Differential response of cumulus cell-enclosed and denuded mouse oocytes in a meiotic induction model system

In this study we have examined the effects of denuded oocyte coculture with dissociated cumulus cells (CC) or intact oocyte-CC complexes on meiotic resumption. When denuded oocytes (DO) or cumulus cell-enclosed oocytes (CEO) were cultured in 40-microl drops of medium under oil, and held in meiotic arrest with 4 mM hypoxanthine plus 25 microM dbcAMP, they underwent germinal vesicle breakdown (GVB) at similar frequencies (34%-35%). Coculture of DO with complexes or dissociated CCs stimulated maturation (50% and 61% GVB, respectively), with no effect of DO on maturation of cocultured CEO (32% GVB). This coculture effect was increased with the number of CCs added to the culture drop. When either glucose or glutamine was eliminated from the medium, no meiotic induction resulted from cocultured CCs. When CEO were cultured alone in microdrops, increasing their number from 10 to 50 significantly lowered the percentage resuming maturation, an effect also reduced by removing glucose and/or glutamine from the medium. This effect was not observed with DO. When inhibitory medium was conditioned overnight with complexes, subsequent culture with DO led to higher maturation percentages than culture in unconditioned medium; however, when CEO were cultured in conditioned medium, there was either no effect or increased inhibition of maturation. Assay of glucose and pyruvate in spent medium showed that DO cultured alone consumed glucose and pyruvate, but under CC coculture conditions more glucose was consumed and significant amounts of pyruvate accumulated in the medium, changes that led to an increase in the maturation of DO. Further experiments showed that DO were more sensitive than CEO to the meiosis-inducing effect of pyruvate. These results demonstrate different responsiveness of DO and CEO to coculture conditions and question the physiological relevance of denuded oocyte/CC coculture to study meiotic induction.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes.

In this study, the effect of different energy sources used in Eagle's minimum essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5.5 mmol 1(-1)), pyruvate (0.23 mmol 1(-1)) and glutamine (2 mmol 1(-1)) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 mumol dibutyryl cAMP 1(-1) during 17-18 h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99-100% germinal vesicle breakdown); all other combinations resulted in < or = 54% germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (< or = 10% GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (> or = 86% viability in pyruvate-containing medium; < or = 35% viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (> or = 89% germinal vesicle breakdown) and viability (> or = 91%). The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5-33% germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon D-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon D-glucose. Uptake and metabolism of D-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of D-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of D-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) D-mannose, a glycolysable sugar, was as effective as D-glucose in supporting the ligand effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 microM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocytecumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 microM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.     

Hypoxanthine (HX) inhibition of in vitro meiotic resumption in goat oocytes

To improve in vitro maturation and to understand the mechanism for meiotic resumption of oocytes, meiotic progression, and its control by hypoxanthine (HX) were studied in goat oocytes. Ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) and follicular fluid were collected from follicles of different surface diameters (SDs). The meiotic competence and progression of oocytes were observed, and the concentration of HX in the follicular fluid and culture media was measured by high-performance liquid chromatography (HPLC). Full meiotic competence of goat oocytes was acquired in follicles of >/=1.5 mm in SD with 90% of the oocytes developing to metaphase II (MII) stage after 24 hr in culture. The HX concentration in follicular fluid decreased with follicle development, from the highest level of 1.16 mM in /=5 mm follicles. HX inhibited meiotic resumption of goat oocytes in a concentration-related manner but this inhibitory effect declined gradually. When we renewed the medium at 4 hr of HX-199 (TCM-199 supplemented with 4 mM HX) culture, the percentage of oocytes with intact germinal vesicle (GV) did not increase but decreased significantly instead. HPLC measurement of HX in the HX-199 culture drops indicated that the HX concentration declined from 0 hr to 4 hr of culture and after medium renewal at 4 hr of culture. By adding dibutyryl cAMP (db-cAMP) at medium renewal, we found that db-cAMP held up the decline of GV percentages. Together, these results were consistent with the possibility that the decline of HX inhibitory effect was not due to HX depletion but rather due to the negative feedback of the metabolites on its further uptake by oocytes. Goat oocytes were capable of normal nuclear maturation and activation after temporal arrest by HX, but prolonged exposure to HX induced spontaneous activation.     

Changes in protein synthesis pattern during in vitro maturation of goat oocytes.

The regulation of meiotic events of goat oocytes from prophase I to metaphase II was studied by inhibiting protein synthesis at different times of the transition and by analyzing the changes in the protein synthesis pattern during maturation. Protein synthesis was required for germinal vesicle breakdown (GVBD). Nevertheless, the concomitant event to the rupture of germinal vesicle, i.e., chromosome condensation, took place even in a cycloheximide-containing medium. The transition from metaphase I to metaphase II was also protein synthesis dependent as evidenced by experiments using this protein synthesis inhibitor. The inhibition was partly reversible, i.e., after removal of the drug, oocytes were able to progress until metaphase I but could not proceed beyond this stage. Changes in the protein synthesis pattern were studied by radiolabelling of oocytes with [35S]methionine. These changes were correlated with the nuclear status of the oocyte: At GVBD, a polypeptide of 25 kD disappeared, while one of 27 kD appeared. At the same time, a polypeptide of 33 kD appeared, whereas concomitantly one of 34 kD became barely detectable and finally disappeared as the maturation progressed. During maturation, the synthesis of a 67 kD polypeptide increased and became predominant at the end of the maturation process. The synthesis of actin decreased after 18 hr of culture from a very high to a low level of synthesis.     

