGR and LHR-1 PGR itself is also a regulator of the antiapoptotic effects of proges­ terone following the LH surge (Friberg et al., 2010; Svensson et al., 2000) and progesterone has additional antiapoptotic effects that are mediated through nonclassical receptors (Peluso, 2003; Peluso and Pappalardo, 1998). In contrast, although LRH-1 regulates granulosa cell proliferation, it has no effect on abundance of cleaved caspase-3 or on a variety of markers of apoptosis and autophagy (Meinsohn et al., 2018), suggesting that LRH-1 and its downstream targets do not alter the sus­ ceptibility of granulosa cells to apoptosis.

7.2. PPARs As noted above, PGR-mediated signaling in the periovulatory follicle induces expression of a wide variety of downstream mediators that regulate the ovulatory event. In murine ovaries, one of the factors upregulated under the influence of PGR is the nuclear receptor PPARG (Kim et al., 2008). Studies using transgenic mice have demonstrated that PPARG signaling participates in ovulation. Cui et al. (2002) reported that ovarian depletion of PPARG with an MMTV-cre, which depletes expression in mammary epithelium, oocytes, megakaryocytes, B- and T-lymphocytes, and salivary gland results in reduced overall fertility (Cui et al., 2002). This was manifest as complete infertility in one third of these mice. Of those that were fertile, all required more than twice as long to become pregnant after being placed with males. The litters in these animals were substantially smaller (Cui et al., 2002). In a similar study, in which PPARG was depleted from granulosa cells after the ovulatory signal using PGR-cre, there was greater than 5-fold reduction in ovulation rates (Kim et al., 2008). The ovaries of these animals demonstrated a phenotype of trapped oocytes, very similar to the models of PGR depletion. In complementary experiments with the PGR germline deletion model, the usual induction of PPARG following the LH signal did not occur, indicating that PPARG is downstream of PGR and mediates some of its functional effects (Kim et al., 2008). Several other genes downstream of PGR also appear to be PPARG-regulated, including IL6, PRKG2, and END1 (Kim et al., 2008), suggesting that PPARG signaling can alter both inflammatory and vascular functions. Interest­ ingly, the expression of each of these PPARG target genes was inhibited in vitro by treatment with a cyclooxygenase inhibitor and rescued by a specific PPARG agonist, suggesting that PTGS2 activity results in the production of long-chain fatty acids that can act as ligands for PPARG (Kim et al., 2008).

6.3. Summary of the role of nuclear receptors in the regulation of cell survival during the ovulatory period A shift in susceptibility of granulosa cells to apoptosis is an important periovulatory event. This event allows granulosa cells, which were previously highly susceptible to cell death, to survive the inflammatory event of ovulation and differentiate into luteal cells. Both progesterone and androgen signaling are important for granulosa cell survival, again highlighting the key role of steroid hormones in the ovulatory process. 7. Nuclear receptors regulate immune cell influx and the inflammatory response during ovulation In a landmark review (Espey, 1980), presented the hypothesis that ovulation has the characteristics of an inflammatory response. Since that time, a number of investigations from laboratories across the globe have confirmed the similarity between these two processes (Duffy et al., 2019). Prostaglandin biosynthesis, a hallmark of inflammation, is a necessary event in ovulation (Davis et al., 1999; Hizaki et al., 1999; Lim et al., 1997). Moreover, cytokines and chemokines are produced during the ovulatory process, and these result in the infiltration of various immune cell types (Duffy et al., 2019), which contributed to vascular remodeling and luteal formation. 7.1. PGR One key regulator of the inflammatory effects during ovulation seems to be the progesterone receptor, both inducing (Akison et al., 2018; Shimada et al., 2007) and resolving (Park et al., 2020) ovarian inflammation. PGR regulates the protein coding gene SNAP25, through which it modulates periovulatory increases in secretion of several cy­ tokines and chemokines, including IL-1beta, IL-6, IL-9 (Shimada et al., 2007) and CXCR4 (Choi et al., 2017a). Among these changes, the in­ duction of IL-6 may be the mechanism by which PGR regulates ovarian prostaglandins (Kim et al., 2009a). This is consistent with the findings of Akison et al. (2018), who reported that PGR regulates changes in cyto­ kines and in other key immune-regulatory transcription factors including NF-κB2, SOCS1, and STAT3. Reports of PGR regulation of immune cell infiltration into the mouse periovulatory follicle are conflicting. Akison et al. (2018) described a decline in cytokine and adhesion molecule abundance in the absence of PGR with the consequence of decreased infiltration of neutrophils. In contrast, Park et al. (2020) report an increased immune cell population in the follicle in the absence of PGR, characterized by increased numbers of T cells, B cells, neutrophils, and monocytes/macrophages (Park et al.,

7.3. Corticoid receptors Glucocorticoid and mineralocorticoid signaling may be another mechanism that modulates the inflammatory response that accompanies ovulation. During ovulation there is an increase in glucocorticoid signaling, and an increase in GR abundance in luteinized granulosa cells (Fru et al., 2006). These effects are not a direct consequence of gonad­ otropin stimulation, as gonadotropins do not induce expression of GR in vitro (Fru et al., 2006) or in vivo (Tetsuka et al., 1999). The glucocor­ ticoid ligands are not locally produced, as de novo synthesis of gluco­ corticoids and mineralocorticoids from steroid precursors requires CYP11B1 and CYP11B2, which are not expressed by cells of the ovarian follicle (Fru et al., 2006; Mukangwa et al., 2020). It would therefore appear that corticoids of adrenal provenance are the factors regulating periovulatory inflammation. Nevertheless, granulosa cells can 13

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

production, with concomitant reduction in gonadotropin receptors. Both PTGS2 and VEGFR2 were present in lower abundance, while IL-6 was substantially elevated, indicating an impaired ability to resolve ovarian inflammation and remodel ovarian vasculature during the ovulatory process (Mouzat et al., 2009).

interconvert these adrenal glucocorticoid forms, as shown by the studies in which hCG induced HSD11B1 and repressed HSD11B2 (Fru et al., 2006; Tetsuka et al., 1999). This results in the ability of granulosa cells to synthesize cortisol, the most potent glucocorticoid, from cortisone. Unlike GR, MR is subject to direct gonadotropin modulation in that its expression is downregulated by superstimulation with eCG, and more profoundly by hCG treatment (Fru et al., 2006). Classical ablation studies could shed more light on the role of cor­ ticoids in periovulatory inflammation and in ovarian function. Unfor­ tunately, germline deletion of GR in mice results in death of neonates within hours of birth, and MR knockout mice die within 1–2 weeks after birth (Berger et al., 1998; Cole et al., 1995). To date, it seems that no ovarian-specific knockout model has been generated for either receptor (Cole and Young, 2017). Germline deletion of the ACTH receptors in the mouse provides some information on the possible roles of these two receptors (Chida et al., 2007, 2009; Matsuwaki et al., 2010). ACTH re­ ceptor null mice lacked corticosterone, the primary glucocorticoid in rodents, and also had substantially reduced aldosterone and epinephrine synthesis and secretion (Chida et al., 2007, 2009, 2011). Although they displayed disrupted estrous cycles, females were fertile, with apparently normal ovarian histology. These mice have normal serum estradiol concentrations during diestrus and although they respond to super­ stimulation, they ovulate approximately 30% fewer oocytes than control counterparts. This suggests that the principle cause of the estrous cycle disruption was not ovarian dysfunction (Matsuwaki et al., 2010). Further exploration revealed that a cause of the reproductive deficiency in these mice was inadequate kisspeptin expression in the anteroventral periventricular nucleus of the hypothalamus (Matsuwaki et al., 2010). Other studies have also shed light on the role of GR in the ovulatory inflammatory condition. FAIREseq-based investigation of open chro­ matin in the mouse granulosa cell nucleus prior to, and after the ovulatory signal indicated that GR transcriptional target motifs were present (Bianco et al., 2019). In that report, GR motifs were associated with those of LRH-1 most frequently prior to the ovulatory signal (Bianco et al., 2019). This, together with the evidence for high con­ centrations of glucocorticoids and mineralocorticoids in the fluid of preovulatory follicles (Amin et al., 2014; Fateh et al., 1989; Fru et al., 2006; Mukangwa et al., 2020; Sneeringer et al., 2011) suggests a role for these steroids in ovulation. In the macaque, glucocorticoids increase in concentration in the follicular fluid following initiation of the ovulatory cascade by hCG (Fru et al., 2006). In this context, it has been suggested that ovarian cortisol may be a regulator of the inflammatory response that accompanies ovulation and may protect the ovary from proteolytic damage (Hillier and Tetsuka, 1998; Rae et al., 2009).

7.5. Summary of the effect of nuclear receptors on ovulatory inflammation The cascade of events that follow the LH ovulatory stimulus include the initiation of an inflammatory response. Evidence from multiple sources implicates the nuclear receptors, among other mediators, in sustaining and resolving this inflammatory state. Although there is abundant evidence for glucocorticoid-mediated regulation of immune cells and inflammation in other physiological contexts (Cain and Cidlowski, 2017), it is not yet clear if the observed alterations in GR abundance during ovulation affect these processes. PGR appears to be essential both early in the process, to provoke inflammation and later, to bring about its ebb and eventual decline. There is also evidence for PGR-regulated invasion of leucocytes and production of cytokines. PPARG appears to be downstream of PGR in several of these processes. Finally, the LXR family, which has received little attention in studies of reproductive biology, appears to be a regulator of the resolution of ovarian inflammation. 8. Nuclear receptors are essential for granulosa cell migration and follicular rupture The dramatic conclusion of the ovulatory process is the rupture of the follicle and release of the oocyte. A coordinated series of events regulate rupture, beginning with matrix remodeling and thinning of the follicular apex, followed by vascular changes, and ultimately, the breakdown of the follicular wall and escape of the oocyte. Nuclear receptors are essential to the process of rupture, which ultimately relies on proteases, primarily of the ADAMTS and MMP families (Curry and Osteen, 2003; Duffy et al., 2019; Richards et al., 2005). 8.1. PGR Although progesterone has a panoply of effects in the ovary, the most distinct of the consequences of the PGR germline null mutation is failure of follicular rupture. Mice lacking PGR develop antral follicles normally, but these follicles are completely anovulatory (Lydon et al., 1995, 1996). Oocytes with expanded cumuli are trapped within the follicle, a condition that cannot be reversed by exogenous ovarian super­ stimulation. This phenotype has since been corroborated in ovary-specific deletion studies in the mouse with an ESR2-cre (Park et al., 2020), in siRNA-mediated PGR silencing studies in the monkey ovary (Bishop et al., 2016), and in studies with specific PGR antagonists ¨m, 1993; Chen in a wide variety of species, including humans (Br¨ annstro et al., 1995; Curry and Nothnick, 1996; Gayt´ an et al., 2003; Iwamasa et al., 1992; Kanayama et al., 1994; Ledger et al., 1992; Loutradis et al., 1991). Indeed, the progesterone antagonist RU486 blocks proteolytic enzyme activity in the ovulatory follicle of the rat, thereby preventing rupture (Iwamasa et al., 1992). The rapid rise and equally rapid decline in expression of the PGR described above calls into question how PGR is regulated. It is known that activation of the G-protein proteins Gαq and Gα11 by LH is required for induction of PGR (Breen et al., 2013). Further, the transcription factors NFIL3 (Li et al., 2011), Sp1/Sp3, GATA4 (Sriraman et al., 2003; Robker et al., 2009), and LRH-1 (Bertolin et al., 2014) have been re­ ported to be involved in the increase in the expression of PGR in response to the LH surge. PGR controls the expression of proteins that are directly responsible for follicular rupture. The first PGR-regulated proteases identified were ADAMTS1 and cathepsin L (Robker et al., 2000) followed by discovery

7.4. LXR The nuclear receptor LXR is a regulator of cholesterol, fatty acid, and glucose homeostasis. It has attracted little attention as an ovarian factor, in spite of its potential significance to resolution of inflammation during the ovulatory process. Germline deletion of either or both of the LXRs, LXRA and LXRB, reduced fertility (Steffensen et al., 2006). The number of follicles did not differ in the ovaries of any of these three genotypes from that observed in wild type controls. The primary consequence of loss of LXR was impairment of the ability of oocytes to resume meiosis. This effect was localized to the oocyte itself, and not the cumulus, as naked wildtype oocytes resumed meiosis in response to an LXR agonist, while cumulus-enclosed oocytes did not (Steffensen et al., 2006). Interestingly, when the mice lacking both LXRs were superstimulated with eCG and hCG, there was a distinct ovarian somatic cell phenotype that shared characteristics with ovarian hyperstimulation syndrome (Mouzat et al., 2009). These mice had enlarged ovaries with increased vascular permeability and multiple hemorrhagic CL. More oocytes were ovulated relative to the counterpart controls, but nearly half of oocytes from mice lacking LXR were nonviable (Mouzat et al., 2009). Consistent with the increase in follicle number, there was increased estradiol 14

