Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes

Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte. Nevertheless, the oocyte's companion somatic cells play crucial roles in the expression of these lesions.     

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.     

Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer

The present study examined the competence of oocytes from bovine ovaries stored at low temperatures for at least 1 day, which is the necessary time period to complete inspection for bovine spongiform encephalopathy. Storage of ovaries at 10 degrees C for 24 h did not affect oocyte maturation (68% versus 68%) or the potential of oocytes to develop into day 8 blastocysts after in vitro fertilization (25% versus 27%), parthenogenetic activation (19% versus 25%), or somatic cell nucleus transfer (27% versus 32%) compared with controls. In vitro-fertilized and parthenogenetic oocytes from ovaries stored at 10 degrees C for 48 h had a significantly decreased maturation rate and developmental potential, but nucleus-transferred oocytes that received cultured cumulus cells did not (27% versus 32%). Thus, bovine ovaries can be stored at 10 degrees C for at least 24 h without decreasing oocyte maturation competence or the developmental potential of in vitro-fertilized, parthenogenetically activated, and somatic cell nucleus-transferred oocytes, at least to the blastocyst stage. The present study provides valuable information with regard to removing bovine ovaries from abattoirs after testing for bovine spongiform encephalopathy.     

Nitric oxide is essential for optimal meiotic maturation of murine cumulus-oocyte complexes in vitro

This study was conducted to determine whether endothelial-derived nitric oxide synthase (eNOS) affects meiotic maturation of mouse oocytes in vitro. Cumulus-oocyte complexes (COC) were isolated from ovarian follicles of 27-day-old PMSG-primed wildtype (WT), and eNOS-knockout (eNOS-KO) females, and cultured in drops of medium under oil at 37 degrees C for 16-18 hr. Experiment 1 was carried out to determine effects of eNOS deficiency on the ability of COC to mature in vitro. To determine whether acute synthesis of nitric oxide (NO) was required for oocyte maturation, COC collected from WT mice were cultured in medium without (control) or with different doses of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS (exp. 2). To assess effects of NO deficiency on the kinetics of germinal vesicle breakdown (GVBD), COC from WT and eNOS-KO females were observed for 3.5 hr. COC from WT females were also incubated in medium without or with L-NAME (exp. 3 and 4). After the culture period, cumulus cells were removed, and oocytes were counted and classified as metaphase II (M II), metaphase I (M I) or showing atypical (degenerative) morphology. To determine viability and nuclear morphology of oocytes, they were stained with fluorescein diacetate or 4,6-diamidine-2'-phenylindole dihydrochloride, respectively. There were no differences in body weights but ovarian weights were lower in eNOS-KO mice compared with WT mice (P < 0.05). Ovaries from eNOS-KO mice contained fewer COC collected relative to WT mice (P < 0.01). Maturation of COC from eNOS-KO mice or WT oocytes treated with L-NAME resulted in a lower percentage of oocytes at M II stage (P < 0.01 and P < 0.05, respectively) and a higher percentage of oocytes at M I or atypical stages compared with those from WT (P < 0.01 and P < 0.05, respectively). Many oocytes that showed either an arrest in M I stage or abnormal morphology were not viable. Several oocytes in M II stage demonstrated abnormalities in distribution of maternal chromosomes. Our data demonstrate that eNOS-derived NO is a key modulator of oocyte meiotic maturation in vitro. These results support our previous observations in vivo and indicate that eNOS/NO has independent functions in both oocyte maturation and follicular/oocyte development.     

In-vivo and in-vitro maturation rate of oocytes from two strains of mice

Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3-5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.     

Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro

Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells.     

Germ cell differentiation and synaptonemal complex formation are disrupted in CPEB knockout mice.

CPEB is a sequence-specific RNA binding protein that regulates translation during vertebrate oocyte maturation. Adult female CPEB knockout mice contained vestigial ovaries that were devoid of oocytes; ovaries from mid-gestation embryos contained oocytes that were arrested at the pachytene stage. Male CPEB null mice also contained germ cells arrested at pachytene. The germ cells from the knockout mice harbored fragmented chromatin, suggesting a possible defect in homologous chromosome adhesion or synapsis. Two CPE-containing synaptonemal complex protein mRNAs, which interact with CPEB in vitro and in vivo, contained shortened poly(A) tails and mostly failed to sediment with polysomes in the null mice. Synaptonemal complexes were not detected in these animals. CPEB therefore controls germ cell differentiation by regulating the formation of the synaptonemal complex.     

Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology

The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.     

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.     

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.     

Interactions between somatic cells and germ cells throughout mammalian oogenesis

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.     

Juvenile and adult immature and in vitro matured ovine oocytes evaluated in relation to membrane electrical properties, calcium stores, IP3 sensitivity and apoptosis occurrence in cumulus cells

The analysis of differences between juvenile and adult oocytes may provide useful information on the acquisition of meiotic and developmental competence of the female gamete. In oocytes collected from either ewes or 40-day-old lambs, we evaluated membrane electrical properties, such as resting potential, conductance, activation ion currents, L-type Ca(2+) currents as well as calcium stores and IP3 sensitivity; in addition, the incidence of apoptosis in cumulus cells in these two age categories was compared. The analysis was carried out in oocytes both prior to and after in vitro maturation. Significant differences were found in all the examined parameters in relation to maturational stages whereas minor differences were recorded in relation to age of the donor. IP3 sensitivity strongly increased after in vitro maturation following a dose-dependent pattern from 1 to 500 micromol/L with a significant interaction (P < 0.01) between dose and maturational stage. The incidence of apoptosis in cumulus cells strongly increased after in vitro maturation and was greater in adult than in juvenile cumulus cells (39.2 +/- 5.8% vs. 21.9 +/- 3.5%; P < 0.01). In conclusion, all the examined parameters were greatly affected by the maturational stage, whereas minor differences were due to age-related oocyte quality, that is, at plasma membrane levels to conductance, activation current peaks and calcium currents, at cytosol level to calcium stores and IP3 sensitivity, and to incidence of apoptosis in cumulus cells. These parameters were compared with previous data in bovine to analyze oocyte quality in juvenile and adult individuals or between species.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

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.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

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.