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PubMed2000

Effect of hyaluronan on monospermic penetration of porcine oocytes fertilized in vitro.

An experimental series was carried out to examine the ability of exogenous hyaluronan (HA) to facilitate monospermic penetration of in vitro-matured (IVM) oocytes during conventional porcine in vitro fertilization (IVF). IVM oocytes were co-cultured in fertilization medium with frozen-thawed, ejaculated boar spermatozoa. Addition of 0.5 mg/mL HA to the fertilization medium increased the rates of monospermic penetration (p < 0.05) on cumulus-denuded oocytes, without detrimentally affecting penetration rates (rates of monospermy were 57% and 78% in 2 mM caffeine-containing fertilization medium without and with HA, respectively). Caffeine and cumulus cells affected the rate of monospermic fertilization (p < 0.02-0.03), and caffeine affected the rate at which oocytes were penetrated (p < 0.02). Moreover, exogenous HA (0.5 mg/mL) in the sperm pre-incubation medium promoted monospermic penetration rates without affecting overall penetration rates. The effects of exogenous HA on sperm penetration and monospermy were also compared among semen batches. After semen was pooled from three fertile boars and held in homologous seminal plasma (SP) for 3 (batch A), 10 (batch B) or 20 (batch C) h prior to being cooled, spermatozoa from each semen batch were co-cultured with IVM oocytes. Monospermy of cumulus-denuded oocytes was increased (p < 0.05) by the addition of exogenous HA within the same semen batch (batches B and C). Furthermore, co-culture with cumulus cells did not mask the effects of HA on batch-B semen. The concentration of exogenous HA (0, 0.05 and 0.5 mg/mL) during sperm-oocyte co-culture differentially affected the rates of penetration and monospermy of cumulus-intact oocytes, depending on the time spermatozoa were held in SP before being frozen. The highest penetration rate was obtained by exposing semen to SP for 20 h prior to being cooled (0.05 mg/mL HA). The results suggest that exogenous HA decreases polyspermy during conventional porcine IVF. Furthermore, it appears that the action of HA is dependent upon the priming action of seminal plasma.

Animals↗
PubMed1992

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.

Animals↗
PubMed1983

Factors affecting successful in vitro fertilization of bovine follicular oocytes.

These experiments were designed to define and optimize the efficiency of a system whereby bovine oocytes could be fertilized in vitro. The frequency of ova penetrated and the stage of fertilization were the end points examined. All experiments utilized cumulus-oocyte complexes from 1- to 5-mm follicles which were matured in vitro prior to fertilization. The experiments were designed to examine the effects of the following factors on fertilization: 1) pretreatment of sperm with ionomycin (a Ca++ ionophore), 2) preincubation of sperm at a high concentration and the presence of hypotaurine and epinephrine during fertilization, 3) the use of either follicle-stimulating hormone (FSH) or cAMP for the induction of cumulus expansion prior to fertilization, and 4) the need for the presence of cumulus cells during fertilization. Sperm exposure to ionomycin or preincubation at high sperm concentrations was not necessary for fertilization. The presence of hypotaurine and epinephrine during fertilization improved (P less than 0.05) the quality of fertilization (i.e., higher frequencies of oocytes with both female and male pronuclei were observed). However, they did not increase the percentage of ova penetrated (P greater than 0.05). Fertilization frequencies were not different (P greater than 0.05) between oocytes with cumulus expansion induced by FSH or cAMP. However, the use of either treatment resulted in higher fertilization rates when compared to untreated controls (P less than 0.05). Finally, while the presence of cumulus cells was not necessary for penetration of ova, increased frequencies of ova with both male and female pronuclei were found when cumuli were present (P less than 0.05).

Animals↗
PubMed2003

Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation.

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.

Acetylation↗
PubMed1998

Immunohistochemical evidence for the localization of bovine viral diarrhea virus, a single-stranded RNA virus, in ovarian oocytes in the cow.