Adenosine blocks hormone-induced meiotic maturation by suppressing purine de novo synthesis

We have examined adenosine (Ado) suppression of FSH-induced germinal vesicle breakdown (GVB) and its relationship to purine de novo synthesis. Oocyte-cumulus cell complexes (OCC) from PMSG-primed, immature mice were cultured 17-18 hr in medium containing 4 mM hypoxanthine (HX) or 300 microM dibutyryl cAMP (dbcAMP) to maintain meiotic arrest, and FSH was added to stimulate meiotic maturation. In the absence of FSH, Ado (1-250 microM) had no effect in dbcAMP-arrested oocytes but dose-dependently suppressed maturation in HX-treated oocytes. FSH-induced maturation was prevented by Ado, though more effectively in dbcAMP-supplemented cultures. Ado affected the magnitude, but not the kinetics pattern, of the response to FSH. Inosine also blocked meiotic induction, but only in dbcAMP-arrested oocytes. Purine de novo synthesis was nearly doubled in OCC by FSH treatment, and this response was completely prevented by Ado. FSH had no effect on HX salvage, although Ado reduced this activity by 98%. Inosine effects on metabolism were intermediate between the control and Ado groups. Experiments with radiolabeled energy substrates showed that Ado suppressed FSH activation of the pentose phosphate pathway but did not prevent significant activation of glycolysis or oxidation of pyruvate. Finally, in cultured follicles from primed mice, hCG-induced maturation was blocked by Ado as effectively as by the purine de novo synthesis inhibitor, azaserine. It is concluded that Ado has an inhibitory action on hormone-induced maturation that is due, at least in part, to suppression of glucose metabolism, leading to compromised purine de novo synthesis.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

Effects of spermatozoa during in vitro meiosis progression in the porcine germinal vesicle oocytes

We have investigated the effect of co-culture with porcine spermatozoa on in vitro maturation of porcine germinal vesicle (GV) oocytes before fertilization. Most oocytes were arrested at the first prophase of meiosis when oocytes were cultured in TCM 199 alone, but the proportion of oocytes that reached metaphase II was significantly elevated by co-incubation with spermatozoa in vitro. The oocyte maturation effect was observed with intact and parts of spermatozoa (head and tail) collected from adult swine (regardless of source). However, gonocytes from the newborn porcine testis were not able to enhance in vitro maturation of porcine germinal vesicle oocytes. Interestingly, the oocyte maturation effect by spermatozoa was not decreased with heat treatment, but the maturation effect of oocyte treatment disappeared with exposure to detergent in sperm suspension. Porcine spermatozoa were also observed to stimulate meiosis of oocytes, which was maintained at meiotic arrest using dibutyryl cyclic AMP or forskolin. The study suggests that (i) membrane of porcine spermatozoa contains a substance(s) that can enhance in vitro maturation of oocytes prior to fertilization, (ii) the putative meiosis-enhancing substance(s) of spermatozoa from adult testes retains the oocyte maturation effect during transportation of spermatozoa through epididymis, and (iii) the putative meiosis-enhancing substance(s) is able to overcome the inhibitory effect of dibutyryl cyclic AMP or forskolin by inducing germinal vesicle breakdown of porcine cumulus-oocyte complexes maintained in meiotic arrest.     

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17β on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17β and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 μg ml⁻¹) of estradiol-17β and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17β), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17β supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.     

Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

Summary In the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA) I. MORPHOLOGICAL STUDY OF OOCYTE MATURATION

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of mciosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The meiosis reinitiating substance induces numerous morphological changes in the oocytes: general (shape), cortical (microvilli retraction, plasma membrane flattening), cytoplasmic (cortical granules repartition) and nuclear modifications (germinal vesicle breakdown, chromosome condensation, formation of a meiotic maturation spindle). A kinetic study of these morphological modifications has been performed.     

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium,glucose, and cumulus cells

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.     

Involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta,orthoptera)

Following their biosynthesis in the follicle cells of vitellogenic ovaries, large amounts of ecdysteroids pass into the oocytes where they accumulate and persist during ovulation and egg-laying. The present paper shows that free ecdysone is unevenly distributed in the oocytes exhibiting the highest concentrations in the region of the posterior pole where the final sequences of nuclear maturation, including germinal vesicle breakdown (GVBD), occur. A correlative study indicates that the concentrations of free ecdysone in this region are particularly high (10 to 20 μM) during two periods of meiotic reinitiation observed in the oocytes: reinitiation I, leading from prophase I to metaphase I with GVBD; and reinitiation II, from metaphase I to the end of meiosis. In vitro incubations of oocytes in meiotic arrest (prophase I) in the presence of exogenous ecdysone demonstrate that complete reinitiation (including GVBD) can be triggered in a dose-dependent manner by this hormone.     

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Requirement for, and patterns of, pyruvate and glutamine metabolism in the domestic dog oocyte in vitro

Supplementation of energy substrates to culture medium is essential for resumption and completion of meiosis in vitro for many mammalian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n=681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0-2.5 mM) and glutamine (0-4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation decreased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF.     

小鼠年龄及卵丘—卵母细胞间联接的解离对卵母细胞体外成熟的影响

实验研究了小鼠卵母细胞体外过程中卵丘－卵母细胞间的相互作用。实验小鼠为雌性Ｂ６Ｄ２杂交一代。激素处理４８小时后分离出卵后天和卵母细胞复合体,并培养在含有次黄嘌呤的培养液中。２４小时后检查卵母细胞核成熟情况。