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PubMed1996

Role of antral follicle development and cumulus cells on in vitro fertilization of mouse oocytes.

The role exerted by antral follicle development and by companion granulosa cells on the ability of mouse oocytes at metaphase II to fuse with spermatozoa (as estimated by the kinetics of sperm-egg fusion), and to support normal fertilization (estimated by the formation of a male pronucleus) was investigated. After 17 h of culture in vitro, nuclear maturation occurred in 82% of oocytes derived from early antral follicles devoid of granulosa cells (denuded oocytes) and in 95% of oocytes derived from preovulatory follicles and cultured cumulus-intact or cumulus-free. Among the matured oocytes, 95% of cumulus-intact oocytes were arrested at metaphase II, while 61% of denuded and 50% of cumulus-free oocytes were arrested at metaphase I. The competence of denuded and cumulus-free oocytes to override the metaphase I stage was not affected by the addition of preantral granulosa, early antral granulosa or cumulus cells to the maturation medium. The kinetics of sperm-oocyte fusion were comparable in cumulus-intact and cumulus-free oocytes, and were more rapid than those of the early antral oocytes for insemination periods up to 60 min. However, when denuded oocytes were matured in medium containing early antral granulosa or cumulus cells, the kinetics of sperm-egg fusion was greatly accelerated to values equivalent to those of preovulatory oocytes. The ability of denuded oocytes to fuse with spermatozoa was unaffected by the addition of preantral granulosa cells to the maturation medium. Sixty minutes after insemination, the fertilization rates were 39% for denuded oocytes, 50% for cumulus-free oocytes and 73% for cumulus-intact oocytes. The fertilization rate of denuded and cumulus-free oocytes was significantly improved by the addition to the maturation medium of cumulus cells, but not by the addition of preantral or early antral granulosa cells. Taken together, the present results show that in mice, the maturation of both oolemma and ooplasm, required for successful fertilization, is acquired together with the development of antral follicles and is positively affected by cumulus cells during meiotic progression.

Animals↗
PubMed2003

Vascular endothelial growth factor and leptin: regulation in human cumulus cells and in follicles.

BACKGROUND: The factors that determine oocyte competency are poorly understood. It is believed that angiogenic factors are crucial. Modulation of these factors is therefore a central consideration. The adjacent association of cumulus cells to oocytes gives these cells a particular importance in relation to oocyte behavior. We report the effects of gonadotropins on the secretion of VEGF and leptin from cumulus cells; and the concentrations of the proteins in follicular fluid and their relationship to fertilization of oocytes in vitro. METHODS: The subjects were women undergoing intracytoplasmic sperm injection (ICSI). Oocytes and follicular fluid were collected. Leptin and vascular endothelial growth factor (VEGF) concentrations in incubation supernatants and follicular fluid and leptin in cell lysates were measured. Fertilization of corresponding oocytes were noted. In the present study, cells from individual follicles were incubated, as well as pooled cells from a woman. RESULTS: For the first time we demonstrated that VEGF release by human cumulus cells was modulated by gonadotropins in a dose-related, time-dependent manner, but no leptin was detected in either the supernatants after cumulus cell incubations or in cell lysates. Mean leptin levels were similar whether from follicles associated with eggs that were fertilized (14.4 +/- 1.1 ng/ml, mean +/- SEM) or not (12.4 +/- 1.1 ng/ml). Mean VEGF levels were also similar (11.1 +/- 1.2 ng/ml; 12.8 +/- 1.3 ng/ml). A greater proportion of VEGF in follicles was derived from follicular activities, compared with transfer from other physiological compartments, than leptin. CONCLUSIONS: Whereas it is possible that VEGF is part of the gonadotropin-mediated network that regulates development of the oocyte, leptin may not be produced in significant quantities by cumulus cells or be related to oocyte competency.

Adult↗
PubMed2007

Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro.