Bovine viral diarrhea virus (BVDV) is a single-stranded RNA virus responsible for enteric disease and reproductive failure in cattle. The virus can pass vertically from cow to fetus, causing abortion, birth of malformed calves, and calves born with persistent and life-long infections. In this study, we investigated the tropism of BVDV in ovarian tissue from persistently infected animals. Three heifers persistently infected with BVDV were euthanatized and their ovaries were recovered. A specimen of each ovary was taken (n = 6) for virus isolation, and the remaining ovarian tissue was stored at -70 C. Cryosections (6 microm) cut from each ovary were analyzed for the presence of BVDV antigens by indirect immunofluorescence. The immunofluorescent analysis employed two monoclonal antibodies, WB103 and WB162, previously raised against the nonstructural protein NS3 and the envelop glycoprotein E2, respectively. High titers (6.97 +/- 0.17 log10 tissue culture infective dose50/ml) of BVDV were recovered from 6/6 ovarian samples; NS3 and E2 were widely distributed within the ovarian stroma, the cumulus cell population, and the oocytes maturing in primordial, primary, and secondary follicles. Overall, 362/1,939 (18.7%) of the oocytes contained BVDV antigens, and there was no significant (P > 0.05) difference in the proportion of BVDV-infected oocytes recorded within the primordial (227/1,247, 18.2%), primary (122/630, 19.4%), and secondary (13/62, 21.0%) follicle populations. Although the developmental potential of the infected oocytes could not be established in the present study, we conclude that bovine oocyte and the cumulus cells are susceptible to BVDV infection.

Animals↗
PubMed1985

Antagonistic actions of estradiol and tamoxifen upon forskolin-dependent meiotic arrest, intercellular coupling, and the cyclic AMP content of hamster oocyte-cumulus complexes.

The effects of estradiol (E2) and the anti-E2 tamoxifen (tam) on forskolin (F)-dependent meiotic arrest in hamster oocytes were investigated. The hypotheses tested were that 1) the arresting action of F is enhanced by E2 and suppressed by tam and 2) the extent of heterologous metabolic coupling and the concomitant transfer of cumulus cell cAMP into the oocyte is increased and decreased by E2 and tam, respectively. E2 was tested with the ID25 F (where ID25 is the dose of F previously shown to arrest the meiosis of 25% cultured oocytes; intact, 3 microM; denuded, 10 microM) and tam was tested with the ID75 F (intact, 10 microM; denuded, 100 microM). E2 induced reversible dose-dependent increases in the percent germinal vesicle (%GV; determined cytogenetically) of both intact and denuded oocytes in the presence of ID25 F (intact: ID50 E2 = 18.0 microM; denuded: ID50 E2 = 17.2 microM) but, in contrast to intact oocytes, E2 also exerted a dose-dependent action on denuded oocytes in the absence of F (ID50 = 26.1 microM). E2 induced dose-dependent increases in the cAMP content (determined by RIA) of intact oocytes (cAMP-oo) and of cumulus masses (cAMP-cm) and in the ratio of cAMPooo:cAMP-cm but failed to elevate F-stimulated cAMP in denuded oocytes. Heterologous metabolic coupling, as assessed by determination of the fraction of radiolabeled uridine marker that was transferred from the cumulus cells to the oocyte, was not significantly enhanced by E2. In contrast to denuded oocytes, tam induced dose-dependent decreases in the %GV and cAMP content of intact oocytes in the presence of ID75 F and significantly depressed heterologous metabolic coupling. While tam failed to antagonize the E2 action on denuded oocytes in the presence of ID25 F, in intact oocytes cultured with E2 and the ID25 F, the anti-E2 significantly decreased the %GV, the cAMP-oo and cAMP-cm, and the extent of heterologous metabolic coupling. These data show that while E2 can directly arrest the maturation of denuded hamster oocytes with no associated elevation of cAMP-oo, E2-enhancement of arrest in intact oocytes is correlated with both elevation of cAMP within the oocyte-cumulus complex and maintenance of heterologous metabolic coupling and is accompanied by an increase in cAMP-oo.

Animals↗
PubMed1998

Mouse oocytes regulate granulosa cell steroidogenesis throughout follicular development.

Mouse oocytes secrete a factor(s) that inhibits progesterone and enhances estradiol production by granulosa cells. This study determined the ability of mouse oocytes to secrete this steroid-regulating factor during oocyte growth and the ability of granulosa cells to respond to the factor during follicular development. Oocyte-granulosa cell complexes from preantral and antral follicles were oocytectomized (OOX; oocytes microsurgically removed) and cultured for up to 48 h with FSH (150 ng/ml) and testosterone (500 nM). At all stages of development examined, OOX complexes produced more progesterone than did intact complexes, from 1.45-fold for early growing follicles to 23-fold for complexes from antral follicles. Significant estradiol production was restricted to intact complexes from late antral follicles. Progesterone accumulation by OOX complexes cocultured with oocytes was inhibited by all stages of oocytes examined, with maximal inhibition by fully grown oocytes. Ovulated complexes produced large quantities of progesterone, even though oocytes secreted progesterone-inhibitory factor, because of a desensitization of cumulus cells to the factor during their terminal differentiation. Even in the presence of abundant pregnenolone, OOX complexes showed reduced ability to produce and/or accumulate progesterone in the presence of oocytes, suggesting that the oocyte-secreted factor, either directly or indirectly, regulates the activity of 3beta-hydroxysteroid dehydrogenase and/or progesterone metabolism. These results demonstrate that oocytes secrete a factor with steroid-regulating activity in increasing amounts and/or potency during follicular development, but responsiveness of cumulus cells to this factor declines during luteinization.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Spermatozoa of the shrew, Suncus murinus, undergo the acrosome reaction and then selectively kill cells in penetrating the cumulus oophorus.

