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PubMed1998

Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway.

In this study we tested the hypothesis that the pentose phosphate pathway (PPP) participates in the meiotic induction of mouse oocytes. The electron acceptors methylene blue, phenazine ethosulfate (PES), and pyrroline-5-carboxylate (P5C) oxidize NADPH to NADP and activate the NADP-dependent enzymes of the PPP. Each of these compounds triggered a dose-dependent increase in meiotic maturation in hypoxanthine-arrested cumulus cell-enclosed oocytes during 17- to 18-h cultures. More than 96% of the oocytes underwent germinal vesicle breakdown (GVB) at the highest concentrations of P5C and PES tested (250 and 1 microM, respectively) as compared to only 45-52% of control oocytes. P5C was also stimulatory to denuded oocytes. Analysis of energy substrates in microdrop cultures revealed a 3.6-fold increase in glucose consumption by PES-treated oocyte-cumulus cell complexes that was associated with stimulation of GVB. On the other hand, 2-deoxyglucose, which interferes with glucose utilization, prevented the induction of maturation brought about by P5C. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase, prevented meiotic maturation in the presence or absence of FSH. Gonadotropin-induced maturation was also prevented by 6-aminonicotinamide (6-AN) and dehydroepiandrosterone (DHEA), inhibitors of the two NADP-dependent enzymes of the PPP, and this was accompanied by suppression of glucose consumption. Phosphoribosyl-pyrophosphate (PRPP) is an important compound required in purine metabolism and can be formed from the end product of the oxidative arm of the PPP, ribose-5-phosphate. Ribose, which can be metabolized to PRPP, increased PRPP synthesis in complexes and induced meiotic maturation when added to hypoxanthine-arrested cumulus cell-enclosed oocytes in glucose-free medium in both the presence and absence of FSH. PRPP levels within complexes were also increased by glucose and FSH, but were reduced by hypoxanthine, 6-AN, and DHEA. In addition, exogenous PRPP stimulated maturation in hypoxanthine-arrested oocytes. These results support the proposition that glucose metabolism through the PPP is important in the meiotic induction mechanism and may involve the generation of PRPP that acts, at least in part, through the purine metabolizing pathways.

Animals↗
PubMed1998

In vitro maturation and fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of oocyte quality and type of serum.

In Experiment 1, to determine the developmental potential of buffalo oocytes of different qualities, compact cumulus-oocyte complexes (COCs) with an unexpanded cumulus mass, and with homogeneous ooplasm were classified as Grade 1 (with > or =5 layers of cumulus cells) and Grade 2 less than 4 layers of cumulus cells). Grade-3 oocytes were either without cumulus cells or with expanded cumulus mass, and with irregular ooplasm. The oocytes were matured for 24 h at 38.5 degrees C, 5% CO2 in air in maturation medium (10% fetal bovine serum (FBS) in TCM-199 supplemented with 5 microg mL(-1) follicle stimulating hormone-P). The nuclear maturation and cleavage rates, and the proportion of cleaved embryos which developed to morula and blastocyst stage were in the order Grade 1>Grade 2>Grade 3 (P < 0.05). For Experiment 2, the maturation medium consisted of TCM-199 supplemented with one of the following sera at 10% concentration: (1) buffalo oestrus serum (BOS), (2) superovulated buffalo serum (SBS), (3) fetal bovine serum (FBS) and (4) steer serum (SS). After in vitro fertilization (IVF), the oocytes were co-cultured with buffalo oviductal epithelial cells in TCM-199 containing the respective sera at 10% concentration for the subsequent 9 days. The extent of cumulus expansion and nuclear maturation were not different among different groups. The cleavage rates were lower (P < 0.05) with FBS than with BOS, SBS and SS. The proportion of cleaved embryos which developed to blastocyst stage was higher (P < 0.05) with SBS than with BOS, FBS and SS.

Animals↗
PubMed1997

Involvement of purine nucleotide synthetic pathways in gonadotropin-induced meiotic maturation in mouse cumulus cell-enclosed oocytes.

