Human chorionic gonadotropin-dependent up-regulation of epiregulin and amphiregulin in equine and bovine follicles during the ovulatory process

General and Comparative Endocrinology 180 (2013) 39–47

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Human chorionic gonadotropin-dependent up-regulation of epiregulin and amphiregulin in equine and bovine follicles during the ovulatory process q Khampoun Sayasith a,⇑, Jacques Lussier a, Monique Doré b, Jean Sirois a a Centre de recherche en reproduction animale and Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6 b Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6

a r t i c l e

i n f o

Article history: Received 9 July 2012 Revised 18 October 2012 Accepted 22 October 2012 Available online 21 November 2012 Keywords: EREG AREG Ovulatory process Equine preovulatory follicles Primary bovine granulosa cells Ovulation-regulated genes

a b s t r a c t Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39 h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0 = day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0 h but increased thereafter (P < 0.05), with maximal levels observed 33–39 h post-hCG. This significant increase was observed in both granulosa and theca cells. Immunohistochemistry and immunoblot analyses confirmed the hCG-dependent induction of EREG protein in both cell types. RT-PCR/Southern blot analyses of ADAM17, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, showed that levels of its transcript were high and remained constant throughout the period studied. Studies on the hCG-dependent regulation of EREG and AREG in bovine preovulatory follicles in vivo showed that the induction of both transcripts was transient, observed predominantly at 6 h post-hCG and localized only in granulosa cells. To characterize the effect of epidermal growth factor receptor (EGFR) activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species, primary cultures of bovine granulosa cells were established. Results from RT-PCR analyses revealed that EREG and AREG mRNAs were induced by forskolin treatment in vitro; but the EGFR inhibitor PD153035 suppressed the forskolin-dependent induction of several ovulation-related transcripts, including PTGS2, PTGER2, TNFAIP6, PGR, MMP1, VEGFA, and CTSL2 mRNAs. Moreover, these transcripts were induced in granulosa cell cultures by EGF, an analog of EREG and AREG. Collectively, this study identifies differences in the temporal and cellular localization of EREG and AREG expression in equine and bovine preovulatory follicles, and underscores the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Ovarian follicular development is accompanied by the differentiation of granulosa cells into two compartments: the mural granAbbreviations: LH, luteinizing hormone; hCG, human chorionic gonadotropin; COC, cumulus–oocyte complex; HAS2, hyaluronan synthase 2; PTGS2, prostaglandin synthase 2; TNFAIP6, tumor necrosis factor-alpha-induced protein 6; EGFR, epidermal growth factor receptor; EREG, epiregulin; AREG, amphiregulin; ADAM17, a disintegrin and metalloprotease 17; PGR, progesterone receptor; CTSL2, cathepsin L type 2; VEGFA, vascular endothelial growth factor; MMP1, matrix metalloprotease type 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. q This study was supported by Natural Sciences and Engineering Research Council of Canada Grant OPG0171135 (to J.S.). ⇑ Corresponding author. Address: Faculté de médecine vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6. Fax: +1 450 778 8103. E-mail address: [email protected] (K. Sayasith).

0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2012.10.012

ulosa cells which form with theca cells the follicular wall, and the cumulus cells which surround and form a compact complex with the oocyte, the cumulus–oocyte complex (COC) [42,43,46]. The preovulatory LH surge triggers the activation of multiple intracellular signaling cascades regulating the expression of genes required for ovulation [42,43,46]. COC expansion is essential for ovulation, and a number of LH-induced genes critical for this process have been identified, including hyaluronan synthase 2 (HAS2), prostaglandin synthase 2 (PTGS2), tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) and epidermal growth factor receptor (EGFR) ligands epiregulin (EREG) and amphiregulin (AREG) [13,36]. However, because cumulus cells and oocytes are known to express very few LH receptors in a number of species, including rodent, cattle and mare [12,17,40], and thus do not respond to direct exposure of LH [37], the molecular and biological mechanisms by which LH exerts its effect on the COC are thought to

