Inhibition Of Germinal Vesicle Breakdown Research Articles

Assessment of nuclear membrane dynamics using anti-lamin staining offers a clear cut evidence of germinal vesicle breakdown in buffalo oocytes

Importance of germinal vesicle breakdown (GVBD) in the development chronology of oocyte development can be gauged from the fact that it has been the target of several studies attempting to improve oocyte competence. Manipulation of this event rests on its precise and explicit documentation. Pertinently, we decided to compare three different methods for their efficacy and precision in assessing GVBD. Immature buffalo oocytes were subjected to GVBD inhibition using Roscovitine (Ros) and Cilostamide (Cil) under different concentrations of each as well as their combination, along with control. After 24 h of inhibition, chromatin assessment was done using aceto-orcein staining, hoechst staining and anti-lamin staining. Hoechst staining and anti-lamin staining proved to be easier and less time consuming when compared to aceto-orcein, which was cumbersome and lengthy. Aceto-orcein and hoechst staining were equally ambiguous, while antilamin staining was most precise, accurate and clear in characterizing an oocyte as germinal vesicle (GV) or GVBD. We conclude by stating that anti-lamin staining is an easy and specific technique for assessing GVBD in buffalo oocytes.

Inhibition of germinal vesicle breakdown in Xenopus oocytes in vitro by a series of substituted glycol ethers.

A 24 hour in vitro Xenopus oocyte maturation (germinal vesicle breakdown [GVBD]) assay developed by Pickford and Morris (Environmental Health Perspectives, 1999, 107, 285-292) was used to screen a series of substituted glycol ethers (GEs). Substituted GEs included: ethylene glycol monomethyl ether (EGME); EG monoethyl ether (EGEE); EG monopropyl ether (EGPE); EG monobutyl ether (EGBE); EG monohexyl ether (EGHE); diethylene glycol monomethyl ether (DGME); triethylene glycol monomethyl ether (TGME); ethylene glycol monophenyl ether (EGPhE); EG monobenzyl ether (EGBeE); EG diphenyl ether (EGDPhE); and propylene glycol monophenyl ether (PGPhE). The GEs inhibited progesterone- or androstenedione-induced GVBD with the following relative potency: EGPhE > PGPhE > EGME >> EGEE ≥ EGBeE > EGPE >> EGBE >EGHE > EGDPhE >> DGME ≥ TGME, or EGPhE >> PGPhE >> EGBeE > EGDPhE > EGEE > EGME > EGPE > EGBE, EGHE, DGME and TGME, respectively. Further, [3 H]progesterone or [3 H]androstenedione binding affinities to the oocyte plasma membrane progesterone receptor (OMPR) or classical androgen receptor (AR) were: EGME > EGPhE ≥ PGPhE ≥ EGEE > EGBeE >> EGPE >> EGBE ≥ EGHE > EGDPhE, TGME, and DGME, or EGPhE > PGPhE >> EGBeE > EGDPhE >> EGEE ≥ EGME >> EGPE, EGBE, and EGHE > DGME and TGME, respectively. Binary joint mixture studies with the GVBD model using flutamide (AR antagonist) and EGPhE indicated that flutamide/EGPhE mixture acted in a concentration additive manner. The effects of substituted GE series, however, may be mediated through the OMPR; the potency of EGPhE may be the result of bimodal inhibition of both the OMPR and AR pathways.

Transient Arrest of Germinal Vesicle Breakdown Improved In Vitro Development Potential of Buffalo (Bubalus Bubalis) Oocytes.

