Glucose utilization during gonadotropin-induced meiotic maturation in cumulus cell-enclosed mouse oocytes

Earlier work from this laboratory has determined that glucose plays an important role in the mechanisms regulating meiotic maturation in mammalian oocytes. In the current study, we have further explored the role of glucose in hormone-induced germinal vesicle breakdown (GVB) in an effort to better understand how glucose utilization and metabolism relate to the control of meiotic maturation in mouse cumulus cell-enclosed oocytes (CEO). When CEO were cultured in medium containing 4 mM hypoxanthine (to maintain meiotic arrest), 5.5 mM glucose, and 0.23 mM pyruvate, follicle-stimulating hormone (FSH) stimulated lactate accumulation in a time-dependent manner. Addition of 2-deoxyglucose (2-DG) to the medium at various times after the initiation of culture resulted in rapid termination of lactate production and suppression of FSH-induced GVB scored after 18 hr of culture, the effectiveness diminishing the longer the delay before addition of 2-DG. By 8 hr, addition of 2-DG was without effect on GVB. Similar effects were seen when FSH-treated CEO were washed free of glucose. In a 2-DG dose-response experiment, gonadotropin-induced lactate production was prevented, but this inhibition did not necessarily prevent GVB. The activities of six metabolic enzymes were measured in extracts of freshly isolated complexes, and in order of increasing activity were: hexokinase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase. Of the six enzymes examined, only hexokinase activity was increased in CEO exposed to FSH. CEO were cultured in microdrops in the presence or absence of FSH, and aliquots from the same microdrop were assayed for glucose, lactate, and pyruvate. In response to FSH, utilization of glucose in microdrop cultures by CEO was markedly increased and was accompanied by comparable lactate production and limited pyruvate production. Cycloheximide and α-amanitin both blocked FSH-induced oocyte maturation, but only cycloheximide prevented the increase in hexokinase activity and glucose consumption. These data suggest that hexokinase is an important rate-limiting enzyme for glucose utilization that is under translational control and participates in the mechanisms controlling the reinitiation of meiosis. However, stimulation of glycolytic activity does not appear to be a necessary concomitant for meiotic induction. © 1996 Wiley-Liss, Inc.     

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

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

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 meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.     

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

To test the hypothesis that culture conditions influence meiotic regulation in mouse oocytes, we have examined the effects of six culture media, four organic buffers, and pH on spontaneous maturation, the maintenance of meiotic arrest and ligand-induced maturation in cumulus cell-enclosed oocytes from hormonally primed immature mice. The media tested were Eagle's minimum essential medium (MEM), Ham's F-10 (F-10), M199, M16, Waymouth's MB 752/1 (MB 752/1), and Leibovitz's L-15 (L-15). All six media supported ≥94% spontaneous germinal vesicle breakdown (GVB) during a 17–18 hr incubation period, but polar body formation was lower in M199 and MB 752/1 than in the other media. The incidence of polar bodies could be increased in these two media by the addition of pyruvate. With the exception of M16 and MB 752/1, 4 mM hypoxanthine maintained a significant number of cumulus cell-enclosed oocytes in meiotic arrest. Inhibition could be restored by the addition of glutamine to M16 and pyruvate to MB 752/1. Folliclestimulating hormone (FSH) and epidermal growth factor (EGF) stimulated GVB in those media in which hypoxanthine was inhibitory. dbcAMP was able to maintain meiotic arrest in all of the media, but was least effective in M16. FSH stimulated GVB in all dbcAMP-arrested groups except L-15, and FSH became stimulatory in L-15 when the pyruvate level was reduced to 0.23 mM and galactose was replaced with 5.5 mM glucose. When MEM was buffered principally with the organic buffers MOPS, HEPES, DIPSO, or PIPES (at 20 mM), high frequencies of GVB and polar body formation were observed in inhibitor-free medium. dbcAMP suppressed GVB in all groups; hypoxanthine also maintained meiotic arrest in all buffering conditions, although this effect was nominal in PIPES-buffered medium. FSH and EGF stimulated GVB in all dbcAMP- and hypoxanthine-treated groups. When the concentration of HEPES was increased from 20 mM to 25 mM, a more pronounced suppressive effect on maturation in both dbcAMP- and hypoxanthine-supplemented groups was observed in the absence of FSH. But whereas HEPES reduced the induction of maturation by FSH in dbcAMP-arrested oocytes, this buffer had no effect on FSH action in hypoxanthine-treated oocytes. When MEM was buffered with HEPES and the pH was adjusted to 6.8, 7.0, 7.2, or 7.4, a dramatic effect of pH on meiotic maturation was observed. pH had no significant effect on hypoxanthine salvage by oocyte-cumulus cell complexes, but FSH-induced de novo purine synthesis was significantly augmented by increased pH, in parallel with increased induction of GVB. The results of this study demonstrate that the use of different culture media, or minor changes in culture conditions, can lead to significant variation in (1) the spontaneous maturation of oocytes, (2) the ability of meiotic inhibitors to suppress GVB, or (3) the efficacy of meiosis-inducing ligands. Furthermore, such observations provide a unique opportunity to examine specific molecules and metabolic pathways that can account for this variation and thereby gain valuable insights into the mechanisms involved in meiotic regulation. Mol. Reprod. Dev. 46:551–566, 1997. © 1997 Wiley-Liss, Inc.     