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depend on paracrine factors secreted by mural granulosa and theca cells which express LH receptors [12,17,23,40]. EREG and AREG are two major EGFR ligands that are structurally and functionally related [18]. They are synthesized as transmembrane protein precursors containing a single bioactive EGF-like sequence in the ectodomain [24,31]. The cleavage of this ectodomain by a member of metalloprotease family, such as a disintegrin and metalloprotease 17 (ADAM17), is known to play a key role in shedding bioactive EREG and AREG, and enabling their interaction with specific EGFRs on target cells [10,21,38,44]. A number of studies have reported that EREG and AREG expression is rapidly induced by LH or human chorionic gonadotropin (hCG) in mural granulosa cells of rodent preovulatory follicles [2,33,50]. Other investigations have provided convincing evidence for a critical role of EREG and AREG in the activation of cumulus expansion and oocyte meiotic resumption, as both processes were induced by EREG, AREG or EGF in cultures of intact COC [2,36]. Moreover, cumulus cells were shown to express EGFR, and its phosphorylation by EGF-like ligands was key for successful ovulation [1,2,33,35,36]. Collectively, these reports suggest that EREG and AREG induced by the LH surge in mural granulosa cells act as endogenous messengers to propagate signals throughout the follicles prior to ovulation [8,36]. The role of EGFR signaling in ovulation was further evidenced by genetic ablation studies in which mice bearing a hypomorphic allele of Egfr, Egfrwa2 (waved 2), showed a significant compromise in COC expansion and oocyte maturation [20]. Interestingly, double mutants Areg/Egfrwa2/wa2 mice generated by the introduction of null alleles Areg into Egfrwa2 background were infertile with a severe impairment of COC expansion and oocyte maturation, leading to ovulation failure [20]. However, single Areg or Ereg knockout mice remained fertile likely because of functional redundancy [20,25,30]. Although the regulation and role of EREG and AREG during ovulation have been studied extensively in rodents, much less is known about their expression and control in ovarian follicles of large monoovulatory animal species like the horse and the cow. A previous report in mares showed a gonadotropin-dependent increase in EREG and AREG mRNAs in granulosa cells collected by follicular aspiration 2–6 h after hCG treatment, as compared to cells collected prior to hCG [28]. In cattle, one study using GnRH as an agonist revealed a significant induction of AREG transcripts in both granulosa and theca cells [27], whereas another investigation using differential display reverse transcription polymerase chain reaction analysis and suppressive subtractive hybridization showed the expression of EREG mRNA in granulosa cells of bovine follicles containing a competent oocyte [41]. The development of the equine preovulatory follicle is of particular interest because of its large size (40–45 mm), relatively long ovulatory process (defined as the interval of time from hCG administration to follicular rupture) and pattern of recruitment and selection, which resembles that observed in women [11,16,53]. Thus, the objectives of the present work were to characterize the gonadotropin-dependent regulation of EREG and AREG in equine follicles prior to ovulation and, using primary cultures of bovine granulosa cells, to document the effect of EGFR activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species. 2. Materials and methods 2.1. Animal tissues and RNA extraction Equine preovulatory follicles and corpora lutea were isolated at specific stages of the estrous cycle from Standardbred and Thoroughbred mares, as previously described [22]. Briefly, when preovulatory follicles reached 35 mm in diameter during estrus, the ovulatory process was induced by injection of hCG (2500 IU, iv), and ovariectomies were performed via colpotomy using an ovario-

tome at 0, 12, 24, 30, 33, 36 and 39 h post-hCG (n = 4–6 mares/time point). The interval of time from hCG administration to ovulation (i.e. the length of the ovulatory process) corresponded to 39–42 h in the group of animals used in the present study [22]. Corpora lutea (n = 3 mares) were isolated on day 8 after ovulation (day 0 = day of ovulation). Preovulatory follicles and corpora lutea were dissected from the surrounding ovarian tissues with a scalpel. Follicles were further dissected into three cellular preparations, including a follicular wall preparation (theca interna with attached granulosa cells), and isolated preparations of granulosa cells and theca interna cells, as described [54]. Testicular tissues were obtained from the Hôpital équin of the Faculté de médecine vétérinaire (Université de Montréal) following a routine castration, whereas other non-ovarian tissues were collected at a local slaughterhouse. For bovine preovulatory follicles, Holstein heifers (2–3 yr old) exhibiting normal estrous cycles were used as previously described [55]. Briefly, luteolysis was induced on day 7 of the estrous cycle (day 0 = day of estrus) with 25 mg PGF2a (Lutalyse, Upjohn), and preovulatory follicles were obtained after intravenous administration of an ovulatory dose of hCG (3000 IU) 36 h after induction of luteolysis. The ovary bearing the preovulatory follicle was isolated by ovariectomy via colpotomy from individual heifers from 0 to 24 h after hCG. The interval of time from hCG administration to ovulation is 28–30 h in this animal model. Bovine preovulatory follicles were dissected into three cellular preparations, as described above [55]. In all cases, total RNAs were extracted from tissues with TRIzol reagent (Invitrogen Canada Inc.), according to manufacturer’s instructions using a Kinematica PT 1200C Polytron Homogenizer (Fisher Scientific, Montréal, Canada). The relative purity of each cellular preparation is estimated to exceed 95% based on the selective expression of P450 17a-hydroxylase-C17–20 lyase (CYP17A1) and P450 aromatase (CYP19A1) mRNAs by theca interna and granulosa cells, respectively [3]. All animal procedures were approved by the Institutional animal Care and Use Committee of the University of Montréal and were consistent with the Guidelines of the Canadian Council of Animal Care. 2.2. Semiquantitative RT-PCR and Southern blot analysis of equine tissues The semiquantitative analysis of EREG, AREG, ADAM17 and RPL7A (internal control gene) mRNA levels in equine tissues was performed using the OneStep RT-PCR System (Qiagen, Mississauga, Canada) as previously described [47,48], and sense (50 -TTC CAT CTT CTC CAA GCA GTT CTC-30 ) and anti-sense (50 -CAG ACT TGT GGC AAC GCT GGA TCC-30 ) primers specific for equine EREG (generating a DNA fragment of 440 bp), sense (50 -GCT GGA TTG GAT GTC AAT GAC ACC-30 ) and anti-sense (50 -TTA CTG TCA ACC ATG CTG TGA GTC-30 ) primers specific for equine AREG (generating a DNA fragment of 493 bp), sense (50 -CAC CGT GTG CTT GGA TCT TGG C-30 ) and anti-sense (50 -CTG TCA ACA CGA CTC TGA CGC-30 ) primers specific for equine ADAM17 (generating a DNA fragment of 624 bp), and sense (50 -ACA GGA CAT CCA GCC CAA ACG-30 ) and anti-sense (50 -GCT CCT TTG TCT TCC GAG TTG-30 ) primers specific for equine RPL7A (generating a DNA fragment of 516 bp). These primers were designed from published sequences of equine EREG (accession number: XM_001490281), equine AREG (accession number: XM_001489471), equine ADAM17 (accession number: XM_001918266) and equine RPL7A (accession number: AF508309). Each reaction was conducted using 100 ng of total RNA, and cycling conditions were one cycle of 50 °C for 30 min and 95 °C for 15 min, followed by a variable number of cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 2 min. The number of PCR cycles used was optimized for each gene to fall within the linear range of PCR amplification and was 22, 25, 30 and 18 cycles