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

of another proteinase, ADAM8 (Sriraman et al., 2008). These proteases are believed to be the direct regulators of the collagen breakdown that takes place at the time of follicular rupture. Several members of the MMP and TIMP family are regulated by progesterone, in diverse species, including ruminants, primates, and rodents (Chaffin and Stouffer, 1999; Iwamasa et al., 1992; Morgan et al., 1994; Murdoch et al., 1986). Interestingly, in a study in which HSD3B was inhibited with trilostane and progesterone signaling was restored by treatment with the progestin R5020, the collagenase MMP1 and the MMP inhibitor TIMP1 were revealed to be progesterone regulated. Other proteases, MMP7, MMP9, and TIMP2 were downregulated by trilostane treatment, but could not be rescued by treatment with progestin, suggesting that these molecules are regulated by steroid hormones other than progesterone (Chaffin and Stouffer, 1999). Regulation of gene expression by progesterone in the periovulatory follicle has been reviewed in depth by others; for more detail and complete lists of PGR-regulated genes, the reader is referred to recent reviews (Duffy et al., 2019; Kim et al., 2009a; Robker et al., 2009). Endothelins (EDN1 and EDN2) are potent vasoactive molecules, the loss of which results in impaired follicular rupture. These molecules are also PGR-regulated, indicating that PGR alters follicular vasculature to promote follicular rupture (Cacioppo et al., 2017; Palanisamy et al., 2006). The transcriptional regulators HIF1A, 2A, and 1B are also induced by PGR and blocking the actions of these factors with echino­ mycin prevents follicular rupture, phenocopying the progesterone re­ ceptor knockout model (Kim et al., 2009b). The inhibition of HIFs is characterized by the failure of induction of important regulators of the process of rupture, including ADAMTS1 and EDN1 (Kim et al., 2009b).

cells causes infertility via interference with a variety of normal ovarian functions, including follicular rupture (Meinsohn et al., 2019). LRH-1 regulates the ADAMTS family member ADAMTS4 (Bertolin et al., 2014). In addition, a recent chromatin immunoprecipitation sequencing experiment revealed an additional mechanism by which LRH-1 may regulate rupture. In this study many of the genes for which binding by LRH-1 was enhanced after the LH surge were regulators of cytoskeletal reorganization and cellular migratory processes (Bianco et al., 2019). In addition to the importance of these changes for cumulus expansion, as previously discussed, these changes are likely contributors to follicular rupture (Grossman et al., 2015). Furthermore, it has been shown that depletion of LRH-1 in granulosa cells throughout follicular development or from antral follicle development forward compromises the transient expression of PGR that follows the ovulatory LH surge (Bertolin et al., 2014). Therefore, it is not clear to what extent mediators of rupture are regulated by LRH-1 via PGR and which may be direct LRH-1 targets. Mice lacking LRH-1 do not display alteration in the abundance ADAMTS1, although abundance of ADAMTS4 is reduced (Bertolin et al., 2014). 8.3. Summary of the regulation of rupture by nuclear receptors The process of follicular rupture pivotal in the sequence of ovulatory events, and without this process, no oocyte is released and ovulation fails. Multiple nuclear receptors are important regulators of this process (Fig. 3). Years of research have clearly implicated the progesterone re­ ceptor as a regulator of many of the proteases that directly regulate rupture. More recently, LRH-1 was identified as a regulator of granulosa cell migration, suggesting that it also likely affects rupture directly. Moreover, LRH-1 regulates the abundance of PGR, suggesting that it may also have an indirect effect on rupture.

8.2. LRH-1 As noted elsewhere, conditional depletion of LRH-1 in granulosa

Fig. 3. Nuclear receptors regulate follicular rupture. A summary of key functions regulated by nuclear receptors during follicular rupture. Steroid hormone receptors are colored green and receptors of the NR5A family are colored blue, while others are black. Functions are arranged temporally. Arrows indicate a positive relationship of a receptor with a function. The heavy grey arrow indicates LH surgemediated alteration of the functions of the indicated transcriptional regulators. LRH-1: liver receptor ho­ molog 1; LH: luteinizing hormone; PGR: Progesterone receptor; ADAM8: ADAM metallopeptidase domain 8; ADAMTS: ADAM metallopeptidase with thrombo­ spondin type 1; EDN:Endothelin; GATA: GATA bind­ ing protein; MMP: Matrix metalloprotease; NFIL3: Nuclear factor, interleukin 3 regulated; SP: Sp tran­ scription factor; PPARG: Peroxisome proliferator activated receptor gamma. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

15

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

9. Overlap of PGR and LRH-1 regulated genes during the periovulatory period

coregulated by LRH-1 and PGR. This analysis indicated that there were 110 mural granulosa cell transcripts that were regulated both by loss of LRH-1 and by loss of PGR in at least one dataset. In the study of Dinh et al. (2019) and that of Park et al. (2020) respectively, 18% and 15% respectively of mRNAs that were changed by loss of PGR were also changed by the loss of LRH-1 (Bianco et al., 2019). Among these mRNA commonly altered by loss of LRH-1 and PGR, 59% were affected by depletion of PGR and LRH-1 in the same direction and nearly all declined in response to knockout. Interestingly, these genes that were coregulated by LRH-1 and PGR, as judged by mutual decrease following ablation of the cognate nuclear receptor, were associated with hormone response and secretion, as well as lipid and steroid transport and metabolism (Fig. 4A) (Zhou et al., 2019). All of these are key processes essential to ovulation. Prominent among these genes were the transcription factor Cebpb and the tran­ scription factor inhibiter Nfkbia, both of which have established roles in ovulation (Fan et al., 2009; Park et al., 2020). Cumulus-specific patterns of gene expression were similar to those identified in mural granulosa cells. Among 56 cumulus transcripts regulated both by loss of LRH-1 and PGR, 55% were regulated in the same direction, and these were, for the most part, factors that modulate metabolic processes and lipid transport. In contrast, 41% of genes modified by loss of both PGR of LRH-1 were regulated in opposite directions. Pathways associated with these included several intracellular signaling transduction pathways, path­ ways associated with immune response, and cell death pathways (Fig. 4B) (Zhou et al., 2019). Some of these may be downstream of PTGS2, as loss of LRH-1 downregulates PTGS2 (Duggavathi et al., 2008), while loss of PGR increases PTGS2 in mice (Park et al., 2020). Most notable among genes that were regulated in opposite directions in LRH-1 and PGR knockout were the regulators of cumulus expansion, Areg and Btc, which were reduced in LRH-1 knockouts, but upregulated in PGR knockouts. This is consistent with the phenotypes of each of these models; LRH-1 knockouts lack cumulus expansion (Bertolin et al., 2017), while PGR knockouts have normal cumulus expansion but failed follicular rupture (Lydon et al., 1996; Robker et al., 2000). In summary, this analysis has demonstrated that key ovarian func­ tions, including functions related to metabolism and steroid hormone transport, may be coregulated by LRH-1 and PGR. Further study of the overlap between these receptors, including investigation of the com­ monalities in their cistromes and in vitro investigation of potential protein-protein interactions, is certainly warranted.

The ovulatory defects observed in mice with ovarian depletion of either LRH-1 or PGR were the most profound of any observed in a nu­ clear receptor knockout model. Mice lacking either of these nuclear receptors failed completely to ovulate. Moreover, in mice with loss of LRH-1 following the ovulatory signal, using PGR-cre, ovulation is more or less normal, but the CL that form are smaller and less able to produce progesterone (Zhang et al., 2013). In other models (for example, the model of PPARG depletion via PGR-cre), reduced ovulation rates are seen when the cre recombinase enzyme is induced by the ovulatory signal. However, this is not the case for LRH-1, indicating that its key role is the ovarian response to the LH surge. There is no evidence for LRH-1-mediated regulation of LHCGR in the ovary (Bianco et al., 2019; Saxena et al., 2004), indicating that the functions that differ in mice lacking LRH-1 likely not regulated by a decreased ability to respond to LH, rather by a decreased ability of LH to elicit downstream effects. Similarly, PGR is induced by the LH surge, so it also does not alter ability of cells to respond to LH, although it dramatically alters LH downstream target expression. In a recent investigation of the PGR cistrome following the ovulatory signal, previous targets of PGR were confirmed, including ADAMTS1 (Dinh et al., 2019). An advantage of ChIP sequencing studies is the ability to investigate the genomic motifs to which a given transcription factor binds. In the ChIPseq investigation of PGR, the investigators revealed that the vast majority of PGR binding sites in granulosa cells (13022 motifs) were not classical PGR/NR3C response elements, while only 1422 were classical PREs. This suggests that in granulosa cells, PGR actions are not limited to genes containing a PRE/NR3C motif, and that PGR likely interacts with coregulators to bind and elicit transcription (Dinh et al., 2019). Moreover, HOMER analysis revealed that LRH-1 motifs were highly enriched in the PGR ChIP, indicating likely PGR/LRH-1 coregulation of targets. In addition, there is evidence for regulation of PGR via depletion of LRH-1 (Bertolin et al., 2014). AN analysis of publicly available transcriptome data was undertaken to investigate the overlap between genes regulated by PGR and those regulated by LRH-1. The basis for the query was granulosa tran­ scriptomes during the periovulatory period in mice lacking LRH-1 or PGR, relative to their wildtype counterparts. The transcripts for LRH-1regulated genes were identified both prior to the LH surge (44 h after eCG) and 4 h after the treatment with hCG (Bianco et al., 2019). In contrast, the PGR regulated genes were assessed 6 and 8 h after the ovulatory signal (Akison et al., 2018; Dinh et al., 2019; Park et al., 2020). In the study of Park et al., single-cell RNAseq was used (Park et al., 2020), while in the studies of Dinh et al. (2019), and Akison et al. (2018), mural granulosa and cumulus granulosa cells were isolated, allowing the separate investigation of these two cell types. Character­ istics of the datasets used can be seen in Table 2. These three datasets were then compared with the dataset of transcriptomic changes result­ ing from loss of LRH-1, with the goal of identifying the genes that may be

10. Conclusion Nuclear receptor regulation of gene expression has fascinated re­ searchers from the discovery of the steroid hormone signaling pathways in the 1970s and the identification of the nuclear receptor family in the 1980s, to the applications of advanced molecular biology and bio­ informatic technology to identify regions of open chromatin and com­ plex regulatory networks of today. Although nuclear receptors are not the only regulators of transcriptional change in the ovulatory follicle,

Table 2 Characteristics of datasets used to compare LRH-1 and PGR regulated genes. Reference

GEO identifier

Model

Platform

Time of collection

Cell type

How were these data accessed?

Bianco et al. (2019)

GSE119508

RNAseq

4 h after hCG

All granulosa cells

Accessed as a supplemental file

Dinh et al. (2019) Park et al. (2020)

GSE92438

LRH-1 f/f, Amhr2-cre (depletion from granulosa cells of all follicles), compared to LRH-1 f/f, AMHR2-cre negative littermates. PGR germline knockout

Microarray

8 h after hCG

Mural granulosa cells

Single cell RNAseq

6 h after hCG

Microarray

8 h after hCG

Cumulus and granulosa cell clusters Cumulus granulosa cells

Accessed as a supplemental file Accessed as a supplemental file

Akison et al. (2018)

GSE145107 GSE92438

PGR f/f, ESR2-cre (depletion from granulosa cells of follicles), compared to PGR f/f, ESR2-cre negative littermates. PGR germline knockout

16

Analysed using Geo2R

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Fig. 4. Functions regulated by LRH-1 (Bianco et al., 2019) and PGR (Park et al., 2020; Dinh et al., 2019) in mural granulosa cells in response to the ovulatory signal. A. Metascape pathway analysis (Zhou et al., 2019) of transcripts that were regulated in the same direction by loss of LRH-1 and PGR, suggesting likely coregulation. B. Metascape pathway analysis of transcripts that were regulated in opposite directions by loss of LRH-1 and PGR. For both A and B, the color of each circle represents the functional cluster, while the edges represent the similarity or relatedness among clusters. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

among members of this family there are receptors that are absolutely required for ovulation, including LRH-1 and PGR. Others that may not be absolutely required for the ovulatory process to occur, but regulate key functions, include the ESRs, AR, and PPARG. Much has been learned about the regulation of ovulation by nuclear receptors, and technology such as ChIPseq and ATACseq have and will continue to allow delin­ eation of the complex network of gene expression changes that induce ovulation. These investigations will continue to lead to an improved understanding of the process of ovulation, resulting in the development

of novel technology to treat infertility. Declaration of competing interest The authors have no competing interests to declare. Acknowledgements The authors express gratitude to Vickie Roussel for her assistance 17

Molecular Aspects of Medicine xxx (xxxx) xxx

C.H.K. Hughes and B.D. Murphy

with preparation of figures and to Olivia E. Smith for critically reading the manuscript.