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

This study was carried out to test the hypothesis that purine nucleotide-generating pathways are required for ligand-stimulated oocyte maturation in meiotically arrested cumulus cell-enclosed oocytes. Oo-cytes from hormonally primed, immature mice were cultured overnight in Eagle's minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (to maintain meiotic arrest), plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these conditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, or adenosine were included during the first 3 hr. Furthermore, preincubation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, could not mimic this protective effect of hypoxanthine. Azaserine and aminopterin, inhibitors of purine de novo synthesis, blocked hormone-triggered maturation in dbcAMP-arrested oocytes, but had little effect on hypoxanthine-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that can be taken up by cells and phosphorylated to form AICAR, which can enter the purine de novo pathway at a point distal to the sites of azaserine inhibition. FSH was stimulatory to purine de novo synthesis, while azaserine, aminopterin, hypoxanthine, and AICA riboside all suppressed de novo synthesis in the presence or absence of FSH, with dbcAMP having no effect. HPLC analysis of ¹⁴C-hypoxanthine metabolism in oocyte-cumulus cell complexes revealed that changes in the pattern of purine metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathways are vital participants in the mechanism(s) regulating hormone-induced meiotic maturation, and that either the de novo or salvage pathway can fulfill this nucleotide requirement. Mol Reprod Dev 46:155–167, 1997. © 1997 Wiley-Liss, Inc.     

EGF-like peptides mediate FSH-induced maturation of cumulus cell-enclosed mouse oocytes

This study was carried out to examine the participation of epidermal growth factor (EGF)-like peptides in the induction of germinal vesicle breakdown (GVB) in mouse cumulus cell-enclosed oocytes (CEO). The EGF-like peptide, amphiregulin (AR), dose-dependently stimulated meiotic resumption in CEO, but not denuded oocytes (DO) maintained in meiotic arrest with 300 microM dbcAMP. The EGF receptor (EGFR) kinase inhibitor, AG1478, blocked meiotic resumption induced by FSH and AR in CEO, but had no effect in DO. FSH-induced maturation was also suppressed by antisera to both EGFR and EGF. Maturation occurred with slightly faster kinetics in AR-stimulated CEO when compared to FSH-stimulated CEO. When CEO were maintained in meiotic arrest with a low level of dbcAMP, FSH was initially inhibitory to maturation and later stimulatory; the stimulatory phase was prevented by AG1478, indicating mediation by EGF-like peptides. Pulsing CEO with high levels of dbcAMP also stimulated GVB and could be blocked by AG1478. Treatment of arrested CEO with PKC agonists stimulated maturation and this was prevented with AG1478 as well as antibodies to EGFR. FSH-induced maturation of dbcAMP-arrested CEO was blocked by bisindolylmaleimide I (BIM-I), an inhibitor of PKC, implicating PKC in FSH action. EGF-stimulated CEO failed to resume maturation in the presence of glycerrhetinic acid, a gap junction inhibitor, suggesting transfer of positive signal through the cell-cell coupling pathway. These data support the idea that EGF-like peptides provide a common pathway mediating the meiosis-inducing influence of FSH, cAMP pulsing, and PKC activation in mouse CEO by a gap junction-dependent process.     

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.     

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.     

