Mammalian ovary differentiation – A focus on female meiosis

Molecular and Cellular Endocrinology 356 (2012) 13–23

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Review

Mammalian ovary differentiation – A focus on female meiosis Adrienne Baillet a, Béatrice Mandon-Pepin b,c,⇑ a

Laboratoire de Génétique et Biologie Cellulaire, EA 4589 Université de Versailles Saint-Quentin-en-Yvelines, Ecole Pratique des Hautes Etudes, F-78035 Versailles cedex, France INRA, UMR1198 Biologie du Développement et Reproduction, F-78352 Jouy-en-Josas, France c ENVA, F-94704 Maisons Alfort, France b

a r t i c l e

i n f o

Article history: Available online 21 September 2011 Keywords: Ovary Development Prophase I Meiosis Small RNA

a b s t r a c t Over the past 50 years, the ovary development has been subject of fewer studies as compare to the male pathway. Nevertheless due to the advancement of genetics, mouse ES cells and the development of genetic models, studies of ovarian differentiation was boosted. This review emphasizes some of new progresses in the research field of the mammalian ovary differentiation that have occurred in recent years with focuses of the period around prophase I of meiosis and of recent roles of small non-RNAs in the ovarian gene expression. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ovarian differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Around prophase I of meiosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Molecular mechanisms involved in germ cell meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Initiation of meiosis I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. New meiotic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small RNAs: a new area of research for ovarian development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction An appropriate set up of the ovary during development allows oocyte generation and hormone production in adulthood. The simplicity of this assertion does not hide the complexity of the pathways for ovary organogenesis where the slightest failure could be fatal to ovarian function. For many years, early mammalian ovarian development has remained mysterious and has been considered as the female default pathway particularly since Jost’s studies in the early 1950s (Jost et al., 1953; Jost, 1972). Testis development has

⇑ Corresponding author at: UMR1198 Biologie du Développement et Reproduction, INRA-Domaine de Vilvert, F-78352 Jouy-en-Josas, France. Tel.: +33 1 64 65 25 39; fax: +33 1 34 65 22 41. E-mail addresses: [email protected] (A. Baillet), [email protected] (B. Mandon-Pepin). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.09.029

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been extensively studied and was believed to be the more active process especially since the discovery of the Sry gene in men and mice (Sinclair et al., 1990; Gubbay et al., 1990). However, during the last decade, greater interest in studies of the mammalian ovary formation has been triggered by the discovery of several ovaryspecific genes expressed during the critical stage of embryogenesis (Nef et al., 2005; Beverdam and Koopman, 2006). Data from studies in humans and from the use of novel technologies, like mouse genetic models, have dramatically increased the number of studies on the factors involved in formation of the mammalian ovary. Thus, the popularity of the hypothesis of the default process of the mammalian female development has decreased and it was pointed out that active mechanisms are required to lead to the differentiated state of the ovary with ovary-determining or maintenance genes (for example Uhlenhaut et al., 2009; Garcia-Ortiz

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et al., 2009; Vainio et al., 1999; Parma et al., 2006; Ottolenghi et al., 2007; Chassot et al., 2008). The goal of this review is to focus on recent advances in our understanding of certain steps in early ovarian development. We have chosen to highlight certain studies involved in these pathways. Several studies of the ovarian formation involve analysis of both sexes, and so in this review we approach the female gonad development by comparison with testis development or function.

2. Ovarian differentiation The reproductive capacity of a mammalian female is the culmination of events starting early in fetal life. Many key events in ovarian development such as germ cell migration, gonadal sex differentiation, germ cell mitosis, atresia and first division of meiosis are initiated during fetal life. Follicular formation is initiated during, at the end, or just after birth according to species. The germline appears when the precursors of primordial germ line differentiate from the somatic lineage of the embryo and migrate to the urogenital ridge, a thickening of the coelomic epithelium. Here, primordial germ cells (PCGs) undergo a period of proliferation (mitosis) to form a so-called undifferentiated or bipo-

tential gonad. Clusters of PGCs with somatic cells come closer to form ovigerous cords. In the fetal ovary, the first recognizable step in the process of ovigerous cord formation is the establishment of an initial contact between germ cells and somatic cells near the surface of the ovarian epithelium. The ovigerous cord becomes a large structure bound by basal laminae, also called germ cell nests or cysts that are loosely surrounded by somatic cells. Through functional genetic analysis, several genes have been identified to be involved in this early ovarian differentiation and are even considered as ovarydetermining genes (see references in Edson et al., 2009). They can be classified into two classes (i) extracellular factors with paracrine and/or autocrine factors (RSPO1, WNT4) (Parma et al., 2006; Chassot et al., 2008; Vainio et al., 1999) and (ii) intracellular factors or transcription factors (FoxL2, b-catenin) (Crisponi et al., 2001; Pailhoux et al., 2001, 2002; Liu et al., 2009). Furthermore, the ovarian structural organization is not really the same across species (Fig. 1). At the time of initiation of the prophase I of meiosis, there is a distinguishable structural organization in the fetal ovary (with a cortex and a medulla) of most mammals (human, ruminant) (Fig. 1A). However, there is little morphological evidence of a cortical/medullary structure in the mouse ovary at the same period (E12.5) (Fig. 1B). In addition,