Betowski, J., Semczuk, A., 2010. Liver X receptor (LXR) and the reproductive system – a potential novel target for therapeutic intervention. Pharmacol. Rep. 62, 15–27. Bianco, S., Rodrigue, S., Murphy, B.D., G´ evry, N., 2015. Global mapping of open chromatin regulatory elements by formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq). Methods Mol. Biol. 1334, 261–272. ´ Beauparlant, C.J., Bertolin, K., Droit, A., Bianco, S., Bellefleur, A.-M., Beaulieu, E., Schoonjans, K., Murphy, B.D., G´ evry, N., 2019. The ovulatory signal precipitates LRH-1 transcriptional switching mediated by differential chromatin accessibility. Cell Rep. 28 (9), 2443–2454 e2444. Bishop, C.V., Hennebold, J.D., Kahl, C.A., Stouffer, R.L., 2016. Knockdown of progesterone receptor (PGR) in macaque granulosa cells disrupts ovulation and progesterone Production1. Biol. Reprod. 94 (5), 109. Bookout, A.L., Jeong, Y., Downes, M., Yu, R.T., Evans, R.M., Mangelsdorf, D.J., 2006. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 126 (4), 789–799. Bortolussi, M., Marini, G., Dal Lago, A., 1977. Autoradiographic study of the distribution of LH(HCG) receptors in the ovary of untreated and gonadotrophin-primed immature rats. Cell Tissue Res. 183 (3), 329–342. Br¨ annstr¨ om, M., 1993. Inhibitory effect of mifepristone (RU 486) on ovulation in the isolated perfused rat ovary. Contraception 48 (4), 393–402. Bridges, P.J., Komar, C.M., Fortune, J.E., 2006. Gonadotropin-induced expression of messenger ribonucleic acid for cyclooxygenase-2 and production of prostaglandins E and F2α in bovine preovulatory follicles are regulated by the progesterone receptor. Endocrinology 147 (10), 4713–4722. Brązert, M., Kranc, W., Nawrocki, M., Sujka-Kordowska, P., Konwerska, A., Jankowski, M., Kocherova, I., Celichowski, P., Jeseta, M., O˙zegowska, K., Antosik, P., Bukowska, D., Skowro´ nski, M., Bruska, M., Pawelczyk, L., Zabel, M., Piotrowska-Kempisty, H., Nowicki, M., Kempisty, B., 2020. New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation. Mol. Med. Rep. 21, 1537–1551. Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., Greenleaf, W.J., 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10 (12), 1213–1218. Buenrostro, J.D., Wu, B., Chang, H.Y., Greenleaf, W.J., 2015. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protocols Mol. Biol. 109 (1). Cacioppo, J.A., Lin, P.P., Hannon, P.R., McDougle, D.R., Gal, A., Ko, C., 2017. Granulosa cell endothelin-2 expression is fundamental for ovulatory follicle rupture. Sci. Rep. 7 (1), 817. Cain, D.W., Cidlowski, J.A., 2017. Immune regulation by glucocorticoids. Nat. Rev. Immunol. 17 (4), 233–247. Carletti, M.Z., Christenson, L.K., 2009. Rapid effects of LH on gene expression in the mural granulosa cells of mouse periovulatory follicles. Reproduction 137 (5), 843–855. Celichowski, P., Nawrocki, M.J., Dyszkiewicz-Konwi´ nska, M., Jankowski, M., Budna, J., Bryja, A., Kranc, W., Borys, S., Knap, S., Ciesi´ ołka, S., Jeseta, M., PiaseckaStryczy´ nska, K., Khozmi, R., Bukowska, D., Antosik, P., Brüssow, K.P., Bruska, M., Nowicki, M., Zabel, M., Kempisty, B., 2018. “Positive regulation of RNA metabolic process” ontology group highly regulated in porcine oocytes matured in vitro: a microarray approach. BioMed Res. Int. 2018, 1–10. Chaffin, C.L., Stouffer, R.L., 1999. Expression of matrix metalloproteinases and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid Regulation1. Biol. Reprod. 61 (1), 14–21. Chaffin, C.L., Vandevoort, C.A., 2013. Follicle growth, ovulation, and luteal formation in primates and rodents: a comparative perspective. Exp. Biol. Med. 238 (5), 539–548. Chaffin, C.L., Hess, D.L., Stouffer, R.L., 1999. Dynamics of periovulatory steroidogenesis in the rhesus monkey follicle after ovarian stimulation. Hum. Reprod. 14 (3), 642–649. Chen, L., Russell, P.T., Larsen, W.J., 1993. Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass. Mol. Reprod. Dev. 34 (1), 87–93. Chen, F., Cooney, A.J., Wang, Y., Law, S.W., O’Malley, B.W., 1994a. Cloning of a novel orphan receptor (GCNF) expressed during germ cell development. Mol. Endocrinol. 8 (10), 1434–1444. Chen, W.S., Manova, K., Weinstein, D.C., Duncan, S.A., Plump, A.S., Prezioso, V.R., Bachvarova, R.F., Darnell, J.E., 1994b. Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos. Genes Dev. 8 (20), 2466–2477. Chen, S.H., Dharmarajan, A.M., Wallach, E.E., Mastroyannis, C., 1995. RU486 inhibits ovulation, fertilization and early embryonic development in rabbits: in vivo and in vitro studies*. Fertil. Steril. 64 (3), 627–633. Chen, L.-M., Wang, R.-S., Lee, Y.-F., Liu, N.-C., Chang, Y.-J., Wu, C.-C., Xie, S., Hung, Y.C., Chang, C., 2008. Subfertility with defective folliculogenesis in female mice lacking testicular orphan nuclear receptor 4. Mol. Endocrinol. 22 (4), 858–867. Chen, H., Chu, G., Zhao, L., Yamauchi, N., Shigeyoshi, Y., Hashimoto, S., Hattori, M.-A., 2012. Rev-erbα regulates circadian rhythms and StAR expression in rat granulosa cells as identified by the agonist GSK4112. Biochem. Biophys. Res. Commun. 420 (2), 374–379. Cheng, L.E.C., Chan, F.K.-M., Cado, D., Winoto, A., 1997. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. 16 (8), 1865–1875. Cheng, X.B., Jimenez, M., Desai, R., Middleton, L.J., Joseph, S.R., Ning, G., Allan, C.M., Smith, J.T., Handelsman, D.J., Walters, K.A., 2013. Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice. Am. J. Physiol. Endocrinol. Metab. 305 (6), E717–E726.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.mam.2020.100937. Funding Research on SF-1 and LRH-1 described herein was supported by grants from the Canadian Institutes of Health Research to BDM. CHKH is the recipient of a Lalor Foundation postdoctoral fellowship. References Aghajanova, L., Lindeberg, M., Carlsson, I.B., Stavreus-Evers, A., Zhang, P., Scott, J.E., Hovatta, O., Skj¨ oldebrand-Sparre, L., 2009. Receptors for thyroid-stimulating hormone and thyroid hormones in human ovarian tissue. Reprod. Biomed. Online 18 (3), 337–347. Akhmedov, N.B., Piriev, N.I., Chang, B., Rapoport, A.L., Hawes, N.L., Nishina, P.M., Nusinowitz, S., Heckenlively, J.R., Roderick, T.H., Kozak, C.A., Danciger, M., Davisson, M.T., Farber, D.B., 2000. A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc. Natl. Acad. Sci. Unit. States Am. 97 (10), 5551–5556. Akison, L.K., Alvino, E.R., Dunning, K.R., Robker, R.L., Russell, D.L., 2012. Transient invasive migration in mouse cumulus oocyte complexes induced at ovulation by luteinizing hormone. Biol. Reprod. 86 (4), 125. Akison, L.K., Robertson, S.A., Gonzalez, M.B., Richards, J.S., Smith, C.W., Russell, D.L., Robker, R.L., 2018. Regulation of the ovarian inflammatory response at ovulation by nuclear progesterone receptor. Am. J. Reprod. Immunol. 79 (6), e12835. Amin, M., Simerman, A., Cho, M., Singh, P., Briton-Jones, C., Hill, D., Grogan, T., Elashoff, D., Clarke, N.J., Chazenbalk, G.D., Dumesic, D.A., 2014. 21-Hydroxylasederived steroids in follicles of nonobese women undergoing ovarian stimulation for in vitro fertilization (IVF) positively correlate with lipid content of luteinized granulosa cells (LGCs) as a source of cholesterol for steroid synthesis. J. Clin. Endocrinol. Metab. 99 (4), 1299–1306. Andr´ e, E., Conquet, F., Steinmayr, M., Stratton, S.C., Porciatti, V., Becker-Andr´e, M., 1998. Disruption of retinoid-related orphan receptor β changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice. EMBO J. 17 (14), 3867–3877. Antal, M.C., Krust, A., Chambon, P., Mark, M., 2008. Sterility and absence of histopathological defects in nonreproductive organs of a mouse ER -null mutant. Proc. Natl. Acad. Sci. Unit. States Am. 105 (7), 2433–2438. Antonson, P., Apolin´ ario, L.M., Shamekh, M.M., Humire, P., Poutanen, M., Ohlsson, C., Nalvarte, I., Gustafsson, J.-Å., 2020. Generation of an all-exon Esr2 deleted mouse line: effects on fertility. Biochem. Biophys. Res. Commun. 529 (2), 231–237. Ashkenazi, H., Cao, X., Motola, S., Popliker, M., Conti, M., Tsafriri, A., 2005. Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146 (1), 77–84. Baena, V., Owen, C.M., Uliasz, T.F., Lowther, K.M., Yee, S.-P., Terasaki, M., Egbert, J.R., Jaffe, L.A., 2020. Cellular heterogeneity of the luteinizing hormone receptor and its significance for cyclic GMP signaling in mouse preovulatory follicles. Endocrinology 161 (7). Bagavandoss, P., Midgley, A.R., 1988. Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro. Life Sci. 43 (20), 1607–1614. Banerjee, J., Komar, C.M., 2006. Effects of luteinizing hormone on peroxisome proliferator-activated receptor gamma in the rat ovary before and after the gonadotropin surge. Reproduction 131 (1), 93–101. Barak, Y., Nelson, M.C., Ong, E.S., Jones, Y.Z., Ruiz-Lozano, P., Chien, K.R., Koder, A., Evans, R.M., 1999. PPARγ is required for placental, cardiac, and adipose tissue development. Mol. Cell 4 (4), 585–595. Berga, S., Naftolin, F., 2012. Neuroendocrine control of ovulation. Gynecol. Endocrinol. 28 (Suppl. 1), 9–13. Berger, S., Bleich, M., Schmid, W., Cole, T.J., Peters, J., Watanabe, H., Kriz, W., Warth, R., Greger, R., Schütz, G., 1998. Mineralocorticoid receptor knockout mice: pathophysiology of Na+ metabolism. Proc. Natl. Acad. Sci. Unit. States Am. 95 (16), 9424–9429. Bertolin, K., Bellefleur, A.-M., C, Z., D, M.B., 2010. Orphan nuclear receptor regulation of reproduction. Anim. Reprod. 3 (3), 146–153. Bertolin, K., Gossen, J., Schoonjans, K., Murphy, B.D., 2014. The orphan nuclear receptor Nr5a2 is essential for luteinization in the female mouse ovary. Endocrinology 155 (5), 1931–1943. Bertolin, K., Meinsohn, M.-C., Suzuki, J., Gossen, J., Schoonjans, K., Duggavathi, R., Murphy, B.D., 2017. Ovary-specific depletion of the nuclear receptor Nr5a2 compromises expansion of the cumulus oophorus but not fertilization by ICSI. Biol. Reprod. 96 (6), 1231–1243. Best, M.W., Wu, J., Pauli, S.A., Kane, M.A., Pierzchalski, K., Session, D.R., Woods, D.C., Shang, W., Taylor, R.N., Sidell, N., 2015. A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells. MHR: Basic Sci. Reprod. Med. 21 (6), 527–534.

