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PubMed1998

Theca cells and theca-cell conditioned medium inhibit the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.

Animals↗
PubMed2003

In vitro and in vivo developmental potential of nuclear transfer embryos using bovine cumulus cells prepared in four different conditions.

We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).

Animals↗
PubMed1998

Bacteria in semen used for IVF affect embryo viability but can be removed by stripping cumulus cells by vortexing.

Bacterial contamination of in vitro vs in vivo produced embryos presents a particular danger because of the alteration of the zona pellucida and the use of various biological products during culture. Our objective was to investigate the effects of semen contaminated with bacteria on IVF of bovine oocytes and to determine if removal of cumulus cells by vortexing as opposed to pipetting would reduce contamination and improve subsequent embryonic development. Semen from 5 bulls of the Native Korean breed (Bulls A, B, C, D, E) was used for IVF of matured oocytes. Preliminary studies had shown that the semen from Bulls A, B, D and E but not Bull C was contaminated with various species of common bacteria. After IVF, the cumulus cells surrounding the oocytes were removed either by pipetting or vortexing. Viability and cleavage rates of the resulting zygotes was assessed after 44 h in culture. When cumulus cells were removed by pipetting, only zygotes derived from oocytes that were fertilized with uncontaminated semen from Bull C developed to morula and blastocyst stages; zygotes derived from oocytes that were fertilized with contaminated semen from Bulls A, B, D and E started to degenerate, and the culture media became noticeably turbid. When cumulus cells were removed by vortexing, zygotes derived from oocytes fertilized with either contaminated or uncontaminated semen showed good rates of development (16 to 32%) to morula or blastocyst stages. From these results it can be concluded that the bacteria introduced with the semen contaminated the in vitro system and severely reduced the viability of the embryos. In contrast, complete removal of the cumulus cells with vortexing, as opposed to pipetting, reduced the contamination of the culture medium, allowing embryonic development to take place.

Animals↗
PubMed1981

Porcine granulosa and cumulus cell properties. LH/hCG receptors, ability to secrete progesterone and ability to respond to LH.

In order to compare the properties of isolated cumulus and granulosa cells, granulosa cells and cumulus cells surrounding oocytes were harvested from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) porcine antral follicles and the number of LH/hCG receptors was measured by the binding of [125I]hCG. The ability of the cells to secrete progesterone in culture was examined in the presence and absence of hCG and LH. In 3 separate experiments of 1-h incubations at 37 degrees C using cells harvested from medium-sized follicles, granulosa cells bound 10--15-fold more iodinated hCG than an equivalent number of cumulus cells. During a 2-day culture period, cumulus cells secreted less progesterone than granulosa cells from medium- and large-sized antral follicles (p less than 0.01). The potential of both cumulus and granulosa cells to secrete progesterone in culture increased as the follicle progressed from small to large size. Also, the ability of the oocyte to mature in culture increased with antral follicle size. Concurrently the ability of cumulus-oocyte complexes to form monolayers in culture decreased as the follicle matured. Cumulus and granulosa cells harvested from small- and medium-sized follicles responded similarly to LH and hCG with a stimulation in progesterone secretion after 2-6 days in culture.

Animals↗
PubMed1985

Steroid secretion by cumulus cells isolated from human preovulatory follicles.

The secretion of progesterone, testosterone, and oestradiol by intact human oocyte-cumulus complexes in vitro was examined in incubations lasting 6-24 h. The complexes were aspirated from preovulatory follicles in 32 women who, due to tubal disease, were participating in an in vitro fertilization program. In 12 of the women follicular maturation was induced with clomiphene and human chorionic gonadotrophin (hCG), in 13 women with human menopausal gonadotrophin (hMG) and hCG and in 7 women with a combination of clomiphene-hMG plus hCG. The net secretion of steroids into the fertilization medium was studied before (0-6 h) and after (6-24 h) the addition of sperm, by RIA of aliquots removed at specific times. A high and sustained secretion of progesterone was found both before and after insemination. Testosterone secretion remained at a low and constant level while a net release of oestradiol was found mainly during the first hours of incubation. The release of steroids, particularly progesterone, varied according to the mode of hormonal stimulation in vivo and was highest in complexes from clomiphene-hMG-treated women, probably reflecting different maturity of the aspirated follicles. In a second series of experiments the dispersed cumulus cells were recovered after fertilization and cultured as monolayers for 2-4 days. The cells underwent spontaneous luteinization and secreted high amounts of progesterone. These results extend previous work in animals showing that also in the human the periovulatory cumulus cells are steroidogenically active. The results also suggest a functional difference in the cumulus cells related to the mode of ovulation induction.

