Oocyte-cumulus Cell Complexes Research Articles

Oocyte-derived growth factors promote development of antrum-like structures by porcine cumulus granulosa cells in vitro.

Oocytes communicate with the surrounding somatic cells during follicular development. We examined the effects of two oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the development of porcine oocyte–cumulus cell complexes (OCCs) in vitro. We collected OCCs from early antral follicles (1.2–1.5 mm) and prepared oocytectomized cumulus cell complexes (OXCs), which were then cultured in a growth medium supplemented with 0–100 ng/ml GDF9 and/or BMP15 for 7 days. In the medium without GDF9 or BMP15, OCCs developed during culture, and approximately 30% of them formed antrum-like structures. GDF9 promoted OCC development and structure formation in a dose-dependent manner. However, OXCs did not form antrum-like structures without growth factors. GDF9 promoted the development of OXCs, and 50 and 100 ng/ml GDF9 promoted the formation of the structures by 8% and 26%, respectively; however, BMP15 did not promote the formation of these structures. OXCs were then cultured with 100 ng/ml GDF9 and various concentrations of BMP15 to investigate their cooperative effects on the formation of antrum-like structures. BMP15 promoted the formation of antrum-like structures in a dose-dependent manner. In conclusion, GDF9 derived from oocytes is probably important for the formation of antrum-like structures in porcine OXCs, and BMP15 cooperates with GDF9 to form these structures.

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro.

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes.

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5–0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.

Grafted polymer brush coatings for growth of cow granulosa cells and oocyte-cumulus cell complexes.

In the present work, three types of grafted brush coatings [P4VP, POEGMA246, and P(4VP-co-POEGMA246)] were successfully fabricated using graft polymerization of monomers "from the surface." The composition, thickness, and morphology of the grafted brush coatings were analyzed by TOF-SIMS, ellipsometry, and AFM, respectively. The chemical nature of the polymer surface plays a crucial role in the growth and development of the cow granulosa cells and, therefore, also oocyte-cumulus complexes. In comparison with other coatings, the P(4VP-co-POEGMA246) copolymer coating enables the formation of dispersed and small but numerous cell conglomerates and high cumulus expansion in oocyte-cumulus complexes with highly homogeneous cumulus layers surrounding the oocytes. Moreover, the cellular oxygen uptake for this coating in the presence of NaF (inhibitor glycolysis) was stimulated. This new (4VP-co-POEGMA246) copolymer nanostructured coating is a promising material for granulosa cell and oocyte-cumulus complex cultivation and possibly will have great potential for applications in veterinary and reproductive medicine.

ID: 1067 The Effect of Oocyte-Cumuls Cell Complexes on Maturation Rates and Pronuclear Formation After Pathenogenetic Activation

Cumulus cells are integral in oocyte growth, maturation, pronuclear formation as well as preimplantation development after fertilization. In this study, we investigated the effect of different cumulus morphology on maturation rates and pronuclear formation after parthenogenetic activation. Oocyte-cumulus cell complexes (OCCs) were classified into three grades: (A) over 3 layers, (B) 2–3 layers, (C) less than 1 layer or incomplete. Our results showed that group A and B achieved high maturation rates (84.4% and 82.5%) whereas group C were 45.7%. In a similar pattern, after parthenogenetic activation, the rate of pronuclear formation of group A and B were namely 65.5% and 52%, while that of group C was only 36.4%. Moreover, we also examined the impact of duration of in vitro maturation on the rate of nuclear formation after activation. We found that 25-hour would result in the highest rate (55.5%) while only 4.44% in group of 30-hour, which was possibly caused by the aging of oocytes. In conclusion, this study found that OCCs with more than 2 layers of cumulus cells could reach the high maturation rates, finally oocytes activated at 25 hours resulted in high rate of pronuclear formation in bovine.

Efficacy of Hyaluronic Acid in The Selection of Human Spermatozoa with Intact DNA by The Swim-up Method.

