Onset Of Germinal Vesicle Breakdown Research Articles

Chromatin Morphology in Human Germinal Vesicle Oocytes and Their Competence to Mature in Stimulated Cycles.

The search for simple morphological predictors of oocyte quality is an important task for assisted reproduction technologies (ARTs). One such predictor may be the morphology of the oocyte nucleus, called the germinal vesicle (GV), including the level of chromatin aggregation around the atypical nucleolus (ANu)-a peculiar nuclear organelle, formerly referred to as the nucleolus-like body. A prospective cohort study allowed distinguishing three classes of GV oocytes among 135 oocytes retrieved from 64 patients: with a non-surrounded ANu and rare chromatin blocks in the nucleoplasm (Class A), with a complete peri-ANu heterochromatic rim assembling all chromatin (Class C), and intermediate variants (Class B). Comparison of the chromatin state and the ability of oocytes to complete meiosis allowed us to conclude that Class B and C oocytes are more capable of resuming meiosis in vitro and completing the first meiotic division, while Class A oocytes can resume maturation but often stop their development either at metaphase I (MI arrest) or before the onset of GV breakdown (GVBD arrest). In addition, oocytes with a low chromatin condensation demonstrated a high level of aneuploidy during the resumption of meiosis. Considering that the degree of chromatin condensation/compaction can be determined in vivo under a light microscope, this characteristic of the GV can be considered a promising criterion for selecting the best-quality GV oocytes in IVM rescue programs.

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

Azole pharmaceuticals induce germinal vesicle breakdown (GVBD) in preovulatory oocytes of zebrafish (Danio rerio): an in vitro study.

Azoles, the antifungal pharmaceuticals are emerging as a new class of water contaminants with a potential to influence the endocrine physiology of surrounding aquatic fauna. In this study, we made an attempt to assess the relative efficacy of widely used azoles belonging to two subclasses, i.e., (i) triazoles (letrozole, fluconazole, itraconazole) and (ii) imidazoles (ketaconazole, ornidazole, clotrimazole), on the onset of germinal vesicle breakdown (GVBD) (an initial step in the final maturation of oocytes) in fully grown preovulatory oocytes of zebrafish (Danio rerio) using an in vitro model. Oocytes (> 650 μm) isolated manually from gravid ovaries were exposed to (i) 0.01 and/or 0.1, 1.0, 5.0, 10, 15, and 20 ng/ml and (ii) 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml of drugs. Zebrafish Ringer's solution (vehicle) and 0.01%ethyl alcohol (solvent) were used as negative controls. 17α, 20 β-Dihydroxy-4-pregnen-3-one (17α-DHP) and diethylstibestrol (DES), potent inducers of GVBD in fish, were used as positive controls. GVBD was scored hourly from 0-6 h. In negative controls, there were no indications of GVBD even at the 6th hour, while in 17α-DHP- and DES-exposed oocytes, GVBD was initiated from the 1st hour, reaching 80% and 76% respectively at the 6th hour. Among azoles, letrozole induced GVBD in 73-85%, fluconazole (30-33%), itraconazole (23-33%), ketaconazole (46-53%), ornidazole (36-40%), and clotrimazole (30-33%) of oocytes. These results suggest that azole pharmaceuticals induce GVBD in fish oocytes that may be attributed to their variable degree of cytochrome P450 enzyme inhibitor activity.

Histone Deacetylation is Required for Orderly Meiosis

Histone acetylation is associated with a diversity of chromatin-related processes in mitosis. However, its roles in mammalian oocyte meiosis are largely unknown. In the present study, we first investigated in detail the acetylation changes during porcine oocyte maturation using a panel of antibodies specific for the critical acetylated forms of histone H3 and H4, and showed meiosis stage-dependent and lysine residue-specific patterns of histone acetylation. By using trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), we further determined that selective inhibition of histone deacetylation (thereby maintaining hyperacetylation) delayed the onset of germinal vesicle breakdown and produced a high frequency of lagging chromosomes or chromatin bridges at anaphase and telophase I (AT-I), suggesting that histone deacetylation is required for orderly meiotic resumption and accurate chromosome segregation in porcine oocytes. In addition, we examined the localization and expression of HDAC1 by performing immunofluorescence and immunoblotting analysis. The results showed that subcellular translocation, expression level and phosphorylated modification of HDAC1 were temporally regulated and likely to co-participate in the establishment of histone acetylation profiles in oocyte meiosis.

Behavior of γ-tubulin during spindle formation in Xenopus oocytes: requirement of cytoplasmic dynein-dependent translocation

Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. gamma-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of gamma-tubulin during spindle formation in oocytes. We first observed sequential localization of gamma-tubulin during spindle formation in Xenopus oocytes. gamma-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and gamma-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, gamma-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of gamma-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with gamma-tubulin for the assembly of microtubules, but not gamma-tubulin, was phosphorylated during oocyte maturation. These results suggest that gamma-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with gamma-tubulin, but not gamma-tubulin itself, is important for microtubule reorganization.

Formation of polar‐body‐like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.