Cell Count↗
PubMed2006

Ovine cumulus cells estradiol-17Beta production in the presence or absence of oocyte.

The objective of the present study was to compare the in vitro production of estradiol-17Beta (E(2)) by cumulus cells in the presence or absence of ovine oocyte. Moreover, the relationship between the concentration of produced estradiol-17Beta and oocyte nuclear maturation was assessed. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30-35 degrees C within 1-3 h after collection. The oocytes of follicles, 2-6 mm in diameter, were recovered by aspiration. The oocytes with evenly granulated cytoplasm and which were surrounded with at least three layers of cumulus cells were selected and subjected to culture in pre-incubated oocyte culture medium (OCM). Before culturing, the selected oocytes were randomly divided into five treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM (Group COCs); Group 2, denuded oocytes cultured in OCM (Group D); Group 3, denuded oocytes co-cultured with a cumulus cell-monolayer in OCM (Group D+M); Group 4, denuded oocytes co-cultured with previously cultured (for 26 h) cumulus cell-monolayer (10(5) cells/ml) in refreshed OCM (Group D+M(26)); Group 5, cumulus cell-monolayer (10(5) cells/ml) cultured in OCM (Group M). After an incubation period (26 h at 38.6 degrees C, 5% CO(2) and 100% humidity), the media were collected and kept at -20 degrees C until hormonal assay. The concentration of E(2) was determined by RIA method. For assessment of nuclear status, the completely denuded oocytes were subjected to DAPI staining. The highest percentage of metaphase II (MII) stage oocytes was observed in Group N (91%) and the lowest percentage was observed in Group D (6%) and Group D+M(26) (6%). The mean production of E(2) was highest and lowest in Group D+M (378.69+/-54.34 pg/ml) and Group D+M(26) (109.15+/-8.24 pg/ml), respectively. The production of E(2) was significantly (P<0.01) higher in Group D+/-M when compared with Groups M and D+/-M(26). Regarding the nuclear maturation, the percentage of MII stage oocytes was significantly (P<0.001) higher in Group COCs compared to the other groups. The results suggest that steroidogenic activity of cumulus cells in in vitro condition can be influenced by the pattern of connection between cumulus cells and the oocyte. Moreover, the nuclear maturation of oocytes is not influenced by the different production levels of E(2).

Animals↗
PubMed1980

Steroidogenesis by the human oocyte-cumulus cell complex in vitro.

The ability of the human oocyte-cumulus cell complex to synthesize progesterone, androgens and estrogens and to modify its endocrine environment in vitro was investigated. Germinal-vesicle stage oocytes with adhering layers of cumulus cells were recovered from human ovaries and maintained for 40--50 h in vitro in a culture medium with or without antral fluid. The results show that oocyte-cumulus (O-C) cell complexes were capable of synthesizing progesterone, androgens and estrogens. Oocytes with the capacity of resuming meiosis in vitro were part of an O-C complex producing significantly more progesterone than those O-C complexes containing oocytes incapable of resuming meiosis. Irrespective of the stage of oocyte maturation at the end of culture, testosterone and estrone were respectively the major androgen and estrogen produced. It is concluded that the oocyte-cumulus compartment of the antral follicle is a steroidogenically competent unit and that it has the capacity to modify the endocrine microenvironment of the follicle.

Adult↗
PubMed1990

Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins.

We have used carbohydrate-binding proteins, or lectins, as tools to investigate the physiological phenomena associated with the preovulatory maturation of the oocyte-cumulus cell complex. Certain lectins are mitogens, and since other mitogenic agents such as growth factors are known to stimulate meiotic maturation and cumulus expansion, we tested the ability of lectins to provoke these physiological responses. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) and treated with one of eleven different lectins. With the exception of pokeweed mitogen (PWM), all of the mitogenic lectins tested were able to induce germinal vesicle breakdown (GVB) in meiotically arrested oocytes, and this action required the presence of the somatic cumulus cells; in fact, either there was no effect or maturation was suppressed when cumulus cell-free oocytes (denuded oocytes; DO) were treated with lectins. None of the nonmitogenic lectins stimulated meiotic maturation in either CEO or DO. The mitogenic lectin concanavalin A (Con A) also induced maturation in CEO when meiotic arrest was maintained with hypoxanthine, guanosine, or 3-isobutyl-1-methylxanthine. The kinetics of spontaneous oocyte maturation in inhibitor-free medium were not altered by Con A. Only the mitogenic lectins that induced meiotic maturation stimulated cumulus expansion, with Con A the most active lectin. The actions of Con A on the maturation of the oocyte-cumulus cell complex were inhibited by methyl-alpha-D-mannopyranoside as predicted by its sugar-binding specificity. These results demonstrate that (1) lectins can stimulate maturation of the mouse oocyte-cumulus cell complex; (2) mitogenicity is associated with the positive activity of the lectins; and (3) cumulus cells mediate the stimulatory action of lectins on oocyte maturation, while inhibition of GVB occurs at the oocyte level. These data support the idea that common signals mediate the mitogenic and maturation-inducing actions of lectins.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes.