Fig. 1. Chronological events of ovarian differentiation of (A) ruminant (sheep) and (B) mouse. Ovaries develop a structural organization as soon as ovigerous cords are formed. Then two parts are recognizable: cortex in the periphery and medulla in the center of ovary. This organization is observed before meiosis in ruminant (A) and after meiosis in mouse (B). Estrogen synthesis occurs in feta ovarian development in ruminant and only after birth in mouse (B). (Adapted from Juengel et al., 2002 (sheep) and from Brennan and Capel, 2004 (mouse)).

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developing fetal ovary is steroidogenically active in ruminants such as goat (Pannetier et al., 2006), sheep (Mauléon et al., 1977; Quirke et al., 2001), and cattle (Shemesh, 1980; Dominguez et al., 1988; Yang and Fortune, 2008). In general in these species, steroidogenic activity is high before the beginning of female fetal meiosis. The gonadal content of estradiol and androgens and the secretion of estradiol decrease when prophase I of meiosis is initiated (Shemesh et al., 1978; Dominguez et al., 1988; Quirke et al., 2001). Estrogens have been also shown to be crucial for proper ovarian differentiation in different marsupial species (Burns, 1955; Fadem and Tesoriero, 1986; Coveney et al., 2001); fishes (Krisfalusi and Cloud, 1999; Guiguen et al., 1999) reptiles (Pieau et al., 1999) and birds (Scheib, 1983; Elbrecht and Smith, 1992; Wade and Arnold, 1996). Taken together, these data support the hypothesis that estrogens play an important role in control of ovarian differentiation during the fetal life of non-rodent mammals. By contrast, the mouse, one of the most studied models in sex determination and gonad development, has no ovarian steroidogenesis during fetal life. Mice with null mutation of Cyp19 (Fisher et al., 1998), StAR (Hasegawa et al., 2000) or estrogen receptor (Couse et al., 1999; Dupont et al., 2000) genes present no severe phenotypes in the ovaries of mouse newborns. 3. Around prophase I of meiosis. . . One important event which initiates in the fetal ovary of all placental mammalian species is the prophase I of meiosis. Meiosis is restricted to germ cells and an achievement of meiosis is crucial

for fertility. Mistakes during meiosis can be fatal for the development of the zygote. In most organisms, homologous recombination during meiosis is required for correct segregation of homologous chromosomes during the first meiotic division. In this review, after a brief overview of meiotic events, we will focus on recently described-genes involved in the initiation or the progression of meiosis. 3.1. Molecular mechanisms involved in germ cell meiosis Our understanding of meiosis has been mainly enriched by studies conducted on model organisms such as yeast and mice. A number of mouse models have helped to identify master meiotic genes active at the prophase I critical period. The findings in these mutant mice are summarized in Table 1. During embryogenesis, the prophase I of meiosis is initiated synchronously (rodents) or asynchronously (human: Skrzypczak et al., 1981, sheep: Sawyer et al., 2002) in the female germline, then stops before birth (except in rabbit where female meiosis arrests a few days after birth) and resumes at periodic intervals after puberty. By contrast, in mammalian testis, meiosis begins only after birth and continues throughout the reproductive lifespan (Fig. 2). The prophase I of meiosis is divided into five stages, namely leptotene (chromosomes start to condense), zygotene (chromosomes become closely paired and synapsis begin), pachytene (synapsis is completed and crossovers occur), diplotene (homologous chromosomes begin to separate but remain attached by the chiasmata) and diakinesis (chromosomes separate until ter-

Table 1 Example of mouse models with defects in prophase I of meiosis. Type of protein

Gene mutation

Meiosis function

Male effects

Female effects

References

Stimulated by Retinoic Acid

Stra8

Meiotic initiation in germ cells

Sterile

Sterile

Baltus et al. (2006)

Synaptonemal proteins

Sycp1/

Pairing homologous chromosome

Sterile

de Vries et al. (2005)

Sycp3/

Pairing homologous chromosome

Fertile (aneuploïdie)

Yuan et al. (2002)

Syce2/

Synaptonemal complex formation

Sterile Meiosis stops in pachytene Sterile Meiosis stops in zygotene Sterile

Subfertile

Bolcun-Filas et al. (2007)

Tex11/

Pairing homologous chromosome

Sterile

Subfertile

Tex15/ Tex19/

Pairing homologous chromosome Pairing homologous chromosome

Sterile Subfertile

Fertile Subfertile

Adelman and Petrini (2008) Yang et al. (2008a) Ollinger et al. (2008)

Spo11/

Double strand breaks (DSB) formation

Sterile

Sterile

Dmc1/

Search for the homologous DNA strand and induced the Sterile synapsis of chromosome

TEX proteins

DSB proteins

MSH–MLH proteins

AtaxiaTelangiectasia protein

Germ cells lost Sterile

Romanienko and Camerini-Otero (2000) Pittman et al. (1998)

