Chromatin remodeling in somatic cells injected into mature pig oocytes

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.     

X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (n=25) were synchronized with 2-3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence of XIAP mRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples. XIAP mRNA was subsequently localized by in situ hybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positive XIAP mRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.     

Alterations in ovarian function of mice with reduced amounts of KIT receptor

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.     

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation

Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.     

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.     

Developmental expression and cellular distribution of Mullerian inhibiting substance in the primate ovary

Ovarian follicle formation during development and follicle maturation in adulthood are crucial determinants of female fertility and disruptions in these processes may result in subfertility or infertility. Among the several factors that are involved in ovarian physiology, Müllerian inhibiting substance (MIS) also known as anti-Müllerian hormone has emerged as an important marker to predict the follicle reserve. However, the roles of MIS in human ovarian physiology are unknown. To gain an insight into the potential roles of MIS in human ovarian differentiation during development and its regulation in adulthood, the expression profiles of MIS mRNA in the developing and adult human and monkey ovaries was examined by in situ hybridization. The results revealed that in the fetal human ovaries, MIS is specifically expressed at low levels in the granulosa cells of the developing primordial follicles; a small subset (approximately 2-3%) of oocytes express high amounts of MIS. In the adult human and monkey ovary, MIS mRNA is expressed at low levels in the primordial follicles, maximally in the primary and secondary follicles, and the expression is downregulated in the antral and atetric follicles. MIS expression is extinguished in the granulosa cells only after ovulation. These observations strongly favor the regulatory roles of MIS in folliculogenesis. MIS in the primate ovary may exert its effect during the primordial follicle formation to the terminal granulosa cell differentiation. The presence of MIS in a small subset of oocytes in the fetal ovary further points towards its additional role during fetal oocyte development.     

Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro

Hepatocyte growth factor (HGF) regulates granulosa cell (GC) steroidogenesis and suppresses apoptosis in non-ovarian cells. The hypothesis was thus developed that intraovarian HGF supports folliculogenesis by mediating steroidogenesis and suppressing apoptosis. To investigate the latter, the anti-apoptotic actions of HGF were tested in GCs and follicles isolated from immature rats. Results showed that HGF suppressed apoptosis in GC and follicle cultures as visualized using apoptosis indicator dye, YO-PRO-1. Immunohistochemistry was used to investigate the distribution of HGF, c-met, and HGF activator (HGFA) protein during folliculogenesis in equine chorionic gonadotropin (eCG)-primed rats. Immunoreactive HGF content was the greatest in GCs within preantral follicles. Following eCG, large antral follicles showed elevated HGF staining in theca and interstitial cells when compared with GCs. Intense c-met staining was observed in GCs within non-primed small preantral follicles; following eCG, the level of c-met was diminished in GCs, but increased within theca and interstitial cells. Theca, interstitium, and GCs in non-primed and primed ovaries contained HGFA. Following eCG, HGFA was more apparent in theca cells and the interstitium when compared to that in GCs within large antral follicles. The presence of HGF, c-met, and HGFA in preantral follicles would potentially enable the anti-apoptotic effects of HGF that were observed in vitro to occur in vivo. Advanced folliculogenesis led to a change in the cellular distribution of the HGF, c-met, and HGFA, suggesting that the ovarian HGF system is hormonally regulated in vivo.     

Localization and temporal regulation of tissue inhibitor of metalloproteinases-4 in mouse ovary

Tissue inhibitors of metalloproteinases (TIMPs) are potential regulators of tissue remodeling in the ovary. The aim of the present study was to examine the localization and temporal regulation of TIMP-4 protein in the mouse ovary. An induced superovulation model (eCG/hCG) was employed in immature mice to evaluate TIMP-4 protein expression profiles in ovaries collected during the follicular phase, the pre ovulatory period, and the luteal lifespan. Immunofluorescence results indicated that TIMP-4 protein was localized to theca of both antral and preovulatory follicles and adjacent ovarian stroma. After the initiation of luteinization with hCG, TIMP-4 was observed within the luteinizing granulosa cells and persisted throughout the lifespan of the corpus luteum. In the cycling ovary, TIMP-4 signaling localized to corpus luteum from previous estrous cycles, the theca of preovulatory follicles, and appeared to be lower in newly forming corpus luteum. Western analysis further showed that the levels of TIMP-4 increased significantly during the luteinization process of granulosa cells, but no significant change was found among all corpus luteum stages. A putative regulatory mechanism of TIMP-4 expression was identified utilizing an in vitro model. Treatment of cultured granulosa cells with hCG significantly augmented TIMP-4 protein expression levels. Together our data indicate that the luteinization process of granulosa cells is associated with up-regulation of TIMP-4 and that TIMP-4 might play an essential role in maintenance of the luteal function during the whole lifespan of corpus luteum.     

