Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cumulus Cells”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed1983

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

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

1-Methyl-3-isobutylxanthine↗
PubMed2002

Role of meiosis activating sterols, MAS, in induced oocyte maturation.

Meiosis of follicle enclosed oocytes is maintained in the prophase of the first meiotic division and oocytes do not spontaneously resume meiosis during oocyte growth and follicle development. Arrest of the meiotic process is most likely secured by the presence of follicular purines, e.g. hypoxanthine, which maintain high levels of cAMP in the oocyte and which also in vitro prevent oocytes from resuming meiosis. Only in response to the mid-cycle surge of gonadotropins will oocytes of preovulatory follicles overcome the meiosis arresting effect of hypoxanthine and resume meiosis proceeding to the metaphase of the second meiotic division. Morphologically, resumption of meiosis is observed by the disappearance of the oocyte's nuclear membrane (germinal vesicle), a process called germinal vesicle breakdown (GVB). The molecular mechanism down-stream to receptor activation by which the mid-cycle surge of gonadotropins induces oocytes to resume meiosis is, however, only partly understood. The oocyte itself lacks gonadotropin receptors and its action is mediated through the attached cumulus cells. In vitro it has been shown that FSH induces synthesis of a signal in the cumulus cells, which overcomes the meiosis arresting effect of hypoxanthine. We have shown that a group of sterols, meiosis activating sterols (MAS), induces oocyte maturation in vitro even in oocytes depleted of cumulus cells. MAS were identified as intermediates in the cholesterol biosynthesis between lanosterol and cholesterol. The two best characterized members of the MAS family are FF-MAS purified from human follicular fluid (4,4-dimethyl-5alpha-cholest-8,14,24-triene-3beta-ol) and T-MAS purified from bull testicular tissue (4,4-dimethyl-5alpha-cholest-8,24-diene-3beta-ol). The synthesis, quantification, localization and tissue-accumulation of MAS are reviewed. Several publications have documented the pharmacological effect of MAS in different species, including oocytes from mouse, rat and human. Conflicting results obtained by the use of sterol synthesis inhibitors, which prevent MAS-accumulation, are also discussed. Whether FSH actually uses MAS as a signal transduction molecule for inducing oocyte maturation and the mechanism by which MAS induce resumption of meiosis is currently unknown, but data to support that MAS is part of the FSH induced signal transduction pathway are presented.

Animals↗
PubMed1993

Species differences in the regulation of cumulus expansion by an oocyte-secreted factor(s).

The expansion of the mouse cumulus oophorus in vitro in response to FSH is dependent upon the presence of an enabling factor secreted by the oocyte. The purpose of this study was to determine whether the expansion of cumulus cells from rats and pigs were similarly dependent upon an oocyte-secreted enabling factor. Mouse and rat oocyte-cumulus cell complexes were isolated from pregnant mares' serum gonadotrophin (PMSG)-stimulated animals; pig oocyte-cumulus cell complexes with the attached piece of mural granulosa were obtained from either prepubertal gilts or cyclic sows. FSH (25-1000 ng ml-1) or dibutyryl cyclic adenosine monophosphate (dbcAMP; 0.05-2 mmol l-1) induced a dose-dependent expansion of the oocyte-cumulus complexes from rats and pigs. The hypothesis that the oocyte plays a role in the regulation of cumulus expansion was tested by microsurgically removing oocytes from oocyte-cumulus complexes and the oocytectomized complexes were tested for their ability to undergo expansion in response to FSH. FSH did not induce cumulus expansion in oocytectomized mouse complexes; however, expansion occurred in rat and pig oocytectomized complexes. The pieces of mural granulosa, detached from the pig complexes, also expanded in response to FSH stimulation. Rat and pig oocytectomized complexes were then held in culture for up to 48 h before stimulation by FSH. The degree of expansion in rat oocytectomized complexes decreased as the delay before FSH stimulation increased such that, with an 8 h delay, oocytectomized complexes did not expand.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta-mercaptoethanol.

Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta-mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co-culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage.

Animals↗
PubMed1997

Differential responses of bovine oocytes and preimplantation embryos to heat shock.

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four-to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41 degrees C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41 degrees C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41 degrees C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39 degrees C), 18% (41 degrees C), 17% (39 degrees C + BSO), and 11% (41 degrees C + BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [35S]methionine and [35S]cysteine at 39 degrees C or 41 degrees C for 12 hr. Exposure of oocytes to 41 degrees C for 12 hr reduced overall synthesis of 35S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function.