This study was carried out to test the hypothesis that purine nucleotide-generating pathways are required for ligand-stimulated oocyte maturation in meiotically arrested cumulus cell-enclosed oocytes. Oocytes from hormonally primed, immature mice were cultured overnight in Eagle's minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (to maintain meiotic arrest), plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these conditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, or adenosine were included during the first 3 hr. Furthermore, preincubation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, could not mimic this protective effect of hypoxanthine. Azaserine and aminopterin, inhibitors of purine de novo synthesis, blocked hormone-triggered maturation in dbcAMP-arrested oocytes, but had little effect on hypoxanthine-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that can be taken up by cells and phosphorylated to form AICAR, which can enter the purine de novo pathway at a point distal to the sites of azaserine inhibition. FSH was stimulatory to purine de novo synthesis, while azaserine, aminopterin, hypoxanthine, and AICA riboside all suppressed de novo synthesis in the presence or absence of FSH, with dbcAMP having no effect. HPLC analysis of 14C-hypoxanthine metabolism in oocyte-cumulus cell complexes revealed that changes in the pattern of purine metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathways are vital participants in the mechanism(s) regulating hormone-induced meiotic maturation, and that either the de novo or salvage pathway can fulfill this nucleotide requirement.

Aminoimidazole Carboxamide↗
PubMed2005

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation.

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.

Animals↗
PubMed2004

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes.

Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the <1 and 1-1.9 mm follicles, but the GV0 pattern disappeared completely in the 2-2.9 and 3-6 mm follicles. As follicles grew, the number of oocytes with GV1 configurations increased and reached a maximum in the preovulatory follicles 4 hr post-hCG injection. During maturation in vivo, the number of GV1 oocytes decreased while oocytes undergoing GVBD increased. The percentage of oocytes with GV3 and GV4 configurations was constant during oocyte growth except at the 2-2.9 mm follicle stage, but these configurations disappeared completely after hCG injection. On the contrary, the in vitro maturing oocytes showed a large proportion of GV3 and GV4 configurations. There was no significant difference in distribution of chromatin configurations between the nonatretic and atretic follicles, and between oocytes with more than two layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.

Animals↗
PubMed1991

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes.

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation.

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.

Animals↗
PubMed1997

Culture conditions affect meiotic regulation in cumulus cell-enclosed mouse oocytes.

To test the hypothesis that culture conditions influence meiotic regulation in mouse oocytes, we have examined the effects of six culture media, four organic buffers, and pH on spontaneous maturation, the maintenance of meiotic arrest and ligand-induced maturation in cumulus cell-enclosed oocytes from hormonally primed immature mice. The media tested were Eagle's minimum essential medium (MEM), Ham's F-10 (F-10), M199, M16, Waymouth's MB 752/1 (MB 752/1), and Leibovitz's L-15 (L-15). All six media supported > or = 94% spontaneous germinal vesicle breakdown (GVB) during a 17-18 hr incubation period, but polar body formation was lower in M199 and MB 752/1 than in the other media. The incidence of polar bodies could be increased in these two media by the addition of pyruvate. With the exception of M16 and MB 752/1, 4 mM hypoxanthine maintained a significant number of cumulus cell-enclosed oocytes in meiotic arrest. Inhibition could be restored by the addition of glutamine to M16 and pyruvate to MB 752/1. Follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) stimulated GVB in those media in which hypoxanthine was inhibitory. dbcAMP was able to maintain meiotic arrest in all of the media, but was least effective in M16. FSH stimulated GVB in all dbcAMP-arrested groups except L-15, and FSH became stimulatory in L-15 when the pyruvate level was reduced to 0.23 mM and galactose was replaced with 5.5 mM glucose. When MEM was buffered principally with the organic buffers MOPS, HEPES, DIPSO, or PIPES (at 20 mM), high frequencies of GVB and polar body formation were observed in inhibitor-free medium. dbcAMP suppressed GVB in all groups; hypoxanthine also maintained meiotic arrest in all buffering conditions, although this effect was nominal in PIPES-buffered medium. FSH and EGF stimulated GVB in all dbcAMP- and hypoxanthine-treated groups. When the concentration of HEPES was increased from 20 mM to 25 mM, a more pronounced suppressive effect on maturation in both dbcAMP- and hypoxanthine-supplemented groups was observed in the absence of FSH. But whereas HEPES reduced the induction of maturation by FSH in dbcAMP-arrested oocytes, this buffer had no effect on FSH action in hypoxanthine-treated oocytes. When MEM was buffered with HEPES and the pH was adjusted to 6.8, 7.0, 7.2, or 7.4, a dramatic effect of pH on meiotic maturation was observed. pH had no significant effect on hypoxanthine salvage by oocyte-cumulus cell complexes, but FSH-induced de novo purine synthesis was significantly augmented by increased pH, in parallel with increased induction of GVB. The results of this study demonstrate that the use of different culture media, or minor changes in culture conditions, can lead to significant variation in (1) the spontaneous maturation of oocytes, (2) the ability of meiotic inhibitors to suppress GVB, or (3) the efficacy of meiosis-inducing ligands. Furthermore, such observations provide a unique opportunity to examine specific molecules and metabolic pathways that can account for this variation and thereby gain valuable insights into the mechanisms involved in meiotic regulation.