It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0h group) were cultured for 24h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20h without supplements (second culture step; 44h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24h group) or partly (H-DO 24h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24h (DO 24h+CC group). The maturation rates of all the cumulus-removed groups (DO 0h, DO 24h, H-DO 24h and DO 24h+CC groups) were lower (34.3-45.0%) than that of the control group (64.5%; P<0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0h, DO 24h and DO 24h+CC groups) were lower (4.03-5.26pmol/oocyte) than that of the control group (9.60pmol/oocyte; P<0.05); however, the H-DO 24h group had an intermediate value (7.0pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4-59.3%) than that of the control group (89.4%; P<0.05), whereas the H-DO 24h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0-4.5%) than that of the control group (19.9%; P<0.05), and the H-DO 24h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation (P<0.01) and blastocyst formation (P<0.01), and also with the number of cells per blastocyst (P<0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.

Animals↗
PubMed1994

Co-culture of human pronucleate oocytes with their cumulus cells.

The aim of this prospective randomized work was to study the value of co-culturing human pronucleate oocytes with their cumulus cells. A total of 550 fertilized oocytes from 95 in-vitro fertilization patients were randomly divided into two groups on the day after insemination. Group A oocytes (n = 260) were left undisturbed with their attached cumulus cells and group B oocytes (n = 290) were dissected from their cumulus cells. Both groups were incubated and examined daily for 3 days. In group A, 78% (202/260) reached the 4-cell stage 48 h after retrieval compared to 69% (200/290) in group B. At 72 h after retrieval, 70% (141/202) had reached the 8-cell stage in group A compared to 56% (112/200) in group B. The percentages of grade 1 embryos at 48 and 72 h after retrieval were 70% (141/202) and 76% (107/141) in group A compared to 50% (100/200) and 43% (48/112) in group B respectively. We concluded that co-culture of human oocytes with their cumulus cells significantly decreased their fragmentation and increased the number of embryos that reached the 4-cell and 8-cell stages with regular blastomeres. The technique is simple and avoids the use of heterogeneous cells.

Culture Techniques↗
PubMed1999

Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro.

Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.

Animals↗
PubMed2005

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation.

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.

Animals↗
PubMed2004

Exposure to L-ascorbic acid or alpha-tocopherol facilitates the development of porcine denuded oocytes from metaphase I to metaphase II and prevents cumulus cells from fragmentation.

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.

Animals↗
PubMed2003

[Cumulus cell apoptosis as a predictor for oocyte quality in artificial reproduction technique].

OBJECTIVE: To be able to predict the success of ART reliable tests for determining the quality of the oocytes are necessary. Apart from a vague morphologic assessment via microscopy a direct analysis of the oocyte quality is not possible. Because of the very close relation between the oocyte and the cumulus cells the analysis of the cumulus cells might give sufficient information on the oocyte quality. In this study we correlate the apoptotic activity of cumulus cells to the outcome of fertilized oocytes after Intracytoplasmic Sperm Injection (ICSI). MATERIAL AND METHODS: 246 cumulus-oocyte-complexes from patients undergoing infertility treatment with the ICSI procedure were individually collected. The comet assay was used to determine the proportion of apoptotic cells within the cumulus population of each oocyte and correlated with oocyte fertilization and oocyte quality as well as with pregnancy outcome in 86 patients. RESULTS: We were able to show that high quality embryos correlate to a low rate of apoptotic cells in their corresponding cumuli. Differences regarding the pregnancy outcome were statistically not significant. CONCLUSIONS: Our results on cumulus cell apoptosis and embryo quality confirm other publications. To arrive at statistically proven criteria for the further development of single oocytes an increase in the number of analyzed patients is necessary.

Apoptosis↗
PubMed1989

Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+-free medium.

Cumulus cell processes remaining in the zona pellucida of mouse oocytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl-phalloidin. If the isolation of the oocytes is performed in Ca2+-free medium, the presence of such processes allows the entry into the cell of low molecular weight molecules (such as 5-6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions. Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+-free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.

Actin Cytoskeleton↗
PubMed2001

Cryopreservation of human cumulus cells for co-cultures and assessment of DNA damage after thawing using the comet assay.

PURPOSE: Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation. METHODS: Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. RESULTS: The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability. CONCLUSIONS: The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw. More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.

Acridine Orange↗
PubMed1994

Effects of cumulus cells on male pronuclear formation and subsequent early development of bovine oocytes in vitro.