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K. Sayasith et al. / General and Comparative Endocrinology 180 (2013) 39–47

for EREG, AREG, ADAM17 and RPL7A, respectively. PCR products were electrophoresed on 2% TAE-agarose gels, transferred to Biotrans nylon membranes (ICN Pharmaceuticals, Montréal, Québec), and hybridized with corresponding radiolabeled EREG, AREG and RPL7A cDNA fragments using QuikHyb hybridization solution (Stratagene, LaJolla, CA). Membranes were exposed to a phosphor screen. Signals were quantified on a Storm imaging system using the ImageQuant software version 1.1 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA). To confirm their identity, PCR products were subcloned into pGEM-T Easy vector, and sequenced. 2.3. Cell extracts and Western blot analysis Protein extracts were prepared as previously described [56,58]. Briefly, tissues were cut into small pieces and homogenized in prechilled TED cell lysis buffer containing 50 mM Tris (pH 8.0), 10 mM EDTA, 1 mM diethyldithiocarbamic acid (DEDTC), and 0.1% Tween20, and centrifuged at 30,000g for 1 h at 4 °C. Supernatants containing cytosolic proteins were removed and stored at 80 °C until ready for use. The crude pellets (membranes, nuclei, and mitochondria) were resuspended in cold TED sonication buffer containing 20 mM Tris (pH 8.0), 50 mM EDTA, 0.1 mM DEDTC, and 1.0% Tween-20, sonicated on ice (three cycles, 8 s/cycle), and centrifuged at 16,000g for 15 min at 4 °C. Supernatants containing solubilized membrane (microsomal) proteins were recovered and stored at 80 °C. Protein concentration was quantified by the method of Bradford (Bio-Rad Protein Assay; [5]). Samples (100 lg/well) were resolved by one-dimensional SDS–PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were incubated with a specific rabbit polyclonal anti-human EREG antibody (1:400; Deciphergen Biotechnology, Cheshire, CT) [26]. Immunoreactive proteins were visualized on Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) after incubation with the horseradish peroxidase-linked donkey anti-rabbit secondary antibody (1:5000) and the enhanced chemiluminescence system (ECL Plus), following the manufacturer’s protocol (Amersham Pharmacia Biotech). 2.4. Immunohistochemical localization of EREG in equine follicles Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), as previously described [54]. Briefly, formalin-fixed tissues were paraffinembedded, and 3 lm-thick sections were prepared and then the paraffin was removed using a series of alcohol concentrations. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum for 20 min at room temperature. The anti-EREG antibody was diluted in PBS (1:150) and applied, and sections were incubated overnight at 4 °C. Control sections were incubated with PBS alone. After rinsing in PBS for 10 min, a biotinylated goat anti-rabbit antibody (1:222; Vector Laboratories, Burlingame, CA) was applied, and sections were incubated for 45 min at room temperature. After washing in PBS for 10 min, sections were incubated with the avidin DH-biotinylated horseradish peroxidase H reagents for 45 min at room temperature, washed with PBS for 10 min, and incubated with diaminobenzidine tetrahydrochloride (DAB; Sigma, St.-Louis, MO) as the chromogen substrate. Sections were counterstained with Gill’s hematoxylin stain and mounted. 2.5. Primary cultures of granulosa cells and RT-PCR analysis of bovine transcripts Primary cultures of bovine granulosa cells were prepared as previously described [29,49]. Briefly, pairs of bovine ovaries bear-

ing a newly formed corpus luteum and a follicle of 8–12 mm in diameter (dominant follicle of the first wave of the estrous cycle) were obtained from a slaughterhouse). Granulosa cells were isolated from largest follicles, seeded in 6-well plates at a density of 1  106 cells per 2 ml of minimal essential medium (MEM) supplemented with L-glutamine, non-essential amino acids, 2% fetal bovine serum (FBS), insulin (1 lg/ml), transferrin (5 lg/ml), and penicillin (100 units/ml)/streptomycin (100 lg/ml), and incubated at 37 °C in a humidified atmosphere of 5% CO2. Confluent cells were serum-starved overnight in MEM and then incubated in serumfree MEM in the absence or presence of variable agonists, including forskolin (FSK; 10 lM; Calbiochem, La Jolla, CA) alone, FSK (10 lM) and PD153035 (PD; 10 lM; an inhibitor of EGFR tyrosine kinase activity; Calbiochem, La Jolla, CA), or soluble recombinant human EGF (10 ng/ml; Sigma–Aldrich, Oakville, Canada). After various times of incubation, cells were harvested, and total RNA was extracted as described previously [49]. The expression of bovine transcripts coding for EREG, AREG, prostaglandin (PG) G/H synthase-2 (PTGS2), PG E2 receptor (PTGER2), tumor necrosis factor-a -induced protein 6 (TNFAIP6), progesterone receptor (PGR), cathepsin L type 2 (CTSL2), vascular endothelial growth factor (VEGFA), matrix metalloprotease type 1 (MMP1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control gene) were analyzed by RT-PCR using RNA extracts (100 ng), the OneStep RT-PCR kit (Qiagen) and appropriate primers (see Table 1). Each reaction was performed using one cycle of 50 °C for 30 min and 95 °C for 15 min followed by 25 cycles for GAPDH or 35 cycles for other genes of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 2 min. PCR products were electrophoresed on 2% Tris–acetate/ EDTA–agarose gels containing ethidium bromide (0.5 lg/ml), visualized under ultraviolet light, digitized and the intensity of signal was quantified by densitometry using the ImageQuant software (Amersham Pharmacia Biotech). 2.6. Statistical analysis One-way ANOVA was used to test the effect of time after hCG on levels of EREG, AREG and ADAM17 mRNAs. Prior to analysis, levels

