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. 2016 Jan 6;94(2):44. doi: 10.1095/biolreprod.115.133058

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice1

Yi A Ren 3, Zhilin Liu 3, Lisa K Mullany 3, Chen-Ming Fan 4, JoAnne S Richards 3,2
PMCID: PMC4787628  PMID: 26740594

Abstract

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1flox/flox mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12–16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Keywords: C/EBP alpha, C/EBP beta, corpus luteum, Gas1, ovulation

INTRODUCTION

The processes of ovulation and luteinization are initiated by the release of the luteinizing hormone (LH) surge from the pituitary. LH binds to its cognate G-protein coupled receptors present in theca cells and granulosa cells of large antral, preovulatory follicles [1] and stimulates a cascade of intrafollicular and intracellular events that lead to ovulation and corpus luteum (CL) formation [2]. Of particular relevance is the induction by LH of the epidermal growth factor (EGF)-like factors amphiregulin, epiregulin, and betacellulin [3] that activate the EGF-receptors and downstream regulatory kinases, MAPK3/1 (also known as ERK1/2) [4–8]. Targets of MAPK3/1 include the transcription factors CCAAT enhancer binding proteins alpha and beta (C/EBPα and C/EBPβ) [3]. Targeted disruption of the Cebpa and Cebpb genes selectively in granulosa cells of antral follicles in mice has documented that they are essential for both ovulation and luteinization [9, 10].

C/EBPα/ or C/EBPβ are activated selectively during inflammatory responses in the liver [11], during adipocyte differentiation [12, 13], in macrophages [12], during the uterine decidual response [14, 15], and by LH during ovulation [10, 16]. Microarray analyses of RNA prepared from granulosa cells of wild-type and Cebpa/bfl/fl;Cyp19a1-Cre mutant mice have identified known and novel genes that are either up- or down-regulated by the deletion of these transcription factors [10], such as down-regulated expression of the prolactin receptor (Prlr) [10], the multidrug transporter Abcb1b1 and plexin D1 (Plxnd1) [10], and up-regulated expression of inhibin (Inha) [17] and aromatase (Cyp19a1) [9]. Putative C/EBP binding sites have been identified in the promoters of these genes, indicating that they are likely to be direct targets of these transcription factors [18, 19]. One novel gene that we identified in the Cebpa/bfl/fl;Cyp19a1-Cre mutant ovary microarray is growth arrest specific-1 (Gas1) [10]. The Gas1 gene also has potential C/EBP binding sites [18, 19] and is markedly reduced in granulosa cells of the Cebpa/bfl/fl;Cyp19a1-Cre mutant mice [10].

GAS1 is a glycosyl phosphatidylinositol-linked membrane protein that is an important coordinator of cell proliferation and survival through multiple mechanisms, such as inhibiting DNA synthesis [20] and increasing apoptosis [21]. Overexpression of GAS1 documents that it has potent tumor suppressor functions and suppresses melanoma metastasis [22–24]. The physiological importance of GAS1 is much more complex as revealed by the phenotype of Gas1 knockout mice [25–28]. During development, GAS1 appears to act, in part, as a coregulator of the morphogenic factors in the hedgehog (HH) signaling cascades [29]. In the developing somite, GAS1 is induced by WNT to attenuate cell response to SHH [30]. GAS1 has also been linked to the glial cell-derived neurotrophic factor (GDNF) and its receptor RET signaling cascade [31, 32]. In endothelial cells, GAS1 is induced by VE-cadherin and VEGF through the PI3K pathway to inhibit endothelial cell apoptosis and may thus facilitate resting endothelium integrity [32]. Considering the wide range of functions of GAS1, it is not surprising that the majority of Gas1 null mice die embryonically with multiple defects in retinal, cerebral, neural tube, limb, and cardiac development [25–28, 33].

Based on our evidence that Gas1 expression was highly increased in granulosa cells of ovulating follicles [10] and that the promoter of the Gas1 gene has putative C/EBPβ binding sites [34, 35], we hypothesized that Gas1 is a C/EBPβ target gene in granulosa cells and might be critical for mediating specific events associated with ovulation and/or luteinization. GAS1 is also expressed in rat CL where it possibly impacts apoptosis during the demise of CL mediated by luteolytic events [36]. To address the potential roles of GAS1 in the mouse ovary, we have analyzed the temporal and spatial expression of Gas1 mRNA and protein in ovarian cells in response to LH/hCG in wild-type and Cebpa/bfl/fl;Cyp19a1-Cre mutant mice and determined the intraovarian localization of GAS1 protein before and following the exposure to an LH/hCG stimulus. Because the Gas1 null mice die shortly after birth [25], Gas1fl/fl mice have been generated [37], allowing us to create granulosa cell- and cumulus cell-specific Gas1 knockout mice with Cyp19a1-Cre mouse strain [38] and to analyze the impact of Gas1 deletion on the processes of ovulation and luteinization.