Meiosis stops in pachytene Letal

Yoshida et al. (1998)

Rad51/

Search for the homologous DNA strand and induced the Letal synapsis of chromosome

Tsuzuki et al., 1996

Msh4/

Pairing of homologous chromosome to avoid illegitimate echange between chromosme

Sterile

Sterile

Kneitz et al. (2000)

Msh5/

Pairing of homologous chromosome to avoid illegitimate echange between chromosme

Sterile

Germ cells lost Sterile

de Vries et al. (1999)

Mlh1/

Crossing over formation

Mlh3/

Crossing over formation

Pms2/

Pairing of homologous chromosome to avoid illegitimate echange between chromosme

Germ cells lost Sterile Germ cells lost Sterile Germ cells lost Sterile

Germ cells lost Stérile Germ cells lost Sterile Germ cells lost Fertile

Atm/

Associated with synapsed meiotic chromosomes

Sterile Stop between zygote and pachytene

Sterile Xu et al. (1996) Disrupted before normal meiotic arrest

Edelmann et al., 1996 Lipkin et al. (2002) Baker et al. (1995)

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minal chiasmata only connect the two chromosomes) (Fig. 3). In the female, the first four stages take place during fetal life, the last one (diakinesis) starts at puberty. Several genes have been shown to be involved in prophase I of meiosis. In pre-leptotene, new sister chromatids are attached to the older by cohesins proteins (REC8, SMC1b, SMC3, STAG3). Then, in leptotene two events are observed: (i) the pairing of homologous chromosomes (or synapsis to pachy-

tene stage) by a process that involves the synaptonemal complex (SC) and (ii) DNA double-strand breaks (DSB) necessary for DNA exchange (genetic recombination). The SC (Fig. 4) is composed of the axial element (SYCP2 and SYCP3, Fig. 4 in green), the transversal element (SYCP1, Fig. 4 in purple) and the central element (SYCE1, SYCE2 and TEX12, Fig. 4 in grey). The DSBs, observed along the sister chromatids, are induced by SPO11 and are required for

Fig. 2. Time course of meiosis initiation in mammals. Comparison of periods when meiosis is initiated in mammalian female or male gonads. In females, prophase I is initiated during the fetal ovarian development, then arrests and resumes at puberty. In male, meiosis is initiated and progresses after birth.

Fig. 3. Description of prophase I of meiosis in mammals. The prophase I of meiosis is divided into five steps: leptotene, zygotene, pachytene, diplotene and diakinesis. In females, the four first stages take place during fetal life, the last one (diakinesis) at puberty. The principal events of each step and the proteins involved are described below each cartoons. The Stra8 gene is expressed just before the leptotene stage and initiates meiosis. This gene is only expressed in vertebrates. (Adapted from http://www.mun.ca/ biology/desmid/brian/BIOL2060/BIOL2060-20/CB20.html)

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Fig. 4. Graphic representation of the synaptonemal complex (SC). The synaptonemal complex is composed of 3 elements: the central, transversal and axial elements. The central one consists of SYCE1-SYCE2 and TEX12 proteins, the transversal element consists of SYCP1 and the axial element consists of SYCP2 and SYCP3. The SC is necessary for the pairing of homologous chromosomes and is present from the leptotene to the pachytene stages.

homologous recombination between chromosomes (Romanienko and Camerini-Otero, 2000). These DSBs induce the phosphorylation of cH2AX histone thus triggering its attachment on the breaks (Mahadevaiah et al., 2001). Afterwards, the RAD50-NBS1-MRE11 complex fixes to cH2AX and allows the formation of DNA single strand (see references in Gerton and Hawley, 2005). The recombinase proteins DMC1 and RAD51, interact with SYCP3 (component of the axial element of the SC) to aid in recognition of the homologous DNA strand and to induce the synapsis of the chromosome (Tarsounas et al., 1999). In the mouse, TEX15 protein is involved in the localization of DMC1 and RAD51 on the DSB (Yang et al., 2008a). At the zygotene stage, the transversal element and the central element of SC are formed. TEX11, a protein only expressed by germ cells, interacts with SYCP2 (axial element) and allows the formation of SC (Yang et al., 2008b). During this step, the protein complex MSH4–MSH5–PMS2 is involved in the pairing of homologous chromosomes to avoid inappropriate exchange between chromosomes (Baker et al., 1995; Kneitz et al., 2000). At the pachytene stage, the SC is totally observed along the chromosome suggesting that the greatest level of synapsis occurs at this stage. At this step, crossovers can start. The complex of MSH4–MSH5–MLH1–MLH3 participates in crossover formation and repairs the DNA doublestrand breaks (Edelmann et al., 1996; Lipkin et al., 2002; Santucci-Darmanin et al., 2000). Another group of genes that are required for progression through meiosis are those involved in repression of transposable genetic elements. Indeed, mutations in genes involved in mediating DNA methylation-dependent transcriptional repression of retrotransposons cause increased expression of transposable elements and defects in chromosome synapsis during meiosis in either male or female germ cells (Bourc’his and Bestor, 2004; De La Fuente et al., 2006). After that, the female germ cells arrest at diplotene stage of prophase I of meiosis during the fetal period and meiosis will resume at puberty in the ovulated-oocytes. 3.2. Initiation of meiosis I Several global approaches using microarray technology have been used to isolate genes involved in meiosis by comparing transcriptional activity in ovaries with or without germ cells in meiotic prophase I (Herrera et al., 2005; Grimmond et al., 2000; Small et al.,