Bovine oocytes were matured in culture with and without cumulus cells. The proportion of oocytes matured to metaphase-II 24 h after culture was not different between those matured with and without cumulus cells. When cultured oocytes were inseminated in vitro, high proportions (84 to 87%) of oocytes were penetrated, with no difference between those matured with and without cumulus cells. However, the proportion of oocytes penetrated at the male pronuclear stage was significantly higher in cumulus-intact than in cumulus-free oocytes (90 vs 31%). The proportion of oocytes cleaved beyond the 8 approximately 32-cell stage was also significantly higher in cumulus-intact than in cumulus-free oocytes. It is concluded that the cumulus cells may have an important function for normal cytoplasmic maturation of bovine oocytes in vitro.

Journal Article↗
PubMed1997

Interactions between the oocyte and cumulus cells in the ovary of the B6.Y(TIR) sex-reversed female mouse.

The XY (B6.Y(TIR)) sex-reversed female mouse is infertile, primarily because of the early death of its embryos. We have previously determined that the XY oocyte itself, not the surrounding somatic cells, is responsible for its failure in postfertilization development. In the present study, we assessed the ability of the XY oocyte to regulate granulosa cell differentiation and functions. Oocyte-cumulus complexes (OCC) were isolated from antral follicles and were cultured in the presence of FSH and testosterone. Microsurgical removal of oocytes prevented cumulus cell expansion and suppressed estradiol production while it promoted progesterone production. Coculture with denuded oocytes from either XX or XY ovaries restored cumulus expansion and the endocrine profile observed in intact OCC. Morphology of oocytes and OCC in the preantral and antral follicles in situ as well as after isolation was compared for XX and XY ovaries. The average area of XY oocytes was smaller by 20% only at the preantral stage, whereas the zona pellucida layer was thinner by 20% at all stages. Furthermore, the XY oocyte was found to be attached to fewer cumulus cells (60% of XX control) in antral follicles and isolated OCC. In conclusion, the XY oocyte develops the normal ability of regulating granulosa cell differentiation despite its inferiority with respect to some morphometric parameters when compared to the XX oocyte.

Animals↗
PubMedDate not supplied

Immunocytochemical localization of transforming growth factor-alpha and epidermal growth factor receptor in human fallopian tubes and cumulus cells.

PROBLEM: Transforming growth factor-alpha (TGF-alpha) has been shown to be a potent stimulant of oocyte maturation and embryonic development. The role of maternal growth factors and their mechanism of action in early mammalian development is not well understood. METHOD: In this study, the presence of TGF-alpha and epidermal growth factor receptor (EGF-R) in human cumulus cells and fallopian tubes was investigated by immunocytochemical techniques. RESULTS: The fallopian tube showed intense staining for TGF-alpha in the apical region of the epithelial cells, and the cumulus cells showed intense staining for EGF-R on cell membranes. CONCLUSION: The presence of TGF-alpha in the fallopian tube epithelium and its receptor on cumulus cells suggest a paracrine mechanism between maternal growth factors and the developing embryo.

Embryonic and Fetal Development↗
PubMedDate not supplied

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells.

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.

Animals↗
PubMed2002

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes.

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.

Animals↗
PubMed1997

Insulin-like growth factor-1, a component of serum that enables porcine cumulus cells to expand in response to follicle-stimulating hormone in vitro.