MATERIALS AND METHODS

Animal Studies and Generation of Genetically Engineered Mouse Models

Gas1fl/fl mice have been generated to allow cell-specific disruption of the Gas1 gene [37]. Cebpa/bfl/fl;Cyp19a1-Cre mutant mice were generated as previously described [10]. For superovulation studies, mice were injected intraperitoneally with 5 IU equine chorionic gonadotropin (eCG) followed 48 h later with 5 IU hCG. Granulosa cells, cumulus cells, or whole ovaries were collected from these mice for subsequent analyses. All the animals were 1) housed under a 14L:10D schedule in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum and 2) maintained according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee at Baylor college of Medicine.

Histology, Immunofluorescence, and Immunohistochemistry

Ovarian tissues were fixed in 4% paraformaldehyde and either embedded in paraffin for routine histological (hematoxylin and eosin [H&E]) and immunofluorescent (IF) analyses or frozen in optimal cutting temperature compound (Ted Pella, Inc.) for other specific IF analyses. Primary antibodies specific for GAS1 (AF2644; R&D system), PTX3 (P0496; Sigma Aldrich), SFRP4 (HPA009712; Sigma Aldrich), PECAM-1 (553373; BD Pharmingen), and collagen IV (ab6586; Abcam) were used in the IF studies. Cell nuclei were counterstained with 5μg/ml Hoechst 33342 dye (Sigma Aldrich). Digital images of H&E and IF staining were captured using a Zeiss AxioPlan2 microscope in the Integrated Microscopy Core, and images of IF staining were captured with Zeiss LSM780 confocal microscope in the Optical Imaging and Vital Microscopy Core at Baylor College of Medicine.

RNA Extraction, Real-Time RT-PCR, and Microarray Analyses

Total RNA was extracted from granulosa cells, cumulus-oocyte-complexes (COCs), ovarian stromal tissue (residual tissues after removing granulosa cells via needle puncture of the ovary), isolated CL, or whole ovaries at different time intervals before and after ovulation. Total RNA extraction, reverse transcription, and real-time PCR were performed using procedure previously described [39]. Primers used for real-time RT-PCR are listed in Supplemental Table S1 (available online at www.biolreprod.org).

Western Blot Analyses

Granulosa, COCs, ovarian stromal tissue, or whole ovaries were lysed in RIPA buffer (Boston BioProducts). Proteins were separated using a 10% Bis-Tris gel and transferred to an immobilon-P membrane (Millipore). Primary antibodies used included GAS1 (1:1000 dilution, AF2644; R&D system) and AKT (1:2000 dilution, 9272; Cell Signaling Technology). Signals were detected using the Peirce ECL 2 Western Blotting Substrate (Thermo Scientific).

Measurement of Serum Progesterone

Blood was collected via heart puncture at 24 h post-hCG stimulation from mice anesthetized with isoflurane and spun down in a BD Microtainer serum collection tube (BD Pharmingen). All the sera were then shipped on dry ice to the University of Virginia Core Laboratory for hormone measuring.

DNA Extraction and PCR Verification of Gas1 Deletion in Gas1fl/fl;Cyp19a1-Cre Mice

DNA was extracted from granulosa cells and COCs collected from wild-type, heterozygous, and homozygous mutant mice at 24 h post-hCG using QIAprep Spin Miniprep Kit (Qiagen). The DNA product from each mouse was then amplified using primers flanking the LoxP sites in the Gas1fl/fl allele (forward primer: 5′-gcggaactcggacaaact-3′; reverse primer: 5′-cctcaacatcccttctccaa-3′). After PCR amplification, the resultant DNA samples were purified with QIAquick Gel Extraction Kit (Qiagen) and submitted for sequencing or were resolved in a 2.5% agarose gel to verify the deletion of Gas1 in the heterozygous and homozygous mice, respectively (wild type: 1458 bp; Gas1 null: 375 bp). Results from sequencing also confirmed successful deletion of Gas1 (data not shown).

