Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Presence of Fas-Fas ligand system and bcl-2 gene products in cells and fluids from gonadotropin-stimulated human ovaries

Apoptosis, or programmed cell death, is an important mechanism for the regulation of embryonic development and tissue homeostasis. It is coordinated by a number of molecules including the Fas-Fas ligand (FasL) system and bcl-2. The purpose of this study was to characterize the expression of these molecules in human oocytes and cumulus cells from gonadotropin-stimulated human ovaries and to determine whether the presence of soluble Fas (sFas), soluble FasL, or interferon-gamma in follicular fluid (FF) correlated with apoptosis in cumulus cells, oocyte maturation, and embryo quality. Levels of sFas were significantly higher in FF containing immature oocytes compared with those containing atretic oocytes (P < 0.05; FF containing mature oocytes had highly variable levels of sFas. Levels of sFas in FF did not correlate with either fertilization, embryo quality resulting from fertilized oocytes, or apoptosis rate in cumulus cells. Fas was expressed in both unfertilized oocytes and cumulus cells, whereas FasL expression was not usually detected in these cell types. Messenger RNA for bcl-2 was detectable in both freshly isolated oocytes and cumulus cells but was not demonstrable following 24 h of culture that coincided with a significant increase of apoptosis in cumulus cells. Our results indicate that soluble forms of the Fas-FasL system are present in FF from gonadotropin-stimulated human ovaries and suggest that this system may play a role in preventing oocyte atresia during folliculogenesis but is probably not important for apoptotic events in cumulus cells and oocytes after fertilization failure. Apoptosis in this case may be facilitated by the downregulation of bcl-2. Further studies on the expression of these molecules in follicles containing atretic oocytes and immature oocytes are needed to confirm this new hypothesis.     

Expression of Olfactory Receptors in Xenopus Oocytes

The rat olfactory epithelium and the amino acid-sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two-electrode voltage clamp, activates stimulus-dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand-activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca(2+)-activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.     

Effects of maturational stage, cumulus cells and coincubation of mature and immature cumulus-oocyte complexes on in vitro penetrability of porcine oocytes

The in vitro penetrability of porcine oocytes is conditioned by several factors, some of which remain unclear. Knowledge of the different effects of the cellular components involved in penetrability would no doubt serve to simplify laboratory IVF methods. This study was designed to evaluate the effects of the following factors on penetrability: oocyte maturational stage, the presence of isolated or oocyte-attached cumulus cells, and coincubation of in vitro-matured and immature oocytes. Immature oocytes and oocytes matured in Waymouth medium were obtained from non atretic follicles and fertilized in TCM 199 medium. Sperm-rich fractions were collected by the gloved hand method and semen was used for IVF at a final concentration of 1 x 10(6) cells/mL in all experiments. Under the same conditions of IVF, the penetrability of the immature cumulus-oocyte complexes (COCs) was significantly lower than that of mature COCs, in terms of penetration rate and mean number of sperm per penetrated oocyte. This difference was abolished when the oocytes were denuded, leading to similar penetration rates. Coincubation of mature and immature COCs reduced the penetrability of immature COCs compared with that observed when these were incubated in isolation. However, neither the addition of isolated cumulus cells from decumulated mature oocytes nor the addition of denuded mature oocytes to immature COCs modified the penetration rate. These findings suggest that the presence of surrounding cumulus cells is mainly responsible for the differences observed in penetrability, regardless of the maturational stage of the oocyte. Moreover, when mature and immature COCs are coincubated, penetrability of immature COCs is diminished by the effects of the mature COC and not by the independent actions of the cellular components.     

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.     

Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.     

Testosterone potentially triggers meiotic resumption by activation of intra-oocyte SRC and MAPK in porcine oocytes

The role of androgen and androgen receptors (ARs) in males has been well established. This steroid and its receptor also exist in follicles, but their functions are still unclear. In this study, using a culture system containing a low dose of hypoxanthine, we revealed the positive contribution of testosterone to oocyte meiotic resumption. By performing ultracentrifugation to allow clear visualization of porcine germinal vesicles, our results provide evidence that mitogen-activated protein kinase (MAPK) in the oocyte itself but not in cumulus cells was activated before germinal vesicle breakdown (GVBD) after testosterone treatment. We further explored the signal cascade of testosterone-triggered GVBD and showed significant contributions of AR to testosterone-induced MAPK activation and GVBD. By using a potent and selective inhibitor of SRC and detecting activation of the kinase, we found that testosterone activated SRC in oocytes but not in cumulus cells and that SRC (as an essential upstream molecule of MAPK) mediated this testosterone- and AR-promoted reinitiation of meiosis. The present findings propose an undefined signaling pathway and suggest the potential competence of testosterone for meiotic resumption in mammalian oocytes.     