Table 1 Primer sets of bovine genes used in RT-PCR analyses. Genes

Primer sequences

PCR product (bp)

EREG

50 -CAACTGTGATTCCTTCATGTATCCC-30 (sense) 50 -CGATTTCTGTACCATCTGC-30 (antisense) 50 -CTGCTGGATTAGACGTCAATGAC-30 (sense) 50 -GCAATAGCTGTGAAGGTCATGG-30 (antisense) 50 -CACAGTGCACTACATACTTACC-30 (sense) 50 -GTCTGGAACAACTGCTCATCGC-30 (antisense) 50 -CTGCTGGATCATTGGAAGTATGC-30 (sense) 50 -TCCATCTCGCTGTTCCACGTG-30 (antisense) 50 -TACCGCAGCTAGAGGCAGCC-30 (sense) 50 -CTTCAAGGTCATGACATTTCCTG-30 (antisense) 50 -TAGAAAGTGCTGTCAGGCTGG-30 (sense) 50 -ATAGAAACGCTGTGAGCTAGG-30 (antisense) 50 -TTGGAGCCTTGCCTTGCTGCTC-30 (sense) 50 -CGGCTTGTCACATCTGCAAGT-30 (antisense) 50 -GATGCACATTGGCACCAGTGG-30 (sense) 50 -ATAGGATTCCTCTGAGTCCAGGC-30 (antisense) 50 -CTTGGACTTGCTCATTCTACTGAC-30 (sense) 50 -GGCATCGATGCTCTTCACCGTTC-30 (antisense) 50 -GTTTCCAGTAGATTCCAC CC-30 (sense) 50 -TCCACCACCCTGTTGCTGTA-30 (antisense)

342

AREG PTGS2 PTGER2 TNFAIP6 PGR VEGFA CTSL2 MMP1 GAPDH

561 735 439 636 742 338 548 467 850

EREG, epiregulin; AREG, amphiregulin; PTGS2, prostaglandin G/H synthase-2; PTGER2, prostaglandin E2 receptor; TNFAIP6, tumor necrosis factor-a-induced protein 6; PGR, progesterone receptor; CTSL2, cathepsin L type 2; VEGFA, vascular endothelial growth factor; MMP1, matrix metalloprotease type 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; bp, base pair.

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K. Sayasith et al. / General and Comparative Endocrinology 180 (2013) 39–47

A

Fo l Br licle a H in e Lu art n Th g y Li mu ve s Ki r d Ad ney r Sp ena le l St en o U ma te ch Te rus s Sk tis i Mn us cl e

of experimental genes were normalized with those of the control gene RPL7A for equine tissues, or those of GAPDH for bovine samples. When ANOVAs indicated significant differences (P < 0.05), the Dunnett’s test was used to compare different time points after hCG with 0 h. Statistical analyses were performed using JMP software (SAS Institute, Inc., Carry, NC).

bp

EREG-

-440

AREG-

-493

RPL7A-

-516

3.2. Cellular localization of EREG and AREG expression in equine preovulatory follicles To determine which cell type was responsible for the follicular expression of EREG and AREG mRNA, RT-PCR/Southern blot were performed with isolated preparations of granulosa cells and theca interna of preovulatory follicles collected 0–39 h after hCG. Results revealed that the expression of EREG and AREG transcripts occurred in both follicular cell types. In granulosa cells, levels of EREG mRNA were almost absent at 0 h, but were increased at 12, 33, 36 and 39 h post-hCG when compared to preparations obtained at 0 h (P < 0.05; Fig. 2A), whereas those of AREG transcripts were increased at all time points obtained after hCG treatment (P < 0.05; Fig. 2B). In theca interna cells, the expression of EREG transcripts was relatively low at 0 h, remained unchanged at 12 and 24, but were increased 30–39 h post-hCG when compared to preparations obtained at 0 h (P < 0.05; Fig. 2C), whereas that of AREG mRNA was relatively low and unchanged from 0 to 30 h, but was increased at 33, 36 and 39 h post-hCG (P < 0.05; Fig. 2D). To determine whether the induction of EREG transcript was related to the regulation of EREG protein in preovulatory follicles, immunoblotting was performed with microsomal (M; membrane fraction) and soluble (C; cytosolic fraction) protein extracts isolated from preovulatory follicles collected at 0, 36 and 39 h post-hCG. Analyses with an anti-human EREG antibody revealed one protein band corresponding to the predicted size of EREG (approximately 28 kDa; Fig. 3A). Levels of EREG protein remained low or undetectable in the microsomal fraction of follicles obtained before and after hCG treatment, but increased markedly in the cytosolic fraction (Fig. 3A), in keeping with the induction of the transcript. Fig. 3B shows that an immunoreactive EREG protein signal of the predicted size was also observed in tissues shown to express EREG transcript (uterus and lung, Fig. 1A),