In the musk shrew, Suncus murinus (and other shrews), the cumulus oophorus is ovulated as a discrete, compact, matrix-free ball of cells linked by specialized junctions. In examining how they penetrate the cumulus, Suncus spermatozoa were observed to first bind consistently by the ventral face over the acrosomal region to the exposed smooth surface of a peripheral cumulus cell. This was apparently followed by point fusions between the plasma and outer acrosomal membranes. Thereafter, spermatozoa without acrosomes were observed within cumulus cells that displayed signs of necrosis, as did some radially neighboring cumulus cells linked by zona adherens and gap junctions. Eventually, penetration of spermatozoa as far as the perizonal space around the zona pellucida left linear tracks of locally necrotic cells flanked by normal cumulus cells. Based on these and previous observations, we conclude that the acrosome reaction in Suncus is always induced by cumulus cells, and that reacted spermatozoa penetrate the cumulus by selective invasion and killing of cumulus cells along a linear track. Loss of the acrosome also exposes an apical body/perforatorium that is covered with barbs that appear to assist reacted fertilizing spermatozoa in binding to the zona pellucida. Because fertilized eggs displayed no other spermatozoa within or bound to the zona, an efficient block to polyspermy must prevent such binding of additional spermatozoa.

Acrosome Reaction↗
PubMed1994

Induction of maturation of cumulus-oocyte complex by gonadotropin-releasing hormone analog is associated with lower progesterone secretion.

Induction of ovulation by CG in women subjected to in vitro fertilization and embryo transfer results in maturation of the cumulus-oocyte complex (COC) in terms of oocyte meiotic maturation, cumulus mucification, and an increase in progesterone (P4) secretion. Recently an alternative approach, in which the ovulatory processes are induced by the administration of GnRH analog (GnRHa), has resulted in COCs yielding viable embryos. In the present study the effect of GnRHa administration on oocyte meiotic maturation and cumulus cell steroidogenesis was evaluated in 27 women undergoing ovarian stimulation with gonadotropin for the purpose of in vitro fertilization and embryo transfer. In GnRHa- or CG-treated women, 79.5 +/- 4.2% and 72.0 +/- 8.4% of the oocytes, respectively, manifested the first polar body. The percentages of atretic oocytes and of oocytes failing to resume meiosis were 5.8 +/- 2.1% and 6.0 +/- 2.4%, respectively, in GnRHa-treated women, and were 9.3 +/- 4.7% and 7.9 +/- 4.1%, respectively, in CG-treated women. P4 was secreted in high quantity in human cumulus cells (CCs) during a 7-day culture period. However, in CCs collected from GnRHa-treated women, P4 secretion was more than 50% less than in CCs of CG-treated women (P < 0.005). Addition of 20 alpha-hydroxycholesterol significantly increased P4 secretion in CCs collected from GnRHa-treated women, similar to P4 secretion in CCs collected from CG-treated women. This study evaluated for the first time the effectiveness of GnRHa administration on COC maturation. It seems that although oocyte meiotic maturation in GnRHa-treated women proceeds as in CG-stimulated women, CC steroidogenic activity is marked by cholesterol deficiency or availability.

Adult↗
PubMed2006

The effect of hyaluronan concentrations in hST-supplemented TCM 199 on in vitro nuclear maturation of bitch cumulus-oocyte complexes.