Animals↗
PubMed1991

The ultrastructure of human cumulus-corona cells at the time of fertilization and early embryogenesis. A scanning and transmission electron microscopic study in an in vitro fertilization program.

We observed the ultrastructure of the cumulus-corona cells (CC cells) surrounding: 1) human preovulatory oocytes unfertilized after in vitro insemination and 2) in vitro-fertilized polypronuclear ova (PO) at the pronuclear stage (3 pronuclei) and during early cleavage, at the 3-8 cell stage (cleaving PO). All the samples were obtained from women who underwent pharmacological hormonal stimulation during in vitro-fertilization procedures. Both cell groups were composed of irregularly rounded CC cells, showing an oval nucleus with one or more reticular nucleoli. Spermatozoa in close contact with CC cells were also seen. Linear and annular gap junctions between neighbouring cells were present, particularly in Group 1. Lipid droplets were present in both groups, appearing slightly more numerous and electron-dense in Group 2. In Group 1, mitochondria were numerous, polymorphic, and provided with cristae varying from lamellar to tubular. In Group 2, mitochondria also showed polymorphism, with bacilliform organelles with tubular cristae being predominant. In both groups cisternae and associated vacuoles and vesicles belonging to the Golgi complex were scattered in the cytoplasm of CC cells. Similarly, tubular and vesicular profiles of smooth endoplasmic reticulum were abundant and uniformly distributed throughout the cytoplasm of CC cells of both groups. In contrast, the abundant rough endoplasmic reticulum in Group 1 was formed by parallel stacks of flattened cisternae, whereas it was less plentiful and not arranged in stacks in Group 2. The CC-cell surface appeared covered by numerous membrane expansions in both groups. The expansions in Group 1 were mainly composed of blebs of various sizes and a few short microvilli, whereas in Group 2 numerous microvilli covered the cell surface. These observations demonstrate that a gradual establishment and maintainance of a steroidosynthetic capability (luteinization) takes place in CC cells, particularly during and shortly after fertilization, as occurs contemporaneously in the granulosa cells of the postovulatory follicle. Our results may be considered as ultrastructural confirmation of the capability of the CC cells to produce small amounts of steroids (estrogens and mainly progesterone). These hormones, alone or together with other substances (proteins, nutrients, growth factors?), might--around the fertilization time--act positively upon the early embryo itself, as well as on the microenvironment in which the embryo develops, both in vivo and in vitro conditions.

Cell Membrane↗
PubMed2004

Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos.

BACKGROUND: The aim of this study was to investigate haploidization of somatic cell nuclei in non-enucleated mature oocytes regarding spindle formation, chromosomes and developmental potential. METHODS: Mouse cumulus cells were injected into metaphase II oocytes. Some injected oocytes were examined for morphological changes of chromosomes and the spindle immediately, and at 30 min, 1 h or 2 h after the injections. The remaining oocytes were activated by Sr(2+) after various incubation periods and observed for formation of a second polar body and pseudo-polar body. Cytogenetic analysis was performed for some of the resulting zygotes. The progress to blastocysts in vitro and the possibility of conception in vivo were assessed. RESULTS: Immediately after injection, the cumulus cell nucleus was still in interphase without spindle formation. The occurrence of premature chromosome condensation (PCC) and spindle formation increased as the incubation time increased. The percentages of activated oocytes increased with the incubation time after nuclear transfer, but the difference was not significant between 1 (58%) and 2 h (62%). The incidence of chromosomal aberrations was high for the derived embryos. Development in vitro was poor, and no procreation of pups occurred after transfer of the 324 embryos. CONCLUSIONS: The PCC and spindle formation induced by cumulus cell nuclei in mature oocytes was time dependent, as was the chance for successful activation. The chromosomal abnormalities from segregation errors presented one obvious cause, apart from the potential epigenetic defects, of developmental failure of the semi-cloned embryos.

Animals↗
PubMed2006

Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes.

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.

Animals↗
PubMedDate not supplied

The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte.