18

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Chida, D., Nakagawa, S., Nagai, S., Sagara, H., Katsumata, H., Imaki, T., Suzuki, H., Mitani, F., Ogishima, T., Shimizu, C., Kotaki, H., Kakuta, S., Sudo, K., Koike, T., Kubo, M., Iwakura, Y., 2007. Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis. Proc. Natl. Acad. Sci. Unit. States Am. 104 (46), 18205–18210. Chida, D., Sato, T., Sato, Y., Kubo, M., Yoda, T., Suzuki, H., Iwakura, Y., 2009. Characterization of mice deficient in Melanocortin 2 receptor on a B6/Balbc mix background. Mol. Cell. Endocrinol. 300 (1–2), 32–36. Chida, D., Miyoshi, K., Sato, T., Yoda, T., Kikusui, T., Iwakura, Y., 2011. The role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in melanocortin receptor 2-deficient mice. Endocrinology 152 (4), 1652–1660. Cho, H., Zhao, X., Hatori, M., Yu, R.T., Barish, G.D., Lam, M.T., Chong, L.W., DiTacchio, L., Atkins, A.R., Glass, C.K., Liddle, C., Auwerx, J., Downes, M., Panda, S., Evans, R.M., 2012. Regulation of circadian behaviour and metabolism by REV-ERBalpha and REV-ERB-beta. Nature 485 (7396), 123–127. Choi, Y., Park, J.Y., Wilson, K., Rosewell, K.L., Br¨ annstr¨ om, M., Akin, J.W., Curry, T.E., Jo, M., 2017a. The expression of CXCR4 is induced by the luteinizing hormone surge and mediated by progesterone receptors in human preovulatory granulosa cells†. Biol. Reprod. 96 (6), 1256–1266. Choi, Y., Wilson, K., Hannon, P.R., Rosewell, K.L., Br¨ annstr¨ om, M., Akin, J.W., Curry, T. E., Jo, M., 2017b. Coordinated regulation among progesterone, prostaglandins, and EGF-like factors in human ovulatory follicles. J. Clin. Endocrinol. Metabol. 102 (6), 1971–1982. Choi, Y., Rosewell, K.L., Brannstrom, M., Akin, J.W., Curry Jr., T.E., Jo, M., 2018. FOS, a critical downstream mediator of PGR and EGF signaling necessary for ovulatory prostaglandins in the human ovary. J. Clin. Endocrinol. Metab. 103 (11), 4241–4252. Chung, A.C., Katz, D., Pereira, F.A., Jackson, K.J., DeMayo, F.J., Cooney, A.J., O’Malley, B.W., 2001. Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation. Mol. Cell Biol. 21 (2), 663–677. Cole, T.J., Young, M.J., 2017. 30 years OF the mineralocorticoid receptor: mineralocorticoid receptor null mice: informing cell-type-specific roles. J. Endocrinol. 234 (1), T83–T92. Cole, T.J., Blendy, J.A., Monaghan, A.P., Krieglstein, K., Schmid, W., Aguzzi, A., Fantuzzi, G., Hummler, E., Unsicker, K., Schutz, G., 1995. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 9 (13), 1608–1621. Collins, L.L., Lee, Y.F., Heinlein, C.A., Liu, N.C., Chen, Y.T., Shyr, C.R., Meshul, C.K., Uno, H., Platt, K.A., Chang, C., 2004. Growth retardation and abnormal maternal behavior in mice lacking testicular orphan nuclear receptor 4. Proc. Natl. Acad. Sci. Unit. States Am. 101 (42), 15058–15063. Couse, J.F., Lindzey, J., Grandien, K., Gustafsson, J.-A.K., Korach, K.S., 1997. Tissue distribution and quantitative analysis of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) messenger ribonucleic acid in the wild-type and ERα-knockout mouse. Endocrinology 138 (11), 4613–4621. Couse, J.F., Bunch, D.O., Lindzey, J., Schomberg, D.W., Korach, K.S., 1999. Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse. Endocrinology 140 (12), 5855–5865. Couse, J.F., Curtis Hewitt, S., Korach, K.S., 2000. Receptor null mice reveal contrasting roles for estrogen receptor α and β in reproductive tissues. J. Steroid Biochem. Mol. Biol. 74 (5), 287–296. Couse, J.F., Yates, M.M., Walker, V.R., Korach, K.S., 2003. Characterization of the hypothalamic-pituitary-gonadal Axis in estrogen receptor (ER) null mice reveals hypergonadism and endocrine sex reversal in females lacking ERα but not ERβ. Mol. Endocrinol. 17 (6), 1039–1053. Couse, J.F., Yates, M.M., Sanford, R., Nyska, A., Nilson, J.H., Korach, K.S., 2004. formation of cystic ovarian follicles associated with elevated luteinizing hormone requires estrogen receptor-β. Endocrinology 145 (10), 4693–4702. Couse, J.F., Yates, M.M., Deroo, B.J., Korach, K.S., 2005. Estrogen receptor-beta is critical to granulosa cell differentiation and the ovulatory response to gonadotropins. Endocrinology 146 (8), 3247–3262. Cui, Y., Miyoshi, K., Claudio, E., Siebenlist, U.K., Gonzalez, F.J., Flaws, J., Wagner, K.U., Hennighausen, L., 2002. Loss of the peroxisome proliferation-activated receptor gamma (PPARgamma ) does not affect mammary development and propensity for tumor formation but leads to reduced fertility. J. Biol. Chem. 277 (20), 17830–17835. Curry Jr., T.E., Nothnick, W.B., 1996. Mifepristone and ovarian function. Clin. Obstet. Gynecol. 39 (2), 486–497. Curry, T.E., Osteen, K.G., 2003. The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle. Endocr. Rev. 24 (4), 428–465. D’Haeseleer, M., Van Poucke, M., Van den Broeck, W., 2005. Cell-specific localization of oestrogen receptor beta (ESR2) mRNA within various bovine ovarian cell types using in situ hybridization. Anat. Histol. Embryol. 34 (4), 265–272. Davis, B.J., Lennard, D.E., Lee, C.A., Tiano, H.F., Morham, S.G., Wetsel, W.C., Langenbach, R., 1999. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1β. Endocrinology 140 (6), 2685–2695. Demczuk, M., Huang, H., White, C., Kipp, J.L., 2016. Retinoic acid regulates calcium signaling to promote mouse ovarian granulosa cell proliferation. Biol. Reprod. 95 (3), 70. Deroo, B.J., Rodriguez, K.F., Couse, J.F., Hamilton, K.J., Collins, J.B., Grissom, S.F., Korach, K.S., 2009. Estrogen receptor beta is required for optimal cAMP production in mouse granulosa cells. Mol. Endocrinol. 23 (7), 955–965.

Deyoung, R.A., Baker, J.C., Cado, D., Winoto, A., 2003. The orphan steroid receptor Nur77 family member nor-1 is essential for early mouse embryogenesis. J. Biol. Chem. 278 (47), 47104–47109. Dinh, D.T., Breen, J., Akison, L.K., DeMayo, F.J., Brown, H.M., Robker, R.L., Russell, D. L., 2019. Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression. Sci. Rep. 9 (1), 11966. Drouineaud, V., Sagot, P., Garrido, C., Logette, E., Deckert, V., Gambert, P., Jimenez, C., Staels, B., Lagrost, L., Masson, D., 2007. Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists. Mol. Hum. Reprod. 13 (6), 373–379. Drummond, A.E., Fuller, P.J., 2010. The importance of ERβ signalling in the ovary. J. Endocrinol. 205 (1), 15–23. Drummond, A.E., Baillie, A.J., Findlay, J.K., 1999. Ovarian estrogen receptor α and β mRNA expression: impact of development and estrogen. Mol. Cell. Endocrinol. 149 (1–2), 153–161. Duffy, D.M., Ko, C., Jo, M., Brannstrom, M., Curry, T.E., 2019. Ovulation: parallels with inflammatory processes. Endocr. Rev. 40 (2), 369–416. Dufour, S., Qu´ erat, B., Tostivint, H., Pasqualini, C., Vaudry, H., Rousseau, K., 2020. Origin and evolution of the neuroendocrine control of reproduction in vertebrates, with special focus on genome and gene duplications. Physiol. Rev. 100 (2), 869–943. Duggavathi, R., Volle, D.H., Mataki, C., Antal, M.C., Messaddeq, N., Auwerx, J., Murphy, B.D., Schoonjans, K., 2008. Liver receptor homolog 1 is essential for ovulation. Genes Dev. 22 (14), 1871–1876. Dupont, S., Krust, A., Gansmuller, A., Dierich, A., Chambon, P., Mark, M., 2000. Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. Development 127 (19), 4277–4291. Dussault, I., Fawcett, D., Matthyssen, A., Bader, J.A., Gigu`ere, V., 1998. Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer. Mech. Dev. 70 (1–2), 147–153. Emmen, J.M.A., Couse, J.F., Elmore, S.A., Yates, M.M., Kissling, G.E., Korach, K.S., 2005. In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER)α and ERβ null mice indicate a role for ERβ in follicular maturation. Endocrinology 146 (6), 2817–2826. Eppig, J.J., 1979. FSH stimulates hyaluronic acid synthesis by oocyte-cumulus cell complexes from mouse preovulatory follicles. Nature 281 (5731), 483–484. Eppig, J.J., Wigglesworth, K., Pendola, F., Hirao, Y., 1997. Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa Cells1. Biol. Reprod. 56 (4), 976–984. Espey, L.L., 1980. Ovulation as an inflammatory reaction—a hypothesis. Biol. Reprod. 22 (1), 73–106. Falender, A.E., Lanz, R., Malenfant, D., Belanger, L., Richards, J.S., 2003. Differential expression of steroidogenic factor-1 and FTF/LRH-1 in the rodent ovary. Endocrinology 144 (8), 3598–3610. Fan, H.Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P.F., Hedrick, S.M., Richards, J.S., 2009. MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science 324 (5929), 938–941. Fateh, M., Ben-Rafael, Z., Benadiva, C.A., Mastroianni, L., Flickinger, G.L., 1989. Cortisol levels in human follicular fluid. Fertil. Steril. 51 (3), 538–541. Fernandez, P.M., Brunel, F., Jimenez, M.A., Saez, J.M., Cereghini, S., Zakin, M.M., 2000. Nuclear receptors Nor1 and NGFI-B/Nur77 play similar, albeit distinct, roles in the hypothalamo-pituitary-adrenal Axis. Endocrinology 141 (7), 2392–2400. Festuccia, N., Owens, N., Navarro, P., 2018. Esrrb, an estrogen-related receptor involved in early development, pluripotency, and reprogramming. FEBS (Fed. Eur. Biochem. Soc.) Lett. 592 (6), 852–877. Flynn, M.P., Fiedler, S.E., Karlsson, A.B., Carr, D.W., Maizels, E.T., Hunzicker-Dunn, M., 2016. Dephosphorylation of MAP2D enhances its binding to vimentin in preovulatory ovarian granulosa cells. J. Cell Sci. 129 (15), 2983–2996. Forrest, D., Hanebuth, E., Smeyne, R.J., Everds, N., Stewart, C.L., Wehner, J.M., Curran, T., 1996. Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor beta: evidence for tissue-specific modulation of receptor function. EMBO J. 15 (12), 3006–3015. Fortune, J.E., Armstrong, D.T., 1977. Androgen production by theca and granulosa isolated from proestrous rat follicles. Endocrinology 100 (5), 1341–1347. Fortune, J.E., Armstrong, D.T., 1978. Hormonal control of 17β-estradiol biosynthesis in proestrous rat follicles: estradiol production by isolated theca versus granulosa. Endocrinology 102 (1), 227–235. Fraichard, A., Chassande, O., Plateroti, M., Roux, J.P., Trouillas, J., Dehay, C., Legrand, C., Gauthier, K., Kedinger, M., Malaval, L., Rousset, B., Samarut, J., 1997. The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO J. 16 (14), 4412–4420. Friberg, P.A., Larsson, D.G.J., Billig, H., 2010. Transcriptional effects of progesterone receptor antagonist in rat granulosa cells. Mol. Cell. Endocrinol. 315 (1–2), 121–130. Fru, K.N., VandeVoort, C.A., Chaffin, C.L., 2006. Mineralocorticoid synthesis during the periovulatory interval in macaques. Biol. Reprod. 75 (4), 568–574. Fujibe, Y., Baba, T., Nagao, S., Adachi, S., Ikeda, K., Morishita, M., Kuno, Y., Suzuki, M., Mizuuchi, M., Honnma, H., Endo, T., Saito, T., 2019. Androgen potentiates the expression of FSH receptor and supports preantral follicle development in mice. J. Ovarian Res. 12 (1), 31. Fül¨ op, C., Salustri, A., Hascall, V.C., 1997. Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Arch. Biochem. Biophys. 337 (2), 261–266. Fulop, C., Sz´ ant´ o, S., Mukhopadhyay, D., B´ ardos, T., Kamath, R.V., Rugg, M.S., Day, A.J., Salustri, A., Hascall, V.C., Glant, T.T., Mikecz, K., 2003. Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development 130 (10), 2253–2261.