Granulosa Cell Culture and In Vitro Deletion of Gas1 Using Cre-Expressing Adenovirus

Granulosa cells were collected from eCG-primed (24 h) 22–23 day-old mice and cultured in Dulbecco-modified Eagle medium nutrient mixture F-12 (DMEM/F12) (Life Technologies) supplemented with penicillin, streptomycin, and 5% fetal bovine serum (Life Technologies). After overnight culture, cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum for 1 h, then transfected with an adenoviral vector expressing CRE recombinase at 7.0 × 1011 plaque-forming units/ml (Ad5-CMV-Cre-eGFP, Vector Development Laboratory, Baylor College of Medicine). After 4 h of transfection, the transfection efficiency was examined under a fluorescent microscope according to eGFP expression (>90% transfection efficiency). Cells were then gently washed with DMEM/F12 and cultured for 1 h in DMEM/F12 supplemented with 5% fetal bovine serum before treatment with forskolin/phorbol 12-myristate 13-acetate (PMA) (Fo/PMA). Cells were harvested for RNA at 0 and 24 h of Fo/PMA treatment.

Statistics

Data are represented as mean ± SEM. Comparisons between two experimental groups were analyzed using unpaired Student t-test. Comparison between multiple experimental groups were analyzed using one way ANOVA and Tukey honest significant difference test. A two-tailed P < 0.05 was considered statistically significant.

RESULTS

LH/hCG Induce Expression of Gas1 mRNA and Protein in Granulosa Cells and Cumulus Cells

To determine the temporal pattern of Gas1 mRNA expression in the murine ovary, granulosa cells (or whole ovaries at 24 h post-hCG) were isolated from ovaries of hormonally primed immature mice prior to and after 5 IU eCG to stimulate preovulatory follicle growth and at selected times after 5 IU hCG to initiate ovulation and luteinization [10]. COCs were also isolated from ovaries and the oviduct of the hormonally primed mice. Following hCG stimulation, Gas1 mRNA was low from 2–8 h but increased markedly between 8–16 h post-hCG (Fig. 1A). Gas1 mRNA exhibited a similar pattern in COCs with marked increases occurring between 8–16 h post-hCG.

FIG. 1.

FIG. 1

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Expression of Gas1 was induced in granulosa cells and COCs around the time of ovulation. Levels of mRNA for Gas1 increased in granulosa cells (A) and COCs (B) at ovulation as measured by real-time quantitative PCR. For both A and B, data shows mean ± SEM; n = 3. Bars without common superscripts are statistically different (P < 0.05). C) Immunofluorescent localization of GAS1 (green) and PTX3 (red) in ovulated COC collected from the oviduct at 16 h post-hCG. Bar = 100 μm. D) Western blot analyses of GAS1 and AKT in granulosa cells, luteal cells and ovarian stromal cells before and after ovulation. GC, granulosa cells; CL, corpus luteum. E) Immunofluorescent staining of GAS1 in the ovary before and after ovulation. Bar = 100 μm. F) Immunofluorescent staining showing GAS1 expression in sporadic granulosa cells (arrows) before ovulation (3 and 10 h post-hCG) and cytoplasmic membrane localization of GAS1 in luteinizing granulosa cells (24 h post-hCG). CL, corpus luteum. Bar = 50 and 10 μm. Expression of GAS1 (green) mostly colocalized with expression of SFRP4 (red) in luteinizing granulosa cells at 24 h post-hCG. CL, corpus luteum; F, follicle.

GAS1 expression and localization in granulosa cells and COCs were examined further by IF staining. At 16 h post-hCG (Fig. 1C), COCs stained positively for GAS1 as well as pentraxin 3 (PTX3), a gene induced in COCs after the LH surge as shown previously [40, 41]. Oocytes also stained positive for GAS1 at this time but not in immature follicles. Western blot analyses demonstrated that while expression of GAS1 in stromal cells of the ovary was evident at all time points examined, GAS1 was detected with this method in luteal cells at 24 h post-hCG but not in granulosa cells at earlier time points (Fig. 1D). However, sporadic granulosa cells expressing GAS1 were detected using IF at 3 and 10 h post-hCG in preovulatory follicles (Fig. 1F). Because only a small number of granulosa cells expressed GAS1 before ovulation, it was not surprising that Western blot analysis did not detect GAS1 protein at these time points (Fig. 1D). In CL, GAS1 distinctly localized to the cytoplasmic membrane of luteal cells that also stain positive for a known luteal cell marker, SFRP4 (Fig. 1F, 24 h post-hCG) [42].