In this study, the effect of hyaluronan (HA) on in vitro nuclear maturation of bitch oocytes was evaluated. Cumulus-oocyte complexes (COCs) were cultured for 48 h at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes with one or more layers of intact cumulus cells and dark cytoplasm were allocated to the following treatments: (A) TCM 199 supplemented with 25 mM Hepes/L (v/v), with 10% heat inactivated estrous cow serum (ECS), 50 microg/mL gentamycin, 2.2 mg/mL sodium bicarbonate, 22 microg/mL pyruvic acid, 20 microg/mL estradiol, 0.5 microg/mL FSH, 0.03 IU/mL hCG and 1 microg/mL human somatotropin (hST) (control medium), (B) Treatment A + 0.5 mg/mL HA and (C) Treatment A + 1.0 mg/mL HA. Supplementation with HA did not increase the number of oocytes that resumed meiosis. Additionally, there were no differences among treatments in the cumulus cell expansion of oocytes. In conclusion, the addition of HA to hST-supplemented TCM 199 did not improve the in vitro nuclear maturation of bitch oocytes, and therefore appeared to be unsatisfactory as supplement for in vitro maturation (IVM) of canine oocytes.

Animals↗
PubMed2001

Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised.

Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.

Aging↗
PubMed1985

Effect of forskolin on the spontaneous maturation and cyclic AMP content of hamster oocyte-cumulus complexes.

Forskolin, a reversible stimulator of the catalytic subunit of adenylate cyclase, has been used to determine: whether an increase in hamster cumulus cell cyclic adenosine monophosphate (cAMP) results in an elevation of intraoocyte cAMP and an accompanying increase in the maintenance of meiotic arrest (%GV where GV is germinal vesicle) when heterologous coupling is maintained, whether the hamster oolemma possesses the catalytic subunit of adenylate cyclase in an amount adequate to stimulate sufficient cAMP synthesis to maintain arrest, and whether release from meiotic arrest is accompanied by a decrease in the content of intraoocyte cAMP. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. While the %GV of both cumulus-enclosed (intact) and cumulus-free (denuded) oocytes was dose-dependent upon forskolin, that of intact oocytes was much more sensitive to the drug (intact: ID50 3.4 microM; denuded: ID50 65.0 microM, where ID50 is the dose of forskolin that inhibits the maturation of 50% of cultured oocytes). Forskolin stimulated a significant, dose-dependent increase in the amount of cAMP within the cumulus mass [(r) = 0.789, P less than 0.001)], the intact oocyte [(r) = 0.715, P less than 0.001], and the denuded oocyte [(r) = 0.673, P less than 0.01)]. The cAMP content of intact oocytes was significantly greater than that of denuded oocytes above 6.25 microM forskolin (25 microM forskolin: 9.28 +/- 1.01 vs. 3.98 +/- 0.15 fmol cAMP, intact and denuded oocytes, respectively; P less than 0.001, paired t test). A highly significant positive correlation was established between the amount of cAMP in groups of cumulus masses and that in the corresponding enclosed oocytes [(r) = 0.635, P less than 0.001]. The enhanced sensitivity of meiotic arrest in intact, as compared to denuded, oocytes was due to the presence of adherent cumulus cells but was not attributable to a significant increase in the cAMP content of intact oocytes (at 6.25 microM forskolin; %GV intact = 73.0 +/- 10.7, denuded = 20.3 +/- 7.4; fmol cAMP intact = 5.02 +/- 1.50; denuded = 4.63 +/- 0.81). The arresting action of forskolin on intact oocytes was transient and fully reversible, but release from arrest was not accompanied by a decrease in either intraoocyte cAMP or heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Survival rate of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants.

The relationship between cryoprotectant and exposure time on the development of IVF-derived embryos in culture after freezing and thawing was examined. Good to excellent quality Day 7 to 8 blastocysts and expanded blastocysts were suspended in 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol, and 1.3 M ethylene glycol monomethyl ether (EME). In Experiment I, after 30 and 120 min of exposure, embryos were placed directly into culture medium and washed three times. Embryos were cocultured with cumulus cells in culture medium. After a 48-h culture, there were no significant differences in fully expanded, hatching, and hatched rates between the 30- and 120-min exposure to all four cryoprotectants except for the fully expanded rate with PG (55 vs 83%). In Experiment II, to compare the cryoprotective effects of the four cryoprotectants, embryos were exposed to each cryoprotectant for variable times from 10 to 120 min, seeded, and cooled to -30 degrees C at 0.3 degree C/min, plunged into liquid nitrogen for storage, and then thawed. Survival was measured by coculturing the embryos with cumulus cells in culture medium. There were no significant differences in development to hatched blastocysts of embryos exposed to either PG (27-42%) or EG (37-56%). The number of embryos developing after exposure to EME was similar to that in PG or EG when exposure time did not exceed 40 min (43-49%). Our findings suggest that the toxicity of different cryoprotectants is related to the exposure time and that PG and EG were relatively nontoxic as is exposure to EME for short periods.