19

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Funahashi, R., Sakamoto, T., Taguchi, N., Naiki, R., Terashima, R., Kawaminami, M., Kurusu, S., 2017. Possible role of PPARgamma in the negative regulation of ovulatory cascade and luteal development in rats. J. Vet. Med. Sci. 79 (6), 1043–1051. Gayt´ an, F., Bellido, C., Gayt´ an, M., Morales, C., S´ anchez-Criado, J.E., 2003. Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat. Biol. Reprod. 69 (1), 99–105. Gerdin, A.K., Surve, V.V., J¨ onsson, M., Bjursell, M., Bj¨ orkman, M., Edenro, A., Schuelke, M., Saad, A., Bjurstr¨ om, S., Lundgren, E.J., Snaith, M., Fransson-Steen, R., T¨ ornell, J., Berg, A.-L., Bohlooly-Y, M., 2006. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-γ receptor knockout mice. Biochem. Biophys. Res. Commun. 349 (2), 825–832. Gigu`ere, V., 1999. Orphan nuclear receptors: from gene to function. Endocr. Rev. 20 (5), 689–725. Gomez-Sanchez, E.P., Gomez-Sanchez, M.T., de Rodriguez, A.F., Romero, D.G., Warden, M.P., Plonczynski, M.W., Gomez-Sanchez, C.E., 2009. Immunohistochemical demonstration of the mineralocorticoid receptor, 11betahydroxysteroid dehydrogenase-1 and -2, and hexose-6-phosphate dehydrogenase in rat ovary. J. Histochem. Cytochem. 57 (7), 633–641. Grossman, H., Chuderland, D., Ninio-Many, L., Hasky, N., Kaplan-Kraicer, R., Shalgi, R., 2015. A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation. Faseb. J. 29 (8), 3206–3216. Gutierrez-Mazariegos, J., Schubert, M., Laudet, V., 2014. Evolution of retinoic acid receptors and retinoic acid signaling. Subcell. Biochem. 70, 55–73. Hasegawa, T., Kamada, Y., Hosoya, T., Fujita, S., Nishiyama, Y., Iwata, N., Hiramatsu, Y., Otsuka, F., 2017. A regulatory role of androgen in ovarian steroidogenesis by rat granulosa cells. J. Steroid Biochem. Mol. Biol. 172, 160–165. Havelock, J.C., Smith, A.L., Seely, J.B., Dooley, C.A., Rodgers, R.J., Rainey, W.E., Carr, B. R., 2005. The NGFI-B family of transcription factors regulates expression of 3betahydroxysteroid dehydrogenase type 2 in the human ovary. Mol. Hum. Reprod. 11 (2), 79–85. Hegele-Hartung, C., Siebel, P., Peters, O., Kosemund, D., Muller, G., Hillisch, A., Walter, A., Kraetzschmar, J., Fritzemeier, K.H., 2004. Impact of isotype-selective estrogen receptor agonists on ovarian function. Proc. Natl. Acad. Sci. Unit. States Am. 101 (14), 5129–5134. Hess, K.A., Chen, L., Larsen, W.J., 1999. Inter-α-Inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse Oocytes1. Biol. Reprod. 61 (2), 436–443. Hickey, T.E., Marrocco, D.L., Gilchrist, R.B., Norman, R.J., Armstrong, D.T., 2004. Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles. Biol. Reprod. 71 (1), 45–52. Hiebl, V., Ladurner, A., Latkolik, S., Dirsch, V.M., 2018. Natural products as modulators of the nuclear receptors and metabolic sensors LXR, FXR and RXR. Biotechnol. Adv. 36 (6), 1657–1698. Hild-Petito, S., Stouffer, R.L., Brenner, R.M., 1988. Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle*. Endocrinology 123 (6), 2896–2905. Hillier, S.G., Tetsuka, M., 1998. An anti-inflammatory role for glucocorticoids in the ovaries? J. Reprod. Immunol. 39 (1–2), 21–27. Hillier, S.G., Tetsuka, M., Fraser, H.M., 1997. Location and developmental regulation of androgen receptor in primate ovary. Hum. Reprod. 12 (1), 107–111. Hinshelwood, M.M., Shelton, J.M., Richardson, J.A., Mendelson, C.R., 2005. Temporal and spatial expression of liver receptor homologue-1 (LRH-1) during embryogenesis suggests a potential role in gonadal development. Dev. Dynam. 234 (1), 159–168. Hiroi, H., Christenson, L.K., Chang, L., Sammel, M.D., Berger, S.L., Strauss, J.F., 2004. Temporal and spatial changes in transcription factor binding and histone modifications at the steroidogenic acute regulatory protein (StAR) locus associated withStAR Transcription. Mol. Endocrinol. 18 (4), 791–806. Hizaki, H., Segi, E., Sugimoto, Y., Hirose, M., Saji, T., Ushikubi, F., Matsuoka, T., Noda, Y., Tanaka, T., Yoshida, N., Narumiya, S., Ichikawa, A., 1999. Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP2. Proc. Natl. Acad. Sci. Unit. States Am. 96 (18), 10501–10506. Hu, Y.C., Wang, P.H., Yeh, S., Wang, R.S., Xie, C., Xu, Q., Zhou, X., Chao, H.T., Tsai, M.Y., Chang, C., 2004. Subfertility and defective folliculogenesis in female mice lacking androgen receptor. Proc. Natl. Acad. Sci. Unit. States Am. 101 (31), 11209–11214. Iwamasa, J., Shibata, S., Tanaka, N., Matsuura, K., Okamura, H., 1992. The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat. Biol. Reprod. 46 (2), 309–313. Jeppesen, J.V., Kristensen, S.G., Nielsen, M.E., Humaidan, P., Dal Canto, M., Fadini, R., Schmidt, K.T., Ernst, E., Yding Andersen, C., 2012. LH-receptor gene expression in human granulosa and cumulus cells from antral and preovulatory follicles. J. Clin. Endocrinol. Metab. 97 (8), E1524–E1531. Jeyasuria, P., Ikeda, Y., Jamin, S.P., Zhao, L., De Rooij, D.G., Themmen, A.P., Behringer, R.R., Parker, K.L., 2004. Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function. Mol. Endocrinol. 18 (7), 1610–1619. Johnson, L.E., Deluca, H.F., 2001. Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J. Nutr. 131 (6), 1787–1791. Kanayama, K., Sankai, T., Nariai, K., Endo, T., Sakuma, Y., 1994. Effects of antiprogesterone compound RU486 on ovulation in immature mice treated with PMSG/ hCG. Res. Exp. Med. 194 (1), 217–220. Kastner, P., Grondona, J.M., Mark, M., Gansmuller, A., Lemeur, M., Decimo, D., Vonesch, J.-L., Doll´ e, P., Chambon, P., 1994. Genetic analysis of RXRα developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell 78 (6), 987–1003.

Kastner, P., Mark, M., Leid, M., Gansmuller, A., Chin, W., Grondona, J.M., Decimo, D., Krezel, W., Dierich, A., Chambon, P., 1996. Abnormal spermatogenesis in RXR beta mutant mice. Genes Dev. 10 (1), 80–92. Kawai, T., Yanaka, N., Richards, J.S., Shimada, M., 2016. De novo-synthesized retinoic acid in ovarian antral follicles enhances FSH-mediated ovarian follicular cell differentiation and female fertility. Endocrinology 157 (5), 2160–2172. Kawai, T., Richards, J.S., Shimada, M., 2018. The cell type–specific expression of lhcgr in mouse ovarian cells: evidence for a DNA-demethylation–dependent mechanism. Endocrinology 159 (5), 2062–2074. ´ Vigneault, C., Blondin, P., Sirard, M.-A., 2016. Khan, D.R., Landry, D.A., Fournier, E., Transcriptome meta-analysis of three follicular compartments and its correlation with ovarian follicle maturity and oocyte developmental competence in cows. Physiol. Genom. 48 (8), 633–643. Khristi, V., Chakravarthi, V.P., Singh, P., Ghosh, S., Pramanik, A., Ratri, A., Borosha, S., Roby, K.F., Wolfe, M.W., Rumi, M.A.K., 2018. ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation. Mol. Cell. Endocrinol. 474, 214–226. Kim, J.W., Peng, N., Rainey, W.E., Carr, B.R., Attia, G.R., 2004. Liver receptor homolog-1 regulates the expression of steroidogenic acute regulatory protein in human granulosa cells. J. Clin. Endocrinol. Metab. 89 (6), 3042–3047. Kim, J., Sato, M., Li, Q., Lydon, J.P., Demayo, F.J., Bagchi, I.C., Bagchi, M.K., 2008. Peroxisome proliferator-activated receptor is a target of progesterone regulation in the preovulatory follicles and controls ovulation in mice. Mol. Cell Biol. 28 (5), 1770–1782. Kim, J., Bagchi, I.C., Bagchi, M.K., 2009a. Control of ovulation in mice by progesterone receptor-regulated gene networks. Mol. Hum. Reprod. 15 (12), 821–828. Kim, J., Bagchi, I.C., Bagchi, M.K., 2009b. Signaling by hypoxia-inducible factors is critical for ovulation in mice. Endocrinology 150 (7), 3392–3400. Kinuta, K., Tanaka, H., Moriwake, T., Aya, K., Kato, S., Seino, Y., 2000. Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology 141 (4), 1317–1324. Kipp, J.L., Golebiowski, A., Rodriguez, G., Demczuk, M., Kilen, S.M., Mayo, K.E., 2011. Gene expression profiling reveals Cyp26b1 to be an activin regulated gene involved in ovarian granulosa cell proliferation. Endocrinology 152 (1), 303–312. Kitasaka, H., Kawai, T., Hoque, S.A.M., Umehara, T., Fujita, Y., Shimada, M., 2018. Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice. PloS One 13 (2), e0192458. Koike, S., Sakai, M., Muramatsu, M., 1987. Molecular cloning and characterization of rat estrogen receptor cDNA. Nucleic Acids Res. 15 (6), 2499–2513. Komar, C.M., Braissant, O., Wahli, W., Curry Jr., T.E., 2001. Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period. Endocrinology 142 (11), 4831–4838. Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., Warner, M., Mahler, J.F., Sar, M., Korach, K.S., Gustafsson, J.A., Smithies, O., 1998. Generation and reproductive phenotypes of mice lacking estrogen receptor. Proc. Natl. Acad. Sci. Unit. States Am. 95 (26), 15677–15682. Krezel, W., Dupe, V., Mark, M., Dierich, A., Kastner, P., Chambon, P., 1996. RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable. Proc. Natl. Acad. Sci. Unit. States Am. 93 (17), 9010–9014. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., Gustafsson, J.A., 1996. Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. Unit. States Am. 93 (12), 5925–5930. Kuiper, G.G.J.M., Carlsson, B., Grandien, K., Enmark, E., H¨ aGgblad, J., Nilsson, S., Gustafsson, J.-A.K., 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology 138 (3), 863–870. Labelle-Dumais, C., Jacob-Wagner, M., Par´e, J.-F., B´elanger, L., Dufort, D., 2006. Nuclear receptor NR5A2 is required for proper primitive streak morphogenesis. Dev. Dynam. 235 (12), 3359–3369. Labelle-Dumais, C., Par´e, J.-F., B´elanger, L., Farookhi, R., Dufort, D., 2007. Impaired progesterone production in Nr5a2+/− mice leads to a reduction in female reproductive function. Biol. Reprod. 77 (2), 217–225. Laird, M., Thomson, K., Fenwick, M., Mora, J., Franks, S., Hardy, K., 2017. Androgen stimulates growth of mouse preantral follicles in vitro: interaction with folliclestimulating hormone and with growth factors of the TGFbeta superfamily. Endocrinology 158 (4), 920–935. Lan, Z.J., Gu, P., Xu, X., Jackson, K.J., DeMayo, F.J., O’Malley, B.W., Cooney, A.J., 2003. GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility. EMBO J. 22 (16), 4070–4081. Lebbe, M., Taylor, A.E., Visser, J.A., Kirkman-Brown, J.C., Woodruff, T.K., Arlt, W., 2017. The steroid metabolome in the isolated ovarian follicle and its response to androgen exposure and antagonism. Endocrinology 158 (5), 1474–1485. Ledger, W.L., Sweeting, V.M., Hillier, H., Baird, D.T., 1992. Inhibition of ovulation by low-dose mifepristone (RU 486). Hum. Reprod. 7 (7), 945–950. Lee, S.L., Wesselschmidt, R.L., Linette, G.P., Kanagawa, O., Russell, J.H., Milbrandt, J., 1995a. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science 269 (5223), 532–535. Lee, S.S., Pineau, T., Drago, J., Lee, E.J., Owens, J.W., Kroetz, D.L., FernandezSalguero, P.M., Westphal, H., Gonzalez, F.J., 1995b. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell Biol. 15 (6), 3012–3022. Li, Q.X., Ke, N., Sundaram, R., Wong-Staal, F., 2006. NR4A1, 2, 3–an orphan nuclear hormone receptor family involved in cell apoptosis and carcinogenesis. Histol. Histopathol. 21 (5), 533–540.

