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PubMed1988

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes.

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.

Animals↗

PubMed1995

Differential regulation of oocyte maturation and cumulus expansion in the mouse oocyte-cumulus cell complex by site-selective analogs of cyclic adenosine monophosphate.

In the present study, we have examined how differential distribution of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase isozymes within the mouse oocyte-cumulus cell complex might influence the physiological response of the complex to cAMP, by determining the actions of site-selective cAMP analogs on oocyte maturation and cumulus expansion. Five different analogs of cAMP were utilized: 8-thiomethyl-cAMP and 8-bromo-cAMP, which bind to site 1 on the type II regulatory subunit (RII) of cAMP-dependent protein kinase A (PKA); 8-aminohexylamino-cAMP, which binds to site 1 on the type I regulatory subunit (RI) of PKA; N6-monobutyryl cAMP, which binds to site 2 on either RI or RII; and 8-piperidino-cAMP, which binds to either site 1 on RII or site 2 on RI. These analogs were tested alone or in paired combinations that synergistically activate either the type I or type II PKA isozyme. When tested alone, analogs that can bind to, and presumably activate, type I PKA were the most potent inhibitors of germinal vesicle breakdown (GVB) in both cumulus cell-enclosed and denuded oocytes. Consistent with this result was the finding that paired combinations of analogs that selectively activate type I PKA were also most effective in preventing GVB. On the other hand, pulsing meiotically arrested cumulus cell-enclosed oocytes with high concentrations of analogs that bind to PKA II, or with paired combinations of analogs that selectively activate type II PKA, led to induction of GVB; stimulation with analogs or combinations thereof that presumably stimulate type I PKA was less effective. Cumulus expansion in response to PKA stimulation showed similar selectivity in that type II PKA-stimulating treatments were considerably more effective in provoking expansion than type I PKA-stimulating treatments. 8-N3-[32P]cAMP photoaffinity labeling of PKA regulatory subunits revealed that only RI was present in oocyte extracts, while extracts from oocyte-cumulus cell complexes contained both RI and RII. These results support the hypothesis that type II PKA mediates cAMP-stimulated cumulus expansion and resumption of meiotic maturation, while direct elevation of type I PKA within the oocyte is instrumental in maintaining meiotic arrest.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

High performance liquid chromatography analysis of hypoxanthine metabolism in mouse oocyte-cumulus cell complexes: effects of purine metabolic perturbants.

This study was undertaken to examine the metabolism of hypoxanthine by mouse oocyte-cumulus cell complexes. Complexes were isolated from immature mice 48 h after priming with 5 IU eCG and culture for 3 h in medium containing 14C-hypoxanthine in the absence or presence of one of three metabolic inhibitors: alanosine, mycophenolic acid, or 6-mercaptopurine. Tissue extracts from complexes were analyzed by HPLC using either a C18 reversed-phase column (for separation of purine bases and nucleosides) or an ion exchange column (for separation of nucleotides). Most of the hypoxanthine taken up by complexes was salvaged to inosine monophosphate (IMP) and then converted to nucleotides. Metabolism favored the synthesis of adenyl nucleotides over guanyl nucleotides. No evidence of metabolism to uric acid via xanthine oxidase was encountered, and metabolism to inosine via purine nucleoside phosphorylase was negligible. A similar pattern of hypoxanthine metabolism was observed in extracts of oocytes that had been denuded after the culture period. Addition of alanosine to the culture medium significantly reduced the synthesis of adenyl nucleotides in complexes and partially shunted metabolism in the direction of guanyl nucleotides. However, neither alanosine nor another inhibitor of adenylosuccinate synthetase, hadacidin, significantly influenced the meiotic arrest maintained by hypoxanthine. Mycophenolic acid eliminated conversion of IMP to guanyl nucleotides but did not appreciably affect metabolism to other nucleotides. 6-Mercaptopurine produced an increase in the hypoxanthine-containing peaks, which was consistent with suppression of purine salvage. These results demonstrate that hypoxanthine is readily salvaged by the murine oocyte-cumulus cell complex and that the inhibitor-induced changes in metabolism are consistent with the presumed mechanism of action of each inhibitor. In addition, whereas metabolism favors conversion of IMP to adenyl nucleotides, synthesis of adenyl nucleotides by this route during the culture period is apparently not required for hypoxanthine-maintained meiotic arrest in vitro.