Induction of Gas1 in Granulosa Cells and COCs by hCG Is Dependent on C/EBPα/β

Our previous studies showed that selective depletion of Cebpa and Cebpb in granulosa cells of the Cebpa/bfl/fl;Cyp19a1-Cre mice prevented ovulation and thus impaired fertility [10]. Microarray analyses further indicated that Gas1 mRNA was reduced markedly in ovaries of the Cebpa/bfl/fl;Cyp19a1-Cre mice [10]. Because ovulation and luteinization fail to occur in the Cebpa/bfl/fl;Cyp19a1-Cre mutant mice, we were able to isolate granulosa cells from these mice and CL from wild-type controls to compare their mRNA and protein expression of selected genes. To verify and extend the above observations, CL (control mice, 24 h post-hCG), granulosa cells (mutant mice, 24 h post-hCG), and COCs (16 h post-hCG) were isolated from hormonally primed immature control and Cebpa/bfl/fl;Cyp19a1-Cre mutant mice (Fig. 2A). Gas1 mRNA was reduced markedly in granulosa cells and COCs of the Cebpa/bfl/fl;Cyp19a1-Cre mutant mice compared to wild-type controls. Immunofluorescent analyses confirmed that GAS1 protein was not detectable in the granulosa cells of follicles that failed to ovulate in the Cebpa/bfl/fl;Cyp19a1-Cre mutant mice (Fig. 2B) [10]. GAS1 was present and not reduced in cells within the stromal ovarian tissue between preovulatory follicles but was not detected in PECAM-1-positive endothelial cells in the ovarian stroma (Fig. 2C). These observations show that GAS1 is expressed in ovarian stromal cells surrounding follicles and luteinizing granulosa cells, and only the latter is dependent on C/EBPα/β.

FIG. 2.

FIG. 2

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Induction of Gas1 expression in luteinizing granulosa cells depends on C/EBPα/β. A) Levels of Gas1 mRNA in periovulatory granulosa cells (mutant mice) and luteinized granulosa cells (wild type) (24 h post-hCG) and COCs (16 h post-hCG) were significantly reduced in Cebpa/bfl/fl;Cyp19a1-Cre mutant mice compared to wild-type controls. Data shows mean ± SEM; n = 3, *P < 0.01. B) Immunofluorescent localization of GAS1 in CL of wild-type controls and preovulatory follicles (POF) of Cebpa/bfl/fl;Cyp19a1-Cre mutant mice at 16 h post-hCG. Bar = 50 μm. C) Immunofluorescent localization of GAS1 and PECAM-1 in POF of Cebpa/bfl/fl;Cyp19a1-Cre mutant mice at 16 h post-hCG. Bar = 50 μm. D) Representative image of IF staining of GAS1 (green) or GAS1 and PECAM-1 (red) in ovaries of wild-type immature unstimulated mice. F, follicles. Bar = 100 (left) and 40 (right) μm. E) Representative image of IF staining of GAS1 (green) in ovaries at 4 h post-hCG stimulation. F, follicles. Bar = 200 μm. F) Representative image of IF staining of GAS1 (green) and collagen IV (red) in ovaries at 12 h post-hCG stimulation. FCL, forming corpus luteum. Bar = 100 (left) and 60 (right) μm. G) Representative image of IF staining of GAS1 and autofluorescent (green, from steroidogenic cells) signals in ovaries at 72 h post-hCG stimulation. F, follicles; CL, corpus luteum. Bar = 100 μm.

We further investigated the ovarian stromal expression of GAS1 in ovaries from wild-type mice during different stages of follicular development using IF staining. In immature mice (Fig. 2D), no GAS1 was detected in granulosa cells, whereas intense staining was detected in stromal cells surrounding growing follicles. Some cells expressing GAS1 were adjacent to, but had minimal overlapping expression with, PECAM-1-positive endothelial cells. A similar pattern of GAS1 expression was also observed at 4 h post-hCG (Fig. 2E). Around the time of ovulation (12 h post-hCG) (Fig. 2F), sporadic GAS1-expressing cells were readily detected in the newly forming CL (FCL) sharing some overlap with collagen IV, a marker of vascular tissue. Strong expression of GAS1 also remained present in the stromal cells. However, at 24 and 48 h post-hCG, GAS1 expression in ovarian stromal cells appeared less prominent (but still detectable via IF staining) due to the very high level of expression for GAS1 in luteal cells (Figs. 1E and 2B). By 72 h post-hCG, GAS1 remained detectable in most luteal cells (steroidogenic cells that emit autofluorescence), but the relative level of GAS1 expression in luteal cells versus stromal cells had decreased significantly (Fig. 2G). Taken together, GAS1 is expressed in ovarian stromal cells surrounding follicles and CL in a persistent and dynamic pattern.