20

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx Minge, C.E., Bennett, B.D., Norman, R.J., Robker, R.L., 2008. Peroxisome proliferatoractivated receptor-gamma agonist rosiglitazone reverses the adverse effects of dietinduced obesity on oocyte quality. Endocrinology 149 (5), 2646–2656. Minkina, A., Lindeman, R.E., Gearhart, M.D., Chassot, A.A., Chaboissier, M.C., Ghyselinck, N.B., Bardwell, V.J., Zarkower, D., 2017. Retinoic acid signaling is dispensable for somatic development and function in the mammalian ovary. Dev. Biol. 424 (2), 208–220. Mitsunaga, K., Araki, K., Mizusaki, H., Morohashi, K., Haruna, K., Nakagata, N., Giguere, V., Yamamura, K., Abe, K., 2004. Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos. Mech. Dev. 121 (3), 237–246. Mohan, M., Thirumalapura, N., Malayer, J., 2003. Bovine cumulus-granulosa cells contain biologically active retinoid receptors that can respond to retinoic aci. Reprod. Biol. Endocrinol. 1 (1), 104. Molinari, E., Bar, H., Pyle, A.M., Patrizio, P., 2016. Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence. Mol. Hum. Reprod. 22 (8), 866–876. Monaghan, A.P., Bock, D., Gass, P., Schwger, A., Wolfer, D.P., Lipp, H.P., Schütz, G., 1997. Defective limbic system in mice lacking the tailless gene. Nature 390 (6659), 515–517. Morgan, A., Keeble, S.C., London, S.N., Muse, K.N., Curry Jr., T.E., 1994. Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells. Fertil. Steril. 61 (5), 949–955. Mosselman, S., Polman, J., Dijkema, R., 1996. ERβ: identification and characterization of a novel human estrogen receptor. FEBS (Fed. Eur. Biochem. Soc.) Lett. 392 (1), 49–53. Mouzat, K., Volat, F., Baron, S., Alves, G., Pommier, A.J., Volle, D.H., Marceau, G., DeHaze, A., Dechelotte, P., Duggavathi, R., Caira, F., Lobaccaro, J.M., 2009. Absence of nuclear receptors for oxysterols liver X receptor induces ovarian hyperstimulation syndrome in mice. Endocrinology 150 (7), 3369–3375. Mukangwa, M., Takizawa, K., Aoki, Y., Hamano, S., Tetsuka, M., 2020. Expression of genes encoding mineralocorticoid biosynthetic enzymes and the mineralocorticoid receptor, and levels of mineralocorticoids in the bovine follicle and corpus luteum. J. Reprod. Dev. 66 (1), 75–81. Mulac-Jericevic, B., Mullinax, R.A., DeMayo, F.J., Lydon, J.P., Conneely, O.M., 2000. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 289 (5485), 1751–1754. Mulac-Jericevic, B., Lydon, J.P., Demayo, F.J., Conneely, O.M., 2003. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc. Natl. Acad. Sci. Unit. States Am. 100 (17), 9744–9749. Murayama, C., Miyazaki, H., Miyamoto, A., Shimizu, T., 2008. Involvement of Ad4BP/ SF-1, DAX-1, and COUP-TFII transcription factor on steroid production and luteinization in ovarian theca cells. Mol. Cell. Biochem. 314 (1–2), 51–58. Murdoch, W.J., Peterson, T.A., Van Kirk, E.A., Vincent, D.L., Inskeep, E.K., 1986. Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the Ewe. Biol. Reprod. 35 (5), 1187–1194. Murray, A.A., Gosden, R.G., Allison, V., Spears, N., 1998. Effect of androgens on the development of mouse follicles growing in vitro. Reproduction 113 (1), 27–33. Olivares, A.M., Han, Y., Soto, D., Flattery, K., Marini, J., Mollema, N., Haider, A., Escher, P., Deangelis, M.M., Haider, N.B., 2017. The nuclear hormone receptor gene Nr2c1 (Tr2) is a critical regulator of early retina cell patterning. Dev. Biol. 429 (1), 343–355. Orisaka, M., Jiang, J.Y., Orisaka, S., Kotsuji, F., Tsang, B.K., 2009. Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis. Endocrinology 150 (6), 2740–2748. O’Malley, B.W., 2005. A life-long search for the molecular pathways of steroid hormone action. Mol. Endocrinol. 19 (6), 1402–1411. Palanisamy, G.S., Cheon, Y.-P., Kim, J., Kannan, A., Li, Q., Sato, M., Mantena, S.R., Sitruk-Ware, R.L., Bagchi, M.K., Bagchi, I.C., 2006. A novel pathway involving progesterone receptor, endothelin-2, and endothelin receptor B controls ovulation in mice. Mol. Endocrinol. 20 (11), 2784–2795. Par´ e, J.-F., Malenfant, D., Courtemanche, C., Jacob-Wagner, M., Roy, S., Allard, D., B´ elanger, L., 2004. The fetoprotein transcription factor (FTF) gene is essential to embryogenesis and cholesterol homeostasis and is regulated by a DR4 element. J. Biol. Chem. 279 (20), 21206–21216. Park, O.-K., Mayo, K.E., 1991. Transient expression of progesterone receptor messenger RNA in ovarian granuiosa cells after the preovulatory luteinizing hormone surge. Mol. Endocrinol. 5 (7), 967–978. Park, J.-I., Park, H.-J., Choi, H.-S., Lee, K., Lee, W.-K., Chun, S.-Y., 2001. Gonadotropin regulation of NGFI-B messenger ribonucleic acid expression during ovarian follicle development in the rat. Endocrinology 142 (7), 3051–3059. Park, J.-I., Park, H.-J., Lee, Y.-I., Seo, Y.-M., Chun, S.-Y., 2003. Regulation of NGFI-B expression during the ovulatory process. Mol. Cell. Endocrinol. 202 (1–2), 25–29. Park, J.Y., Su, Y.-Q., Ariga, M., Law, E., Jin, S.-L.C., Conti, M., 2004. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303 (5658), 682–684. Park, J.I., Kim, S.G., Chun, J.S., Seo, Y.M., Jeon, M.J., Ohba, M., Kim, H.J., Chun, S.Y., 2007. Activation of protein kinase Czeta mediates luteinizing hormone- or forskolininduced NGFI-B expression in preovulatory granulosa cells of rat ovary. Mol. Cell. Endocrinol. 270 (1–2), 79–86. Park, C.J., Lin, P.C., Zhou, S., Barakat, R., Bashir, S.T., Choi, J.M., Cacioppo, J.A., Oakley, O.R., Duffy, D.M., Lydon, J.P., Ko, C.J., 2020. Progesterone receptor serves the ovary as a trigger of ovulation and a terminator of inflammation. Cell Rep. 31 (2), 107496.

Lim, H., Paria, B.C., Das, S.K., Dinchuk, J.E., Langenbach, R., Trzaskos, J.M., Dey, S.K., 1997. Multiple female reproductive failures in cyclooxygenase 2–deficient mice. Cell 91 (2), 197–208. Lim, J.J., Lima, P.D.A., Salehi, R., Lee, D.R., Tsang, B.K., 2017. Regulation of androgen receptor signaling by ubiquitination during folliculogenesis and its possible dysregulation in polycystic ovarian syndrome. Sci. Rep. 7 (1), 10272. Lin, S.J., Yang, D.R., Yang, G., Lin, C.Y., Chang, H.C., Li, G., Chang, C., 2017. TR2 and TR4 orphan nuclear receptors: an overview. Curr. Top. Dev. Biol. 125, 357–373. Liu, Y.-X., Hsueh, A.J.W., 1986. Synergism between granulosa and theca-interstitial cells in estrogen biosynthesis by gonadotropin-treated rat ovaries: studies on the two-cell, two-gonadotropin hypothesis using steroid Antisera1. Biol. Reprod. 35 (1), 27–36. Liu, Y., Johnson, B.P., Shen, A.L., Wallisser, J.A., Krentz, K.J., Moran, S.M., Sullivan, R., Glover, E., Parlow, A.F., Drinkwater, N.R., Schuler, L.A., Bradfield, C.A., 2014. Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice. Proc. Natl. Acad. Sci. Unit. States Am. 111 (39), 14295–14300. Liu, W., Xin, Q., Wang, X., Wang, S., Wang, H., Zhang, W., Yang, Y., Zhang, Y., Zhang, Z., Wang, C., Xu, Y., Duan, E., Xia, G., 2017. Estrogen receptors in granulosa cells govern meiotic resumption of pre-ovulatory oocytes in mammals. Cell Death Dis. 8 (3), e2662. Liu, Z., Sun, Y., Jiang, Y., Qian, Y., Chen, S., Gao, S., Chen, L., Li, C., Zhou, X., 2018. Follicle-stimulating hormone (FSH) promotes retinol uptake and metabolism in the mouse ovary. Reprod. Biol. Endocrinol. 16 (1). Lohnes, D., Kastner, P., Dierich, A., Mark, M., Lemeur, M., Chambon, P., 1993. Function of retinoic acid receptor γ in the mouse. Cell 73 (4), 643–658. Loutradis, D., Bletsa, R., Aravantinos, L., Kallianidis, K., Michalas, S., Psychoyos, A., 1991. Preovulatory effects of the progesterone antagonist mifepristone (RU486) in mice. Hum. Reprod. 6 (9), 1238–1240. Lubahn, D.B., Moyer, J.S., Golding, T.S., Couse, J.F., Korach, K.S., Smithies, O., 1993. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc. Natl. Acad. Sci. Unit. States Am. 90 (23), 11162–11166. Lufkin, T., Lohnes, D., Mark, M., Dierich, A., Gorry, P., Gaub, M.P., Lemeur, M., Chambon, P., 1993. High postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice. Proc. Natl. Acad. Sci. Unit. States Am. 90 (15), 7225–7229. Luo, X., Ikeda, Y., Parker, K.L., 1994. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77 (4), 481–490. Luo, J., Pasceri, P., Conlon, R.A., Rossant, J., Gigu`ere, V., 1995. Mice lacking all isoforms of retinoic acid receptor β develop normally and are susceptible to the teratogenic effects of retinoic acid. Mech. Dev. 53 (1), 61–71. Luo, J., Sladek, R., Bader, J.A., Matthyssen, A., Rossant, J., Gigu`ere, V., 1997. Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388 (6644), 778–782. Luo, J., Sladek, R., Carrier, J., Bader, J.A., Richard, D., Giguere, V., 2003. Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha. Mol. Cell Biol. 23 (22), 7947–7956. Lydon, J.P., Demayo, F.J., Funk, C.R., Mani, S.K., Hughes, A.R., Montgomery, C.A., Shyamala, G., Conneely, O.M., O’Malley, B.W., 1995. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 9 (18), 2266–2278. Lydon, J.P., Demayo, F.J., Conneely, O.M., O’Malley, B.W., 1996. Reproductive phenotypes of the progesterone receptor null mutant mouse. J. Steroid Biochem. Mol. Biol. 56 (1–6), 67–77. Makris, A., Ryan, K.J., 1975. Progesterone, androstenedione, testosterone, estrone, and estradiol synthesis in hamster ovarian follicle cells. Endocrinology 96 (3), 694–701. Maneix, L., Antonson, P., Humire, P., Rochel-Maia, S., Casta˜ neda, J., Omoto, Y., Kim, H.J., Warner, M., Gustafsson, J.-Å., 2015. Estrogen receptor β exon 3-deleted mouse: the importance of non-ERE pathways in ERβ signaling. Proc. Natl. Acad. Sci. Unit. States Am. 112 (16), 5135–5140. Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., Evans, R.M., 1995. The nuclear receptor superfamily: the second decade. Cell 83 (6), 835–839. Matsuwaki, T., Nishihara, M., Sato, T., Yoda, T., Iwakura, Y., Chida, D., 2010. Functional hypothalamic amenorrhea due to increased CRH tone in melanocortin receptor 2deficient mice. Endocrinology 151 (11), 5489–5496. Meinsohn, M.C., Morin, F., Bertolin, K., Duggavathi, R., Schoonjans, K., Murphy, B.D., 2018. The orphan nuclear receptor liver homolog receptor-1 (Nr5a2) regulates ovarian granulosa cell proliferation. J. Endocr. Soc. 2 (1), 24–41. Meinsohn, M.C., Smith, O.E., Bertolin, K., Murphy, B.D., 2019. The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiol. Rev. 99 (2), 1249–1279. Mendelson, C.R., Jiang, B., Shelton, J.M., Richardson, J.A., Hinshelwood, M.M., 2005. Transcriptional regulation of aromatase in placenta and ovary. J. Steroid Biochem. Mol. Biol. 95 (1–5), 25–33. Minegishi, T., Karino, S., Tano, M., Ibuki, Y., Miyamoto, K., 1996. Regulation of midkine messenger ribonucleic acid levels in cultured rat granulosa cells. Biochem. Biophys. Res. Commun. 229 (3), 799–805. Minegishi, T., Hirakawa, T., Kishi, H., Abe, K., Ibuki, Y., Miyamoto, K., 2000a. Retinoic acid (RA) represses follicle stimulating hormone (FSH)-Induced luteinizing hormone (LH) receptor in rat granulosa cells. Arch. Biochem. Biophys. 373 (1), 203–210. Minegishi, T., Hirakawa, T., Kishi, H., Abe, K., Tano, M., Abe, Y., Miyamoto, K., 2000b. The mechanisms of retinoic acid-induced regulation on the follicle-stimulating hormone receptor in rat granulosa cells. Biochim. Biophys. Acta Mol. Cell Res. 1495 (3), 203–211.

