Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.

The injection of Xenopus laevis females with human chorionic gonadotropin (HCG) leads to ovulation (and maturation) of oocytes whose diameters are 1.2 mm or larger. However, when Xenopus oocytes are removed from their follicular investments by manual dissection and exposed to the steroid, progesterone, in vitro, they exhibit maturation down to about 0.90 mm in diameter with the majority larger than 1.0 mm showing a positive response. Within each female the larger of the oocytes undergo maturation earlier than smaller ones.The response of oocytes also was shown to depend on the length of time since females were last stimulated to ovulate. Similar-sized oocytes from recently ovulated (stimulated) females matured much faster than those of untreated, unstimulated females. Indeed, even the smaller oocytes from stimulated females often matured before the largest oocytes of females without previous HCG injection.The experiments demonstrate that the physiological state of an oocyte cannot be accurately deduced solely from its size nor response to gonadotropins; unresponsiveness presumably being due to inability of follicular elements to respond to the trophic hormones or transfer the stimulus to the oocyte via the appropriate steroid.     

Evidence for steroid metabolism during the in vitro induction of maturation in oocytes of Rana pipiens

Oocytes of Rana pipiens exposed to exogenous progesterone in order to induce maturation have been observed to extensively metabolize this hormone. When progesterone was injected directly into the oocytes, they did not mature, but similar metabolism of progesterone occurred. The metabolites have been tentatively identified as the 5α-reduced derivatives, 5α-pregnanedione, 5α-pregnan-20α-ol-3-one, and 5α-pregnan-3β, 20α-diol, and the pathway of conversion has been examined. Samples of these steroids obtained from commercial sources and those extracted from progesterone-treated oocytes were effective in inducing maturation when added to the medium. Evidence is presented which suggests that steroid metabolism is not a prerequisite for maturation and that the metabolites like progesterone must interact with the oocyte surface to be effective.     

STEROID INHIBITION OF PROTEIN INCORPORATION BY ISOLATED AMPHIBIAN OOCYTES

The relationship between blood protein (vitellogenin) incorporation and nuclear maturation was studied in individual amphibian oocytes after in vitro exposure to desoxycorticosterone acetate (DOCA). Isolated Rana pipiens oocytes were incubated in vitro with radioactively labeled oocyte yolk precursor ([³H]vitellogenin) obtained from estrogenized Xenopus laevis. Incorporation of labeled vitellogenin into the oocytes continued over a 24-h period. Oocytes simultaneously exposed to DOCA and to labeled vitellogenin exhibited both inhibition of vitellogenin incorporation and stimulation of nuclear maturation and cortical changes. Inhibition of vitellogenin incorporation was observed after approximately 9 h of incubation and was correlated with the time of nuclear breakdown. Preincubation of oocytes in steroid for 9 h essentially terminated vitellogenin incorporation. Incorporation of vitellogenin occurred after removal of follicle cells from the oocyte by a short treatment with EDTA. These results demonstrate the macromolecular vitellogenin transport system remains operative in oocytes which can undergo nuclear maturation and that the steroid DOCA can affect its function. Evidence suggests that the mechanism of steroid inhibition is in part the result of inhibition of the micropinocytotic process in the oocyte cortex.     

Steroid-Cell Surface Interactions in the Induction of Meiotic Maturation in Amphibian Oocytes

The involvement of steroid-cell surface interactions, in the induction of oocyte maturation, was investigated. A simple in vitro method was developed for the application of steroids or other substances to isolated portions of the surface of individual amphibian ovarian follicles or oocytes. The technique involved placing follicles in conical tubes between two reservoirs containing culture media. With the use of dye or radioactive tracer studies, the extent of exchange between reservoirs could be monitored and the proportion of the surface treated could be calculated.
Follicular oocytes remained viable and underwent nuclear and cytoplasmic maturation while maintained in conical tubes after a short exposure to progesterone over their total surface. Restriction of the percentage of the oocyte surface exposed to progesterone markedly reduced the incidence of meiotic maturation. Reduced responsiveness to locally applied progesterone occurred even in the presence of elevated steroid concentrations. The results suggest that the amount of follicle surface exposed to steroid is an important determinant in initiation of nuclear-cytoplasmic interactions associated with the meiotic maturation process. The procedures and results described should provide a new approach to investigating regional differences in oocyte and follicle surfaces and to studying their differentiation.     