0 12 24 36

C Hrs post-hCG 0 12 24 36 -EREG

-AREG

-RPL7A

-RPL7A

*

40

AREG/RPL7A

40

*

30 20

* 0

12

20 10

*

*

0 24

36

Hrs post-hCG

CL (D 8)

10

30

*

0 0

12

24

36

Hrs post-hCG

CL (D 8)

Hrs post-hCG

EREG/RPL7A

The relative expression of EREG and AREG mRNA in various equine tissues was determined by RT-PCR/Southern blot. Results showed that EREG and AREG transcripts were expressed at different levels in equine tissues. Levels of EREG mRNA were very high in the wall of preovulatory follicles isolated 36 h after hCG, moderate in uterus and muscle, low in lung, kidney and adrenal, and very low or undetectable in other tissues tested (Fig. 1A). Levels of AREG transcripts were relatively high in preovulatory follicle and testis, low in lung, kidney, uterus and skin, and very low or undetectable in other tissues (Fig. 1A). To study the regulation of EREG and AREG transcripts during the ovulatory process, RT-PCR/Southern blot analyses were performed using total RNA extracted from the wall of equine preovulatory follicles isolated between 0 and 36 h post-hCG, and corpora lutea obtained on day 8 of the estrous cycle. Results showed that levels of EREG and AREG mRNAs were very low or undetectable before hCG treatment (0 h) but significantly increased thereafter to reach a peak at 36 h post-hCG (P < 0.05; Fig. 1B and C). Levels of both transcripts were very low or undetectable in corpora lutea.

B

CL (D 8)

3.1. Distribution of EREG and AREG mRNA in equine tissues and their up-regulation in equine preovulatory follicles by hCG

CL (D 8)

3. Results

Fig. 1. Expression of EREG and AREG transcripts in equine tissues and their gonadotropin-dependent upregulation in equine preovulatory follicles. Total RNAs were extracted from various tissues (A), preovulatory follicles isolated 0, 12, 24 and 36 h after hCG administration, and corpora lutea (CL) obtained on day 8 of the estrous cycle (B and C). Samples (100 ng) were analyzed by semiquantitative RTPCR/Southern blot for EREG, AREG and RPL7A (control gene) content, as described in Section 2. The number of PCR cycles for EREG, AREG and RPL7A was 22, 25 and 18 cycles, respectively, and was optimized to fall within the linear range of PCR amplification. Numbers on the right indicate the size of the PCR fragment (A). Representative results of EREG, AREG and RPL7A mRNA levels are presented from one follicle/time point (B and C). Each EREG (B) and AREG (C) signal was normalized with that of the control gene, RPL7A, and results are presented as ratios of EREG or AREG to RPL7A (mean ± SEM; n = 4–6 distinct follicles (i.e. mares) or n = 3 corpora lutea per time point). Bars marked with an asterisk are significantly different from 0 h (P < 0.05).

but not in tissues in which no EREG transcript was detected (testis and CL; Fig. 1A and B). Attempts to study the expression of AREG protein in preovulatory follicles were unsuccessful as the specificity of the antibody on equine tissues could not be established. To define the cellular localization of the EREG protein expression, immunohistochemistry was performed on sections of equine preovulatory follicles isolated before and after hCG treatment. Results showed that follicles obtained before hCG (0 h) were characterized by a compact granulosa cell layer and a very low or undetectable EREG immunoreactivity in granulosa and theca layers (Fig. 3C). However, treatment with hCG induced a characteristic expansion of the equine granulosa cell layer, and a marked increase of EREG immunohistochemical staining was observed in granulosa and theca layers of follicles isolated at 33 h post-hCG (Fig. 3E and F), as compared with 0 h (Fig. 3C), in accordance with the induction of EREG transcripts in these follicular cells detected at this time point (Fig. 2). As a negative control, no staining was observed when a section of the follicle presented in Fig. 3E (33 h post-hCG) was incubated with PBS without the primary antibody (Fig. 3D).