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.

Animals↗
PubMed2004

Peroxiredoxin 6 is upregulated in bovine oocytes and cumulus cells during in vitro maturation: role of intercellular communication.

Peroxiredoxins are peroxidases involved in antioxidant defense and intracellular signaling. Expression of transcripts coding for peroxiredoxin 6 (PRDX6) has been previously described to be upregulated in oocytes after in vitro maturation, a period during which general transcription decreases dramatically in oocytes. The aim of the present work was to evaluate PRDX6 regulation in bovine cumulus-oocyte complexes in relation to maturation and intercellular communication. PRDX6 expression was analyzed by reverse transcription-PCR and Western blotting in oocytes and cumulus cells before and after in vitro maturation. PRDX6 was found to be upregulated at the mRNA and protein levels in both cell types after maturation. The effect of paracrine and gap junctional communication on PRDX6 expression was then assessed by culturing cumulus clusters in the presence or absence of denuded oocytes. While PRDX6 upregulation in oocytes required intact cumulus-oocyte junctions, the presence of denuded oocytes was necessary but sufficient for the upregulation to occur in cumulus cells. Finally, the influence of recombinant mouse growth differentiation factor-9 (GDF-9) on PRDX6 expression in cumulus cells was studied. GDF-9 induced cumulus expansion and PRDX6 upregulation in bovine cumulus clusters. Altogether, our data suggest that PRDX6 upregulation in cumulus-oocyte complexes during in vitro maturation is mutually regulated by both cell types: PRDX6 upregulation in oocytes would require gap junctions with cumulus cells, while upregulation in cumulus would depend on secretion of oocyte paracrine factor(s) with GDF-9 being a likely candidate.

Animals↗
PubMed1994

A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

Animals↗
PubMed2003

Role of intracellular cyclic adenosine 3',5'-monophosphate concentration and oocyte-cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte.

The present study was designed to address the physiological role played by cAMP on gap junction (GJ) mediated communications between oocyte and cumulus cells during in vitro maturation. Cyclic AMP was stimulated by different collection and maturation media known to induce different rates of nuclear maturation and developmental competence as well as different levels of cumulus expansion. Cumulus-oocyte complexes (COCs) were matured for 0, 3, 7, 12, 18, and 24 h in the absence of stimulation or in the presence of serum and gonadotropins (fetal bovine serum+human menopausal gonadotropins [FCS+hMG]) or 0.01 microg/ml of invasive adenylate cyclase (iAC). For each time point, intracellular cAMP concentration ([cAMP]i) was determined either in the whole COC or oocyte after cumulus cell removal. GJ functional status was analyzed by microinjection of Lucifer yellow fluorescent dye in cumulus-enclosed oocytes and by immunohistochemical localization of connexin 43 (Cx43). In the absence of stimulation, [cAMP]i in COC and oocyte was lower than in other groups, and communications declined after 3 h of culture. In the FCS+hMG group, [cAMP]i increased significantly in COC, with a peak between 3 and 7 h that was temporally correlated with the beginning of the cumulus expansion process, which occurred only in this group and with the termination of the communications. COC matured in the presence of iAC showed a moderate increase of [cAMP]i during all of the maturation times as well as a prolongation of oocyte-cumulus cell communications. The immunohistochemical localization of Cx43 confirmed the delay in connexons protein turnover in iAC-treated COCs. Our results show that cumulus expansion and oocyte developmental competence are induced by different levels of cAMP and that its intracellular concentration may affect cell coupling between oocyte and cumulus cells. We hypothesize that the higher developmental competence of COCs matured in the presence of iAC could be achieved through a moderate increase of intracellular cAMP, which in turn determines a prolongation of communications between the two cell types.

Animals↗
PubMed1999

Tumor necrosis factor alpha regulates collagenolytic activity in preovulatory ovine follicles: relationship to cytokine secretion by the oocyte-cumulus cell complex.