Adenylosuccinate Synthase↗

PubMed2003

Physiological changes in oocyte-cumulus cell complexes from diabetic mice that potentially influence meiotic regulation.

We have previously shown that the type I diabetic condition significantly alters meiotic regulation in mouse oocytes. In the present study, possible physiological deficiencies underlying such meiotic dysfunction were examined in oocyte-cumulus cell complexes (OCC) from type I diabetic mice. Whereas the diabetic condition did not affect glycolysis or the tricarboxylic acid cycle, the increased flux of glucose through the pentose phosphate pathway in response to FSH treatment was suppressed. De novo purine synthesis was also compromised, and ATP levels were reduced in freshly isolated OCC. Additionally, diabetes resulted in a reduction in FSH-mediated cAMP synthesis. The responsiveness of the oocyte to cAMP was also affected; fewer oocytes were induced to resume maturation after a stimulatory pulse with cAMP analogs. Meiotic induction triggered by FSH was significantly reduced, but that stimulated by phorbol ester or epidermal growth factor was affected to a much lesser extent. In addition to metabolic deficiencies, the cell-cell communication between the oocyte and the cumulus cells was reduced in diabetic mice as determined by coupling assays. Thus, numerous physiological parameters are affected by type I diabetes, and these changes may collectively contribute to altered meiotic regulation.

Animals↗

PubMed2005

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione.

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.

Animals↗

PubMed2000

Developmental regulation of effect of epidermal growth factor on porcine oocyte-cumulus cell complexes: nuclear maturation, expansion, and F-actin remodeling.

Epidermal growth factor (EGF) efficiently stimulates expansion of mouse and rat oocyte-cumulus complexes (OCC). Contradictory data have been published by several laboratories about the ability of EGF to stimulate expansion of porcine OCC. We assumed that these contradictions may have resulted from heterogeneous conditions used for isolation, culture, and assessment of OCC. The present experiments were designed to test the hypothesis that porcine OCC acquire the ability to synthesize hyaluronic acid (HA) and undergo expansion following EGF-stimulation gradually during the growth of follicles. For this reason, we isolated OCC from follicles of different sizes and assessed quantity of produced HA and proportions of expanding OCC after stimulation by EGF. In addition, we assessed in those OCC changes in morphology of cumulus cells and assembly of F-actin microfilaments, which are necessary for expansion to occur. Finally, nuclear maturation of EGF-stimulated OCC was assessed and its relationship with occurrence of expansion was evaluated. In all experiments, OCC stimulated with FSH were used as positive controls. The results showed that EGF did not stimulate production of HA, rearrangement of F-actin and expansion in OCC isolated from small follicles (<4 mm in diameter). OCC isolated from large preovulatory follicles (6-7 mm in diameter and PMSG-stimulated follicles) underwent efficient expansion when stimulated by EGF (93% and 100%, respectively). EGF dramatically stimulated total production of HA in these OCC and its retention in extracellular matrix of the expanding cumulus. Cumulus cells of the large OCC underwent essential changes of their morphology and extensive rearrangement of F-actin microfilaments following stimulation with EGF. Interestingly, EGF enhanced nuclear maturation of OCC isolated from both small and large follicles, which suggest diversity of signaling pathways controlling maturation and expansion. FSH caused cumulus expansion, F-actin remodeling, and enhancement of oocyte nuclear maturation in OCC originated from both small and large follicles. We conclude that EGF can stimulate expansion of porcine OCC in vitro; however, only of those isolated from large follicles. This indicates that EGF may have a physiological role in regulation of porcine cumulus expansion in preovulatory follicles, presumably as a mediator of signals elicited by the LH surge.

Actins↗

PubMed2004

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos.

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 degrees C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p < 0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p < 0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p > 0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p < 0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p > 0.05) among methods 1, 3 and 4, but method 2 produced a higher (p < 0.05) blastocyst rate than method 3.