K. Sayasith et al. / General and Comparative Endocrinology 180 (2013) 39–47

A. Granulosa cells

C.Theca interna

Hrs post-hCG

Hrs post-hCG 0 12 24 30 33 36 39

-EREG

-EREG

-RPL7A

-RPL7A

40

*

30

*

*

20 10

*

EREG/RPL7A

EREG/RPL7A

0 12 24 30 33 36 39

0

40

*

*

20

*

10 0

0

12

24

30

33 36

0

39

12

Hrs post-hCG

30 33

Hrs post-hCG

0 12 24 30 33 36 39

0 12 24 30 33 36 39

-AREG

-RPL7A

-RPL7A

*

*

30 20

*

*

39

-AREG

AREG/RPL7A

*

36

D. Theca interna

Hrs post-hCG

40

10

24

Hrs post-hCG

B. Granulosa cells

AREG/RPL7A

*

30

*

0

40

*

*

30

*

20 10 0

0

12

24

30 33

36

Hrs post-hCG

39

0

12

24

30 33

36

39

Hrs post-hCG

Fig. 2. Gonadotropin-dependent regulation of EREG and AREG mRNA in equine granulosa and theca interna cells. Total RNA was extracted from individual preparations of granulosa cells and theca interna cells obtained from equine preovulatory follicles isolated 0–39 h post-hCG. Samples (100 ng) were analyzed for EREG, AREG and RPL7A mRNA content by semiquantitative RT-PCR/Southern blot, as described in Section 2. Representative results of EREG (A and C), AREG (B and D) and RPL7A mRNA levels are presented from a representative granulosa (A and B) and theca interna (C and D) sample per time point. EREG and AREG signals were normalized with those of the control gene, RPL7A, and results are presented as ratios of EREG or AREG to RPL7A (mean ± SEM; n = 4 samples per time point). Bars marked with an asterisk are significantly different from 0 h (P < 0.05).

3.3. Expression of ADAM17 transcripts in equine preovulatory follicles To examine the expression and regulation of ADAM17 mRNA, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, RT-PCR/Southern blot were performed on extracts of equine preovulatory follicles obtained before and after hCG, and corpora lutea, as described above. Results revealed that levels of ADAM17 mRNA were high prior to hCG (0 h), and remained relatively constant after hCG treatment (Fig. 4). Similarly, levels of ADAM17 mRNA were high and remained unchanged in isolated preparations of granulosa and theca cells of preovulatory follicles from 0 to 39 h post-hCG (data not shown), indicating that both cell types constitutively expressed ADAM17 throughout the period studied. High levels of ADAM17 mRNA were also observed in corpora lutea (Fig. 4). 3.4. Effect of EGFR activation on the expression of ovulation-related genes in granulosa cells Equine granulosa cells could not be used in vitro to study the effect of EGFR activation on the expression of ovulation-related genes because of difficulties associated with the establishment

43

and maintenance of primary cultures of equine cells. Thus, bovine granulosa cells were used as a model to study in vitro the effect on EGFR activation in a monoovulatory species. Prior to studies in vitro, the regulation of EREG and AREG transcripts was characterized in vivo by RT-PCR in bovine preovulatory follicles obtained before and after hCG treatment. Results showed that levels of both transcripts were very low or undetectable at 0 h, increased markedly at 6 h (0 h; P < 0.05) and returned to baseline at 12 h (AREG) or 24 h (EREG) after hCG (Fig. 5A–C). Analyses performed on isolated preparation of granulosa and theca interna obtained from bovine follicles isolated 6 h post-hCG revealed that expression of both transcripts was high in granulosa cells but very low or undetectable in theca cells (Fig. 5D–F). To determine whether the hCG-dependent up-regulation of EREG and AREG observed in vivo could be recapitulated in vitro, granulosa cells isolated from dominant follicles (8–12 mm in diameter) of the first wave of the estrous cycle were cultured in the absence and presence of forskolin (FSK; 10 lM) for 0–24 h. Results from RT-PCR analyses showed that levels of EREG mRNA were low or undetectable before FSK treatment (0 h) but markedly increased from 6 to 24 h after FSK (P < 0.05; Fig. 6A and B), whereas those of AREG mRNA were low or undetectable at 0 h, significantly increased at 6 and 12 h but declined thereafter (Fig. 6A and C). To study the potential role of EGFR activation in the regulation of ovulation-related genes, two approaches were used. First, bovine granulosa cells were cultured for 24 h in the absence (Ctl) or presence of FSK and PD153035 (PD; 10 lM), an inhibitor of EGFR tyrosine kinase activity. Results revealed the induction of numerous ovulation-related genes by FSK, as expected (Fig. 6D). Interestingly, treatment with PD153035 blocked most of the FSK-dependent induction, suggesting an important contribution of EGFR tyrosine kinase activity (Fig. 6D). As a second approach, bovine granulosa cells were cultured in the presence of soluble EGF, an analog of EREG and AREG. Results showed that, although different kinetics were observed among transcripts, soluble EGF induced the expression of different mRNAs tested, again in keeping with a potential involvement of EGFR activation in the induction of ovulation-related genes. In contrast, levels of bovine GAPDH mRNA (control gene) remained relatively constant. 4. Discussion The preovulatory LH/gonadotropin surge triggers a remarkable change in follicle gene expression, which ultimately leads to cumulus expansion, oocyte meiotic resumption, follicular rupture and luteinization [42,43,46]. Cumulus expansion is required for ovulation and, since cumulus cells and oocytes express very low levels of LH receptors, expansion is thought to be induced indirectly by factors secreted by mural granulosa and theca cells [44]. EREG and AREG produced by mural follicular cells have been proposed as paracrine transducers of LH signal. Indeed, cultures of preovulatory follicles or isolated germinal vesicle-stage COCs with EREG or AREG induced cumulus expansion and oocyte meiotic resumption in a manner similar to that observed by LH/hCG in ovarian follicles in vivo [2,35,36]. To increase our understanding of EREG and AREG regulation in follicular cells of large monoovulatory animal species, the equine preovulatory follicle was selected as a model because of its large size (40–45 mm in diameter), protracted ovulatory process (39–42 h interval from hCG to ovulation) and ability to monitor preovulatory follicular development by ultrasonography [53]. The current study revealed the presence of a marked gonadotropin-dependent induction of EREG and AREG mRNAs in equine follicle prior to ovulation, with an induction observed in both granulosa and theca cells. A previous report investigating the expression of EREG and AREG mRNAs in equine granulosa cells collected by follicular aspiration

