Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.     

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.     

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.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Evidence that Meishan and Large-White hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles ≥4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P<0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P<0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentrations of oestradiol and progesterone in the conditioned media did not differ between the breeds (P>0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration inDrosophila melanogaster

Summary It is known from previous work that juvenile hormone (JH) is required to initiate vitellogenin uptake into maturing oocytes ofDrosophila melanogaster, but additional requirements for this hormone during oocyte maturation have not been fully understood. To determine if early vitellogenic oocytes (stages 8 and 9) require JH for continued development, these oocytes were transplanted toDrosophila female and male hosts which were rendered deficient in JH by three methods. Implanted stage 9 and usually stage 8 oocytes were found to degenerate in JH-deficient hosts unless ZR-515, a JH analogue, was applied to the host shortly after implantation.These results were confirmed during in situ ovary development. JH deficiency was produced in gravid females, and ovaries examined at subsequent time intervals were found to be deficient in stage 8–10 oocytes as early as 6 h after treatment. Degenerating oocytes corresponding to these stages were commonly found. ZR-515 prevented oocyte degeneration during at least the first 8 h and continued to support stage 8–10 oocyte development 24 h after application to these females. The results suggest that JH is required not only for initiation but also for continuation of vitellogenin uptake and oocyte development.     

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34^cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5μMROS for 6 h, (3) oocytes that were treated with 50μMBL-I for 6 h and (4) oocytes that were treated with 6.25 μMROS+25 μMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34^cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34^cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Expression of the zebrafish intermediate neurofilament Nestin in the developing nervous system and in neural proliferation zones at postembryonic stages

Background

At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca²⁺ ([Ca²⁺]_i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca²⁺]_i oscillations through the generation of inositol 1,4,5-triphosphate (IP₃), which causes Ca²⁺ release by binding to IP₃ receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.

Results

Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca²⁺]_i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP₃ receptors.

Conclusion

Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca²⁺]_i transients, and to be activated after completion of maturation.     

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.     

Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes

This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 μM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture. Mol. Reprod. Dev. 51:339–345, 1998. © 1998 Wiley-Liss, Inc.     

Activation of the progesterone-signaling pathway by methyl-beta-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Galphas

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685-1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gα_s is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gα_s protein in cortices and increased internal levels of 45-kDa Gα_s in cortical-free extracts. Dose-dependent increases in internal Gα_s after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gα_s after hormone treatment was comparable to the decrease in cortical Gα_s. These results are consistent with a model in which release of Gα_s from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Inhibition of protein synthesis elicits early grey crescent formation in the axolotl oocyte

Summary In the axolotl (Ambystoma mexicanum Shaw), it was recently shown that cycloheximide (CH) could induce early grey crescent formation (EGC) in non-activated oocytes, maturing in vitro (Grinfeld and Beetschen 1982). Since it was not proved that EGC was a consequence of protein synthesis inhibition rather than a side-effect of the drug, experiments were performed using microinjections of a quite different inhibitor, diphtheria toxin (DT). This toxin also appeared to elicit EGC. Incorporation of (³H) leucine into oocyte proteins in the presence of increasing DT concentrations (10^–11 to 10^–6 M) was then investigated. The frequency of EGC closely parallels the level of protein synthesis inhibition, which is higher in symmetrized oocytes. The lowest CH concentration which can still elicit EGC also exerts a fairly strong inhibition of (³H) leucine incorporation into proteins. It is concluded that protein synthesis inhibition in the late maturing oocyte actually creates specific conditions which allow cytoplasmic rearrangements to occur, leading to grey crescent formation. These results support the interpretation that (a) proteinic inhibitor (s) of symmetrization could be synthesized in the normal maturing oocyte.     

Leucine uptake and incorporation at three stages of mouse oocyte maturation

Mouse oocytes at the dictyate, metaphase I and metaphase II stages of maturation were incubated with and without cumulus cells in ¹⁴C-leucine (1.7 mM, spec. act. = 35 μCi/μmole) for a 1 h pulse after which the total amount of radioactivity taken up during this time and the amount present in the acid-insoluble fraction were measured. In the absence of cumulus cells there was a peak in total leucine uptake at metaphase I. Cumulus cells resulted in increased oocyte uptake at the dictyate stage, decreased uptake at metaphase I with no effect at metaphase II. The variations in uptake with maturation stage and cumulus cells were not paralleled by similar changes in the acid-insoluble radioactivity. There was no change in the acid-insoluble incorporation with progressive maturation, but there was a positive effect of cumulus cells at the dictyate and metaphase I stages. Small and large dictyate oocytes took up and incorporated similar amounts of leucine even though the smaller oocytes did not mature in vitro. The results are discussed in relation to the changing association between the oocyte and cumulus cells with progressive maturation.     

Effect of vitrification on human oocyte maturation rate during in vitro maturation procedure: A systematic review and meta-analysis

Combination of in vitro maturation (IVM) and cryopreservation offers new opportunities for women with contraindication in ovarian stimulation, and females who desire to postpone the childbearing due to different problems. There are still controversies regarding IVM procedure and its impact on oocytes fertilization capability. This systematic review and meta-analysis were conducted to evaluate the impact of vitrification on human oocyte maturation rate during IVM procedure. In this review, we searched Medline, Embase, Scopus and ISI web of science to identify English-language studies. The last search was implemented on 3 February 2018. The original articles which assessed maturation rate after vitrification of MI or GV oocytes were included. Animal trials and the studies that performed cryopreservation using slow-freeze method were excluded. Bias and quality assessments were performed. 2476 articles were screened primarily. After duplication removing and the application of inclusion and exclusion criteria, 14 studies included for the analysis. All studies compared maturation rate between the oocytes that were vitrified at the GV or MI stage before maturation and oocytes which were matured in vitro without vitrification. Meta-analysis showed that oocyte vitrification at GV stage had a significant negative impact on maturation rate (RR = 0.76, 95% CI: 0.66–0.88); I² = 85.2%; P = 0.000). Finally, based on our results, oocyte vitrification decreases the maturation rate by 24%.