2005; Nef et al., 2005) or by comparing fetal ovaries at different stages of development in sheep (Baillet et al., 2008). One of the first factors necessary to engage germ cells toward the process of meiosis is the RNA-binding protein DAZL (Deleted in AZospermia-like). DAZL is expressed by male and female post-migratory germ cells and has a function upstream of meiotic initiation by turning cells into ‘‘meiosis-competent’’ germ cells (Lin et al., 2008). DAZL has another downstream role in germ cell meiosis (Reynolds et al., 2007). Indeed, DAZL is also required for efficient translation of the Sycp3 (synaptonemal complex protein 3) mRNA in vivo as shown by the DAZL knockout mouse model (Reynolds et al., 2007). In the mouse, once germ cells are made ‘‘meiosis-competent’’ by this intrinsic factor DAZL, several in vitro studies have indicated that the entry of female germ cells into meiosis is actively mediated by the signaling molecule retinoic acid (RA), the active derivative of vitamin A (Bowles et al., 2006; Koubova et al., 2006). Thus, RA could be one of the meiosis inducing factors. By in vitro experiments, this meiosis-inducing role of RA has been demonstrated also in other species like rats (Li and Clagett-Dame, 2009), humans (Le Bouffant et al., 2010), chickens and amphibians (Smith et al., 2008; Wallacides et al., 2009). In the mouse, RA is produced by the mesonephros and the actual hypothesis is that RA diffuses into the adjacent gonad (Bowles et al., 2006). It was shown that supraphysiological levels of RA are responsible for the induction of meiosis in the female mouse developing gonad (Bowles et al., 2006; Koubova et al., 2006; Menke and Page, 2002). Chemical inhibition of all RA receptors (RAR) in ex vivo mouse fetal ovaries cultures leads to inhibition of the known retinoic acid target Stra8 (Stimulated by retinoic acid gene 8) and to inhibition of meiosis entry (Koubova et al., 2006). A vitamin A-deficient rat model (late VAD rat) also highlights RA as an essential extrinsic inducer of meiotic initiation in females. Indeed, in this rat model, in spite of a normal number of germ cells, most of these cells failed to enter meiosis (Li and Clagett-Dame, 2009). The environment of the gonad with respect to RA seems to be the same in both sexes. But in male, gonads produce one or more factors that inhibit the entry into meiosis of male germ cells. One of these factors is cytochrome P450 enzyme 26B1 (Cyp26B1), which has a RA-degrading action (McLaren and Southee, 1997; Bowles et al., 2006; Li et al., 2009; MacLean et al., 2007). The sexually dimorphic meiosis pathway takes place only in fetal ovary because of different regulation of CYP26B1 in the male and female