Specific binding of progesterone to the cell surface and its role in the meiotic divisions in Rana oocytes

Progesterone is believed to act at the cell surface to induce the resumption of the meiotic divisions in amphibian oocytes. Analysis of [³H]- and [¹⁴C]progesterone uptake and exchange by the plasma-vitelline membrane complex, nucleus and cytoplasm of the isolated Rana oocyte indicates that progesterone uptake by the plasma membrane is saturable, specific and temperature-dependent, and has a slow off-rate. Estradiol (a noninducer) did not compete with progesterone, whereas testosterone (an inducer) blocked progesterone uptake by the membrane complex. Scatchard-type plots indicate an apparent K_d of 5.1·10⁻⁷ M over the [progesterone]_o range of 0.01–1.0 μM with maximum binding at about 70 fmol per oocyte. Membrane uptake at higher [progesterone]_o (2–40 μM) indicates apparent cooperative binding, with saturation up to 10 pmol per oocyte. Cytoplasmic uptake was apparently nonspecific and less temperature-dependent than membrane uptake and steroid concentrations (progesterone and pregnanediones) exceeded water solubility by 30–60 min. Nuclear uptake was saturable and specific but uptake was independent of temperature. A comparison of membrane binding and a physiological response (nuclear breakdown) indicated only about 10% of the membrane sites need be filled to initiate a 50% response.     

The role of follicle cells in Rana pipiens oocyte maturation induced by delta 5-pregnenolone.

Previous studies have indicated that pituitary-initiated oocyte maturation in the amphibian is mediated by steroidogenesis in the somatic portion of the follicle. This study compares the ability of (1) oocytes surrounded by a single layer of follicle cells, (2) denuded oocytes, and (3) isolated follicle cells to metabolize Δ⁵-pregnenolone, the common precursor of the steroids. Use of radiolabeled compounds demonstrates that the conversion of Δ⁵-pregnenolone to progesterone is confined to the follicle cells, while further reduction of progesterone takes place in the oocyte. The follicle cells also convert Δ⁵-pregnenolone to a form more potent in inducing meiotic maturation. Thus, the behavior of follicle cells in isolation is consistent with the suggested site of pituitary action leading to meiotic maturation as proposed by an earlier theory.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

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.     

Melatonin supplementation during in vitro maturation of oocyte enhances subsequent development of bovine cloned embryos

Oocyte quality, which is directly related to reprogramming competence, is a major important limiting factor in animal cloning efficiency. Compared with oocytes matured in vivo, in vitro matured oocytes exhibit lower oocyte quality and reprogramming competence primarily because of their higher levels of reactive oxygen species. In this study, we investigate whether supplementing the oocyte maturation medium with melatonin, a free radical scavenger, could improve oocyte quality and reprogramming competence. We found that 10⁻⁹ M melatonin effectively alleviated oxidative stress, markedly decreased early apoptosis levels, recovered the integrity of mitochondria, ameliorated the spindle assembly and chromosome alignment in oocytes, and significantly promoted subsequent cloned embryo development in vitro. We also analyzed the effects of melatonin on epigenetic modifications in bovine oocytes. Melatonin increased the global H3K9 acetylation levels, reduced the H3K9 methylation levels, and minimally affected DNA methylation and hydroxymethylation. Genome-wide expression analysis of genes in melatonin-treated and nontreated oocytes was also conducted by high-throughput RNA sequencing. Our results indicated that melatonin ameliorates oocyte oxidative stress and improves subsequent in vitro development of bovine cloned embryos.     

Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro

The present experiment was to investigate the effect of gonadotropins (LH and hCG), steroids (estradiol and progesterone) and culture media (TCM 199, Ham-F-12, BMOC-3 and modified KRB) on in vitro maturation of cumulus-enclosed bovine oocytes. Oocytes isolated from follicles of 相似文献   

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

Effects of cAMP forskolin and cyanoketone on invitro oocyte maturation in the catfish,Clarias batrachus