Germinal vesicle breakdown (GVBD) is the first milestone that an oocyte needs to achieve toward completing the maturation and gaining potential to fertilize. Significantly lower in vitro embryo production rate in buffaloes can be attributed to heterogeneity of GVBD occurrence among oocytes obtained from abattoir derived ovaries. Evidence from our earlier work had suggested that different qualities of buffalo oocytes differ significantly in their timing of GVBD. Besides, these oocytes also differ in terms of volume of Akt phosphorylation, which initiates the process of GVBD. With objective of synchronizing the oocytes for GVBD, immature buffalo oocytes were subjected to a two-step culture protocol, initially in the presence of GVBD inhibitors and subsequently, in vitro maturation (IVM) with added SC79 (activates Akt). Expression of developmentally important genes was assessed along with embryo development rate and blastocyst health to interpret the consequences. Oocytes subjected to a short GVBD inhibition period of 6 h followed by IVM with SC79 resulted in improved cleavage and blastocyst rates. Resultant blastocysts also possessed higher ICM: TE ratio. Further, GVBD inhibited oocytes displayed a sustained cytoplasmic maturation status in terms of reorganization of cortical granules (CGs), mitochondrial membrane potential, and glutathione levels during the period of inhibition. We conclude that a temporary GVBD arrest of buffalo oocytes and modulation of Akt improves the in vitro embryo development rate as well as quality of resultant embryos. Besides, our meiotic arrest protocol does not affect the cytoplasmic maturation. J. Cell. Biochem. 119: 278-289, 2018. © 2017 Wiley Periodicals, Inc.

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.

Ghrelinergic system in fish ovaries and ghrelin inhibition of germinal vesicle breakdown in zebrafish oocytes

Ghrelin, the only known orexigenic gut hormone has been proposed to integrate energy balance and reproduction in mammals. There is a large set of data available on the orexigenic and LH stimulatory roles of ghrelin in fish. Ghrelin and ghrelin receptor mRNAs are expressed in the gonads of several fishes. However, the direct roles of ghrelin on fish gonads remain unclear. Our objective was to identify the ghrelinergic system in fish ovaries, at the protein level in the cross sections of paraffin fixed ovaries, and test the direct effects of ghrelin on oocyte maturation. Both ghrelin and ghrelin receptor like immunoreactivity was detected in the follicle cells in the cross section of goldfish and zebrafish ovaries. This agrees with the mRNA expression data and further confirms the presence of the ghrelinergic system in fish ovaries. We found that native ghrelin at 10, 50 and 100ng/mL concentrations inhibited both basal and maturation-inducing hormone stimulated stage IV germinal vesicle breakdown (GVBD) of zebrafish oocyte maturation in vitro. This result indicates that ghrelin acts directly on zebrafish follicles. When zebrafish follicles were co-incubated with ghrelin and a well-characterized ghrelin receptor antagonist, d-lys3-GHRP-6, the inhibitory effects of ghrelin on stage IV GVBD was abolished. This result indicates that ghrelin inhibits stage IV GVBD via its receptor(s). Collectively, our results for the first time indicate a direct role for ghrelin in the ovarian physiology of fish.

Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm

In mammalian oocytes, cAMP elevations prevent the resumption of meiotic maturation and thereby block nuclear disassembly (germinal vesicle breakdown (GVBD)), whereas nitric oxide (NO) and its downstream mediator cGMP can either inhibit or induce GVBD. Alternatively, some invertebrate oocytes use cAMP to stimulate, rather than inhibit, GVBD, and in such cases, the effects of NO/cGMP signaling on GVBD remain unknown. Moreover, potential interactions between NO/cGMP and AMP-activated kinase (AMPK) have not been assessed during GVBD. Thus, this study analyzed intraoocytic signaling pathways related to NO/cGMP in a marine nemertean worm that uses cAMP to induce GVBD. For such tests, follicle-free nemertean oocytes were stimulated to mature by seawater (SW) and cAMP elevators. Based on immunoblots and NO assays of maturing oocytes, SW triggered AMPK deactivation, NO synthase (NOS) phosphorylation, and an NO elevation. Accordingly, SW-induced GVBD was blocked by treatments involving the AMPK agonist AICAR, antioxidants, the NO scavenger carboxy-PTIO, NOS inhibitors, and cGMP antagonists that target the NO-stimulated enzyme, soluble guanylate cyclase (sGC). Conversely, SW solutions combining NO/cGMP antagonists with a cAMP elevator restored GVBD. Similarly, AICAR plus a cAMP-elevating drug reestablished GVBD while deactivating AMPK and phosphorylating NOS. Furthermore, sGC stimulators and 8-Br-cGMP triggered GVBD. Such novel results indicate that NO/cGMP signaling can upregulate SW-induced GVBD and that cAMP-elevating drugs restore GVBD by overriding the inhibition of various NO/cGMP downregulators, including AMPK. Moreover, considering the opposite effects of intraoocytic cAMP in nemerteans vs mammals, these data coincide with previous reports that NO/cGMP signaling blocks GVBD in rats.

