Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells

Mammalian homologues of genes that control oogenesis in other organisms may play similar roles in mammalian ovarian development. In Drosophila melanogaster, GUSTAVUS (GUS) protein physically interacts with and is necessary for the proper posterior localization of VASA protein, and thus is required for specification of germ cells. We identified two mouse genes, SSB-1 and SSB-4 (SPRY domain SOCS box protein), whose protein products share 75% identity and are each approximately 70% identical to Drosophila GUS. Both SSB-1 and SSB-4 mRNA were detectable in mouse ovaries by Northern blotting of total and poly(A) + RNA, but were expressed in few other tissues. SSB-1 was detectable in testes, although the 3'-untranslated region of the mRNA was considerably shorter than the ovarian mRNA. In situ hybridization and RT-PCR analysis of ovaries revealed that both genes were expressed in granulosa cells at all stages of follicular development. In contrast, expression was barely detectable in in oocytes. Immunoblotting analysis revealed that SSB-1 protein was present in follicles at different stages of growth, and immunocytochemistry confirmed that SSB-1 and SSB-4 were detectable in granulosa cells of primary and subsequent stage follicles and that they were present in both mural and cumulus granulosa cells of antral follicles. These results establish that GUS-related proteins, which in Drosophila are restricted to the germ cells, are in the mouse instead expressed in the granulosa cells and are present throughout folliculogenesis. Based on their tissue-restricted pattern of expression and apparent abundance in granulosa cells, we propose that SSB-1 and SSB-4 play key roles in regulating granulosa cell physiology.     

Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.     

Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus

Fertility and fecundity decline with advancing age in female mammals, but reproductive aging was decelerated in Siberian hamsters (Phodopus sungorus) raised in a short-day (SD) photoperiod. Litter success was significantly improved in older hamsters when reared in SD and the number of primordial follicles was twice that of females held in long days (LD). Because anti-Müllerian hormone (AMH) appears to inhibit the recruitment of primordial follicles in mice, we sought to determine whether the expression patterns of AMH differ in the ovaries and serum of hamsters raised in SD versus LD. Ovaries of SD female hamsters are characterized by a paucity of follicular development beyond the secondary stage and are endowed with an abundance of large eosinophilic cells, which may derive from granulosa cells of oocyte-depleted follicles. In ovaries from 10-week-old SD hamsters, we found that the so-called 'hypertrophied granulosa cells' were immunoreactive for AMH, as were granulosa cells within healthy-appearing primary and secondary follicles. Conversely, ovaries from age-matched LD animals lack the highly eosinophilic cells present in SD ovaries. Therefore, AMH staining in LD was limited to primary and secondary follicles that are comparable in number to those found in SD ovaries. The substantially greater AMH expression in SD ovaries probably reflects the abundance of hypertrophied granulosa cells in SD ovaries and their relative absence in LD ovaries. The modulation of ovarian AMH by day length is a strong mechanistic candidate for the preservation of primordial follicles in female hamsters raised in a SD photoperiod.     

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.     

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.     

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.     

NR5A1 is required for functional maturation of Sertoli cells during postnatal development

The orphan nuclear receptor steroidogenic factor 1 (NR5A1 (SF-1)) is expressed in both Sertoli and Leydig cells in the testes. This study investigates the postnatal development of the testes of a gonad-specific Nr5a1 knockout (KO) mouse, in which Nr5a1 was specifically inactivated. The KO testes appeared histologically normal from postnatal day 0 (P0) until P7. However, disorganized germ cells, vacuoles, and giant cells appeared by P14 in the seminiferous tubules of KO but not control mice. Expression of NR5A1 and various factors was examined by immunohistochemistry (IHC). The number of NR5A1-positive Sertoli cells in the KO testes was lower compared with controls at all the developmental stages and decreased to nearly undetectable levels by P21. IHC for anti-Müllerian hormone and p27, immature and mature Sertoli cell markers, respectively, indicated a delay in Sertoli cell maturation in the KO testes. The number of Sertoli cell-expressing factors involved in Sertoli cell differentiation including WT1, SOX9, GATA4, and androgen receptor were lower in the KO testes compared with controls. Furthermore, fewer proliferating cell nuclear antigen-positive proliferative germ cells were observed, and the number of TUNEL-labeled cells was significantly higher in the KO testes compared with controls at P14 and P21, indicating impaired spermatogenesis. IHC for CYP11A1 (SCC) indicated the presence of steroidogenic Leydig cells in the interstitium of the KO testes at all stages examined. These results suggest that NR5A1 is essential for Sertoli cell maturation and therefore spermatogenesis, during postnatal testis development.     

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.     

Incidence of apoptosis in granulosa cells from immature human follicles

The aim of this study was to investigate the incidence of apoptosis in granulosa cells from immature human follicles undergoing in vitro maturation (IVM) and to compare the incidence of apoptotic granulosa cells (i) between FSH-primed and unprimed normal ovaries and (ii) between polycystic and normal ovaries. Furthermore, the incidence of apoptosis was related to maturation and subsequent fertilization and cleavage of the oocytes from the corresponding ovary. Seventy women undergoing 70 IVM cycles were included. Group 1 consisted of patients with normal ovaries (n = 52) and group 2 consisted of patients with polycystic ovaries (n = 18). Patients in group 1 were subdivided into two groups according to priming with FSH before aspiration. In group 1a (n = 27 cycles) oocytes were obtained in unstimulated cycles. In group 1b (n = 25 cycles) oocytes were obtained after priming with recombinant FSH for 3 days initiated on day 3 after spontaneous menstruation. In group 2 all patients were primed with recombinant FSH for 3 days before aspiration. Aspiration was performed transvaginally and cumulus-enclosed oocytes were matured for 28-30 h before fertilization. Granulosa cells were collected from follicular aspirates. An APOPTAG detection kit was used to stain the granulosa cells and to detect apoptosis. The incidence of apoptosis in granulosa cells was decreased in follicles from FSH-primed normal ovaries compared with follicles from unprimed normal ovaries and FSH-primed polycystic ovaries. No difference was found between granulosa cells from FSH-primed polycystic ovaries and granulosa cells from unstimulated normal ovaries. No differences in maturation rate, fertilization rate, cleavage rate and implantation rate were observed when oocytes from a polycystic ovary were compared with oocytes from an unstimulated normal ovary. In unstimulated cycles, the ovaries were grouped according to the presence of a dominant follicle. The incidence of apoptosis was significantly higher in granulosa cells from an ovary without a dominant follicle compared with granulosa cells from an ovary with a dominant follicle. The rates of maturation, fertilization and cleavage did not differ between the two groups.     

E- and N-cadherin expression and distribution during luteinization in the rat ovary

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.     

Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle

The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle.     

Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.