Animals↗

PubMed1999

Production of steroids from human cumulus cells treated with different concentrations of gonadotropins during culture in vitro.

OBJECTIVE: To evaluate the output of E2 and progesterone produced by cumulus cells, derived from mature and immature oocytes, in culture medium. DESIGN: Prospective randomized study. SETTING: McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. PATIENT(S): Twenty-one women, <38 years of age and with normal menstrual cycles, who were undergoing intracytoplasmic sperm injection for assisted reproduction. INTERVENTION(S): Culture medium with or without fetal bovine serum (FBS) supplemented with either a physiologic (75 mIU/mL) or a supraphysiologic (7,500 mIU/mL) concentration of gonadotropins. MAIN OUTCOME MEASURE(S): Comparison of steroid levels in culture medium. RESULT(S): Estradiol secretion was significantly increased in the culture medium with FBS supplemented with both concentrations of FSH alone compared with control. However, E2 secretion was inhibited by both concentrations of FSH with LH. The level of E2 was undetectable in the medium without FBS even after supplementation with both concentrations of FSH alone, hCG alone, and FSH with LH. Progesterone production was increased in the medium with FBS supplemented with FSH alone, hCG alone, and FSH with LH compared with control. There was no difference in progesterone levels in the culture medium without FBS supplemented with both concentrations of FSH alone and hCG alone compared with control. However, progesterone secretion was increased in the medium without FBS supplemented with a physiologic concentration of FSH with LH. CONCLUSION(S): Culture medium with FBS supplemented with a physiologic and a supraphysiologic concentration of FSH stimulates E2 secretion from cumulus cells derived from mature and immature oocytes. This suggests that it may be not necessary to add E2 to the culture medium for maturation in vitro of immature human oocytes retrieved from patients undergoing stimulated cycles.

Adult↗

PubMed1996

Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.

Amino Acid Sequence↗

PubMed2001

Kinetics of acetyl coenzyme A: arylamine N-acetyltransferase from human cumulus cells.

PURPOSE: N-acetyltransferase (NAT) activity is involved in the detoxification of exogenous amines. We aimed to evaluate the kinetics of acetyl coenzyme A (AcCoA): arylamine NAT for human cumulus cells. METHODS: Thirty infertile women who were undergoing controlled ovarian hyperstimulation (COH) and oocyte retrievals were recruited. Human cumulus cells were obtained during oocyte retrievals. Using 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) as substrates, NAT activity and Michaelis-Menten kinetics constants of all samples were determined by using high-pressure liquid chromatography. RESULTS: There were 6 rapid, 10 intermediate, and 14 slow acetylators. 2-AF-NAT and PABA-NAT activities were 0.97 +/- 0.74 and 0.89 +/- 0.77 nmol/min/mg protein, respectively. Km/Vmax of rapid and slow acetylators for 2-AF were (161 +/- 55)/(15.6 +/- 2.9) and (27.8 +/- 11.4)/(2.6 +/- 0.9), respectively. Km/Vmax of rapid and slow acetylators for PABA were (104 +/- 36)/(13.2 +/- 2.8) versus (20.0 +/- 10)/(2.0 +/- 0.7), respectively. Compared to slow acetylators, the rapid acetylators exhibited higher Km/Vmax values for 2-AF (5.8-/6-fold) and PABA (6-/6.6-fold), respectively. CONCLUSION: Human cumulus could acetylate arylamine carcinogen (2-AF) and noncarcinogen drug (PABA). Higher percentage of rapid acetylators established in the cumulus during COH. It provides a model for monitoring the effects of pollution or carcinogenesis upon the oocyte during COH and oocyte retrievals.

4-Aminobenzoic Acid↗

PubMed2005

Apoptosis in rabbit embryos produced by fertilization or nuclear transfer with fibroblasts and cumulus cells.