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developing gonad. Thus, several studies were performed in the male developing gonads to prove that meiosis is sex-dimorphic. In mouse E12.5 testis, Cyp26B1 is up-regulated in somatic cells of embryonic testes (Abu-Abed et al., 2002) and more particularly in Sertoli cells and some unidentified interstitial cells (Bowles et al., 2006) allowing the degradation of the RA and then the non-accessibility of the male germ cells to RA, thus preventing meiosis. With the Cyp26B1 knockout mouse model, it was shown that testes of these male animals were exposed to supraphysiological levels of RA and as a result, Stra8 and the meiotic marker Sycp3 (Synaptonemal complex protein 3) were also up-regulated like in XX germ cells. In addition, the use of synthetic retinoid which is not degraded by Cyp26B1 (Am580) in cultured gonads reproduces a meiosis induction in normal XY germ cells (MacLean et al., 2007). These data showed that the non-degradation of RA engaged XY germ cells towards the meiosis pathway and that CYP26B1 is a male-specific meiosis-preventing factor (Bowles et al., 2006; MacLean et al., 2007) by opposition of RA which has in vitro properties of a meiosis-inducing factor. Moreover, the hypothesis that Cyp26B1 may have an additional function in male gonad as a activator of a meiosis inhibiting factor need to be evaluated. Another factor, Nanos2, is involved in the male to prevent meiosis and more particularly to inhibit Stra8 in the mouse fetal testis (Suzuki and Saga, 2008). Nanos2 is expressed exclusively in fetal male germ cells (Tsuda et al., 2003). Loss of Nanos2 expression in the fetal testis increased Stra8 expression and meiosis in male germ cells. Ectopic expression of Nanos2 in the female germ cells decreases Stra8 expression and inhibits germ cell meiosis (Suzuki and Saga, 2008). Moreover, it has been proposed that others factors and especially a secreted factor could contribute to prevent meiosis in XY fetal germ cells. Indeed, when all cellular secretions are blocked in cultured mouse testes (by Brefeldin A) some XY germ cells (those located at the end of the gonad) enter into meiosis (Best et al., 2008) suggesting that somatic cells may secrete a meiosis-preventing substance in addition to the role of Cyp26B1. Furthermore it was report that epigenetic modification like histone deacetylation may play a role to prevent entry into meiosis in males (Wang and Tilly, 2010). Also, FGF9 (Fibroblast Growth Factor 9) was proposed to be involved in meiosis entry (Bowles et al., 2010). This factor was previously known to be expressed in the gonads of both sexes and is upregulated early in the developing testes (Colvin et al., 2001; Nef et al., 2005). The Fgf9 null mutation leads to male-to-female sex reversal in mouse (Colvin et al., 2001; Kim et al., 2006; Schmahl et al., 2004) with the death of most of the XY germ cells (DiNapoli et al., 2006). The meiotic gene cH2AX is expressed in the surviving E 14.5 XY germ cells of the mutated mouse as is expected also in the case of XX germ cells. These results suggested that the absence of FGF9 allows an available RA environment in the XY Fgf9 null gonads and adds another role for FGF9 in germ cell sexual fate. In a recent study FGF9 negatively regulated meiosis entry in opposition to RA by promoting male germ cell fate at the same time (Bowles et al., 2010). To prove that, the authors of this study compared germ cells from XY Cyp26b1 null testes with a wild type or one mutated allele of Fgf9 gene. In this way, the authors showed that Stra8 expression is significantly elevated in Fgf9+/ as compared with Fgf9+/+ testes knock-out for Cyp26b1. FGF9 acts directly on XY germ cells to make them less responsive to RA and to actively promote male germ cell fate (Bowles et al., 2010). Indeed FGF9 is considered as a diffusible meiosis-inhibiting factor in mouse. Thus, FGF9 could be the secreted factor hypothesized as an inhibitor of cellular secretions in cultured mouse testes (Best et al., 2008). Thus it was proposed that RA and FGF9 act antagonistically to determine germ cell fate in the mouse with a crucial sex-dimorphic pathway and sex-specific timing (Bowles et al., 2010) (Fig. 5). Moreover, it was also suggested that the manner in which RA regulates meiosis

initiation in fetal ovary may vary between mice and humans (Le Bouffant et al., 2010). In fact, in the human fetal ovary, production of RA from the fetal ovary itself is required for meiosis initiation and the level of RA is unaffected by inhibition of CYP26B1 (ketoconazole). The authors of this study hypothesized that ALDH1A1, a RA-synthesizing enzyme, could be a central regulator of RA in human fetal ovary and the role of CYP26B1 is much less important by comparison with the mouse (Le Bouffant et al., 2010). Recently, a new study has weakened the case for the role of RA as the meiosis inducer in mouse fetal ovary (Kumar et al., 2011). These authors produced Raldh2/ mouse embryos, lacking RA synthesis. These mutants were produced with a complementary maternal dietary of RA to avoid early death of mutant embryos. Beforehand, the authors verified that the low dose of RA diet did not result in a RA activity in the mutant mesonephros/ovary prior the induction of Stra8 (E12.0) and at the entry into meiosis (E13.5). Once this verification was performed, they demonstrated that fetal ovaries of these RA-signaling devoid-mutant embryos had ovarian Stra8 expression unaffected by loss of endogenous synthesis of RA (Kumar et al., 2011). Moreover, this study showed that physiological RA concentrations (25–100 nM) are able to induce the RARE element, and so too RA signaling, in the whole wild-type ovary culture. These findings demonstrated that the mouse fetal ovary has an operational cellular machinery to engage RA signaling and suggested that the RA synthesized in the mesonephros of normal embryos is not able to diffuse into the ovary in an amount sufficient to stimulate RA signaling in the ovary. Consequently, these authors concluded that Stra8 does not require RA for its expression in the mouse fetal ovary. Thus, the role of RA as a meiosis inducing factor is currently a source of debate. If in vivo experiments using supraphysiological levels of RA had demonstrated RA as a meiosis inducing factor, this recent in vivo study suggested that endogenous RA synthesis in the vicinity of the gonad is fully dispensable to meiosis initiation (Kumar et al., 2011). 3.3. New meiotic genes Most meiotic genes were first described in studies on yeast and usually these genes had been conserved during evolution. Even recently, some new genes involved in vertebrate meiosis have been described. One of them is the Vaccinia related kinase-1 (VRK1) which was recently shown to be involved in coordinating proper chromosomal configuration in mammalian female meiosis (Schober et al., 2011). It was known that VRK1 is an evolutionarily conserved serine/threonine kinase implicated in gametogenesis in different species (invertebrate and vertebrate species). Indeed, VRK1 was shown to be involved in female meiotic progression in Drosophila (the Drosophila homolog of VRK1 is named NHK-1, Nucleosomal Histone Kinase-1) (Cullen et al., 2005; Lancaster et al., 2010) and mouse proliferation of spermatogonia (Choi et al., 2010). In this species, VRK1 is present in both the somatic Sertoli cells and the spermatogonia (Choi et al., 2010; Wiebe et al., 2010). In males, the reduction of Vrk1 results in a progressive loss of spermatogenesis leading to the sterility of the mouse without an effect on germ cell meiosis, but rather by inducing a progressive loss of germ cells at all stages of differentiation (Wiebe et al., 2010). In the homozygous state, both male and female mice carrying a gene trap insertion within the Vrk1 locus were infertile. Recently, the effects of reduction of VRK1 were described in mouse females (Schober et al., 2011). In the ovary, Vrk1 expression is restricted to the cytoplasm of the developing oocyte (only shown for postnatal ovaries) and the reduction of VRK1 causes a delay in meiotic progression resulting in an abnormality of chromosomes during metaphase II in post-ovulation oocytes (Schober et al., 2011), leading to female sterility. VRK1 is involved in meiotic progression during oogenesis