Generation of Genetically Engineered Mice with Conditional Deletion of Gas1 in Granulosa Cells and COCs

GAS1 appears to regulate numerous cell processes that are context specific and signaling-pathway dependent [21]. Moreover, the Gas1 knockout mice die shortly after birth [30]. Therefore, we sought to determine if selective depletion of Gas1 in granulosa and cumulus cells would alter fertility and recapitulate any of the phenotypes of granulosa cells and COCs in Cebpa/bfl/fl;Cyp19a1-Cre mutant mice. To accomplish this, we bred Gas1fl/fl mice to Cyp19a1-Cre mice to generate Gas1fl/fl;Cyp191a-Cre mice [37] (Fig. 3A). Reduction and deletion of Gas1 by Cyp19a1-Cre expression was confirmed using PCR of DNA extracted from granulosa cells and COCs from Gas1fl/fl controls, Gas1fl/+;Cyp191a-Cre, and Gas1fl/fl;Cyp191a-Cre mutant mice (Fig. 3B). Gas1 was reduced in the granulosa cells of heterozygous mutant and absent in the homozygous mutant granulosa cells. In COCs, Gas1 was expressed in heterozygous mutant and absent in homozygous mutant. Compared to Gas1fl/fl controls, GAS1 protein was not detected by IF staining in CL of Gas1fl/fl;Cyp191a-Cre mutant mice at 24 and 48 h post-hCG (Fig. 3C), consistent with its absence in isolated CL from Gas1 mutant females as detected using Western blot analyses (Fig. 3D).

FIG. 3.

FIG. 3

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Generation of mice with conditional deletion of Gas1 in granulosa cells and COCs. A) Breeding scheme. B) PCR amplification of Gas1 gene in wild-type, heterozygous, and homozygous Gas1 mutant granulosa cells and COCs obtained at 24 h post-hCG. C) Immunofluorescent localization of GAS1 in ovaries of control and Gas1fl/fl;Cyp191a-Cre mutant mice at 24 and 48 h post-hCG. F, follicles; CL, corpus luteum. Bar = 100 μm. D) Western blot analyses of GAS1 in isolated CL from Gas1fl/fl control and Gas1fl/fl;Cyp191a-Cre mutant adult female mice. Each sample represents CL isolated from an individual mouse.

Deletion of Gas1 in Granulosa Cells and COCs Enhanced Ovulation Rates

Fertility of female Gas1fl/fl;Cyp191a-Cre mutant mice was tested by mating control and mutant females to proven fertile Gas1fl/fl males for 6 mo. The mutant females produced slightly, but not significantly, more pups than the Gas1fl/fl control mice throughout the 6-mo breeding assay (Fig. 4A). However, when stimulated with a superovulatory eCG/hCG treatment, Gas1fl/fl;Cyp191a-Cre mutant females ovulated significantly more oocytes than the Gas1fl/fl control mice (Fig. 4B).

FIG. 4.

FIG. 4

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Conditional deletion of Gas1 in granulosa cells and COCs promotes ovulation. A) Breeding assay of Gas1fl/fl;Cyp191a-Cre mutant mice and Gas1fl/fl controls; n = 5. B) Superovulation studies of Gas1fl/fl;Cyp191a-Cre mutant mice and Gas1fl/fl controls; n = 8, *P < 0.01. C) Levels of mRNA for Gas1 and ovulation/luteinization-associated genes in granulosa cells from Gas1fl/fl;Cyp191a-Cre mutant mice and wild-type controls at 5 h post-hCG. Data shows mean ± SEM; n = 4, *P < 0.05. D) Levels of mRNA for Gas1 and ovulation-associated genes in COCs from Gas1fl/fl;Cyp191a-Cre mutant mice and wild-type controls at 5 h post-hCG. Data shows mean ± SEM; n = 4, *P < 0.05.

To analyze changes in the expression of selected genes in the Gas1 mutant mice, we collected granulosa cells and COCs from Gas1fl/fl;Cyp191a-Cre mutant mice and Gas1fl/fl control mice at 5 h post-hCG, a time interval when both C/EBPα and C/EBPβ protein levels were elevated and rapid changes in genes controlling ovulation are first observed. As expected, Gas1 mRNA was significantly reduced in the Gas1fl/fl;Cyp191a-Cre mutant mice compared to the Gas1fl/fl control mice in both granulosa cells and COCs. In these same granulosa cell samples, the expression of two genes, Pgr and Ptgs2, critical for ovulation did not change whereas mRNA levels of Lhcgr and Areg were significantly increased in the Gas1fl/fl;Cyp191a-Cre mutant mice compared to controls (Fig. 4, C and D). The level of mRNA for Areg was also increased in COCs from the mutant mice compared to the controls (Fig. 4D). These differences in Lhcgr and Areg mRNA levels between wild-type and mutant mice were not detected before hCG stimulation (Fig. 5, A and B, 48 h post-eCG) or after ovulation (Fig. 5, C and D, 16 h post-hCG). Together these observations suggest that the conditional deletion of Gas1 in granulosa cells and COCs promotes ovulation and the expression of selected genes associated with ovulation.