The present study was aimed at determining whether insulin-like growth factor-1 (IGF-1) is a component of fetal bovine serum (FBS) that enables porcine cumulus cells to expand in response to FSH in vitro. Cumulus-oocyte complexes (COC) obtained from 4- to 6-mm follicles of prepubertal gilts were cultured at 39 degrees C for 24 h in media that contained human recombinant IGF-1 (50 ng/ml), FBS (15% v:v), or their combination, with or without FSH (1.5 microg/ml), and cumulus expansion was scored microscopically. Expansion was FSH dependent and was observed only when IGF-1, FBS, or both were present. The proportion of FSH-stimulated COC exhibiting full expansion in response to IGF-1 alone did not differ significantly (p > 0.05) from the proportion in those cultured with FBS or IGF-1+FBS (79 +/- 2.8% vs. 84 +/- 2.2% or 76 +/- 6.2%, respectively). In a concentration-response study, FSH-stimulated expansion was observed in a significant proportion of COC (32 +/- 2.8% vs. 0% control) at 1 ng/ml IGF-1, with the proportions increasing dose-dependently to maximal values between 10 and 75 ng/ml IGF-1, and decreasing at higher IGF-1 concentrations. Exposure of COC to an IGF-1 receptor (IGF-1R)-neutralizing antibody (Ab) for 90 min before addition of FSH and FBS dose-dependently inhibited cumulus expansion, with maximal inhibition at 10 microg/ml (1 +/- 1.0% vs. 68 +/- 1.1% control). In the absence of FBS or IGF-1, some COC had a tendency toward slight expansion when cultured with FSH, and the Ab completely inhibited that effect, suggesting that this may be due to endogenous IGF-1 production by the COC. The Ab effect was reversible and was eliminated by washing twice with fresh medium followed by culture for an additional 24 h in the presence of FSH and FBS. Expression of IGF-1R mRNA in the isolated oocyte and cumulus cells was determined by reverse tramscriptase polymerase chain reaction using sequence-specific primers. The IGF-1R message was detected in both the oocyte and cumulus cells. Collectively, these observations suggest that IGF-1 is a component of serum that enables cumulus cells to expand in response to FSH in vitro, and that the effect is receptor mediated. Since IGF-1 is present in the follicle in vivo, it may have a physiological role during gonadotropin-induced cumulus expansion.

Animals↗
PubMed2001

Hamster oocyte membrane potential and ion permeability vary with preantral cumulus cell attachment and developmental stage.

BACKGROUND: In vitro maturation of mammalian oocytes is an area of great interest due to its potential application in the treatment of infertility. The morphological and physiological changes that occur during oocyte development are poorly understood, and further studies are needed investigating the physiological changes associated with oocyte maturation. In this study we evaluated the membrane potential and the sodium/potassium permeability ratio of oocytes acutely isolated, and cumulus-oocyte complexes in metaphase II and preantral follicle stages. RESULTS: Intracellular electrical recordings revealed that cumulus-enclosed oocytes have a membrane potential significantly more negative at the preantral follicle stage than at metaphase II stage (-38.4 versus -19.7 mV, p < 0.0005). The membrane potential of the cumulus-free oocytes was not different between the preantral and metaphase II stages. The membrane potential of the cumulus cells forming preantral stage follicles was shown to be significantly different from that of the oocyte within the follicle (-28.6 versus -38.4 mV, p < 0.05). The sodium/potassium permeability measured in cumulus-enclosed oocytes at the preantral stage equaled a mean value of 0.33. The ratio was significantly lower when measured in oocytes denuded of cumulus cells or cumulus-enclosed metaphase II oocytes, 0.76, 0.79, 0.77 respectively (p < 0.001). CONCLUSIONS: These data show a change in the membrane potential and Na+/K+ permeability ratio during ooycte development from the preantral stage oocyte to the metaphase II stage. We have also demonstrated a change in the preantral oocyte membrane potential when surrounding cumulus cells are removed; either due to membrane changes or loss of cumulus cells.

Animals↗
PubMed1991

Cleavage capability of water buffalo follicular oocytes classified by cumulus cells and fertilized in vitro.

Water buffalo (Murrah) oocytes were collected from ovaries obtained from the slaughter house. They were classified according to the character of the cumulus cells under a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO2 in air at 39 degrees C. After 20-24 hr of in vitro maturation, the oocytes were fertilized using capacitated sperm obtained from 4 different bulls. For cleavage the oocytes were cultured at 39 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO2 in air. The good oocytes, with compact and dense cumulus cells cleaved significantly higher (p less than 0.01, 67.3%), than those of fair. partially naked oocytes with thin cumulus layers (27.5%, 25/91) or small remnants of cumulus cells and poor naked oocytes (3/100). A substantial variation cumulus layers (27.5% 25/91) or small remnants of cumulus cells and poor naked oocytes (3/100). A substantial variation in fertilization and developmental rates (16.0% to 43.8%) was observed among 4 different bulls. Late non-surgically into 14 buffalo recipients on day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by rectal palpation on day 60 and confirmed to be so on day 90 post-estrus.

Animals↗