K. Sayasith et al. / General and Comparative Endocrinology 180 (2013) 39–47

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Fig. 3. Regulation and immunohistochemical localization of EREG protein in equine preovulatory follicles. (A) Microsomal (M) and cytosolic (C) protein extracts of preovulatory follicles collected at 0, 36 and 39 h post-hCG were prepared, and analyzed by one-dimensional SDS–PAGE and immunoblotting using a rabbit anti-human EREG polyclonal antibody (1:400 dilution), as described in Section 2. (B) Immunoblotting of protein extracts isolated from uterus, lung, testis and corpora lutea (CL) obtained on day 8 of the estrous cycle was performed for the specificity of EREG antibody against equine proteins. Results from one representative protein sample (100 lg/well) per time point are shown (n = 4 [i.e. 4 mares] per time point). A band corresponding to the EREG protein (28 kDa) is indicated. (C–F) Immunohistochemical staining was performed on formalin-fixed sections of preovulatory follicles isolated at 0 (C) and 33 h (E) post-hCG using an EREG antibody (1:150 dilution), as described in Section 2. (D) As a negative control, a follicle isolated at 33 h post-hCG presented in E was incubated with PBS in the absence of a primary antibody. (F) A magnification of framed region presented in E to show evidence of EREG protein expression in mural granulosa cells. Limits of granulosa cell (GC) and theca interna (TI) layers are indicated (C and E). The region above TI corresponds to a loosening (i.e. expansion) of a granulosa cell (GC) layer (D–F) induced by hCG. Results of one representative follicle/time point are shown (n = 3 [i.e. 3 mares] per time point). Magnification, 100 (C) and 200 (D and E).

between 0 and 6 h after hCG showed an increase of both transcripts 2–6 h post-hCG [28]. Although these results were of interest, the experimental approach precluded gathering information during most of the equine ovulatory process and regarding the potential contribution of different follicle cell types. In the present report, results with follicle wall preparations showed that an induction of EREG and AREG mRNAs was first observed at 12 h and maintained throughout the period studied, with maximal levels observed at 36 h post-hCG. However, although the highest levels of transcripts were observed just prior to ovulation, increases in gene expression observed at 12 h (this study) or earlier [28] could be equally important in regulating follicular and oocyte maturation. The pattern of induction observed in equine follicles differs from the one observed in monkey preovulatory follicles, where a transient increase of both transcripts observed at 12 h was followed by a decline at 24 and 36 h post-hCG [15,59]. Likewise, the sustained and progressive induction in equine follicles contrasts with that observed in bovine follicles where EREG and AREG mRNAs were only transient induced 6–12 h post-hCG (this study; ovulation occurs around 28–30 h post-hCG in this species). Interestingly, both granulosa and theca cells contributed to the elevated expression of EREG and AREG in equine follicles, again diverging from results obtained in rodents [2,36] and cattle (from this study)

in which the induction was observed only in mural granulosa cells. However, a previous study in cattle using GnRH as an agonist reported an induction of EREG and AREG in both granulosa and theca cells [27]. Collectively, these studies and others [2,15,27,39, 50,59] show that, while the gonadotropin-dependent regulation of EREG and AREG appears highly conserved, its temporal of induction and cellular localization vary greatly across species and may be affected by the experimental approach/agonist used. EREG and AREG are initially synthesized as precursor proteins spanning the cell membrane, and their release as soluble and active ligands is controlled by a proteolytic shedding activity required for ovulation [18,24,31]. ADAM17 is a known EGF-like ligand sheddase thought to play a critical role in preovulatory follicles [36]. Although the regulation of ADAM17 expression in ovarian cells remains largely unresolved, FSH was shown to rapidly induce ADAM17 mRNA porcine granulosa cells in vitro [60], whereas ADAM17 mRNA was downregulated in granulosa cells of bovine preovulatory follicles between 12 and 24 h after GnRH treatment in vivo [27]. The present study documents that levels of ADAM17 mRNA were high and remained constant in equine follicles throughout the period studied, suggesting that ADAM17 availability was not limiting. Thus, the induction of EREG and AREG appears as the predominant LH/hCG regulatory step in the produc-

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Fig. 4. Constitutive expression of ADAM17 in equine preovulatory follicles. RNA extracts prepared from equine preovulatory follicles isolated 0–36 h post-hCG, and corpora lutea (CL) obtained on day 8 of the cycle, were analyzed by semiquantitative RT-PCR/Southern blot for the expression of ADAM17 and RPL7A transcripts, as described in Section 2. The number of PCR cycles used for ADAM17 and RPL7A was 30 and 18 cycles, respectively, and was optimized to fall within the linear range of the PCR amplification. The ADAM17 signal was normalized with that of the control gene, RPL7A, and results are presented as ratios of ADAM17 to RPL7A (mean ± SEM; n = 4 distinct follicles or n = 3 CL per time point).