The relationship between factors external to the human oocyte and the developmental potential of the female gamete after meiotic maturation in vivo is discussed. The findings indicate very different intrafollicular oxygen contents exist among follicles from the same and different patients, and marginally hypoxic conditions in some follicles are associated with a comparatively low adenosine triphosphate content and a reduction in intracellular pH for the corresponding metaphase II-stage oocyte. For such oocytes, the finding of an increased frequency of cytoplasmic pathology and chromosomal scattering (aneuploidy) suggests a compromised developmental potential. Three distinctly different patterns of cumulus cell attachment and proliferation during the first 24 hours of culture after insemination are described. As reported earlier, different cumulus cell phenotypes observed in vitro are independent of the presence of the oocyte or fertilized egg and unrelated to the degree of expansion of the cumulus at retrieval. However, developmental viability and implantation potential after in vitro fertilization-embryo transfer appear to be related to an intrinsic pattern of cumulus cell behavior in vitro. The results are discussed with respect to the role of factors external to the human oocyte that may influence or determine the ability of the oocyte to develop progressively after fertilization.

Adult↗
PubMed2005

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism.

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.

Animals↗
PubMed2004

Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes.

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.

Alpha-Globulins↗
PubMed1994

Influence of epidermal growth factor and transforming growth factor-alpha on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium.

This study was carried out to determine the effects of growth factors (epidermal growth factor, transforming growth factors-alpha and -beta 1, basic fibroblast growth factor, insulin), gonadotrophins (LH, FSH), and fetal bovine serum added to TCM199 medium on cumulus expansion and fertilization during in vitro maturation, and on subsequent embryonic development of bovine cumulus cell-enclosed oocytes. Epidermal growth factor, transforming growth factor-alpha, LH and FSH enhanced cumulus expansion and oocyte fertilizability. No significant effect was achieved with transforming growth factor-beta 1 nor with basic fibroblast growth factor. No additive stimulation on cumulus expansion and oocyte fertilizability was observed when epidermal growth factor was combined with LH or FSH. The addition of either epidermal growth factor or transforming growth factor-alpha to the maturation medium increased the number of fertilized ova that developed to the blastocyst stage. These results demonstrate the potential use of epidermal growth factor and transforming growth factor-alpha in obtaining high quality mature bovine oocytes for in vitro fertilization.

Animals↗
PubMed1986

The secretions of the cumulus-oocyte complex in relation to fertilization and early mouse embryonic development: a histochemical study.

A study has been made of the histochemical composition of the murine cumulus-oocyte complex and zona pellucida following treatment of immature females with exogenous gonadotrophins. Selected developmental stages were studied in detail, namely the ovulated and unfertilized egg, the fertilized oocyte and the preimplantation embryo. In addition, the histochemical features observed in normal fertilized embryos have been compared with those of haploid and diploid parthenogenetic embryos at comparable stages following activation. Shortly after fertilization, glycosaminoglycans, which form a major component of the extracellular matrix surrounding the cumulus cells, become incorporated into the zona pellucida of the fertilized egg. In oocytes with few or no attendant cumulus cells, there appeared to be a diminished uptake of glycosaminoglycans and a reduced intensity of the zona staining reaction to Alcian Blue. In these oocytes, uptake of glycosaminoglycans appeared to be from the secretions lining the oviduct. There was little incorporation of the glycosaminoglycans from the extra-cellular matrix of the surrounding cumulus cells into the zona pellucida in unfertilized or parthenogenetic eggs despite the activation stimulus. After fertilization or activation, the zona pellucida became increasingly PAS-positive. Enzymic studies clearly indicate that the composition of the zona pellucida of the early embryo is histochemically different from the zona that surrounds the oocyte in the preovulatory follicle. These findings are discussed in relation to the decreased viability of embryos from oocytes which have been ovulated.

Alcian Blue↗
PubMed2006

Incomplete denudation of oocytes prior to ICSI enhances embryo quality and blastocyst development.

BACKGROUND: Granulosa cells are essential mediators of oocyte maturation and fertilization. Because of the denudation of oocytes in preparation for ICSI, any potential positive effect of surplus cumulus cells (CCs) on further development would be unable to exert further effect. In order to evaluate the actual influence of adhering cumulus cells on further preimplantation development, this prospective study was carried out. METHODS: Sibling cumulus-oocyte complexes for 57 ICSI patients were split into a study group (incomplete denudation, n = 314) and a control group (complete denudation, n = 336). According to the cumulus cell pattern after partial denudation, mature gametes from the study group were further subdivided into type A oocytes, which showed several prominent CC clusters (n = 202), and type B (n = 75), which showed a more homogeneous pattern with CC covering the whole surface of the gamete. RESULTS: In immature oocytes, presence of adhered CCs led to a significant increase in resumption of meiosis (P < 0.01). Fertilization rate (P < 0.05) and ability to cleave (P < 0.01) was impaired in the study group, because of difficulties in ICSI of type B oocytes. By contrast, embryo morphology on days 2 (P < 0.01) and 3 (P < 0.05), as well as blastocyst formation, was better (P < 0.05) in the study group (55 blastocysts out of 88 zygotes) as compared to that in the control group (49/105). CONCLUSION: These data indicate that co-culture of oocytes with attached CCs may enhance preimplantation development.