In 2014, enrolled 20 patients who applied to the Unit of Assisted Reproduction Techniques, Konya Necmettin Erbakan University. Based on the presence of hyaluronic acid (HA) in the oocyte-cumulus cell complex, sperm attached to HA in vivo were modeled in vitro. Available healthy sperm obtained in the swim-up procedure using HA were investigated. This observational cohort study, a routine analysis was conducted on the ejaculation samples obtained from 20 patients. We divided each sample into two groups and the swim-up method was applied. Human serum albumin (HSA, 0.5%) was added to samples from the first group. HA (10%) was added to samples from the second group. We determined the floating linear and non-linear sperm concentrations of both groups annexin V was used to determine the rate of apoptosis of these sperm. Following swim-up, linear and non-linear sperm concentrations were higher in the group that contained HA compared to the group with HSA. However, there was a significantly higher apoptosis rate in the HSA group compared to the HA group. The addition of HA to the medium in the swim-up procedure positively affected sperm parameters. Thus, healthier sperm cells were obtained without DNA damage and with high motility.

Mitochondrial DNA copy number in cumulus cells is a strong predictor of obtaining good-quality embryos after IVF

The aim of this study was to establish a simple tool to predict good-quality embryos in in vitro fertilization (IVF) by using cumulus cells (CCs) or peripheral blood cells (PBCs). Mitochondrial DNA was extracted from CCs and PBCs in patients undergoing IVF. Using real-time polymerase chain reaction, mtDNA copy number in a single cell was calculated. Embryo quality was assessed when it was transferred or frozen. CCs were obtained from 60 oocyte cumulus-cell complexes (OCCCs) in 30 women, and PBCs were collected from 18 women. For the 30 women in the study, the median age was 37 years old (range, 24-43), and the mean body mass index was 21.4 (standard error, 2.0). mtDNA content of CCs and PBCs was highly correlated (Pearson's r = 0.900, p < 0.0001). The median mtDNA content of CCs for good- and poor-quality embryos was 140 and 57, respectively (p < 0.0001). The median mtDNA content of PBCs for good- and poor-quality embryos was 36 and 13, respectively (p = 0.604). The logistic regression model indicated that mtDNA content in CCs was the only parameter that predicted good-quality embryos (p = 0.020). The receiver operating characteristic curve for obtaining good-quality embryos by mtDNA copy number in CCs had an area under the curve of 0.823, and using a threshold of 86, positive and negative predictive values were 84.4 and 82.1 %, respectively. The determination of mtDNA content in CCs can be used to predict good-quality embryos.

Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte–cumulus cell expansion and steroidogenesis

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.

Offspring from Heterotopic Transplantation of Newborn Mice Ovaries

This study is aimed at investigating the developmental potential of the primordial follicles from ovaries of newborn mice after cryopreservation in liquid nitrogen for long-term storage, thawing, and heterografting into the kidney capsules of ovariectomized adult female mice. After stimulation of recipient mice with pregnant mare serum gonadotropin on day-19 after heterografting, the primordial follicles of the transplanted ovaries could develop into antral follicles. When the oocyte-cumulus cell complexes were retrieved from these antral follicles, they could mature after in vitro culture for 16–17 h. After in vitro fertilization, the rates of embryos derived from these oocytes that developed into the two-cell stage and the blastocyst stage after 16–17 h and after day-4, respectively,in the culture medium were 55.40% (55/107) and 9.09% (5/55),respectively. In the ovarian transplantation groups, no pups were derived from the 410 embryos that were transferred into 10 pseudopregnant mothers at the pronuclear stage. However,of the 10 surrogate mothers in whom 570 embryos were transferred at the two-cell stage, four achieved pregnancy and gave birth to 20 live offspring. These results demonstrated that primordial follicles in newborn mice ovaries were capable of sustaining their developmental potential after freezing and thawing. Once transplanted into the kidney capsules of ovariectomized adult female mice, these primordial follicles could develop and respond to gonadotropin stimulation and reach the antral stage; further, live offspring could be derived from these follicles.

Synthesis of Tumor Necrosis Factor Alpha-Induced Protein 6 in Porcine Preovulatory Follicles: A Study with A38 Antibody1

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.

Meiotic Maturation of Incompetent Prepubertal Sheep Oocytes Is Induced by Paracrine Factor(s) Released by Gonadotropin-Stimulated Oocyte-Cumulus Cell Complexes and Involves Mitogen-Activated Protein Kinase Activation

In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation.