Comparative Analysis of Protein Synthesis, Transcription and Cytoplasmic Polyadenylation of mRNA during Maturation of Bovine Oocytes in vitro

In many species, large numbers of macromolecules are accumulated during oocyte growth. The messenger and ribosomal RNAs produced in these cells are far in excess of those necessary to support protein synthesis. Thus, the aim of this study was to elucidate the processes of translational regulation during meiotic maturation. The relationship between transcription, translation and polyadenylation of mRNA during in-vitro maturation (IVM) of bovine oocytes was investigated. The results presented here show that overall protein synthesis is stimulated during meiotic maturation (approximately three times) concomitantly with the onset of germinal vesicle breakdown after 6 to 10 h of IVM. However, in metaphase II, the incorporation of [35S]methionine into proteins showed only basal levels, as in the germinal vesicle (GV) stage. Furthermore, in the course of IVM, de-novo transcription strongly declines as determined by measuring the incorporation of [3H]uridine into RNA. In contrast to this finding, the incorporation of [3H]adenosine increased and showed a peak during the time interval from 6 to 10 h of IVM, parallel with the onset of germinal vesicle breakdown (GVBD) and translation. In the further course of maturation, only a moderate decrease of [3H]adenosine incorporation was observed. These results indicate that (i) translation increased at the time of GVBD; (ii) these processes were accompanied by polyadenylation of mRNA; and (iii) although transcription declines, polyadenylated mRNA is accumulated until metaphase II (as shown by poly(U)-hybridization), in which protein synthesis is low. The correlation of these processes is discussed here. A detailed knowledge of the biochemical and molecular processes which occur during oocyte maturation can be useful for the improvement of IVM conditions.

Hormonal Regulation and Cellular Distribution of Connexin 32.2 and Connexin 32.7 RNAs in the Ovary of Atlantic Croaker

The in vitro effects of human chorionic gonadotropin (hCG) on ovarian connexin (Cx) 32.2 and 32.7 RNA levels and ovarian follicle maturation were assessed, and the cellular distribution of Cx transcripts in the ovary was determined. hCG caused a concentration-dependent induction of Cx32.2 RNA, which peaked coincidentally with the appearance of morphological indices of oocyte maturational competence (OMC). Cx32.2 RNA levels declined thereafter in all treatment groups, although this decline was not accompanied by the onset of germinal vesicle breakdown (GVBD) at the lowest hCG concentration used. The levels of Cx32.7 RNA initially declined and subsequently increased to preincubation values after hCG treatment, but these changes were not dependent on hCG concentration. In a separate experiment, the decline in Cx32.7 RNA occurred in the presence or absence of hCG and was prevented by low (physiological) concentrations of estradiol-17β (E2) or by protein kinase C (PKC) inhibitor, but was enhanced in the presence of high E2 concentrations or of PKC activator. These changes in Cx32.7 RNA abundance were not associated with any indices of oocyte maturation. In situ hybridization of tissue sections showed the presence of Cx32.2 and Cx32.7 RNA in somatic cells of the ovarian follicle but not in oocytes. Cx32.2 RNA seemed to be present in granulosa and thecal cells, but the assay resolution was insufficient to reliably determine the distribution of Cx32.7 transcript by somatic cell type. In view of earlier findings that Cx32.2-based (but not Cx32.7-based) connexons can form functional homotypic channels, these results indicate that Cx32.2 gene expression in granulosa cells is sufficient for the formation of homologous gap junctions (GJ). Northern blot of RNA extracts from ovulated eggs, which are free of follicle cells, showed the presence of relatively low levels of both Cx RNAs. Thus, it is possible that Cx32.2 is present in oocytes and that it participates in heterologous (homotypic) GJ formation between the oocyte and the granulosa cells. In conclusion, Cx32.2 RNA levels in somatic cells of the ovarian follicle correlated positively with morphological indices of OMC acquisition, but subsequently declined during GVBD. These changes in Cx32.2 RNA may function in the regulation of GJ contacts during follicular maturation.

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish.

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17alpha,20beta-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes.

Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes.

The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase-defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD-RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.

Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle

The objective of the present studies was to assess whether hormone induction of oocyte maturation in isolated intact follicles may be linked to desensitization of follicle-stimulating hormone (FSH) in the oocyte-cumulus complex (OCC). Incubation of follicles with chorionic gonadotropin (hCG), FSH or epidermal growth factor (EGF) produced a marked inhibition of FSH-dependent cyclic AMP accumulation in OCC with a time-course coincident with the onset of germinal vesicle breakdown (GVBD). These effects were evident within 3 h for both hCG and FSH, but with EGF a reduced response to FSH was seen within 1 h of treatment followed by an increase in GVBD. In contrast, no inhibition of cyclic AMP accumulation was seen in response to cholera toxin, forskolin or LH in OCC derived from follicles incubated with hCG for 3 h. The time-course for induction of oocyte maturation by incubation of the intact follicle with hCG was also coincident with production of prostaglandin (PG) F 2α, an indirect marker of cyclooxygenase induction. No effect on metabolic coupling between the oocyte and cumulus cells was seen until 9 h after hCG treatment. Retinoic acid caused a marked decrease in metabolic coupling between the oocyte and cumulus cells but inhibited oocyte maturation both in denuded oocytes and OCC. Since FSH desensitization in OCC, the resumption of meiosis, and production of arachidonic acid-derived products were coincident, it is suggested that abrogation of FSH action in cumulus cells by the ovulatory surge of gonadotropins may initiate oocyte maturation.

Changes in membrane potential, membrane resistance, and intracellular H +, K +, Na +, and Cl − activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H +, K +, Na +, and Cl − ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential ( E m) and electrical resistance ( R m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R m gradually increased. By the end of maturation of Pleurodeles oocytes E m had stabilized at about −10 mV and R m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E m spontaneously shifted from about −15 to about +30 mV; simultaneously R m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K + activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na + activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na +, accompanied by an increase in Cl − activity, was observed as soon as E m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E m.

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.