Adrenomedullin: A possible regulator of germinal vesicle breakdown

Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.

Temporary Inhibition of Germinal Vesicle Breakdown by Rho Kinase Inhibitor Y-27632 is Detrimental to Oocyte Maturation

Abstract: In the present study, we investigated the effects of a specific Rho kinase (ROCK) inhibitor, Y-27632, on germinal vesicle breakdown (GVBD), changes of cytoskeletal organization, and the resumption of porcine oocyte maturation after its removal. In Experiment 1, cumulus-oocyte complexes (COCs) cultured in medium containing Y-27632 for 24 h showed GVBD of 98, 88, 84, and 4% (P<0.01) of the oocytes treated with 0, 1, 10, and 100 µM of the drug, respectively. In addition, Y-27632 treatment from 24 to 44 h of incubation significantly inhibited maturation of the oocytes. The metaphase II (MII) rates were 84, 78, 65, and 10% (P<0.01) for the corresponding groups, and 0, 2, 8, and 45% of the oocytes failed to show emission of the first polar body (PB1). In Experiment 2, resumption of meiotic maturation was examined after removal of Y-27632. COCs incubated with Y-27632 for 18 h and then cultured in Y-27632-free medium for an additional 44 h showed a significantly lower MII rate (86 vs. 33%, P<0.01). Afte...

Interactive effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes

The objective of this article was to study the effects of low temperature and roscovitine (ROS) on meiotic resumption and developmental potential of goat oocytes. Goat oocytes were cultured at different temperatures in medium containing different concentrations of ROS, and at the end of culture, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were activated chemically or fertilized in vitro for embryo development. Meiotic arrest was successfully maintained for 24 hr with 0, 50, and 200 microM ROS at 5, 20, and 38.5 degrees C, respectively. Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 hr at 5 degrees C without ROS (Protocol 5C) or at 20 degrees C with 50 microM ROS (Protocol 20C) or for 8 hr at 38.5 degrees C with 200 microM ROS (Protocol 8 hr), but no blastulation was observed after oocytes were inhibited at 38.5 degrees C with 200 microM ROS for 24 hr. Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8 hr developed into morulae, due to a high incidence of polyspermy. Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8 hr, leading to a precocious germinal vesicle breakdown (GVBD) during subsequent maturation. Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8 hr completed CG migration after maturation. In conclusion, goat oocytes were tolerant to chilling and culture at lower temperatures with less ROS was better than culture at higher temperatures with more ROS for oocyte GVBD inhibition.

Effect of dehydroleucodine on meiosis reinitiation inBufo arenarumdenuded oocytes

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.

Developmental competence of bovine oocytes after specific inhibition of MPF kinase activity: Effect of estradiol supplementation and follicle size

In the bovine, the concentration of 17β-estradiol (E2) in the follicular fluid of the dominant follicle is high, indicating a possible role of E2 on the cytoplasmic maturation that occurs before the LH surge. The aim of this study was to investigate the role of E2 on the developmental competence of bovine oocytes originating from different sized follicles and temporarily maintained at the germinal vesicle stage with roscovitine (ROS). First, the efficiency of ROS to inhibit germinal vesicle breakdown (GVBD) in oocytes harvested from small (3–4 mm diameter) and medium (5–8 mm diameter) sized follicles was demonstrated. Next, the effect of E2 during temporary inhibition of GVBD by ROS on the subsequent nuclear maturation was evaluated. Oocytes from small and medium sized follicles were cultured in the presence of ROS, FSH and with or without E2 for 24 h. After this period, oocytes were cultured for another 24 h with FSH but without ROS and E2, after which the nuclear stages and the developmental competence of oocytes were assessed. In conclusion, it is demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small and medium sized follicles. It might be that in vivo, the increase of E2 during follicle growth is more related to selection of the dominant follicle than to the cytoplamsic maturation of the oocyte as such.