Animals↗
PubMed2004

Cumulus-oocyte complex interactions during oocyte maturation.

In most mammals, the oocyte in the Graafian follicle is surrounded by tightly packed layers of cumulus cells, forming the cumulus-oocyte complex. During the preovulatory period, cumulus cells change from a compact cell mass into a dispersed structure of cells for the synthesis and deposition of a mucoid intercellular matrix, a process referred to as cumulus expansion. Cumulus expansion is thought to influence a variety of fundamental developmental changes during oocyte maturation. Volumetric expansion of the cumulus-oocyte complex correlates, at least in pig, with the outcome of oocyte maturation, fertilization, and embryo development. Therefore, detailed functional studies of cumulus expansion seem to be required to elucidate the mechanism of oocyte maturation. We summarize the current knowledge about (1) morphological changes of cumulus-oocyte complexes during oocyte maturation, (2) follicle factors inducing cumulus expansion, (3) the role of cumulus expansion in oocyte maturation, (4) cytoplasmic regulators of oocyte maturation, and (5) possible roles of cumulus expansion.

Animals↗
PubMed2004

Specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha-trypsin inhibitor to hyaluronan: implications for the assembly of the cumulus extracellular matrix.

The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.

Alpha-Globulins↗
PubMed2003

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro.

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.

Alanine Transaminase↗
PubMed1997

Oocyte control of granulosa cell development: how and why.

Interaction between oocytes and granulosa cells is complex and involves both gap junctions and paracrine signalling factors. Oocyte development in antral follicles is highly dependent on communication with cumulus cells, a subset of granulosa cells that is intimately associated with oocytes. Cumulus cells express characteristics distinct from the mural granulosa cells of preovulatory follicles. The thesis of this paper is that, without the influence of oocytes, the pathway of granulosa cell differentiation in antral follicles leads to the establishment of the mural granulosa cell phenotype. Oocytes in antral follicles abrogate that pathway of granulosa cell differentiation and promote the development of the cumulus cell phenotype. Oocytes may do this in order to control their own microenvironment by regulating differentiation of the supporting cells that are in direct communication with them. Possibly, some aspects of the mural granulosa cell phenotype are antagonistic to, or insufficient for, supporting the final stages of oocyte development. We present evidence that oocytes control their environment by suppressing differentiation of the mural granulosa cell phenotype and promoting differentiation of the cumulus cell phenotype. They achieve this suppression via the secretion of labile paracrine signalling factors. Errors in this regulatory mechanism, whether instigated by defects in the production of oocyte-derived ligands or granulosa cell responses to them, may result in the production of oocytes unable to undergo embryo development or that undergo abnormal follicular development.

Cell Differentiation↗
PubMed2002

In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report.

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.

Animals↗
PubMed1990

High-resolution localization of hyaluronic acid in the golden hamster oocyte-cumulus complex by use of a hyaluronidase-gold complex.