Animals↗
PubMed1994

Completion of first meiosis by sperm penetration in vitro of bovine oocytes inhibited at metaphase-I with dimethylsulphoxide.

The effects of dimethylsulphoxide (DMSO) on immature oocytes during maturation in culture and following penetration by spermatozoa were examined. Germinal vesicle breakdown (GVBD) was observed in all oocytes cultured in the maturation medium supplemented with 2, 4 and 8% DMSO. When the oocytes were cultured in medium with 8% DMSO, 95% (57 60 ) of them were inhibited at prometaphase-I. Cumulus cells were significantly (P<0.05) beneficial for resumption of oocyte nuclear maturation during further culture in the maturation medium for 4, 8 and 24 h after DMSO treatment. When the oocytes were additionally cultured for 4 and 8 h in the maturation medium after DMSO treatment, the proportions of oocytes reaching metaphase-II were significantly (P<0.05) higher in those cultured with spermatozoa than without (68 vs 49% and 84 vs 56%, respectively). These results indicate that 8% DMSO does not affect GVBD of oocytes, but conversely it inhibits oocytes at prometaphase-I, and that cumulus cells are important for recovery from DMSO inhibition and for the resumption of nuclear maturation of oocytes. Sperm penetration was also found to stimulate the completion of meiotic maturation of oocytes inhibited at metaphase-I with 8% DMSO.

Journal Article↗
PubMed2002

Survival of oocytes recovered from vitrified sheep ovarian tissues.

The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P<0.05) oocytes obtained from vitrified ovarian tissues (70%) reached metaphase II compared to vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3-5 layers of cumulus cells were considered from each of the three groups, no differences (P>0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.

Animals↗
PubMed1994

Effects of transforming growth factors and activin-A on in vitro porcine oocyte maturation.

Growth factors are known to regulate ovarian function. In the present study, effects of these growth factors, TGF-alpha, TGF-beta, and activin-A were tested on spontaneous porcine oocyte maturation. Cumulus-oocyte complexes (COC) were cultured in the presence of TGF-alpha, TGF-beta, and activin-A for 48 hr. Stages of meiotic maturation were assessed by staining with acetic orcein. Among these factors, only TGF-alpha significantly enhanced the maturation rate, whereas TGF-beta suppressed the spontaneous maturation rate. The site of action of TGF-alpha on COC and the interaction between TGF-alpha and EGF receptor was also examined. Denuded oocytes, alone or in coculture with cumulus cells, were cultured in the presence of TGF-alpha for 48 hr. TGF-alpha did not have any significant effect on denuded oocyte maturation. Heptanol was employed to investigate the role of gap junctions on TGF-alpha-induced oocyte maturation in COC. Although heptanol did not have any significant effect in the control medium, heptanol reversed the stimulatory effect of TGF-alpha on porcine oocyte maturation. TGF-alpha was able to displace 125I-EGF binding on COC. In conclusion, TGF-alpha enhances the spontaneous maturation of porcine oocytes by generating positive signal(s) in cumulus cells that are transferred to the oocyte via gap junctions. TGF-alpha shares the same receptor with EGF on porcine COC. TGF-beta, in contrast, inhibits porcine oocyte maturation.

Activins↗
PubMed1985

Factors affecting human in vitro fertilization: a multifactorial study.

The influence of four factors on the cleavage rate of 705 mature oocytes submitted for in vitro fertilization (IVF) was assessed with the use of a multifactorial method, i.e., logistic regression. The studied factors were (1) the regimen for cycle stimulation, (2) cumulus cell mass appearance, (3) semen quality, and (4) the time of incubation of oocytes before insemination. The logistic function permitted testing the respective influence of each factor on the cleavage rate, with the level of the other factors taken into account. The most important factor was the stimulation treatment, the association of human menopausal gonadotropin with clomiphene citrate (CC) giving better results than CC alone. Time of incubation was demonstrated to have no influence on the cleavage rate, whereas semen quality had an influence. A problem was raised by the existence of an interaction between the stimulation treatment used and the appearance of the cumulus cell mass. It can be concluded that adequate cycle treatment and eventually couple selection with respect to the semen quality of the male partner are to be considered in view of obtaining better results in IVF attempts.

Adult↗
PubMed2005

Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes.

This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17-18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.

Acetyl-CoA Carboxylase↗