In this study, we investigated the development, the cell number of the blastocyst, and apoptosis in rabbit nuclear transfer (NT) embryos derived from adult fibroblasts and cumulus cells as compared with embryos derived from in vivo fertilization and in vitro culture. The developmental rate and the total cell number of the blastocyst were significantly lower in NT embryos than in fertilized embryos (FEs). The type of donor cells did not affect the embryonic developmental rate and the total cell number of blastocysts in NT groups. The present study investigated the onset and the frequency of apoptosis in NT embryos and FEs by using a terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The earliest positive TUNEL signals were detected at the eight-cell stage in NT embryos and at the morula stage in FEs. The apoptotic index of the total blastocysts, the inner cell mass and the trophoderm was greatly higher in the NT embryos than in FEs. Moreover, the apoptotic index of the blastocyst from fibroblasts was significantly higher than that of the blastocyst from cumulus cells.

Animals↗

PubMed1999

Are cumulus cells necessary for the spontaneous maturation of germinal vesicle-stage oocytes to metaphase II.

PURPOSE: In this study we investigated the need of the support from cumulus cells for germinal-vesicle (GV) oocytes collected from stimulated ovaries to complete their maturation to metaphase II (MII). METHODS: We compared the maturation rate of GV oocytes after coculture with cumulus cells (study group) with their spontaneous maturation in culture medium alone (control group). RESULTS: Sixty-four and nine-tenths percent of the GV oocytes matured to metaphase II in the coculture group, and of these, 43.5% gave normal 2pn zygotes following intracytoplasmic sperm injection (ICSI), while 73.8% of the GV oocytes spontaneously matured to the MII stage and 30% of these reached the zygote stage after ICSI. CONCLUSIONS: It is probable that a follicular factor is responsible for this arrested maturation in the human and that maturation occurs spontaneously when the oocytes are separated from their follicular fluid environment after collection.

Cell Culture Techniques↗

PubMed1998

Use of a plant enzyme preparation (Coronase) instead of hyaluronidase for cumulus cell removal before intracytoplasmic sperm injection.

The aim of this study was to compare the efficiency of a plant enzyme preparation (Coronase) with animal extracted hyaluronidase to remove cumulus cells before intracytoplasmic sperm injection (ICSI). The first part of the study was performed on mouse oocytes and embryos. Coronase displayed a similar efficiency to that of hyaluronidase for removing cumulus cells and the same percentage of activated oocytes was obtained with both techniques. However, prolonged incubation in Coronase, 120 min, led to a degeneration of oocytes. Incubation of 2-cell mouse embryos for 10 min with Coronase did not affect their subsequent in-vitro development to blastocyst. Coronase was then compared to hyaluronidase in the treatment of human oocytes prior to ICSI. The time required for total denudation was slightly longer using Coronase (98 s +/- 25 s versus 84 s +/- 24 s respectively for Coronase and hyaluronidase; P < 0.01). However, the two pronuclear (2PN) fertilization rate (70/103 versus 63/107 respectively for Coronase and hyaluronidase, not significant) and the percentage of embryos with a good morphology (39/74 versus 32/67 respectively for Coronase and hyaluronidase, not significant) were identical with both treatments. In conclusion, Coronase displays an efficiency close to that of hyaluronidase, without any adverse effect on oocytes, and may be preferable for human use.

Animals↗

PubMed1990

Histochemical demonstration of a delta 5,3 beta-hydroxysteroid dehydrogenase activity of cumulus cells related to the maturity and developmental potential of recovered oocytes.

A simple and rapid histochemical technique is described for demonstration of delta 5,3 beta-hydroxysteroid dehydrogenase activity in cumulus cells from preovulatory follicles aspirated for in vitro fertilization (IVF) of corresponding oocytes. Histochemical activity of delta 5,3 beta-hydroxysteroid dehydrogenase was demonstrated in samples of cumulus obtained from 62 oocytes recovered from 24 women. Patients were treated with clomiphene citrate in combination with human menopausal gonadotropins and human chorionic gonadotropin injections. The cumulus was found to contain small and large cell types. Small cells possessed more delta 5,3 beta-hydroxysteroid dehydrogenase activity predominantly in the area near the oocyte. Cytoplasmic vacuolation has been noted in large, pale cells with moderate or low enzyme activity. The most active cells were predominant in cumulus from which oocytes were fertilized. Significant differences have been found between high and low delta 5,3 beta-hydroxysteroid dehydrogenase activity of cumulus cells from mature oocyte-corona-cumulus complexes leading to a successful fertilization and cleavage of oocytes and between groups with different histochemical activity when aspirated complexes were scored immature and the IVF of oocytes has failed.