Simulation of intrafollicular conditions prevents GVBD in bovine oocytes: A better alternative to affect their developmental capacity after two-step culture

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2-3, 3-4, and 4-6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20-22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%-8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6-7 mul per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6-7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies.

Developmental capacity of bovine cumulus oocyte complexes after transcriptional inhibition of germinal vesicle breakdown

Oocytes cultured in the presence of FSH and the transcriptional inhibitor, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), remain in meiotic arrest at the germinal vesicle (GV) stage. The objectives of this study were to assess the kinetics of maturation and the developmental capacity of bovine cumulus oocyte complexes (COC) following release from prolonged meiotic arrest by DRB. In Experiment I, COC were cultured for 20 h in Tissue culture medium (TCM)-199 supplemented with 10% estrus cow serum (ECS), 5 μg/ml FSH and 1 μg/ml estradiol in the presence of 120 μM DRB. COC were then released from meiotic arrest and cultured for 20 h in DRB-free medium. Control COC were cultured for 20 h in DRB-free medium, with culture initiated concomitant to the release of DRB-treated COC from meiotic arrest. Nuclear maturation was assessed after 0, 5, 10, 15, and 20 h of culture in DRB-free medium. The proportion of DRB-arrested oocytes reaching metaphase II (MII) following 20 h culture in DRB-free medium was not significantly different from controls (96±4% versus 99±4%). In Experiment II, COC were cultured for 20 h in TCM-199 supplemented with 10% ECS, 10 μg/ml LH, 5 μg/ml FSH, and 1 μg/ml estradiol in the presence or absence of 120 μM DRB. COC in the DRB-treated group were then washed and matured coincident with a second group of control COC for 20 h in DRB-free medium. COC in both groups were fertilized and then randomly assigned to one of two culture systems: TCM-199+10%ECS or mSOF+0.6% fatty acid-free BSA. Development was assessed at 72 h post insemination (hpi), 168 hpi (Day 7) and 216 hpi (Day 9). In this experiment, culture with DRB-arrested oocyte maturation at the GV stage (DRB, 85±3% GV; Control, 2±3% GV; P<0.001). Following release from arrest, maturation and fertilization, the proportion of COC that cleaved by 72 hpi was decreased by treatment with DRB (DRB: 78±3% versus Control: 90±3%; P<0.05). However, no effect of DRB was found on the proportion of cleaved zygotes that reached the blastocyst stage on either Day 7 or Day 9 of culture (Day 7: DRB 16±2% versus Control, 21±2%; Day 9: DRB 23±3% versus Control, 31±3%). More embryos reached the blastocyst stage in the TCM-199/serum culture system compared to the mSOF/BSA system on both Days 7 and 9 (Day 7: TCM-199, 23±2% versus mSOF, 13±2%, P<0.05; Day 9: TCM-199, 32±3% versus mSOF, 22±3%, P<0.05). In summary, bovine COC maintained in meiotic arrest for 20 h by culture in the presence of the transcriptional inhibitor DRB retained their capacity to develop to the blastocyst stage after fertilization in vitro.

Roles of protein kinase C in oocyte meiotic maturation and fertilization*

Abstract Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis release, the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian ...

Cyclic AMP level and phosphodiesterase activity during 17α,20β-dihydroxy-4-pregnen-3-one induction and theophylline inhibition of oocyte maturation in the catfish, Clarias batrachus

This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17α,20β-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 μg/ml 17α,20β-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.