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Fig. 5. Sexually dimorphic initiation of meiosis in mouse germ cells: antagonistic action of RA and FGF9. In the mouse fetal ovary, the down-regulation of FGF9 and the absence of CYP26B1 allow high levels of RA in somatic cells. The complex formed by binding of RA with the RAR receptor acts as a transcription factor in female germ cells to promote the Stra8 expression. STRA8 protein is essential to meiotic initiation. Other inducing meiosis factor could be involved in this process. Inthe mouse fetal testis, CYP26B1 and FGF9 are highly expressed. CYP26B1 degrades endogenous RA. FGF9 antagonizes Stra8 expression in male germ cells and induces Nanos2. Somatic cells may secrete a meiosis-preventing substance in addition to the role of Cyp26B1 in the fetal testis.

to proper chromosomal orientation during both prophase I of meiosis and metaphase arrest (meiosis II). So, these Vrk1 mutant mice seem to present a sexually dimorphic phenotype. In testis, deletion of Vrk1 does not disturb meiosis but Vrk1 does have a function in proliferation and differentiation of male germ cells. On the contrary, Vrk1 has a specific role during meiosis in ovary (Schober et al., 2011). Meiosis I requires different factors that are unique to germ cells and meiosis. Germ cell–specific factors are known to play crucial roles during specific step of meiosis I. Some of them are specific to synaptonemal complex formation with for example synaptonemal complex proteins SYCP1, SYCP2, and SYCP3. Recently, HORMAD1, a mammalian homologue to the yeast Hop1 was described as a new germ cell-specific protein involved in SC formation (Shin et al., 2010; Daniel et al., 2011). Previously, it was shown that Hormad1 expression was sexually dimorphic. In testis, Hormad1 expression was detected postnatally from day 10 and was found to be abundant in the adult testis. In ovary, HORMAD1 protein is identified prenatally during fetal ovarian development (leptotene stage at E14.5) and until the arrest of meiosis in diplotene (E18.5); its expression is weak in the newborn ovary and really limited in the adult ovary (Pangas et al., 2004). Recently, Hormad1 was shown to be essential for mammalian gametogenesis as knockout male and female mice are both infertile (Shin et al.,

2010; Daniel et al., 2011). In males deficient for Hormad1, adult testes were significantly smaller than the wild-type testes. The meiosis is arrested at the early pachytene stage with a partial completion (Daniel et al., 2011) or an absence (Shin et al., 2010) of synaptonemal complexes (SC) according to the strain backgrounds. HORMAD1 protein contains a HORMA domain essential for synaptonemal complex formation and the pairing of homologous chromosomes during leptotene (Shin et al., 2010; Daniel et al., 2011). In female deficient for Hormad1, ovaries showed no distinguishable morphologic differences compared to the wildtype mice (from E14.5 to adult ovaries) and thus, the ovarian development are largely normal in Hormad1/ mice (Shin et al., 2010). The early embryonic death of the pups, due to a default of implantation, leads to infertility in Hormad1/ females. So, the absence of a clear meiotic effect in mutant females is in contrast to males, where spermatocytes are eliminated due to pachytene defect. Same drastic phenotypic differences are found in other mutant mice for example in Sycp3/, Smc1B/ mice (Wang and Hoog, 2006; Hodges et al., 2005). Mammalian females, where meiosis is characterized by an extensive arrest, should have specific protective mechanisms. This could explain how ovarian development is normal in the absence of certain meiotic proteins in knockout mice. It is admitted that oocytes tolerate meiosis I errors (Hassold and Hunt, 2001) but the molecular basis of the synapsis

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checkpoint, which is either more sensitive in males or different between the sexes remains unknown (Kouznetsova et al., 2009; Hunt and Hassold, 2002). In the case of Hormad1, it was also hypothesized that this gene is required only for pathological non-homologous synapsis (Daniel et al., 2011). Oocytes arrested in diplotene stage enter in the process of early folliculogenesis. The functional unit of the ovary is the follicle. Follicle formation takes places at the end of gestation for many species (human, sheep, cattle, pig and goat) or just after birth (rodents and rabbit). Oocytes become surrounded by somatic cells (pre-granulosa cells) and enlarge into different stages of growth to become primordial, primary, secondary and tertiary follicle. All these different steps are all under the influence of specific genes which have been largely described (see references in Edson et al., 2009). The resting pool of primordial follicles, that have the potential to be recruited into the growing follicle pool, constitute the reserve of gametes for all the reproductive life of the female.