The distribution of hyaluronic acid in the oocyte-cumulus complexes collected from the oviduct ampulla of superovulated hamsters was revealed by use of hyaluronidase coupled to colloidal gold. On thin sections of Lowicryl-embedded oocyte-cumulus complexes, gold particles were associated specifically with interconnecting fibrillar materials that make up the cumulus matrix. Inside the cumulus cells, gold particles were found over the cisternal membrane of the rough endoplasmic reticulum, in the contents of lysosomes and multivesicular bodies, and over Golgi vesicles of some cumulus cells. A high concentration of gold labeling was observed over the peripheral condensed chromatin and perinucleolar components in the nucleus. The cell surface of the cumulus cells also appeared to be labeled. Gold particles, however, were absent over the mitochondria and lipid vacuoles. In the oocytes, labeling was found to be associated mainly with rough endoplasmic reticulum and arrays of lamellar structures; cortical granules, mitochondria, and coated vesicles were essentially devoid of gold particles. Gold particles were also seen along the plasma membrane of the oocytes and within the perivitelline space. The zona pellucida was not labeled by hyaluronidase-gold. Different control experiments confirmed the specificity of the labeling. Digestion of thin sections with hyaluronidase prior to incubation with hyaluronidase-gold abolished the initial reaction, whereas treatment of thin sections with chondroitinase did not prevent labeling of oocyte-cumulus complexes by hyaluronidase-gold. Although the function of hyaluronic acid in the oocyte-cumulus complex at the time of ovulation and fertilization is not known, the high concentration of this particular compound in the cumulus matrix and the cumulus cells and its specific locations in the perivitelline space and in the superovulated oocytes implicate the significance of its presence and warrant future investigations.

Animals↗
PubMed2002

Role of growth hormone and growth hormone receptor in oocyte maturation.

Near the completion of growth, mammalian oocytes acquire the competence to resume and complete meiosis. In vivo the preovulatory LH surge triggers the resumption of meiosis in the oocyte contained in preovulatory follicles. When immature oocytes and the surrounding cumulus cells are released from their follicular environment, resumption of meiosis is induced spontaneously. Culture of bovine cumulus oocyte complexes (COCs) obtained from antral follicles results in blastocyst formation following in vitro maturation, in vitro fertilisation and in vitro embryo culture. Addition of growth hormone (GH) to the maturation medium accelerates nuclear maturation of cumulus enclosed bovine oocytes, induces cumulus expansion and promotes early embryonic development following in vitro fertilisation. The effect of GH is exerted through the cumulus cells and not mediated by IGF-I. Cumulus cells and the oocyte express mRNA for GH receptor. Using specific inhibitors it has been shown that the effect of GH on oocyte maturation and cumulus expansion is mediated by the cyclic AMP signal transduction pathway. Within COCs both cumulus cells and oocyte show GH immunoreactivity while expression of GH mRNA is only found in the oocyte. These observations point to a paracrine and/or autocrine action of GH in oocyte maturation.

Animals↗
PubMed2006

[Effects of different donor cells and passages on the development of nuclear transfer porcine embryos].

This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5 microg/mL cytochalasin B. Fibroblast cells (FC), oviduct epithelial cells (OEC), granulosa cells (GC) and cumulus cells(CC) after 3-9 passages in TCM + 10% FBS were treated by serum starvation (0.5% FBS for 2-9 days), 0.1 microg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion (100 V/mm, 30 micros and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1 microg/mL APD + 0.5% FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC and FC, OEC pretreated with 0.1 microg/mL APD + 0.5% FBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection (P<0.05), but no difference was found in their proportion developing to blastocysts. 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype, which did not show significant difference among them (P>0.05). When GC at G3, G4, G5 and G6 of passages were used as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1 microg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is higher than that by microinjection, but there is no difference in the total efficiency between the two methods.

Adenine↗
PubMed2002

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo.

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.

Animals↗
PubMed2005

Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation.

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocyte in vitro maturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs during in vitro maturation has been investigated. Results indicate that exposure of cumulus-oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.

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