TI

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tion of soluble and bioactive EGF-like ligands in equine preovulatory follicles. The molecular control of EREG and AREG gene expression remains the subject of active investigations. In Ptgs2 and Ptger2 knockout mice, levels of LH-induced Areg and Ereg are markedly reduced in granulosa cells, suggesting an important role for PTGS2/PGE2/PTGER2 signaling [51,57]. Interestingly, the timing of PTGS2 induction in equine mural granulosa cells (30 h post-hCG; [4]) slightly precedes the marked increase of EREG and AREG mRNAs observed in these cells 33–39 h post-hCG (this study), thereby supporting a potential role of prostaglandins in enhancing EREG and AREG expression in equine follicles. The LH/hCG surge induces several genes that are essential for proper cumulus expansion and ovulation, as evidence from mice lacking Ptgs2 [9,32], Ptger2 [13,19], Tnfaip6 [14,34], Pgr [7,45], and Adamts1 [6,52]. Studies in rodents have revealed that the expression of a number of ovulation-related genes is dependent on LH-induction of Ereg and Areg [2,20,36,51]. Findings from the present study provide evidence for a functional link between induction of EGF-like ligands and the expression of ovulationrelated genes in bovine preovulatory follicles. Such evidence includes the ability of an EGFR tyrosine kinase inhibitor PD153035 to inhibit or repress the expression of forskolin-induced genes involved in prostaglandin synthesis and action (PTGS2, PTGER2), extracellular matrix and tissue remodeling (TNFAIP6, CTSL2 and MMP1), progesterone action (PGR) and angiogenesis (VEGFA). Furthermore, soluble EGF (an AREG and EREG analog) was able to stimulate mRNA expression of these genes in primary granulosa cell cultures. Interestingly, the time-course of induction of EGF-like ligands (6 h post-hCG) observed in the present study precedes that previously observed for PTGS2 (18 h post-hCG; [56]) in bovine preovulatory follicles in vivo, suggesting a potential link between the EGFR activation and induction of follicular prostaglandin synthesis in the cow. The demonstration of such functional relationship will require further investigations.

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Fig. 5. Gonadotropin-dependent regulation of EREG and AREG in bovine preovulatory follicles. Total RNA extracts (100 ng) prepared from bovine preovulatory follicles collected 0, 6, 12 and 24 h after hCG treatment (A–C), and from isolated preparations of granulosa cells (GC) and theca interna cells (TI) of bovine follicles collected at 6 h post-hCG (D–F) were analyzed by RT-PCR for the mRNA content of bovine genes, EREG, AREG and GAPDH, as described in Section 2. Representative results are presented from two follicles per time point (A and D). EREG and AREG signals were normalized with those of GAPDH, and results are presented as ratios of EREG (B and E) or AREG (C and F) to GAPDH (mean ± SEM; n = 4 distinct follicles [i.e. animals] per time point; except n = 2 at 6 h post-hCG). Bars marked with an asterisk are significantly different from 0 h (P < 0.05).

In summary, this study characterizes the presence of an hCGdependent induction of EREG and AREG transcripts in equine and bovine follicles prior to ovulation, with marked differences observed between the two species. The induction occurred in both granulosa and theca interna cells in the horse, but only in granulosa cells in the cow. Once induced, the expression of EREG and AREG transcripts was maintained throughout the period studied, with a strong up-regulation just before follicular rupture (33– 39 h post-hCG). In contrast, the induction of both transcripts was transient in nature in bovine preovulatory follicles and observed predominantly at 6 h post-hCG, suggesting distinct regulatory mechanisms. Studies using primary cultures of bovine granulosa cells revealed the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species, as evidenced by the ability of EGF to induce, and an EGFR inhibitor to suppress, expression of these genes. Collectively, these results indicate that, while the gonadotropin-dependent

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PTGS2PTGER2TNFAIP6CTSL2MMP1PGRVEGFAGAPDHFig. 6. Forskolin-dependent regulation of EREG and AREG transcripts, and effect of EGFR activation on the expression of ovulation-related genes, in bovine granulosa cells in vitro. A, B and C, granulosa cells isolated from bovine dominant follicles were cultured in the absence or presence of forskolin (FSK; 10 lM) for 0–24 h, as described in Section 2. Total RNAs were isolated and analyzed by RT-PCR for EREG, AREG and GAPDH mRNA content. Representative results from one experiment are shown (A). Signals of EREG and AREG transcripts were normalized with those of GAPDH (control gene), and results are presented as ratios of EREG (B) or AREG (C) to GAPDH (mean ± SEM; n = 3 independent experiments per time point). Bars marked with an asterisk are significantly different from 0 h (P < 0.05). (D) bovine granulosa cells were cultured for 24 h in the absence (Ctl) or presence of FSK (10 lM), without () or with (PD) an inhibitor of EGFR tyrosine kinase activity, PD153035 (10 lM). (E) cultures of granulosa cells were treated 0 to 24 h with EGF (10 ng/ml). (D and E) total RNAs were isolated and analyzed by RT-PCR for various ovulation-related mRNAs (PTGS2, PTGER2, TNFAIP6, CTSL2, MMP1, PGR, VEGFA) and GAPDH (control gene) mRNA content. Representative results from one of three independent experiments are shown.

induction of EREG and AREG is a mechanism conserved in preovulatory follicles of the horse and cow, important differences appear to reside in the precise molecular control and cellular localization of EREG and AREG expression across species. Acknowledgments The authors would like to thank Danielle Rannou for technical assistance with the immunohistochemistry. Natural Sciences and Engineering Research Council of Canada Grant OPG0171135 supported this work.

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