FIG. 5.

FIG. 5

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Levels of mRNA for Areg and Lhcgr were not altered in granulosa cells or COCs of Gas1fl/fl;Cyp191a-Cre mutant mice compared to controls before hCG stimulation (A and B) (48 h post-eCG) or after ovulation (C and D) (16 h post-hCG). Data shows mean ± SEM; n = 3, *P < 0.05.

Deletion of Gas1 in Granulosa Cells Alters Expression of Key Genes in CL

We next sought to determine if the depletion of Gas1 in granulosa cells would also alter luteinization because 1) GAS1 mRNA and protein were maximally increased between 12–24 h post-hCG, 2) Gas1 is a target of C/EBPα/β and appears to be selectively increased during luteinization when granulosa cells cease dividing and become terminally differentiated, and 3) GAS1 has been reported to be a regulator of proliferation and differentiation [29, 43]. We first confirmed the reduced expression of Gas1 in whole ovaries of the Gas1fl/fl;Cyp191a-Cre mutant mice at 24 and 48 h post-hCG (Fig. 6, A and B, left). In addition, the expression of several luteinization-related genes was reduced in the Gas1 mutant ovaries compared to Gas1fl/fl control mice at 24 h post-hCG (Fig. 6A). These included genes involved in luteinization (Prlr), steroidogenesis (Star and Cyp11a1), WNT signaling (Wnt4), angiogenesis (Sema3a), and cumulus/immune cell functions (Cd52) [44, 45]. Cell cycle-related genes (Ccnd1, Ccnd2, Cdkn1a, and Cdkn1b) were not altered (Fig. 6A; data not shown for Ccnd2). Importantly, most of these luteinization-related genes are also targets of C/EBPα/β, supporting the observations that Gas1 mediates some processes associated with luteinization and that are downstream of C/EBPα/β [10]. Interestingly, by 48 h post-hCG, differences in mRNA levels of the above-mentioned luteal genes in ovarian samples were no longer detectable, with the exception of Cdkn1b that was reduced (but not statistically significant) (Fig. 6B). Because compensation from redundant mechanisms or pathways may rescue phenotypes caused by in vivo deletion of Gas1, we next tested whether acute in vitro deletion could cause similar effects by exposing Gas1fl/fl granulosa cells to Cre-expressing adenovirus (Fig. 6C). Fo/PMA treatment of granulosa cells collected from eCG-primed immature mice (24 h post-eCG) induced elevated mRNA expression of key genes in CL (Sema3a, Wnt4, Prlr, Cd52, Star, and Cyp11a1) at 24 h of treatment as expected whereas depletion of Gas1 using Cre-expressing adenovirus efficiently suppressed induction of Gas1 expression (data not shown) as well as significantly dampened the induction of Sema3a, Prlr, Cd52, and Star. In vitro depletion of Gas1 also reduced expression of Wnt4 and Cyp11a1, but the reduction was not statistically significant. Thus, depletion of Gas1 does impact induction of selected luteal cell genes during the initial stages of luteinization.

FIG. 6.

FIG. 6

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Levels of mRNA for key genes in CL were altered in Gas1fl/fl;Cyp191a-Cre mutant mice compared to controls. A) Levels of mRNA for Gas1 and luteal genes in whole ovaries from Gas1 mutant and control mice at 24 h post-hCG. Data for luteal genes are presented as the relative values in the mutants divided by those in controls; n = 4, *P < 0.05. B) The mRNA of Gas1 and luteal genes in whole ovaries of Gas1 mutant and control mice at 24 h post-hCG. Data for luteal genes are presented as the relative values in the mutants divided by those in controls; n = 5, *P < 0.05. C) Deletion of Gas1 in vitro in cultured granulosa cells reduced mRNA levels of luteal genes. Fo/PMA treatment was given for 24 h.

Disruption of the Gas1 in Granulosa Cells Does Not Impair CL Vascularization

Based on the reduced mRNA levels of critical luteal cell steroidogenic genes Cyp11a1 and Star in Gas1fl/fl;Cyp191a-Cre mutant mice compared to controls, we hypothesized that the serum progesterone levels might also be reduced in these mutant mice. However, this was not the case (Fig. 7A). The gross histological appearance of CL was also comparable between the mutant and control ovaries (Fig. 7B). Because the circulating levels of progesterone are determined not only by its synthesis and secretion, but also by its transportation from CL to the circulation via the luteal vasculature, we investigated whether there was enhanced vasculature in the CL of Gas1fl/fl;Cyp191a-Cre mutant mice (Fig. 7C). Immunofluorescent staining confirmed the depletion of GAS1 protein in CL of the Gas1fl/fl;Cyp191a-Cre mutant mice, but staining with vascular markers collagen IV and PECAM-1 demonstrated similar temporal and spatial vascularization processes in the CL of Gas1 mutant and wild-type control ovaries. At 24 h post-hCG, deposits of collagen IV were visible throughout the newly FCL, and endothelial cells (PECAM-1 positive) were branching in between luteinizing granulosa cells in both Gas1 mutant and wild-type control ovaries. Furthermore, a closer look revealed that GAS1 colocalized with some collagen IV in the newly FCL but not with PECAM-1 (Fig. 7D), indicating that GAS1 is selectively expressed in luteinizing granulosa cells and not endothelial cells during the neovascularization processes after ovulation.