21

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

Pelusi, C., Ikeda, Y., Zubair, M., Parker, K.L., 2008. Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa Cells1. Biol. Reprod. 79 (6), 1074–1083. Peluso, J.J., 2003. Progesterone as a regulator of granulosa cell viability. J. Steroid Biochem. Mol. Biol. 85 (2–5), 167–173. Peluso, J.J., Pappalardo, A., 1998. Progesterone mediates its anti-mitogenic and antiapoptotic actions in rat granulosa cells through a progesterone-binding protein with gamma aminobutyric AcidA receptor-like features. Biol. Reprod. 58 (5), 1131–1137. Peng, X.-R., Hsueh, A.J.W., Lapolt, P.S., Bjersing, L., Ny, T., 1991. Localization of luteinizing hormone receptor messenger ribonucleic acid expression in ovarian cell types during follicle development and ovulation*. Endocrinology 129 (6), 3200–3207. Peng, N., Kim, J.W., Rainey, W.E., Carr, B.R., Attia, G.R., 2003. The role of the orphan nuclear receptor, liver receptor homologue-1, in the regulation of human corpus luteum 3beta-hydroxysteroid dehydrogenase type II. J. Clin. Endocrinol. Metab. 88 (12), 6020–6028. Peng, J., Li, Q., Wigglesworth, K., Rangarajan, A., Kattamuri, C., Peterson, R.T., Eppig, J. J., Thompson, T.B., Matzuk, M.M., 2013. Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions. Proc. Natl. Acad. Sci. Unit. States Am. 110 (8), E776–E785. Pereira, F.A., Qiu, Y., Zhou, G., Tsai, M.J., Tsai, S.Y., 1999. The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. Genes Dev. 13 (8), 1037–1049. Pereira, F.A., Tsai, M.J., Tsai*, S.Y., 2000. COUP-TF orphan nuclear receptors in development and differentiation. Cell. Mol. Life Sci. 57 (10), 1388–1398. Peters, J.M., Lee, S.S., Li, W., Ward, J.M., Gavrilova, O., Everett, C., Reitman, M.L., Hudson, L.D., Gonzalez, F.J., 2000. Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol. Cell Biol. 20 (14), 5119–5128. Petit, F.G., Jamin, S.P., Kurihara, I., Behringer, R.R., Demayo, F.J., Tsai, M.J., Tsai, S.Y., 2007. Deletion of the orphan nuclear receptor COUP-TFII in uterus leads to placental deficiency. Proc. Natl. Acad. Sci. Unit. States Am. 104 (15), 6293–6298. Plant, T.M., 2015. 60 years OF neuroendocrinology: the hypothalamo-pituitary–gonadal axis. J. Endocrinol. 226 (2), T41–T54. Ponnio, T., Burton, Q., Pereira, F.A., Wu, D.K., Conneely, O.M., 2002. The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of the mouse inner ear. Mol. Cell Biol. 22 (3), 935–945. Preitner, N., Damiola, F., Luis Lopez, M., Zakany, J., Duboule, D., Albrecht, U., Schibler, U., 2002. The orphan nuclear receptor REV-erbα controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110 (2), 251–260. Puttabyatappa, M., Vandevoort, C.A., Chaffin, C.L., 2010. hCG-induced down-regulation of PPARgamma and liver X receptors promotes periovulatory progesterone synthesis by macaque granulosa cells. Endocrinology 151 (12), 5865–5872. Qiu, Y., Pereira, F.A., Demayo, F.J., Lydon, J.P., Tsai, S.Y., Tsai, M.J., 1997. Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization. Genes Dev. 11 (15), 1925–1937. Rae, M.T., Price, D., Harlow, C.R., Critchley, H.O.D., Hillier, S.G., 2009. Glucocorticoid receptor-mediated regulation of MMP9 gene expression in human ovarian surface epithelial cells. Fertil. Steril. 92 (2), 703–708. Read, C.C., Dyce, P.W., 2019. All-trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes. Mol. Reprod. Dev. 86 (12), 1865–1873. Repa, J.J., Mangelsdorf, D.J., 2000. The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu. Rev. Cell Dev. Biol. 16 (1), 459–481. Richards, J.S., Ireland, J.J., Rao, M.C., Bernath, G.A., Midgley, A.R., Reichert, L.E., 1976. Ovarian follicular development in the rat: hormone receptor regulation by estradiol. Follicle Stimulating Hormone and Luteinizing Hormone 1 (6), 1562–1570. Richards, J.S., Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Teuling, E., Lo, Y., Boerboom, D., Falender, A.E., Doyle, K.H., Lebaron, R.G., Thompson, V., Sandy, J.D., 2005. Regulated expression of ADAMTS family members in follicles and cumulus oocyte complexes: evidence for specific and redundant patterns during Ovulation1. Biol. Reprod. 72 (5), 1241–1255. Robker, R.L., Richards, J.S., 1998. Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biol. Reprod. 59 (3), 476–482. Robker, R.L., Russell, D.L., Espey, L.L., Lydon, J.P., O’Malley, B.W., Richards, J.S., 2000. Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases. Proc. Natl. Acad. Sci. Unit. States Am. 97 (9), 4689–4694. Robker, R.L., Akison, L.K., Russell, D.L., 2009. Control of oocyte release by progesterone receptor-regulated gene expression. Nucl. Recept. Signal. 7 (1), 07012. Robker, R.L., Hennebold, J.D., Russell, D.L., 2018. Coordination of ovulation and oocyte maturation: a good egg at the right time. Endocrinology 159 (9), 3209–3218. Rodriguez, K.F., Couse, J.F., Jayes, F.L., Hamilton, K.J., Burns, K.A., Taniguchi, F., Korach, K.S., 2010. Insufficient luteinizing hormone-induced intracellular signaling disrupts ovulation in preovulatory follicles lacking estrogen receptor-beta. Endocrinology 151 (6), 2826–2834. Rosenfeld, C.S., Murray, A.A., Simmer, G., Hufford, M.G., Smith, M.F., Spears, N., Lubahn, D.B., 2000. Gonadotropin induction of ovulation and corpus luteum formation in Young estrogen receptor-α knockout Mice1. Biol. Reprod. 62 (3), 599–605. Rovani, M.T., Gasperin, B.G., Ilha, G.F., Ferreira, R., Bohrer, R.C., Duggavathi, R., Bordignon, V., Gonçalves, P.B.D., 2014. Expression and molecular consequences of inhibition of estrogen receptors in granulosa cells of bovine follicles. J. Ovarian Res. 7 (1). Ruiz-Cort´ es, Z.T., Kimmins, S., Monaco, L., Burns, K.H., Sassone-Corsi, P., Murphy, B.D., 2005. Estrogen mediates phosphorylation of histone H3 in ovarian follicle and

mammary epithelial tumor cells via the mitotic kinase. Aurora B. Mol. Endocrinol. 19 (12), 2991–3000. Rumi, M.A.K., Singh, P., Roby, K.F., Zhao, X., Iqbal, K., Ratri, A., Lei, T., Cui, W., Borosha, S., Dhakal, P., Kubota, K., Chakraborty, D., Vivian, J.L., Wolfe, M.W., Soares, M.J., 2017. Defining the role of estrogen receptor β in the regulation of female fertility. Endocrinology 158 (7), 2330–2343. Russell, D.L., Ochsner, S.A., Hsieh, M., Mulders, S., Richards, J.S., 2003. Hormoneregulated expression and localization of versican in the rodent ovary. Endocrinology 144 (3), 1020–1031. Ryan, K.J., Petro, Z., Kaiser, J., 1968. Steroid formation by isolated and recombined ovarian granulosa and thecal cells. J. Clin. Endocrinol. Metabol. 28 (3), 355–358. Sablin, E.P., Woods, A., Krylova, I.N., Hwang, P., Ingraham, H.A., Fletterick, R.J., 2008. The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1. Proc. Natl. Acad. Sci. Unit. States Am. 105 (47), 18390–18395. Salustri, A., Garlanda, C., Hirsch, E., De Acetis, M., Maccagno, A., Bottazzi, B., Doni, A., Bastone, A., Mantovani, G., Peccoz, P.B., Salvatori, G., Mahoney, D.J., Day, A.J., Siracusa, G., Romani, L., Mantovani, A., 2004. PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. Development 131 (7), 1577–1586. Sar, M., Welsch, F., 1999. Differential expression of estrogen receptor-β and estrogen receptor-α in the rat ovary. Endocrinology 140 (2), 963–971. Sato, Y., Suzuki, T., Hidaka, K., Sato, H., Ito, K., Ito, S., Sasano, H., 2003. Immunolocalization of nuclear transcription factors, DAX-1 and COUP-TF II, in the normal human ovary: correlation with adrenal 4 binding protein/steroidogenic factor-1 immunolocalization during the menstrual cycle. J. Clin. Endocrinol. Metabol. 88 (7), 3415–3420. Saunders, P.T.K., Millar, M.R., Williams, K., Macpherson, S., Harkiss, D., Anderson, R.A., Orr, B., Groome, N.P., Scobie, G., Fraser, H.M., 2000. Differential expression of estrogen receptor-α and -β and androgen receptor in the ovaries of marmosets and humans. Biol. Reprod. 63 (4), 1098–1105. Saxena, D., Safi, R., Little-Ihrig, L., Zeleznik, A.J., 2004. Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 145 (8), 3821–3829. Schreiber, J.R., Nakamura, K., Erickson, G.F., 1982. Rat ovary glucocorticoid receptor: identification and characterization. Steroids 39 (5), 569–584. Schweigert, F.J., Siegling, C., 2001. Immunolocalization of retinol-binding protein, cellular retinoic acid-binding protein I and retinoid X receptor beta in the porcine reproductive tract during the oestrous cycle. Reprod. Fertil. Dev. 13 (5–6), 421–426. Sen, A., Hammes, S.R., 2010. Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Mol. Endocrinol. 24 (7), 1393–1403. Sewer, M.B., Li, D., 2008. Regulation of steroid hormone biosynthesis by the cytoskeleton. Lipids 43 (12), 1109. Shen, W.-J., Zaidi, S.K., Patel, S., Cortez, Y., Ueno, M., Azhar, R., Azhar, S., Kraemer, F.B., 2012. Ablation of vimentin results in defective steroidogenesis. Endocrinology 153 (7), 3249–3257. Shimada, M., Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Richards, J.S., 2006. Paracrine and autocrine regulation of epidermal growth factor-like factors in cumulus oocyte complexes and granulosa cells: key roles for prostaglandin synthase 2 and progesterone receptor. Mol. Endocrinol. 20 (6), 1352–1365. Shimada, M., Yanai, Y., Okazaki, T., Yamashita, Y., Sriraman, V., Wilson, M.C., Richards, J.S., 2007. Synaptosomal-associated protein 25 gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells. Mol. Endocrinol. 21 (10), 2487–2502. Shimizu, T., Sudo, N., Yamashita, H., Murayama, C., Miyazaki, H., Miyamoto, A., 2009. Histone H3 acetylation of StAR and decrease in DAX-1 is involved in the luteinization of bovine granulosa cells during in vitro culture. Mol. Cell. Biochem. 328 (1–2), 41–47. Short, R.V., 1962. Steroids in the follicular fluid and the corpus luteum of the mare. A ’two-cell type’ theory of ovarian steroid synthesis. J. Endocrinol. 24, 59–63. Silva, C.C., Knight, P.G., 2000. Effects of androgens, progesterone and their antagonists on the developmental competence of in vitro matured bovine oocytes. J. Reprod. Fertil. 119 (2), 261–269. Simon, J.M., Giresi, P.G., Davis, I.J., Lieb, J.D., 2012. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nat. Protoc. 7 (2), 256–267. Sinal, C.J., Tohkin, M., Miyata, M., Ward, J.M., Lambert, G., Gonzalez, F.J., 2000. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 102 (6), 731–744. Sneeringer, R., Penzias, A.S., Barrett, B., Usheva, A., 2011. High levels of mineralocorticoids in preovulatory follicular fluid could contribute to oocyte development. Fertil. Steril. 95 (1), 182–187. Sriraman, V., Eichenlaub-Ritter, U., Bartsch, J.W., Rittger, A., Mulders, S.M., Richards, J. S., 2008. Regulated expression of ADAM8 (a disintegrin and metalloprotease domain 8) in the mouse ovary: evidence for a regulatory role of luteinizing hormone, progesterone receptor, and epidermal growth factor-like growth Factors1. Biol. Reprod. 78 (6), 1038–1048. Steffensen, K.R., Robertson, K., Gustafsson, J.A., Andersen, C.Y., 2006. Reduced fertility and inability of oocytes to resume meiosis in mice deficient of the Lxr genes. Mol. Cell. Endocrinol. 256 (1–2), 9–16. Su, Y.Q., Sugiura, K., Wigglesworth, K., O’Brien, M.J., Affourtit, J.P., Pangas, S.A., Matzuk, M.M., Eppig, J.J., 2007. Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells. Development 135 (1), 111–121. Sugiura, K., Su, Y.Q., Diaz, F.J., Pangas, S.A., Sharma, S., Wigglesworth, K., O’Brien, M. J., Matzuk, M.M., Shimasaki, S., Eppig, J.J., 2007. Oocyte-derived BMP15 and FGFs

