Specific inhibition of mouse oocyte nuclear protein phosphatase-1 stimulates germinal vesicle breakdown

Okadaic acid (OA)-induced germinal vesicle breakdown (GVBD) and localization of protein phosphatase-1 (PP1) in oocyte nuclei are suggestive of PP1's role in regulating oocyte GVBD. To explore this possibility, we microinjected protein phosphatase (PP) inhibitors OA, anti-PP1 antibody (anti-PP1), PP1 inhibitor I2, and anti-PP2A antibody (anti-PP2A) into nuclei of roscovitine (ROSC)-arrested mouse oocytes. Oocytes were also injected with recombinant PP1 in the absence of ROSC. Oocytes were assessed for GVBD and metaphase II (MII) development at 2 and 18 hr post-injection. Data were analyzed using Cochran-Mantel-Haenszel Statistics adjusted for time. Microinjection of OA significantly enhanced GVBD in comparison to controls at 2 and 18 hr (P < 0.01), yet had no effect on MII development. Similarly, microinjection of anti-PP1 resulted in significantly higher levels of GVBD compared to controls at 2 and 18 hr (P < 0.01). Interestingly, anti-PP1 microinjection also tended to enhance MII development at 18 hr in comparison to controls (P < 0.09). Microinjection of I2, anti-PP2A, and PP1 had no effect on GVBD or MII development. If reduction of PP1 activity was important for GVBD, one would anticipate an endogenous means of regulating PP1 activity at this developmental stage. In somatic cells, phosphorylation of PP1 at Thr320 causes PP1 inactivation. Germinal vesicle-intact oocytes did not contain phosphorylated PP1, as determined using a specific Thr320-Phospho-PP1 antibody, Western blot analysis, and confocal immunocytochemistry. At or around the time of GVBD, oocyte PP1 became phosphorylated at Thr320, which remained phosphorylated through MII development. These data indicate that inhibition of intra-nuclear PP1, through specific antibody neutralization, mimics OA-stimulated GVBD, providing the first direct evidence that nuclear PP1 is involved in regulation of oocyte nuclear membrane integrity. In addition, phosphorylation of PP1 occurs at/or around GVBD indicating that inactivation of PP1 is an important intracellular event in regulation of nuclear envelope dissolution at GVBD.     

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

A dramatic loss of cumulus cell gap junctions is correlated with germinal vesicle breakdown in rat oocytes

Interactions between the cumulus-oophorus and the oocyte have been implicated in the regulation of meiotic maturation. Quantitative analysis of freeze-fractured rat cumulus-oocyte complexes reveals that the net area of cumulus cell gap junction membrane decreases about 15-fold, 2-3 hr following an ovulatory stimulus. This dramatic loss of gap junctions is temporally correlated with germinal vesicle breakdown and cumulus expansion, and is discussed with respect to meiotic maturation and ovulation of the mammalian oocyte.     

Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte

Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium.     

Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: effects of a cyclic AMP phosphodiesterase inhibitor.

The turnover of [32P]orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in [32P]phosphatidic acid (PA) and an increase in [32P]-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in [32P]PC and [32P]phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in [32P]PC and [32P]PE which accompanied GVBD. The increase in [32P]phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid. The third is associated with GVBD, and is cAMP-sensitive, and may represent stimulation of de novo synthesis of phospholipid, thereby permitting disruption of the nuclear membrane.     

Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes

Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment 1, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum.     

Signaling pathways in ascidian oocyte maturation: effects of various inhibitors and activators on germinal vesicle breakdown

The Ascidiacea, the invertebrate chordates, includes three orders; the Stolidobranchia is the most complex. Until the present study, the onset of oocyte maturation (germinal vesicle breakdown) had been investigated in only a single pyurid (Halocynthia roretzi), in which germinal vesicle breakdown (GVBD) begins when the oocyte contacts seawater (SW); nothing was known about internal events. This study strongly suggests the importance of protein phosphorylation in this process. Herdmania pallida (Pyuridae) functions like H. roretzi; GVBD occurs in SW. Oocytes of Cnemidocarpa irene (Styelidae) do not spontaneously undergo GVBD in SW but must be activated. Herdmania oocytes are inhibited from GVBD by pH 4 SW and subsequently activated by mastoparan (G-protein activator), A23187 (Ca2+ ionophore) or dimethylbenzanthracene (tyrosine kinase activator). This requires maturation promoting factor (MPF) activity; cyclin-dependent kinase inhibitors roscovitine and olomoucine are inhibitory. It also entails dephosphorylation as demonstrated by the ability of the phosphatase inhibitor vitamin K3 to inhibit GVBD. GVBD is also inhibited by the tyrosine kinase inhibitors tyrphostin A23 and genistein, and LY-294002, a phosphatidylinositol-3-kinase inhibitor previously shown to inhibit starfish GVBD. LY-294002 inhibits strongly when activation is by mastoparan or ionophore but not when activated by dimethylbenzanthracene (DMBA). The DMBA is hypothesized to phosphorylate a phosphatase directly or indirectly causing secondary activation, bypassing inhibition.     

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.     

Time sequence of germinal vesicle breakdown in pig oocytes after cycloheximide and p-aminobenzamidine block

All porcine oocytes cultured 20 hr in medium with 10 μg/ml cycloheximide rested in the germinal vesicle (GV) stage but with the highly condensed bivalents in nucleoplasm. When these oocytes were washed and cultured in the control medium for 2, 4, and 6 hr, germinal vesicle breakdown (GVBD) was completed in 0, 86, and 100% of them, respectively. When similarly inhibited oocytes cultured successively only 2.5 hr in the control medium were given again in cycloheximide enriched medium (3.5 hr), nearly all of them reached late diakinesis stage again. It means that oocytes cultured for 20 hr and washed free of this inhibitor of protein synthesis completed GVBD rapidly (4 hr) and protein synthesis crucial for nuclear membrane disintegration occurred already during the first 2 hr after washing of inhibitor. All oocytes cultured for 20 hr in medium with 1 mM p-aminobenzamidine rested in GV with chromatin around the compact nucleolus. The successive culture in cycloheximide (20 hr) and p-aminobenzamidine (10 hr) prevented GVBD in all oocytes, too. In contrast, when the oocytes washed after cycloheximide block (20 hr) were cultured in p-aminobenzamidine enriched medium 2 and 3 hr and again for 6 hr in cycloheximide medium, the nuclear membrane dissolved in 62 and 68% of oocytes, respectively. These data suggest that inhibition of protein synthesis in pig oocytes does not prevent the high condensation of bivalents in GV. However, nuclear membrane breakdown requires the successive protein synthesis and proteolysis.     

Signaling pathways in ascidian oocyte maturation: the role of cyclic AMP and follicle cells in germinal vesicle breakdown

Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 n m in a reversible manner.     

Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro

The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3, an important regulator of UPP. RAC1, a UPP-controlled regulator of actin polymerization was maintained at steady levels throughout cumulus expansion. We conclude that proteasomal proteolysis has multiple functions in the progression of oocyte meiosis beyond GV and metaphase I stage, polar body extrusion, and cumulus expansion.     

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.     

Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed.