The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a widely distributed protein kinase that regulates numerous physiological functions. Inhibitors of CaMKII are useful tools for investigating the CaMKII functions. Here we identified a novel CaMKII inhibitor protein (CaM-KIIN) from the human dendritic cell cDNA library by large-scale random sequencing. Human CaM-KIIN contains 79 amino acids, which shares 98% identity and 98% positives with rat CaMKII inhibitor protein beta and 65% identity and 78% positives with rat CaMKII inhibitor alpha. Human CaM-KIIN mRNA expression was detectable in various tissues and cell lines by Northern blot and RT-PCR. To investigate its biological functions, full-length human CaM-KIIN was overexpressed in colon adenocarcinoma LoVo cells. When expressed in LoVo cells, it could inhibit cell proliferation, block cell growth, and decrease the viable cell number. These results characterize a potential cellular inhibitor protein of CaMKII that plays an important role in the regulation of cell growth.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

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.     

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.     

Nuclear envelope dynamics in oocytes: from germinal vesicle breakdown to mitosis

We have recently gained new insight into the mechanisms involved in nuclear envelope breakdown, the irreversible step that commits a cell to the M phase. Results from mammalian cell and starfish oocyte studies suggest that mechanical forces of the cytoskeleton, as well as biochemical disassembly of nuclear envelope protein complexes, play important roles in this process.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Inhibition of ras-induced germinal vesicle breakdown in Xenopus oocytes by rap-1B

A cDNA clone (Krev-1) has recently been identified that possesses the ability to reverse the transformed phenotype when introduced into a K-ras-transformed NIH/3T3 cell line. The Krev-1 protein, also known as rap-1A, was found to share 50% homology with the ras proteins. The rap-1A protein has also been shown to block the interaction of ras with its GTPase activating protein in vitro, leading to speculation regarding its role in vivo. A closely related protein, rap-1B, has also been identified in platelets, human erythroleukemia cells, neutrophils, and aortic smooth muscle cells. Unlike rap-1A, rap-1B has been shown to be phosphorylated in platelets. Given the high degree of similarity between the amino acid sequences of rap-1A and rap-1B, we sought to investigate the effect of microinjected rap-1B on H-ras(Val12)-induced germinal vesicle breakdown in Xenopus laevis oocytes. In this assay system, equimolar concentrations of rap-1B were found to block germinal vesicle breakdown triggered by the oncogenic ras protein. However, in the presence of IGF-1, this inhibition was not observed. Moreover, rap-1B is readily phosphorylated in the oocytes.     

Reconstruction of enucleated mouse germinal vesicle oocytes with blastomere nuclei

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4- or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About one-third of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.     

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes’ internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.     

Relationship between ectopic germinal vesicle breakdown in Xenopus oocytes and dorsal development of the embryo

The early development of several species involves the segregation of cytoplasmic components into different regions of the egg. In Xenopus zygotes, a 30° rotation displaces the central animal cytoplasm to the future dorsal side of the embryo. To elucidate the role of the central animal cytoplasm in dorsal determination, we induced germinal vesicle breakdown (GVBD) closer to the equator by cold/centrifugation treatment of oocytes. Centrifugation moved the germinal vesicle to the centripetal side; eggs with such displaced GVBD fertilized and began to develop normally. Dorsal embryonic structures tended to develop on the GVBD side of the egg, but displacement of the GVBD was insufficient to rescue dorsal structures in axis-deficient embryos. The labeling of yolk platelets of oocytes with Trypan Blue revealed similar cytoplasmic patterns in control and treated eggs. Furthermore, 67% of treated eggs had Danilchik's swirl, indicative of the dorsal side, on the GVBD side. In conclusion, both the swirl and dorsal development tend to occur on the GVBD side of cold/centrifuged eggs; however, displaced GVBD cannot by itself determine dorsality.     

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.     

Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and oxidant-dependent apoptosis in Xenopus laevis oocytes

Ceramide regulates many cellular processes, including cell growth, differentiation, and apoptosis. Although the effects of exogenous bacterial neutral sphingomyelinase (SMase) in Xenopus laevis oocytes have been investigated, its microinjection into oocytes has not been reported previously. Thus, we compared the incubation versus microinjection of the neutral Bacillus cereus sphingomyelinase (bSMase) to examine whether the topology of ceramide generation determines its effects on the fate of oocytes. In agreement with previous findings, incubation of mature stage VI oocytes with bSMase increased ceramide levels in oocyte extracts over time, causing the germinal vesicle breakdown indicative of maturation, without evidence of cytotoxicity. In contrast, bSMase microinjection, which increased ceramide levels in a time- and dose-dependent manner, resulted in oocyte apoptosis characterized by reactive oxygen species (ROS) generation, reduced glutathione (GSH) depletion in cytosol and mitochondria, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Microinjection of acidic SMase from human placenta recapitulated the apoptotic effects of bSMase microinjection. Preincubation of oocytes with GSH-ethyl ester before bSMase microinjection prevented ROS generation and mitochondrial downstream events, thus protecting oocytes from bSMase-induced death. These findings show a divergent action of bSMase-induced ceramide on oocyte maturation or apoptosis depending on the intracellular site where ceramide is generated.     

Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.     

The role of germinal vesicle in maturation of Pleurodeles waltlii oocytes induced by steroids

The maturation of oocytes from unstimulated Pleurodeles waltlii females can be induced in vitro. All full-grown oocytes covered by follicular envelopes mature under the influence of progesterone, testosterone, and a synthetic steroid (CDMT). Enucleation of oocytes prior to maturation induction resulted in a significantly lower percentage of maturation. Irradiation, or the treatment of oocytes with actinomycin D, did not affect their maturation. Therefore the lower percentage of maturation after enucleation must be due to the lack of karyoplasm. Karyoplasm appears to be necessary for the production of the maturation-promoting factor in P. waltlii oocytes.     

Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes

The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (H1K) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1% to 13.3%. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P < 0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity.     

Oncogenic Ras-induced germinal vesicle breakdown is independent of phosphatidylinositol 3-kinase in Xenopus oocytes.

A number of reports have identified phosphatidylinositol 3-kinase as a downstream effector of Ras in various cellular settings, in contrast to others supporting the notion that phosphatidylinositol 3-kinase acts upstream of Ras. Here, we used Xenopus oocytes, a model of Ras-mediated cell cycle progression (G2/M transition) to analyze the contribution of phosphatidylinositol 3-kinase to insulin/Ras-dependent signaling pathways leading to germinal vesicle breakdown and to ascertain whether phosphatidylinositol 3-kinase acts upstream or downstream of Ras in those signaling pathways. We analyzed the process of meiotic maturation induced by progesterone, insulin or micro-injected oncogenic Ras (Lys12) proteins in the presence and absence of specific inhibitors of phosphatidylinositol 3-kinase activity. As expected, the progesterone-induced maturation was independent of phosphatidylinositol 3-kinase since similar rates of germinal vesicle breakdown were produced by the hormone in the presence and absence of wortmannin and LY294002. In contrast, insulin-induced germinal vesicle breakdown was completely blocked by pre-incubation with the inhibitors prior to insulin treatment. Interestingly, similar rates of germinal vesicle breakdown were obtained in Ras (Lys12)-injected oocytes, independently of whether or not they had been pre-treated with phosphatidylinositol 3-kinase inhibitors. The effect of wortmannin or LY294002 on MAPK and Akt activation by progesterone, insulin or Ras was also analyzed. Whereas insulin activated those kinases in a phosphatidylinositol 3-kinase-dependent manner, progesterone and Ras were able to activate those kinases in the absence of phosphatidylinositol 3-kinase activity. Since Ras is a necessary and sufficient downstream component of insulin signaling pathways leading to germinal vesicle breakdown, these observations demonstrate that phosphatidylinositol 3-kinase is not a downstream effector of Ras in insulin/Ras-dependent signaling pathways leading to entry into the M phase in Xenopus oocytes.