FIG. 7.

FIG. 7

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GAS1 does not directly regulate vascular development in CL. A) Serum levels of progesterone in Gas1 mutant and control mice at 24 h post-hCG. Data represents mean ± SEM, n = 5. B) Representative images of H&E staining on ovarian sections from Gas1 mutant and control mice at 24 h post-hCG. Bar = 100 μm. C) Representative images of IF staining of GAS1, collagen IV, and PECAM-1 on ovarian sections from Gas1 mutant and control mice. Bar = 100 μm. D) Representative images of IF localization of GAS1, collagen IV, and PECAM-1 on wild-type ovarian sections. Bar = 10 μm. CL, corpus luteum.

DISCUSSION

The glycosyl phosphatidylinositol-linked GAS1 protein appears to have specific functions in many tissues at different stages of development [29]. These include cell cycle arrest, apoptosis, morphogenesis, and differentiation [26, 46, 47]. These diverse functions of GAS1 may be related to tissue-specific transcription factors that can activate the Gas1 gene (such as p53) [22, 48] as well as to diverse signaling pathways (including WNT, HH, and GDNF) with which GAS1 is known to interact [29].

Our studies on the expression and function of GAS1 in ovarian follicles provide additional, novel evidence that 1) the expression of the Gas1 gene is dependent, at least in part, on the C/EBPα/β transcription factors as revealed by its impaired expression in granulosa, cumulus, and luteal cells of the Cebpa/bfl/fl;Cyp19a1-Cre mice (Fig. 2) and 2) GAS1 protein localizes to granulosa, cumulus, and luteal cells and impacts the expression of specific genes involved in ovulation and the initiation of luteinization (Fig. 5). Specifically, we show that GAS1 mRNA and protein are low in granulosa cells of pre-ovulatory follicles but are selectively increased in granulosa and cumulus cells of ovulating follicles following LH/hCG, and in luteal cells of newly FCL, suggesting its involvement in ovulation and early-stage CL function.

The physiological relevance to and impact of GAS1 in ovulation is clearly demonstrated by the increased the rate of ovulation in the Gas1 conditional knockout mice during normal breeding and, more dramatically, in response to exogenous gonadotropins. Moreover, Gas1 depletion in granulosa cells in vivo leads to the increased expression of the LH receptor (Lhcgr) and the EGF-like factor Areg in the granulosa cells and cumulus cell in vivo, both of which are known to be essential for ovulation and luteinization [7, 49]. Because GAS1 protein levels are low in granulosa cells of preovulatory follicles, these results suggest that GAS1, even at low levels of expression, may suppress the responsiveness of granulosa cells to signals that both promote preovulatory follicular development and ovulation. The mechanisms that mediate the effect of GAS1 on follicular development and ovulation are not clear, but based on the cytoplasmic/membrane localization of GAS1, it may directly or indirectly interact with the LH receptor or other membrane-related factors. After hCG stimulation and especially in the immediate postovulatory period (12–16 h post-hCG), expression of GAS1 was dramatically induced in both luteinizing granulosa cells and COCs, a time point corresponding to a further increase in the protein level of C/EBPβ at 12–16 h post-hCG [50], and presumably a time when C/EBPα/β bind to the promoter of the Gas1 gene and directly regulate its transcription in the ovary, inducing Gas1 mRNA expression in both granulosa cells and COCs. Because AREG is transiently induced during ovulation and the LHCGR is down-regulated following the LH surge, the rapid induction of GAS1 may also act to prevent prolonged expression of AREG and LHCGR that would be predicted to alter the ovulation and luteinization processes.