Luteinizing hormone receptors expression in cumulus cells closely related to mouse oocyte meiotic maturation

In the process of oocyte maturation, gonadotrophins are believed as main stimulators for oocyte meiosis resumption. However, which gonadotrophin (i.e. FSH or LH) is the key hormone in this process is still unknown. This study indicated a close relationship between LH and FSH on activating meiotic maturation of oocyte in vitro. FSH efficiently induced oocyte meiosis at concentration of 50 IU/L, while LH alone had no effect on oocyte meiotic initiation. Using RT-PCR and in situ hybridization, a tight corelationship between FSH-stimulated oocyte meiotic resumption and LHR mRNA expression in cumulus cells was found. LHR mRNA was present in cumulus cells of oocyte-cumulus cell complexes. Except the expression of LHRs was present in cumulus cells surrounding all maturing oocytes, low level expression was also detected in cells associated with oocytes that were still at GV stage. Its expression was enhanced by FSH stimulation before oocyte maturation. However, LHRs did not express in cumulus cells associated with oocytes that were completely arrested at GV stage by IBMX. Furthermore, increased progesterone concentration was found in the medium in which CEOs were cultured with FSH and LH, but not in those with FSH or LH alone. LHR expression in cumulus cells increases with time in culture, and the levels of expression are enhanced in the presence of FSH. Oocyte meiotic resumption may create conditions favorable for LHR expression. LHR expression may be considered as a potential marker for oocytes maturation initiation.

The Histone Deacetylase Inhibitor Trichostatin A Induces Retrogressive Chromatin Decondensation in the Germinal Vesicle of Porcine Cumulus-enclosed Oocytes

In porcine growing oocytes, chromatin remains diffuse. As the oocytes approach their final size, 120 μm, the chromatin becomes partly condensed and forms a perinucleolar sheath. These changes occur simultaneously with a decrease in transcriptional activity. In many other cell types, it has been shown that the state of acetylation of nucleosome core histones is essential in chromatin remodeling and transcription so that partial chromatin condensation in oocytes may involve the recruitment of histone deacetylases. In order to test this hypothesis, porcine oocyte-cumulus cell complexes were treated with a specific inhibitor of histone deacetylases, trichostatin A (TSA). The perinucleolar sheath loosened or disappeared after 24 hr culture with 100 nM TSA, but after further culture in TSA-free medium, about 40% developed the perinucleolar sheath again. In the presence of 4 mM hypoxanthine, the decondensation induced by TSA progressed rather slowly, but continuously, for 72 hr. When oocytes were denuded before culture, spontaneous maturation occurred in the presence of TSA. Thus, the inhibitor-induced decondensation is not attributed to the inhibition of the maturation-promoting factor. These results suggest that deacetylation of histones may be involved in chromatin remodeling in oocytes near the end of the growth phase.

Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte–cumulus cell complex

Luteinizing hormone (LH) induces maturational processes in oocyte–cumulus cell complexes (OCC) of preovulatory follicles that include both resumption of meiosis in the oocyte and expansion (mucification) of the cumulus oophorus. Both processes require activation of mitogen-activated protein kinase (MAPK) in granulosa cells. Here, it is reported that inhibition of MAPK activation prevented gonadotropin-stimulated resumption of meiosis as well as the rise in expression of two genes whose products are necessary for normal cumulus expansion, Has2 and Ptgs2. However, inhibition of MAPK did not block gonadotropin-induced elevation of granulosa cell cAMP, indicating that the activation of MAPK required for inducing GVB and cumulus expansion is downstream of cAMP. Moreover, activation of MAPK in cumulus cells requires one or more paracrine factors from the oocyte to induce GVB and cumulus expansion; MAPK activation alone is not sufficient to initiate these maturational processes. This study demonstrates a remarkable interaction between the oocyte and cumulus cells that is essential for gonadotropin-induced maturational processes in OCC. By enabling gonadotropin-dependent MAPK activation in granulosa cells, oocytes promote the generation of a return signal from these cells that induces the resumption of meiosis. It also appears that an oocyte-dependent pathway downstream from oocyte-enabled activation of MAPK, and distinct from that promoting the resumption of meiosis, governs cumulus expansion.

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue.

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting.