Effects of follicle-stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17β was less effective in this regard, and 5α-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium

The present experiments were conducted to examine the hypothesis that follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, and then transferred into a new culture medium supplemented with FSH but without BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. However, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to resume meiosis, and at high concentration of 10(-5) M , it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM +FSH for 60 min could significantly inhibit the effect of FSH in a dose-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Maintenance of murine oocyte meiotic arrest: uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes

To analyze the potential mechanisms by which hypoxanthine and adenosine maintain meiotic arrest in mouse oocytes this study focused on: the uptake and metabolism of hypoxanthine and adenosine; the effect of inhibitors of inosine monophosphate (IMP) dehydrogenase on purine-mediated meiotic arrest; and the role of adenosine metabolism on the maintenance of meiotic arrest. Although the denuded oocyte can take up radiolabeled hypoxanthine and adenosine, an intact cumulus oophorus greatly augments uptake of these molecules (and/or metabolites). Both of these compounds were completely metabolized during incubation in vitro: hypoxanthine was apparently metabolized to uric acid and adenosine was metabolized to ADP; a small amount of each compound was also converted to inosine by cumulus cells and transferred to the oocyte. The IMP dehydrogenase inhibitors, bredinin and mycophenolic acid (MA), induced, in a dose-dependent manner, the resumption of maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine but had no effect on denuded oocytes. MA did not induce maturation when meiotic arrest was maintained by guanosine. Nor did MA alter the uptake of hypoxanthine by cumulus cell-enclosed oocytes. The poorly metabolized analog of adenosine, 2-chloroadenosine, was as effective as adenosine in its synergistic action with hypoxanthine in maintaining meiotic arrest. It is concluded that hypoxanthine and adenosine are metabolized within the oocyte-cumulus cell complex; xanthyl and/or guanyl compounds are produced by oocyte-cumulus cell complexes in the presence of hypoxanthine and play an important role in the maintenance of meiotic arrest; and adenosine need not be metabolized to act synergistically with hypoxanthine in maintaining meiotic arrest.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

Differential effects of testosterone and dibutyryl cyclic AMP on the meiotic maturation of mouse oocytes in vitro

Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of glucocorticoids on spontaneous and follicle-stimulating hormone induced oocyte maturation in mouse oocytes during culture

Several studies have indicated that glucocorticoids are involved in maturation of mammalian oocytes. Recently, maturation of porcine oocytes in culture was shown to be inhibited by glucocorticoids in a time- and dose-dependent manner. In addition, levels of cortisol available for biological action in fluid of preovulatory follicles are higher than that present in circulation. The present study evaluates the effect of cortisol and dexamethasone on mouse cumulus enclosed oocytes (CEO) undergoing spontaneous- and FSH-induced maturation during a 24 h culture period using breakdown of the germinal vesicle (GVBD) as end-point. FSH-induced oocyte maturation was studied using media containing 4.5 mM hypoxanthine to maintain levels of cAMP elevated, whereas spontaneous oocyte maturation was studied in a medium without hypoxanthine. In the presence of FSH (25 IU/l) the rate of GVBD was significantly elevated compared to the control. Dexamethasone (1–20 μg/ml) in combination with FSH resulted in a rate of GVBD similar to FSH alone. Cortisol (0.1–10 μg/ml) resulted in a significant higher rate of GVBD in combination with a physiological concentration of FSH (10 IU/l) as compared to the control but similar to that caused by FSH alone. Nearly all CEO that matured spontaneously resumed meiosis irrespective of whether or not cortisol was present. In conclusion, these results indicate that glucocorticoids have little or no influence on the regulation of oocyte maturation in the mouse. Species differences between mouse and pig oocytes may exist.     

Effect of follicle-stimulating hormone on metabolism and maturation in Bufo arenarum oocytes

In the fully grown Bufo arenarum oocyte, carbohydrate breakdown during the autumn-winter season is accomplished mainly through the glycolytic pathway followed by the krebs cycle. During the breeding season (spring-summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle. The metabolism operating in the oocytes was determined using the following paramenters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PEK) and glucose-6-phosphate dehydrogenase (G-6-PDH); and 3) cirate and ATP compartmentalization. The present paper shows that follicle-stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractios obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding-season animals from dose-response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 μg FSH/ml of incubation medium and 30 min treatment, respecitively. The metabolic effect of FSH upon oocytes could be mediaated by the adenylate cyclase cAMP system, since treatment of ovric fractions with cAMP 10-³ M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracyline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the same hormonal treatment.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.