22

C.H.K. Hughes and B.D. Murphy

Molecular Aspects of Medicine xxx (xxxx) xxx

cooperate to promote glycolysis in cumulus cells. Development 134 (14), 2593–2603. Sugiura, K., Su, Y.-Q., Eppig, J.J., 2009. Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression. Mol. Reprod. Dev. 76 (6), 537–547. Sugiura, K., Su, Y.Q., Li, Q., Wigglesworth, K., Matzuk, M.M., Eppig, J.J., 2010. Estrogen promotes the development of mouse cumulus cells in coordination with oocytederived GDF9 and BMP15. Mol. Endocrinol. 24 (12), 2303–2314. Sun, Z., Unutmaz, D., Zou, Y.-R., Sunshine, M.J., Pierani, A., Brenner-Morton, S., Mebius, R.E., Littman, D.R., 2000. Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288 (5475), 2369–2373. Svensson, E.C., Markstr¨ om, E., Andersson, M., Billig, H., 2000. Progesterone receptormediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin. Biol. Reprod. 63 (5), 1457–1464. Szoltys, M., Slomczynska, M., Duda, M., Sakiewicz, A., Otak, A., 2005. Distribution of androgen receptor in rat ovarian follicles undergoing atresia at the beginning of pregnancy. Acta Histochem. 107 (5), 357–364. Takae, K., Nakata, M., Watanabe, T., Sasada, H., Fujii, H., Tomioka, I., 2019. Evidence for the involvement of FXR signaling in ovarian granulosa cell function. J. Reprod. Dev. 65 (1), 47–55. Takamoto, N., Kurihara, I., Lee, K., Demayo, F.J., Tsai, M.-J., Tsai, S.Y., 2005. Haploinsufficiency of chicken ovalbumin upstream promoter transcription factor II in female reproduction. Mol. Endocrinol. 19 (9), 2299–2308. Tanida, T., Matsuda, K.I., Yamada, S., Hashimoto, T., Kawata, M., 2015. Estrogen-related receptor β reduces the subnuclear mobility of estrogen receptor α and suppresses estrogen-dependent cellular function. J. Biol. Chem. 290 (19), 12332–12345. Tao, T., Wang, Y., Xu, B., Mao, X., Sun, Y., Liu, W., 2019. Role of adiponectin/ peroxisome proliferator-activated receptor alpha signaling in human chorionic gonadotropin-induced estradiol synthesis in human luteinized granulosa cells. Mol. Cell. Endocrinol. 493, 110450. Tatone, C., Benedetti, E., Vitti, M., Di Emidio, G., Ciriminna, R., Vento, M.E., Cela, V., Borzi, P., Carta, G., Lispi, M., Cimini, A.M., Artini, P.G., Italian Society of Embryology, R., Research, 2016. Modulating intrafollicular hormonal milieu in controlled ovarian stimulation: insights from PPAR expression in human granulosa cells. J. Cell. Physiol. 231 (4), 908–914. Tetsuka, M., Milne, M., Simpson, G.E., Hillier, S.G., 1999. Expression of 11βhydroxysteroid dehydrogenase, glucocorticoid receptor, and mineralocorticoid receptor genes in rat Ovary1. Biol. Reprod. 60 (2), 330–335. Tsai, E.M., Chan, T.F., Chen, Y.H., Hsu, S.C., Chuang, C.Y., Lee, J.N., 2008. Mifepristone attenuates human chorionic gonadotropin-induced extracellular signal-regulated kinase 1/2 phosphorylation, cyclooxygenase-2, and prostaglandin E2 production in human granulosa luteal cells. Fertil. Steril. 89 (5 Suppl. l), 1522–1529. Vander Borght, M., Wyns, C., 2018. Fertility and infertility: definition and epidemiology. Clin. Biochem. 62, 2–10. Varani, S., Elvin, J.A., Yan, C., Demayo, J., Demayo, F.J., Horton, H.F., Byrne, M.C., Matzuk, M.M., 2002. Knockout of Pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility. Mol. Endocrinol. 16 (6), 1154–1167. Velez, L.M., Abruzzese, G.A., Motta, A.B., 2013. The biology of the peroxisome proliferator-activated receptor system in the female reproductive tract. Curr. Pharmaceut. Des. 19 (25), 4641–4646. Walters, K.A., Allan, C.M., Jimenez, M., Lim, P.R., Davey, R.A., Zajac, J.D., Illingworth, P., Handelsman, D.J., 2007. Female mice haploinsufficient for an inactivated androgen receptor (AR) exhibit age-dependent defects that resemble the AR null phenotype of dysfunctional late follicle development, ovulation, and fertility. Endocrinology 148 (8), 3674–3684. Walters, K.A., Allan, C.M., Handelsman, D.J., 2008. Androgen actions and the ovary. Biol. Reprod. 78 (3), 380–389. Walters, K.A., McTavish, K.J., Seneviratne, M.G., Jimenez, M., McMahon, A.C., Allan, C. M., Salamonsen, L.A., Handelsman, D.J., 2009. Subfertile female androgen receptor knockout mice exhibit defects in neuroendocrine signaling, intraovarian function, and uterine development but not uterine function. Endocrinology 150 (7), 3274–3282. Walters, K.A., Middleton, L.J., Joseph, S.R., Hazra, R., Jimenez, M., Simanainen, U., Allan, C.M., Handelsman, D.J., 2012. Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Biol. Reprod. 87 (6), 151. Wang, L., Lee, Y.-K., Bundman, D., Han, Y., Thevananther, S., Kim, C.-S., Chua, S.S., Wei, P., Heyman, R.A., Karin, M., Moore, D.D., 2002. Redundant pathways for negative feedback regulation of bile acid production. Dev. Cell 2 (6), 721–731. Wang, R., Kim, B.V., van Wely, M., Johnson, N.P., Costello, M.F., Zhang, H., Ng, E.H., Legro, R.S., Bhattacharya, S., Norman, R.J., Mol, B.W., 2017. Treatment strategies for women with WHO group II anovulation: systematic review and network metaanalysis. BMJ 356, j138. Wang, F., Chang, H.M., Yi, Y., Lin, Y.M., Li, H., Leung, P.C.K., 2020. TGF-beta1 promotes vitamin D-induced prostaglandin E2 synthesis by upregulating vitamin D receptor expression in human granulosa-lutein cells. Am. J. Physiol. Endocrinol. Metab. 318 (5), E710–E722.

Warnecke, M., Oster, H., Revelli, J.P., Alvarez-Bolado, G., Eichele, G., 2005. Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception. Genes Dev. 19 (5), 614–625. Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., Moore, D.D., 2000. The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407 (6806), 920–923. Weick, R.F., Dierschke, D.J., Karsch, F.J., Butler, W.R., Hotchkiss, J., Knobil, E., 1973. Periovulatory time courses of circulating gonadotropic and ovarian hormones in the rhesus monkey. Endocrinology 93 (5), 1140–1147. Wikstr¨ om, L., Johansson, C., Salt´ o, C., Barlow, C., Campos Barros, A., Baas, F., Forrest, D., Thor´en, P., Vennstr¨ om, B., 1998. Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1. EMBO J. 17 (2), 455–461. Wu, X., Matzuk, M.M., 2002. GDF-9 and BMP-15: oocyte organizers. Rev. Endocr. Metab. Disord. 3 (1), 27–32. Wu, Y., Ghosh, S., Nishi, Y., Yanase, T., Nawata, H., Hu, Y., 2005. The orphan nuclear receptors NURR1 and NGFI-B modulate aromatase gene expression in ovarian granulosa cells: a possible mechanism for repression of aromatase expression upon luteinizing hormone surge. Endocrinology 146 (1), 237–246. Wu, S., Sun, H., Zhang, Q., Jiang, Y., Fang, T., Cui, I., Yan, G., Hu, Y., 2015. MicroRNA132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1. Reprod. Biol. Endocrinol. 13, 94. Xie, W., Barwick, J.L., Downes, M., Blumberg, B., Simon, C.M., Nelson, M.C., Neuschwander-Tetri, B.A., Brunt, E.M., Guzelian, P.S., Evans, R.M., 2000. Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406 (6794), 435–439. Xing, W., Danilovich, N., Sairam, M.R., 2002. Orphan receptor chicken ovalbumin upstream promoter transcription factors inhibit steroid factor-1, upstream stimulatory factor, and activator protein-1 activation of ovine follicle-stimulating hormone receptor expression via composite cis-elements1. Biol. Reprod. 66 (6), 1656–1666. Xu, J., Lawson, M.S., Xu, F., Du, Y., Tkachenko, O.Y., Bishop, C.V., Pejovic-Nezhat, L., Seifer, D.B., Hennebold, J.D., 2018. Vitamin D3 regulates follicular development and intrafollicular vitamin D biosynthesis and signaling in the primate ovary. Front. Physiol. 9. Xue, K., Liu, J.-Y., Murphy, B.D., Tsang, B.K., 2012. Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth. Mol. Endocrinol. 26 (12), 2004–2015. Yazawa, T., Kawabe, S., Kanno, M., Mizutani, T., Imamichi, Y., Ju, Y., Matsumura, T., Yamazaki, Y., Usami, Y., Kuribayashi, M., Shimada, M., Kitano, T., Umezawa, A., Miyamoto, K., 2013. Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells. Mol. Cell. Endocrinol. 369 (1–2), 42–51. Yoshizawa, T., Handa, Y., Uematsu, Y., Takeda, S., Sekine, K., Yoshihara, Y., Kawakami, T., Arioka, K., Sato, H., Uchiyama, Y., Masushige, S., Fukamizu, A., Matsumoto, T., Kato, S., 1997. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat. Genet. 16 (4), 391–396. Yu, R.N., Ito, M., Saunders, T.L., Camper, S.A., Jameson, J.L., 1998. Role of Ahch in gonadal development and gametogenesis. Nat. Genet. 20 (4), 353–357. Zeleznik, A.J., Midgley, A.R., Reichert, L.E., 1974. Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormonein Vivo11. Endocrinology 95 (3), 818–825. Zetterstr¨ om, R.H., Solomin, L., Jansson, L., Hoffer, B.J., Olson, L., Perlmann, T., 1997. Dopamine neuron agenesis in Nurr1-deficient mice. Science 276 (5310), 248–250. Zhang, Y., Dufau, M.L., 2001. EAR2 and EAR3/COUP-TFI regulate transcription of the rat LH receptor. Mol. Endocrinol. 15 (11), 1891–1905. Zhang, S., Carrillo, A.J., Darling, D.S., 1997. Expression of multiple thyroid hormone receptor mRNAs in human oocytes, cumulus cells, and granulosa cells. Mol. Hum. Reprod. 3 (7), 555–562. Zhang, C., Large, M.J., Duggavathi, R., DeMayo, F.J., Lydon, J.P., Schoonjans, K., Kovanci, E., Murphy, B.D., 2013. Liver receptor homolog-1 is essential for pregnancy. Nat. Med. 19 (8), 1061–1066. Zhang, Y., Xu, Y., Kuai, Y., Wang, S., Xue, Q., Shang, J., 2016. Effect of testosterone on the Connexin37 of sexual mature mouse cumulus oocyte complex. J. Ovarian Res. 9 (1), 82. Zhao, Y., Bruemmer, D., 2010. NR4A orphan nuclear receptors: transcriptional regulators of gene expression in metabolism and vascular biology. Arterioscler. Thromb. Vasc. Biol. 30 (8), 1535–1541. Zhao, F., Franco, H.L., Rodriguez, K.F., Brown, P.R., Tsai, M.-J., Tsai, S.Y., Yao, H.H.C., 2017. Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice. Science 357 (6352), 717–720. Zhou, Y., Zhou, B., Pache, L., Chang, M., Khodabakhshi, A.H., Tanaseichuk, O., Benner, C., Chanda, S.K., 2019. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10 (1), 1523. Zhuang, Y.H., Ylikomi, T., Lindfors, M., Piippo, S., Tuohimaa, P., 1994. Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus. J. Steroid Biochem. Mol. Biol. 48 (1), 61–68.

23