Previous studies have demonstrated development of antral follicles in cryopreserved human ovarian tissue after autografting and xenografting, thus indicating successful preservation of follicular function. The study aim was to assess whether these follicles could also undergo periovulatory changes in response to hCG. Ovarian tissue from three patients were dehydrated in propanediol (PROH)/sucrose and cryopreserved using the slow cooling/rapid thaw procedure. Thawed tissue was placed under the kidney capsule in immunodeficient mice. Following growth (>20 weeks) in the presence of gonadotrophin, hCG was administered and ovarian tissue examined histologically. Thirty-two antral follicles (diameter range 0.6 to 5 mm) were examined. Histological evidence of a response to hCG was evident in all follicles. Disruption of the concentric layers of mural granulosa and theca cells was apparent in all antral cavities. In 17 (53%) follicles the exterior follicular wall had reduced to a few cells thick, and in eight (25%) the wall had ruptured. Mucified oocyte-cumulus cell complexes were present in 32 follicles, 17 of which had begun to detach from the pedicle. Resumption of meiosis had occurred in over half the oocytes (five metaphase II and seven metaphase I oocytes, eight germinal vesicle breakdown). Two corpora lutea were also detected. Follicles cryopreserved within human ovarian tissue using the PROH procedure, can develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus.

Bovine oocytes in early antral follicles grow in serum-free media: effect of hypoxanthine on follicular morphology and oocyte growth.

Some culture systems have been shown to support oocyte growth in mice, although there has been little success in applying these systems to other species. In the present study, we compared three culture conditions for growing bovine oocytes and examined the effect of hypoxanthine on oocyte growth. In the first experiment, early antral follicles, 0.4-0.7 mm in diameter were collected, and oocyte-cumulus-granulosa cell complexes (OCGs) and oocyte-cumulus cell complexes (OCs) were dissected from the follicles. Follicles (Fs), OCGs and OCs were embedded in collagen gels and cultured in serum-supplemented medium for 16 days. In the Fs, OCGs and OCs cultured in hypoxanthine-free medium, 21%, 9% and 4% of the oocytes showed normal morphology, respectively, and hypoxanthine (4 mM) increased the percentages in all the groups (Fs, 37%; OCGs, 29%; OCs, 10%). In the second experiment, Fs were cultured in serum-free medium with or without hypoxanthine for 16 days. Histological examination demonstrated that hypoxanthine maintained the integrity of the follicular basement membrane. After a growth culture, 91% of the oocytes showed normal morphology, and 87% of the oocytes were at the germinal vesicle stage in serum-free, hypoxanthine-supplemented medium. The mean diameters of the oocytes were significantly larger (117.6 +/- 5.7 microm) than they were in the other groups and than they had been before the culture (approximately 95 microm). After a subsequent maturation culture of the oocytes, 85% underwent germinal vesicle breakdown and 23% reached the second metaphase. These results demonstrate that growing bovine oocytes from early antral follicles grow efficiently in follicles cultured in serum-free, hypoxanthine-supplemented medium and acquire meiotic competence.

Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse.

This study investigated the participation of MAPK in the resumption of meiosis [germinal vesicle breakdown (GVB)] in oocytes and cumulus expansion using oocyte-cumulus cell complexes (OCC) from Mos-null mice (Mos(tm1Ev)/Mos(tm1Ev), hereafter Mos(-/-)). MAPK activity was not detected in Mos(-/-) oocytes whether they matured in vivo or in vitro, with or without gonadotropin stimulation. Therefore, there are no pathways independent of MOS that activate MAPK during gonadotropin-induced maturation. In contrast, MAPK activity was always detected coincident with GVB in Mos(+/+) oocytes. Moreover, MAPK activity was detected in cumulus cells before gonadotropin-induced GVB in OCC regardless of genotype. A specific inhibitor (U0126) of MEK, a MAPKK required for MAPK activity, inhibited gonadotropin-induced GVB in OCC of both Mos(+/+) and Mos(-/-) mice. Activation of MAPK was downstream of elevation of cAMP. U0126 also inhibited cumulus expansion stimulated by FSH, epidermal growth factor, 8-bromo-cAMP, and recombinant growth differentiation factor-9. It is concluded that under the in vitro conditions used here, gonadotropin-induced GVB requires the participation of MAPK activity in the cumulus cells, but not in the oocyte. Moreover, the induction of cumulus expansion also requires the participation of MAPK, and this action is downstream of both elevation of cAMP and growth differentiation factor-9.