In vitro exposure to inhibitors of phosphodiesterase (PDE) 3, but not PDE 4, prevent spontaneous resumption of meiosis in rhesus macaque oocytes.

Objective: To determine whether Phosphodiesterase (PDE) 3 inhibitors selectively prevent the resumption of meiosis in primate oocytes. Design: Nonhuman primate, in vitro experiment. Materials/Methods: Oocytes were obtained surgically from rhesus macaques stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins (r-hFSH, r-hLH, Ares Advanced Technologies, Inc), plus a gonadotropin releasing hormone antagonist (Antide, AAT) to prevent a spontaneous gonadotropin surge. Oocytes with intact germinal vesicles were incubated under oil in TALP with bovine serum albumin and pyruvate with or without various doses of one of three PDE 3 inhibitors; Cilostamide (Sigma), Milrinone (Sigma) or ORG 9935 (Organon), or a selective PDE 4 inhibitor Rolipram (Sigma). An inverted microscope at 200× was used to assess the meiotic maturation of oocytes at 24 and 48 hours. Results: At 24 hours, 72 of 121 (59.5%) control oocytes progressed to germinal vesicle breakdown (GVBD) compared with 0/28 (0%, p < 0.01), 4/36 (11.1%, p < 0.01) and 9/34 (26.5%, p < 0.01) oocytes incubated with Org 9935 at 1.0 μM, 0.5 μM, and 0.1 μM, respectively. Whereas the inhibition of GVBD at 24 hours produced by 1.0 μM Cilostamide (2/24 oocytes, 8.3%, p < 0.01) was similar to 1.0 μM ORG 9935, a higher concentration of Milrinone (100 μM) was necessary to achieve comparable results (2/32, 6.3%, p < 0.01). By 48 hours, additional control (82/121, 67.8%) and 1.0 μM Cilostamide (4/24, 16.7%, p < 0.01) treated oocytes progressed to GVBD, but not oocytes in 1.0 μM ORG 9935 or 100 μM Milrinone. In contrast, no significant difference in GVBD was noted between control oocytes and those incubated with up to 100 μM Rolipram for 24 hours (43/58, 74.1%) or 48 hours (44/58, 75.9%). Conclusions: These experiments establish the specificity and dose-dependent ability of PDE 3, but not PDE 4, inhibitors to block resumption of meiosis in macaque oocytes in vitro. Since different forms of PDE are present in the oocyte (PDE 3) and follicular cells (PDE 4), PDE inhibitors have potential as oocyte specific contraceptives without altering other events in the ovarian cycle. Supported By: (NICHD/NIH SCCPRR HD18185-17, HD01243, RR00163).

Bovine cumulus-oocyte-complex-quality is reflected in sensitivity for α-amanitin, oocyte-diameter and developmental capacity

The aim of the present study was to find more parameters to define developmental competence of cumulus-oocyte-complexes (COCs). Bovine COCs were divided into five groups based on their morphology. In order of increasing level of atresia: COC-A had a bright, compact cumulus investment; COC-B1 also had a compact cumulus investment, but was darker than COC-A; the color of COC-B2 was comparable with COC-B1 but the corona radiata appeared to dissociate from the rest of the cumulus investment; the cumulus of COC-B3 was almost black and the corona radiata was almost completely dissociated from the rest of the cumulus investment; COC-C had a strongly expanded cumulus investment with dark spots of degenerated cells. An increasing level of atresia was accompanied by: (1) an increasing zona pellucida diameter (147.6, 150.8, 151.0, 154.3 and 155.1 μm, respectively, for COC-A, COC-B1, COC-B2, COC-B3 and COC-C); (2) an increasing oocyte diameter except for COC-C (120.9, 122.8, 122.8, 123.9 and 118.4 μm, respectively); (3) an increasing developmental competence except for COC-C (13.9, 14.7, 17.4, 19.1 and 11.5%, respectively, development to morula and blastocyst after in vitro embryo production (IVP)) and (4) by a increasing percentage of oocytes exhibiting germinal vesicle breakdown (GVBD) after 24 h culture with α-amanitin and FSH, except for COC-A (34.4, 68.6, 51.0, 22.0 and 4.8%, respectively, oocytes arrested in GV). In general, embryo-quality, expressed in nuclei-count, was significantly affected by COC-quality ( P<0.05) with B3>B1>B2>A>C. However, if the developmental stages were compared separately, this effect was less evident. It was found that, in absence of FSH, α-amanitin was unable to inhibit GVBD and that the success of GVBD inhibition was positively correlated to the amount of cumulus cells surrounding the oocyte. COCs that had been exposed to α-amanitin and FSH during maturation retained the ability to cleave after IVF, but were unable to develop any further.