Blastocyst↗
PubMed2003

Effect of growth hormone (GH) on in vitro nuclear and cytoplasmic oocyte maturation, cumulus expansion, hyaluronan synthases, and connexins 32 and 43 expression, and GH receptor messenger RNA expression in equine and porcine species.

The aim of this study was to investigate the role of growth hormone (GH) on in vitro cumulus expansion and oocyte maturation in equine and porcine cumulus-oocyte complexes (COCs), and to approach its way of action. Equine COCs were cultured in a control medium (TCM199, 5 mg/ml BSA, 1 microg/ml estradiol, and antibiotics) supplemented with either 0.5 microg/ml equine GH or 5 microg/ml equine LH. Porcine COCs were cultured in a basal medium (TCM199 with 570 microM cysteamine) supplemented with 0, 0.1, 0.5, or 1 microg/ml porcine GH or in a control medium (basal medium with 10 ng/ml epidermal growth factor and 400 ng/ml FSH) supplemented with 0 or 0.5 microg/ml porcine GH. After culture, cumulus expansion and nuclear stage were assessed. The cytoplasmic maturation of porcine oocytes was evaluated by in vitro fertilization and development for 7 days. The modifications of the expression of proteins implicated in cumulus expansion were analyzed in equine COCs by SDS-PAGE with antibodies against connexins 32 and 43 and hyaluronan synthases (Has) 1, 2, and 3. The expression of GH receptor mRNA was studied in oocytes and cumulus cells of the two species using reverse transcription-polymerase chain reaction with specific primers. The addition of GH in maturation medium increased cumulus expansion in equine but not porcine COCs. It improved nuclear maturation in equine and porcine, but had no effect on porcine fertilization and embryo development. The GH receptor mRNA was detected in equine and porcine oocytes and cumulus cells. GH did not influence the expression of Has 1, Has 3, and connexin 43 in equine cumulus cells.

Animals↗
PubMed2006

Factors affecting the efficiency and reversibility of roscovitine (ROS) block on the meiotic resumption of goat oocytes.

Goat oocytes from 2 to 4 and 0.8 to 1.2-mm follicles were freed (DOs) or not (COCs) of cumulus cells and cultured for different times in an inhibition medium supplemented with different concentrations of roscovitine (ROS). At the end of culture, oocytes were either cultured in a maturation medium for 24 hr and activated chemically for embryo development, or examined for GV chromatin configurations. Nuclear status was checked at different time points during maturation culture. Although both 200 and 250 microM ROS maintained 78-85% of oocytes at the GV stage for 24 hr, only oocytes blocked with 200 microM ROS developed to MII stage at a high rate after maturation culture. While few oocytes blocked with 200 microM ROS for 24 hr developed into morulae and none into blastocysts after activation, percentages of oocytes developing into morulae and blastocysts increased to the level of the control oocytes when the block time was reduced to 8 hr. While the GV and pMI stages were shortened with MI, and A/TI unaffected after oocytes were blocked for 8 hr, all the stages but A/TI were shortened after 24 hr of block. The sizes of nucleoli diminished with time and the GV chromatin configuration changed during ROS block. Significantly more DOs than COCs were blocked with 200 microM ROS, but none of the blocked DOs matured after drug withdrawal. However, maturation of the DOs improved significantly when ROS concentration was reduced to 150 microM or DOs were co-inhibited with COCs. The GV intact percentages of DOs did not differ after ROS inhibition with or without eCG, but those of COCs decreased significantly after ROS inhibited in the presence of eCG. When MII-incompetent oocytes from 0.8 to 1.2-mm follicles were inhibited with ROS for 8 and 24 hr prior to maturation culture, nuclear maturation improved significantly, activation rates were as high as that of the control oocytes, and some of the activated developed to 4- or 8-cell stages. It is concluded that (i) the efficiency and reversibility of ROS block was both drug concentration and exposure-time dependent; (ii) cumulus cells alleviated the toxicity of ROS on goat oocytes; (iii) eCG released goat oocytes from ROS block through the mediation of cumulus cells; (iv) ROS block quickened the nuclear maturation of goat oocytes and improved the developmental competence of meiosis-incompetent oocytes, possibly due to a sustained nuclear activity during inhibition culture; (v) oocyte nuclear maturation and activation did not depend upon cumulus expansion, but the embryo development occurred in association with cumulus expansion.