4. Small RNAs: a new area of research for ovarian development The discovery of small non-coding RNAs has emphasized a new dimension in gene expression regulation and function in many tissues (Bartel, 2004; Fazi and Nervi, 2008; Fabian et al., 2010) including the developing ovary (Torley et al., 2011; Tripurani et al., 2011) and oocytes (Watanabe et al., 2008). Small non-coding RNAs include 21–23 nt microRNAs (miRNAs), 21-nt small interfering RNAs (siRNAs), and 24–30 nt Piwi-interacting RNAs (piRNAs). It has been shown that microRNAs down-regulate target gene expression by blocking mRNA translation and/or destabilizing and degrading the mRNA transcript (Bartel, 2004). SiRNAs repress host genes, transposable elements (TEs), and viruses, and piRNAs repress TEs. The biogenesis of miRNAs occurs both in the nucleus and cytoplasm of cells. First, miRNAs are transcribed by RNA Polymerase II generating a long transcript called primary-miRNAs and containing hairpin loop organization. These structures are cleaved by the micro-processor complex (Drosha-DGCR-8) in pre-miRNAs (70–90 nt length) before being exported from the nucleus to the cytoplasm by Exportin 5. In this compartment, pre-miRNAs are cleaved by another RNase called DICER to produce a 21–23 nt mature miRNAs. Either one or both of these individual 22 nt strands are incorporated into the RNA-induced silencing complex (RISC, composed partially by specific Argonaute family proteins) guiding mature miRNAs to 30 UTR of target mRNAs. The perfect complementary sequence between miRNA and its mRNA target leads to down-regulation by translational repression or transcript degradation (see references in Ghildiyal and Zamore, 2009; Katahira and Yoneda, 2011). SiRNAs are also processed by the DICER endonuclease from precursors with double-stranded RNA (dsRNA) features. PiRNAs biogenesis is distinct from that of miRNAs and siRNAs. Indeed, piRNAs are longer small RNAs and their precursors, in general, lack dsRNA features. Their production does not require Dicer (Ghildiyal and Zamore, 2009) and they are specifically expressed in the germ cells of the testis (Aravin et al., 2006; Girard et al., 2006; Grivna et al., 2006; Watanabe et al., 2006) suggesting an essential role in spermatogenesis. PiRNAs interact with mouse piwi-family proteins (Miwi, Mili, Miwi2 named PIWIL1, PIWIL2 and PIWIL4

respectively in human or bovine; in these last species a fourth piwi-family protein has been described as PIWIL3) (Sasaki et al., 2003). Up to now, only one of them (MILI protein) has been detected in oocytes at their early stages of growth (Watanabe et al., 2008). In mice, the suppression of any of these individual proteins causes activation of transposable elements, spermatogenesis arrest and male sterility (Carmell et al., 2007; Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004) suggesting a potential role of piRNAs to suppress transposons mobilization in male germline. One structural characteristic observed in the germline is an electron-dense cytoplasmic domain called germinal granules or ‘‘nuage’’ (Eddy, 1975). Theses germinal granules constitute an RNA and protein rich structure in germ cells of divergent species (several are listed in Table 2) and molecular compositions are conserved suggesting that they possess a common and essential role(s) in the germline. Several small RNAs were identified to have a role in female gonad development. First, in the mouse ovary, around 400 small RNAs were identified with different lengths (Ro et al., 2007), including miRNAs and piRNAs. Some of them have an ovarian-specific expression suggesting a role of these small RNAs in the control of folliculogenesis and female fertility. In growing mouse oocytes, piRNAs and siRNAs were also described corresponding to mRNAs or retrotransposons (Watanabe et al., 2008). On the other hand, although MILI is the only Piwi-family protein detected in oocytes (Watanabe et al., 2008) its suppression has no effect on mouse female fertility (Kuramochi-Miyagawa et al., 2004). If piRNAs seem to have a role in the protection of the male germline by avoiding transposon activation, similar mechanisms to protect female germline have not been demonstrated. In females, piRNA should not be the only factors to maintain stability of female germline. An interesting study was published in early 2011, identifying the expression of several miRNAs in ovine fetal gonads (Torley et al., 2011). One hundred twenty eight miRNAs with mature sequence perfectly homologous between human, mouse bovine and/or goat were selected in public database (miRBase, http:// www.mirbase.org/) and their relative expression was studied in the female and the male gonads of fetal sheep between two developmental stages. The selected stages were 42dpc corresponding to the ovigerous cord development in females and testicular cord differentiation in males and 75dpc where primordial follicles form in females. Significant differences of miRNAs expression were shown between both sexes and developmental stages. Then, with the help of several bioinformatics tools (Targetscanhuman 5.1: http:// www.targetscan.org/; MetaMir: http://mami.med.harvard.edu/ and miRGator: http://genome.ewha.ac.kr/miRGator/), several genes were identified to be targets of these differentially expressed miRNAs including ESR1 (miRNA: miR-22), CYP19A1 (Let 7a, c, d, e, g), FST (miR-410), WNT4 (miR-211) and SOX9 (miR-101) genes (Torley et al., 2011). Moreover, in situ hybridization of miR-22 revealed specific localization of this small RNA in testicular cords of fetal testis in agreement with its up-regulation during testicular development and down-regulation during fetal ovarian development. Estrogen signaling plays an important role during ruminant fetal ovarian development as we already mentioned in this review (Pannetier et al., 2006; Mauléon et al., 1977; Quirke et al., 2001; Shemesh, 1980; Dominguez et al., 1988; Yang and Fortune, 2008) and miR-22 seems to be involved in repressing estrogen signaling within sheep fetal testis. Moreover, miR-196a levels which were

Table 2 Protein components of mammalian germinal granules. Name

Function/domain

Mutant phenotypes

References

Maelstrom Miwi/Piwil1, Mili/Piwil2 Tdrd1/MTR-1, Tdrd6, Tdrd7 Mov10L1

HMG box Paz and PIWI domains Tudor domains repeat DExD-box helicase

Male Male Male Male

Costa et al. (2006) and Soper et al. (2008) Grivna et al. (2006) and Kuramochi-Miyagawa et al. (2004) Chuma et al. (2003), Chuma et al. (2006), Hosokawa et al. (2007) Frost et al. (2010) and Zheng et al. (2010)

sterile sterile sterile (Tdrd1) sterile

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shown to be increased in ovine 42dpc ovary versus 75jpc (Torley et al., 2011) were shown be increased in bovine four-cell and eight-cell stage embryos and declines at morula and blastocyst stages (Tripurani et al., 2011). Recent research in the world of small RNA has pointed out new dimensions of gene expression regulation through translational suppression, mRNA destabilization, and chromatin remodeling. Numerous small RNAs are expressed in gonads and research will probably increase in the next few years because these small RNAs are likely play a critical role in controlling the male gonadal development as initially described, as well as ovarian development. 5. Concluding remarks Historically, research on the ovary has been less developed as compared to that on the testis. However, investigations on ovarian differentiation have expanded rapidly in the last decades with the improvement of genetic inactivation techniques. The generation of mouse models has provided lot of knowledge about mechanisms underlying early ovarian development. In this way, these mutant animals have pointed out some genes that could explain defective reproductive conditions in humans. However, data obtained from mouse are not always applicable to other mammalian species such as humans. While components of certain pathways are conserved between species, many elements of the regulation of ovogenesis may be species-specific and studies of early ovarian development in species other than mouse are of certain interest. New techniques of gene targeting in species other than mice (with Zinc finger nucleases) could help in the understanding of regulation of mammalian ovarian development. Finally, research on small RNAs (miRNA, pi-RNA and their associated proteins) present new and greater understanding of the developing gonad and will highlight new regulating pathways in gametogenesis. The new technologies of high-throughput cDNA sequencing (RNA-Seq) should increase our knowledge in this research area. Acknowledgements We are grateful to our colleagues Drs. Corinne Cotinot and Eric Pailhoux for their critical reading of the manuscript. Special thanks to Jacques Migeon (from Seattle) for English corrections. We apologize to colleagues whose work could not be cited because of space limitations. References Abu-Abed, S., MacLean, G., Fraulob, V., Chambon, P., Petkovich, M., Dolle, P., 2002. Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis. Mech. Dev. 110, 173–177. Adelman, C.A., Petrini, J.H., 2008. ZIP4H (TEX11) deficiency in the mouse impairs meiotic double strand break repair and the regulation of crossing over. PLoS Genet. 4, e1000042. Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., Chien, M., Russo, J.J., Ju, J., Sheridan, R., Sander, C., Zavolan, M., Tuschl, T., 2006. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203–207. Baillet, A., Mandon-Pepin, B., Cabau, C., Poumerol, E., Pailhoux, E., Cotinot, C., 2008. Identification of transcripts involved in meiosis and follicle formation during ovine ovary development. BMC Genom. 9, 436. Baker, S.M., Bronner, C.E., Zhang, L., Plug, A.W., Robatzek, M., Warren, G., Elliott, E.A., Yu, J., Ashley, T., Arnheim, N., Flavell, R.A., Liskay, R.M., 1995. Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell 82, 309–319. Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Baltus, A.E., Menke, D.B., Hu, Y.C., Goodheart, M.L., Carpenter, A.E., de Rooij, D.G., Page, D.C., 2006. In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nat. Genet. 38, 1430–1434. Best, D., Sahlender, D.A., Walther, N., Peden, A.A., Adams, I.R., 2008. Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline–soma interactions in mice. Development 135, 1415–1425.

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