Identification of a C-terminal cdc25 sequence required for promotion of germinal vesicle breakdown

Glutathione S-transferase (GST)-cdc25B(31-566) induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. Purified, N-terminally truncated forms of cdc25B did not induce GVBD, even though many had phosphatase activity and activated cdc2 in vitro. N-terminally truncated forms of cdc25B inhibited induction of GVBD by longer forms of the enzyme suggesting a direct interaction in vivo. cdc25B(356-556), but not cdc25B(364-529), inhibited GVBD induction by GST-cdc25B(31-566) suggesting that a region of cdc25B near to the C-terminus was responsible for the inhibition. To determine the region of peptide sequence that was inhibitory, cdc25B(356-556) was subjected to proteolysis with endoproteinase lys-C. Following a demonstration that the resulting peptide mixture inhibited GST-cdc25B-dependent GVBD, a series of peptides spanning amino acids at the C-terminus were synthesized. The peptide TRSWAGERSR inhibited GVBD induced by GST-cdc25B. An alanine scan of the peptide revealed residues critical for GVBD inhibition, and site-directed mutagenesis of the corresponding residues in GST-cdc25B(31-566) eliminated its ability to induce GVBD. These results demonstrate that a cdc25B C-terminal domain, involved in dominant-negative inhibition of GVBD-competent cdc25B, is required for induction of GVBD following microinjection into oocytes.

Eicosatetraynoic and eicosatriynoic acids, lipoxygenase inhibitors, block meiosis via antioxidant action.

We previously showed that nordihydroguaiaretic acid (NDGA) and other antioxidants inhibit the resumption of meiosis in oocyte-cumulus complexes (OCC) and denuded oocytes (DO). Because NDGA is well known to be an inhibitor of lipoxygenases (LOX), we assessed whether other LOX inhibitors influence spontaneous germinal vesicle breakdown (GVBD) in OCC and DO. Spontaneous GVBD in rat OCC obtained from preovulatory follicles was significantly and reversibly inhibited by the minimum effective doses of 80 and 100 microM 5,8,11, 14-eicosatetraynoic acid (ETYA) and 5,8,11-eicosatriynoic acid (ETI), respectively. In DO, GVBD was significantly inhibited by 100 microM ETYA or ETI. The minimum effective concentrations of ETYA and ETI for inhibition of GVBD in either OCC or DO are approximately 30- to 50-fold higher than the concentrations necessary to inhibit LOX activity by 50% in intact cells. Because we previously showed that NDGA and other antioxidants inhibit the spontaneous resumption of meiosis, we assessed whether ETYA and ETI may act similarly as scavengers of reactive oxygen species (ROS). Luminol-amplified chemiluminescence showed that 50 microM of either ETYA or ETI markedly and significantly reduced ROS generated with 10 mM 2, 2'-azobis(2-amidinopropane)dihydrochloride (AAPH). Moreover, incubation of DO with 30 mM AAPH reversed the inhibition of GVBD produced by 100 microM ETYA or ETI. These findings support the conclusion that ETYA and ETI inhibit oocyte maturation by acting as antioxidants rather than by inhibiting LOX.

Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes.

In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.