Animals↗
PubMed1996

In vitro penetration assay of boar sperm fertility: effect of various factors on the penetrability of immature pig oocytes.

The present study was designed 1) to examine the influence of cumulus cells, ovary storage time and oocyte size on the penetrability of immature pig oocytes, and 2) to investigate the effect of 2 methods of treating the semen from different boars on the inter-assay variability of homologous in vitro penetration tests of boar sperm fertility. In Experiment 1, cumulus oocyte complexes, oocytes with spontaneous loss of the cumulus cells during collection, and oocytes mechanically stripped of cumulus cells were used. No differences were observed in oocyte penetrability among the 3 types of oocyte, although mechanical removal of the cumulus caused an increase (P < 0.005) in the degeneration rate compared with the other oocyte types. In Experiment 2, the oocytes were recovered from ovaries kept in PBS (30 degrees C) for 2, 4 or 6 h after slaughter of prepuberal gilts. Ovary storage did not modify the penetrability of oocytes but increased (P < 0.02) their degeneration rates. In Experiment 3, the diameters of fresh oocytes were determined after co-incubation with spermatozoa. They were classified into 4 groups according to diameter: A) < 105 microm, B) 105-115 microm, C) 116-120 microm and D) > 120 microm. Oocytes from Groups C and D exhibited higher (P < 0.05) penetrability than oocytes from the other groups. In Experiment 4, stored, diluted spermatozoa from 4 boars were pretreated by centrifugation at 50 x g for 3 min and subsequent concentration of the supernatants at 1,200 x g for 3 min. The pellets were treated (washed twice and preincubated for 40 minutes) before co-incubation with immature oocytes or used directly as untreated samples (unwashed and non-preincubated). A boar effect (P < 0.001) was evident for the parameters of in vitro penetration, independently of sperm treatment. When the oocytes were inseminated with untreated spermatozoa, the effects of the replicate and the boar-by-replicate interaction on the variability in oocyte penetrability were not significant. The results of this study indicate that the use of standardized immature pig oocytes and stored untreated, diluted spermatozoa can provide a useful method for optimizing the homologous in vitro penetration (hIVP) assay of boar fertility.

Journal Article↗
PubMed1998

Improved development of in vitro-derived bovine embryos by use of a nitric oxide scavenger in a cumulus-granulosa cell coculture system.

This study was conducted to examine the hypothesis that nitric oxide (NO) affects prehatching development of bovine oocytes fertilized in vitro. In experiment 1, inseminated oocytes were cultured in a cumulus-granulosa cell (CG) coculture system to which 0.008 or 0.04 mM of sodium nitroprusside (SNP), a spontaneous NO releaser, was added at 18 or 60 hr postinsemination. Embryo development was greatly (P < 0.001) inhibited by the addition of SNP, regardless of time of addition or SNP concentration. In experiment 2, eight-cell embryos were cultured singly in a defined medium, to which 0.0016, 0.008, or 0.04 mM of SNP was added. Development to the blastocyst stage was greatly (P < 0.001) decreased after addition of SNP compared with no addition. Higher (P < 0.02) concentration of NO metabolites was found in developmentally arrested embryos than in developing embryos at 144 hr postinsemination (experiment 3). In experiment 4, blastocyst formation of oocytes cocultured with CGs was significantly (P < 0.02) increased after addition of hemoglobin (Hb, 1 microgram/ml), an NO scavenger. Prehatching development of oocytes was significantly (P < 0.05) increased after addition of Hb at different time intervals (18, 60, or 144 hr postinsemination) in experiment 5. Embryo development was not enhanced by Hb addition to the culture medium in the absence of CGs (experiment 6). Prehatching development of eight-cell embryos derived from a Hb-containing culture system was not promoted by the further addition of Hb after transfer of the embryos to a defined and CG-free single-embryo culture system (experiment 7). In conclusion, NO, which may be secreted from CGs, has an inhibitory role in prehatching development of bovine oocytes fertilized in vitro, and use of an NO scavenger, Hb, in a coculture system enhances blastocyst formation.

Animals↗