The pleiotropic cytokine tumor necrosis factor (TNF)-alpha has been implicated in the mechanism of ovulation. Experiments were designed to test the hypothesis that TNF-alpha secreted from the oocyte-cumulus cell complex stimulates follicular collagenase production and thereby contributes to ovarian wall degradation and ovulatory rupture. Proestrous ewes were treated with GnRH to synchronize the onset of the gonadotropin surge; ovulation occurs approximately 24 h later. There was an increase in TNF-alpha (immunoassay) in antral fluid of preovulatory follicles at 18 h after GnRH, which was related to tissue collagenolytic bioactivity (radiolabeled type I substrate digestion by enzymatic extract) and collagen (hydroxyproline) depletion. Intrafollicular injection of TNF-alpha antibodies at 12 h after GnRH negated the rise in follicular collagenolytic bioactivity (and is known to block ovulation in the sheep). Moreover, collagenase production was enhanced when follicular tissues (0 h GnRH) were incubated (6 h) with recombinant TNF-alpha; this effect was abolished by the transcriptional inhibitor actinomycin D. Secretion of TNF-alpha by oocyte-cumulus cell complexes isolated from preovulatory follicles simulated the in vivo circumstance. Immunostaining indicated that TNF-alpha was confined mainly to the oocyte before GnRH administration, accumulated in cumulus cells during the mid-to-late preovulatory period, and was expended with the imminent approach of ovulation. To our knowledge, this is the first report specifying that up-regulation of collagenase expression is a target mode of TNF-alpha action in preovulatory follicles. The oocyte-cumulus cell complex is an apparent source of soluble TNF-alpha.

Animals↗
PubMed1998

Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.

Animals↗
PubMed2000

Periovulatory human oocytes, cumulus cells, and ovarian leukocytes express type 1 but not type 2 11beta-hydroxysteroid dehydrogenase RNA.

OBJECTIVE: To further elucidate cortisol metabolism in the follicular microenvironment at the time of oocyte retrieval, the presence of 11beta-hydroxysteroid dehydrogenase (HSD) messenger (m)RNA transcripts in oocytes; cumulus cells; granulosa cells; and CD45(+), CD15(+) leukocytes was assessed semiquantitatively. DESIGN: Controlled study using semiquantitative assessment of 11beta-HSD mRNA. SETTING: University IVF center. PATIENT(S): Twenty-six patients undergoing controlled ovarian hyperstimulation for assisted conception. INTERVENTION(S): Metaphase II oocytes; cumulus cells; granulosa cells, and CD45(+), CD15(+) leukocytes from individual follicular fluid aspirates. MAIN OUTCOME MEASURES: Semiquantitative analysis of PCR products after total RNA extraction and complementary DNA synthesis. RESULT(S): Periovulatory human oocytes; cumulus cells; CD45(+), CD15(+) leukocytes; and granulosa cells consistently express type 1 but not type 2 11beta-HSD mRNA. Expression of mRNA is greatest in cumulus cells. Type 1 11beta-HSD mRNA expression varies considerably in all cell types and among individual follicles and patients. CONCLUSION(S): These studies of mRNA expression suggest that the enzymes present both in and around the periovulatory oocyte will favor a high-cortisol environment.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1980

Ribonucleoside metabolism by mouse oocytes: metabolic cooperativity between the fully grown oocyte and cumulus cells.

Ribonucleoside uptake and metabolism were studied in mouse oocytes arrested at dictyate and metaphase II, with or without attached cumulus cells. Uptake of uridine and guanosine decreased after meiotic maturation, whereas that of adenosine and cytidine remained unaltered. Dictyate oocytes free of cumulus cells (denuded) phosphorylated all of the radiolabeled ribonucleosides. Little radiolabeled ribonucleoside-5'-triphosphate was found. This is in contrast to the early mouse embryo, which rapidly converts ribonucleosides to the 5'-triphosphate (Daentl and Epstein, "71; Clegg and Piko, "77). Following meiotic maturation, the major metabolite present when metaphase II oocytes were cultured with cytidine or uridine was the ribonucleoside, whereas the 5'-monophosphate was still the major species for oocyte cultured with guanosine or adenosine. Ribonucleoside uptake was increased and the distribution of phosphorylated metabolites was different for cumulus-enclosed dictyate oocytes compared to their denuded counterparts. This increase in uptake and alteration in metabolite distribution was not observed for cumulus-enclosed metaphase II oocytes. These results are discussed in terms of gap junction-mediated metabolic cooperatively between the cumulus cells and the fully grown oocyte and the possible role this type of cell-to-cell interaction may serve during oocyte growth.

Adenosine↗
PubMed2000

Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells.

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.

Animals↗
PubMed2006

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts.

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.

Animals↗