This study investigated the interactive effects of cyanoketone (CK), an inhibitor of 3β-hydroxysteroid dehydrogenase on the effects of cAMP and forskolin (FK) on oocyte maturation inClarias batrachus using an in vitro incubation technique. When the oocytes were incubated in the presence of 1 Μg/ml 17α, 20β-dihydroxy-4-pregnen-3-one[l7α, 20Β-DP, the maturation-inducing steroid (MIS) of this species] for 6h, they matured [85.3 + 1.36% germinal vesicle breakdown (GVBD)] normally after additional incubation for 20–30 h in plain medium. On the other hand, exposure to 1.0 and 8 0 mM of cAMP after MIS stimulation caused significant inhibition of GVBD but lower concentrations (0.1 and 0.5 mM) of cAMP were noninhibitory. However, when the oocytes were preincubated for 1 h with 1 μg/mI CK, a significant inhibition in the percentage of GVBD was recorded including the lower concentrations of cAMP. FK, an activator of adenylate cyclase, could significantly induce GVBD at all of its concentrations (0.1, 0.5, 1.0 and 10.0 μM) in a dose- and time-dependent manner. However, when the oocytes were exposed to 1 μg/ml CK for 1 h, prior to FK stimulation, a complete inhibition of GVBD occurred but when CK treatment was given after the FK stimulation, only a partial inhibition of maturation was observed. Taken together, these data indirectly suggest that FK induces catfish oocyte maturation probably by stimulating follicular production of Δ⁴ steroid ( 17α,20 β-DP)through an adenylate cyclase-c AMP-mediated pathway, a mechanism identical to the gonadotropin-induced oocyte maturation.     

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3′ untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3′ UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin

Prostaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro-cultured ovarian follicles. Exposure of follicles to PGF₂α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF₂α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF₂α to FPH-treated follicles. PGE, in contrast to PGF₂α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF₂α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF₂α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH-induced ovulation and maturation. PGF₂α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH-induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF₂α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.     

Nuclear requirement of post-maturational cortical differentiation of amphibian oocytes: effects of cycloheximide.

Cycloheximide induced a complex series of alterations in the cortical cytoplasm of amphibian (Rana pipiens) oocytes undergoing steroid induced nuclear and cytoplasmic maturation in vitro. The morphological changes were described and the role of nuclear-cytoplasmic interactions in the induction of these changes was investigated in intact, enucleated and enucleated-reinjected oocytes. Three stages of cortical changes were ascertained on the basis of: localized alterations at the animal pole, redistribution of pigment and localized contractility (furrow formation) primarily along the animal:vegetal pole axis. The extent and type of cortical alterations varied depending upon the time at which oocytes were examined following hormonal stimulation and cycloheximide treatment. Cycloheximide did not produce cortical alterations in non-hormone treated oocytes nor in steroid treated oocytes until after germinal vesicle breakdown. Nuclear and cytoplasmic maturation and the appearance of cortical alterations were all inhibited when cycloheximide was added to oocytes at the time of steroid treatment. Cycloheximide induction of cortical alterations occurred only after the inhibitor was no longer effective in preventing germinal vesicle breakdown. Enucleated oocytes underwent cytoplasmic maturation in response to the steroid but exhibited no cortical alterations following the delayed addition of cycloheximide. Simultaneous administration of cycloheximide and steroid to enucleated oocytes inhibited cytoplasmic maturation and all observable cortical alterations. Reinjection of nuclear material into enucleated oocytes restored the ability of cycloheximide to induce cortical alterations following steroid induction of cytoplasmic maturation. Without steroid treatment, such reinjected oocytes did not exhibit cortical changes in response to cycloheximide. The data demonstrate that the nucleus is required for and contains a factor(s) which controls the cycloheximide response and post-maturation differentiation of the oocyte. The maturational changes in the cortical cytoplasm appear to be dependent on the intermixing of the germinal vesicle nucleoplasm materials with mature cytoplasm following germinal vesicle breakdown. The results further suggest that the cortical effects of cycloheximide are dependent upon the initiation of protein synthesis during this period of oocyte development. The significance of these observations and experimental studies are discussed in relation to current understanding of the molecular mechanisms controlling meiosis induction and the composition of the germinal vesicle.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Metabolism of glucose,pyruvate, and glutamine during the maturation of oocytes derived from pre‐pubertal and adult cows

The aim of the study was to compare the energy metabolism of oocytes from pre‐pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre‐pubertal oocytes. The metabolism of [5‐³H] glucose, [2‐¹⁴C] pyruvate, and [G‐³H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre‐pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre‐pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre‐pubertal cows at 12 hr (P < 0.05). Oocytes from pre‐pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre‐pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre‐pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre‐pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post‐aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre‐pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre‐pubertal and adult cows that may account for the poor developmental potential of many pre‐pubertal oocytes. Mol. Reprod. Dev. 54:92–101, 1999. © 1999 Wiley‐Liss, Inc.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.