Implication of cortisol and 11beta-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts

This study investigated cortisol inactivation by 11beta-hydroxysteroid dehydrogenase (11beta HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP(+)-dependent 11beta-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11beta HSD, this studyalso investigated whether levels of 11beta HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11beta HSD1 activity in rat kidney homogenates decreased progressively from 50 +/- 8% inhibition by FF from small antral follicles (P < 0.001) to 23 +/- 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11beta HSD1 activity by 59 +/- 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35-48% inhibition, P < 0.05) and cyst fluid (45-75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11beta HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11beta HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.     

Gap junctions and ovarian folliculogenesis

Gap junctions are collections of intercellular membrane channels that allow adjacent cells to share small molecules (< 1 kDa). Gap junction channels are composed of connexins, a homologous family of more than 20 proteins. In developing follicles, gap junctions couple the growing oocyte and its surrounding follicle cells into a functional syncytium. This review summarizes evidence on the expression of various connexins in developing follicles and the likely roles that some of the connexins play, on the basis of findings from gene targeting experiments in mice. Gap junctions between cumulus cells contain predominantly connexin43, and this connexin has also been detected using immunoelectron microscopy in a small minority of gap junctions at the oocyte surface. The importance of connexin43 for granulosa cell function is demonstrated by the fact that follicles lacking this connexin arrest in early preantral stages and produce incompetent oocytes. Connexin37 appears to be the only connexin contributed by oocytes to the gap junctions coupling them with granulosa cells, and loss of this connexin interferes with the development of antral follicles. The expression of multiple connexins in developing follicles is thus likely to reflect the multiple functions served by gap junctional communication in folliculogenesis.     

Immunohistochemical localization of active caspase-3 in the mouse ovary: growth and atresia of small follicles

Caspase-3 belongs to a family of highly conserved cysteine proteases that mediate the course of apoptotic cell suicide. It is recognized that ovarian follicular atresia is associated with apoptosis, a process that has been characterized mainly in larger antral follicles. The aims of this study were to investigate the expression of caspase-3 in the mouse ovary, and determine whether active caspase-3 is present within smaller follicles, which may constitute the resting pool. The inactive enzyme was expressed as a 32 kDa band on a western blot of tissue extracts, whereas the active form was localized immunohistochemically. Bromodeoxyuridine (BrdU) was administered to mice (n = 7) during a 12 h period and subsequently localized to identify potentially quiescent follicles. Measurements of BrdU-positive cells in the mouse ovary were extrapolated with data obtained by morphometric analyses of small follicles using the nucleator technique. BrdU was incorporated into the granulosa cells of follicles regardless of size and the number of cells they contained, but was absent in a large proportion (89%) of small, single layered follicles. Active caspase-3 was localized to both the oocyte and granulosa cells of follicles that were considered to be undergoing atresia, but was not localized to the granulosa cells of any small, single layered follicles. The results of this study indicate that, in small follicles, granulosa cell proliferation occurs independently of the size of follicles and the number of constituent cells, and that follicles of this type may be inherently less susceptible to the normal physiological factors that induce atresia.     

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta ) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, BMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. In addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.     

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.     

Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.     

Heat stress diminishes gonadotropin receptor expression and enhances susceptibility to apoptosis of rat granulosa cells

Heat stress inhibits ovarian follicular development in mammalian species. We hypothesized that heat stress inhibits the function of follicular granulosa cells and suppresses follicular development. To test this, immature female rats were injected with pregnant mare serum gonadotropin (PMSG) at 48 h after the start of temperature treatment (control: 25 degrees C, 50% RH; heat stress: 35 degrees C, 70% Relative Humidity). The ovaries and granulosa cells of follicles at different developmental stages were analyzed for gonadotropin receptor levels and aromatase activity; estradiol levels were measured in follicular fluid. Before injection, heat stress diminished only the amount of FSH receptor on granulosa cells of antral follicles. During PMSG-stimulated follicular development, heat stress strongly inhibited gonadotropin receptor levels and aromatase activity in granulosa cells, and estradiol levels in the follicular fluid of early antral, antral and preovulatory follicles. To examine apoptosis and mRNA levels of bcl-2 and bax in granulosa cells, follicles harvested 48 h after PMSG injection were cultured in serum-free conditions. Heat-stressed granulosa cells showed a time-dependent increase in apoptosis. The bcl-2 mRNA levels were similar in control and heat-stressed granulosa cells; bax mRNA levels were increased in heat-stressed granulosa cells. According to these results, heat stress inhibits expression of gonadotropin receptors in granulosa cells and attenuates estrogenic activity of growing follicles, granulosa cells of heat-stressed follicles are susceptible to apoptosis, and the bcl2/bax system is not associated with heat-stress-induced apoptosis of granulosa cells. Our study suggests that decreased numbers and function of granulosa cells may cause ovarian dysfunction in domestic animals in summer.