3-Hydroxysteroid Dehydrogenases↗

PubMed1999

Metabolism of radiolabeled glucose by mouse oocytes and oocyte-cumulus cell complexes.

This study was carried out to examine the metabolism of [1-14C]-, [6-14C]-, and [5-3H]glucose by oocyte-cumulus cell complexes (OCC) and denuded oocytes (DO) and to test the hypothesis that metabolism of glucose through the pentose phosphate pathway is associated with meiotic induction. OCC or DO were cultured in hanging drops suspended from the cap of a microfuge tube, with NaOH serving as a trap to collect released 3H2O or 14CO2. Preliminary experiments established that this culture system supports both spontaneous and ligand-induced meiotic maturation. An initial time course experiment (1.5-6 h) showed that hypoxanthine-treated OCC from eCG-primed animals metabolized glucose principally via glycolysis, with an increase to 2.7-fold in response to FSH. Though more [1-14C]glucose was oxidized than [6-14C]glucose, its metabolism was about two orders of magnitude less than that of [5-3H]glucose. Also, FSH significantly increased oxidation of [1-14C]glucose but not [6-14C]glucose, indicating a preferential activation of the pentose phosphate pathway. Pyrroline carboxylate, an activator of the pentose phosphate pathway, increased the activity of this pathway to over 2-fold but failed to affect glucose oxidation through the tricarboxylic acid cycle. Glycolytic metabolism was increased by 25%. The addition of pyruvate to pyruvate-free medium resulted in significant reduction in the metabolism of all three glucose analogues. In OCC retrieved from hCG-injected, primed mice and cultured under hormone-free conditions, metabolic responses were similar to those in FSH-treated complexes cultured in hypoxanthine. DO metabolized glucose, but at a much reduced rate when compared to OCC. Pyruvate reduced the consumption of all three glucose analogues by DO. Pyrroline carboxylate reduced [5-3H]glucose metabolism by DO but had little effect on [1-14C]- and [6-14C]glucose oxidation. These data demonstrate metabolism of glucose by both DO and OCC, but reveal that cumulus cells are more active than the oocyte in this regard. In addition, induction of maturation by FSH, hCG, or pyrroline carboxylate was accompanied by a significant increase in the oxidation of [1-14C]glucose but not [6-14C]glucose by OCC, supporting a proposed role for the pentose phosphate pathway in meiotic induction.

Animals↗

PubMed2001

Partial characterization of the factor in theca-cell conditioned medium that inhibits the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to the membrana granulosa.

A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as compared to culture in CMt, but progression of meiosis was clearly inhibited as demonstrated by a significant higher proportion of the oocytes blocked in the M1 stage after resumption of meiosis. In general, with regard to meiotic inhibition, bFF showed the same pattern as CMt following the various treatments. It is concluded that the theca cell secreted factor which inhibits the FSH-induced resumption of meiosis in COCGs is a small, stable, polar molecule which is not a peptide.

Animals↗

PubMed2000

In vitro developmental potential of bovine nuclear transfer embryos derived from primary cultured cumulus cells.

The in vitro development and the quality of blastocysts produced from the nuclear transfer (NT) embryos reconstituted from primary cultured cumulus cells (NT-cumulus) were examined compared to in vitro fertilized embryos (IVF) and NT embryos reconstituted from the embryonic blastomeres (NT-blastomere). The cleavage rate, and the development to blastocyst were the same for all three sets of embryos. The time required for blastocoel formation starting from the time of the initial cleavage was shorter for NT embryo groups than IVF ones. All experimental groups produced morphologically similar and normal blastocysts containing the same cell number. The percentage of the blastocysts with normal chromosomal complements were the same for NT-cumulus and IVF.

Animals↗

PubMed2005

Cumulus cells accelerate aging of mouse oocytes.

The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.

Anaphase↗