The physiological relevance of GAS1 to luteal cell function and the formation of the CL is shown by its elevated expression and localization to luteal cells 24 h post-hCG and to the impaired expression of specific genes during the initial phase of luteinization. Specifically, once the ovulation and luteinization programs have been set in motion by the LH surge, GAS1 increases markedly and appears to be a downstream target of C/EBPα/β [18, 19]. Genes with altered mRNA levels in the CL of Gas1fl/fl;Cyp191a-Cre mice, such as Sema3a, Cyp11a1, Star, Prlr, and Wnt4, are also targets of C/EBPβ, thus supporting GAS1 as a mediator of at least some effects of C/EBPβ on the luteinization process. Disruption of Gas1 expression in cultured granulosa cells with adenoviral-Cre further confirms a role for GAS1 in regulating these luteal-related genes as well as the immune-related gene Cd52 [51]. Although a plethora of data demonstrate that two major functions of GAS1 are to cause cell cycle arrest and apoptosis [24, 43, 46], disruption of the Gas1 gene in granulosa cells does not impair granulosa cell cycle arrest or prevent their terminal differentiation to nondividing luteal cells in response to LH/hCG. This could be dependent, in part, on the degree of Gas1 depletion in granulosa cells because our data show that even low levels of Gas1 mRNA and protein appear critical for regulating induction of genes such as Areg. Additionally, the effects of GAS1 are likely context specific and related to in vivo versus in vitro analyses, the cell type being analyzed (NIH-3T3 and COMMA D cells, adipocytes, or endothelial cells), the isoforms of C/EBP present or other transcription factors being expressed, and the cell-type-specific pathways that are coordinately activated.

GAS1 exerts a myriad of functions through its interactions with multiple pathways during development, including the HH-, WNT-, and GDNF/RET-signaling pathways. However, although GAS1 can interact with the G-protein-coupled HH-signaling receptor SMO and well as CDO and BOC [28, 52–54], the HH-signaling pathway is suppressed to a minimal level in granulosa cells after LH surge [55, 56]. Thus, it is unlikely that the HH-signaling pathway is a key interacting factor with GAS1 in the preovulatory follicles and CL. However, it is possible that ovarian stromal cell GAS1 may interact with the HH-signaling pathway to impact early stages of follicular development, perhaps by regulating differentiation of α-smooth muscle actin-positive cells. The transient impact of GAS1 depletion in the newly FCL on the other hand, is likely to be partially through other G-protein-coupled receptors, such as the LH receptor itself or the WNT/FZD-signaling pathway, both of which play key roles in the formation and function of CL [42, 57–60], including regulation of the expression of steroidogenesis genes Cyp11a1, Star, and Cyp19a1, as well as CL vascularization [10].

Although a previous study reported that GAS1 is regulated by VE-cadherin and VEGF in endothelial cells [32], this does not seem to be the case in growing follicles or CL because GAS1 does not colocalize or exhibit close proximity with the endothelial marker PECAM-1. Expression of GAS1 in CL appears to be specific for luteal cells because it colocalized with SFRP4, a known marker for luteal cells, and because GAS1 was no longer detectable using IF staining when depleted with granulosa cell/luteal cell-specific Cyp19a1-Cre. Moreover, different from SFRP4, which is a secreted protein and can exhibit diffusive localization and is even detected in serum, the distinct membrane localization of GAS1 makes it an easily identifiable new marker for luteal cells as compared to granulosa cells.

In summary, based on the rapid induction but transient expression of GAS1 in ovulating follicles and CL and the impact of its depletion in granulosa, cumulus, and luteal cells, GAS1 is a C/EBP-target gene that is involved in follicular development, ovulation, and luteinization. The granulosa cell-specific depletion of Gas1 enhanced the rate of ovulation both in regular breeding assays and upon superovulatory hormone stimulation, and this was associated with enhanced expression of Areg and Lhcgr, key factors known to mediate preovulatory follicle responses to the LH surge [7, 61]. Conversely, depletion of Gas1 reduced the expression of luteal cell-related genes, suggesting that it may also impact luteal cell functions.

Supplementary Material

Supplemental Data
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ACKNOWLEDGMENT

The authors thank Dr. Victor Kostyuk for statistical analyses.

Footnotes

1

This work has been supported by NIH-CA-181808 (J.S.R.), NIH-HD-16229 (J.S.R.), U54-HD07945 (Project 2, J.S.R.), SCCPIR (to J.S.R.), Specialized Cooperative Centers Program in Reproduction and Infertility Research (Bert W. O'Malley, B.W.O.), NIH-T32-HD-001765-36 (B.W.O.), and NIH-RO1-DK084963 (C.M.F.). This project was also supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from the NIH (HD007495, DK56338, and CA125123), the Dan L. Duncan Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics as well as the Pathology and Histology Core at Baylor College of Medicine with funding from the NIH (NCI P30-CA125123). Presented as a poster in part at the 46th Annual Meeting of the Society for the Study of Reproduction, 22–26 July 2013, Montréal, Québec, Canada.

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