Proviral DNA of caprine arthritis encephalitis virus (CAEV) is detected in cumulus oophorus cells but not in oocytes from naturally infected goats

The aim of this study was to determine whether oocytes taken from ovarian follicles in 123 naturally infected goats were carrying the proviral CAEV genome. Examination of DNA isolated from 190 batches of oocytes with intact cumulus cells and 190 batches of oocytes whose cumulus cells had been removed, taken from follicles of the same ovaries, demonstrated that 42/190 batches of oocytes with intact cumulus cells had the proviral CAEV genome, whereas none of the 190 batches of oocytes without cumulus cells were positive for the provirus. To confirm that the proviral genome was present in the cumulus cells and not in the oocyte cells, 586 oocytes from 56 different ovaries, were separated from their cumulus cells. The DNA was then extracted from each fraction and examined. The purity of the oocyte fraction was verified by searching for granulosa cell-specific mRNA, using RT-PCR; this was negative in all the batches of oocytes in which the cumulus cells had been removed. PCR analysis demonstrated that none of the oocytes without cumulus cells were positive, whereas 22/56 of the batches with cumulus cells were found to be positive. This study clearly demonstrates that despite being surrounded by infected cumulus cells, the oocytes are not infected, and that the enzymatic and mechanical technique for removing the cells surrounding the oocyte, as used in this study, is effective, thus enabling CAEV-free oocytes to be obtained from infected goats.     

Role of cumulus cells for rat oocyte maturation and metabolism

Oocytes collected from immature PMSG-treated rats on the morning of proestrus were allowed to mature in culture either surrounded by their cumulus cells or after denudation. It was found that the time course of oocyte nuclear maturation was similar whether the cumulus cells were present or not. The oxygen consumption of noncultured oocytes was 0.12 nl/hr/oocyte and increased by 40% after four to eight hours in culture with intact cumulus. Respiration of oocytes cultured without cumulus remained constant throughout the culture, except for a transient decrease after four hours. It is concluted that the cumulus cells do not affect the spontaneous nuclear maturation in vitro, but that the metabolism in oocytes cultured with intact cumulus is different from that of cultured denuded oocytes. Furthermore, it appears that the rise in oocyte oxygen consumption is not a prerequisite for nucler maturation.     

Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased (3)H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [(3)H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.     

Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI).

The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.     

Lanosterol 14alpha-demethylase expression in the mouse ovary and its participation in cumulus-enclosed oocyte spontaneous meiotic maturation in vitro

The expression of lanosterol 14alpha-demethylase (LDM) in the mouse ovary after gonadotrophin administration was examined and the action of follicle fluid meiosis activating sterol (FF-MAS), derived from lanosterol by the action of LDM, on oocyte spontaneous maturation was also evaluated in cumulus cell enclosed oocytes (CEOs). Expression of LDM was primarily in oocytes in primordial and secondary follicles prior to administration of gonadotrophins, but obvious LDM expression was apparent in ovarian somatic cells 48 h after administration of equine chorionic gonadotrophin (eCG), especially in luteal and cumulus cells 54 h after eCG or 48 h after eCG plus 6 h after human chorionic gonadotrophin (hCG). The LDM expression in oocytes was only slightly elevated in larger growing follicles after eCG treatment. On the contrary, 48 h after hCG treatment, the elevated expression of LDM was only detected in interstitial cells. Therefore, eCG may be the primary gonadotrophin for LDM expression, and furthermore for production of FF-MAS in mouse cumulus cells (which are indispensable for oocyte maturation in vivo). Conversely, inhibitors of LDM, either 40 microM azalanstat or 50 microM RS-21745, significantly inhibited oocyte germinal vesicle breakdown (GVB) after 4h of in vitro culture; GVB rates decreased to 14 or 20%, compared to 90% in spontaneous maturation, respectively. There was no significant increase in GVB in CEOs following specific inhibitor of sterol Delta14-reductase and Delta7-reductase, AY9944-A-7 (5-100 microM), until marked oocytes degeneration appeared (50 microM). The phenomena may be ascribed to slow, passive accumulation of FF-MAS by AY9944-A-7, which cannot be associated with fast spontaneous progression. Furthermore, in spontaneous-matured CEOs, LDM was expressed preferentially in cumulus cells instead of oocytes. Therefore, FF-MAS may have a positive role in the spontaneous maturation of CEOs. In conclusion, there was an eCG-dependent dual LDM expression pattern on both oocytes and somatic cells in growing follicles in vivo, which may increase LDM expression and FF-MAS production in cumulus cells for oocyte maturation. For the first time, the inhibitory effect of LDM inhibitors on spontaneous maturation, together with the strong LDM expression in spontaneous matured CEOs, indicated that FF-MAS produced by cumulus cells might participate in spontaneous maturation of mouse CEOs.     

The effect of periovulatory imbalance of prolactin on the expression of prolactin receptors in rat ovary cells

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in the prolactin level in the blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.