Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.

Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes-associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P-MST/MST1 and upregulated the ratio of P-YAP1/YAP1 significantly, whereas Yap1-treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo-YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development.

Degradation of securin in mouse and pig oocytes is dependent on ubiquitin-proteasome pathway and is required for proteolysis of the cohesion subunit, Rec8, at the metaphase-to-anaphase transition.

Although securin/separase/cohesion pathway was reported to regulate chromosome segregation during meiotic metaphase-to-anaphase transition, little biochemical evidence was provided. We recently found that oocytes could not progress beyond meiotic metaphase when ubiquitin-proteasome pathway was inhibited, but the mechanisms remain unclear. In the present study, we investigated the quantity of securin and Rec8 protein and the localization of securin, a cohesion subunit, during oocyte meiosis providing data in support of the hypothesis that the effect of ubiquitin-proteasome pathway on metaphase-to-anaphase transition was mediated by regulating securin and Rec8 degradation in mouse and pig oocytes. In germinal vesicle-stage oocytes, immunostaining of securin was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, immunoreactive securin accumulated around the condensed chromosomes at prometaphase I. At metaphase I and metaphase II, when chromosomes were organized at the equatorial plate, immunoreactive securin was concentrated around the aligned chromosomes, putatively associated with the position of the metaphase spindle. The accumulation of securin could not be detected at anaphase I and anaphase II. In both mouse and pig oocytes, Western blot analysis showed that securin protein was low at germinal vesicle stage, reached the highest level at metaphase I, while decreased at anaphase I. Securin was increased again at metaphase II, while it was decreased at anaphase II. Rec8 protein was present in germinal vesicle-stage oocytes and remained until metaphase I, while it was decreased at anaphase I. Like securin, Rec8 was increased at metaphase II, while it was decreased again at anaphase II. The inhibition of the ubiquitin-proteasome pathway inhibited the decrease in securin and Rec8 at metaphase-to-anaphase transitions in both mouse and pig oocytes. Microinjection of securin antibody into MII-arrested oocytes leads to the degradation of Rec8. In conclusion, these results suggest that the proteolysis of securin is dependent on ubiquitin-proteasome pathway and is necessary for the degradation of Rec8 during meiotic metaphase-to-anaphase transitions in mouse and pig oocytes.

The Nuclear Mitotic Apparatus (NuMA) protein is contributed by the donor cell nucleus in cloned porcine embryos.

The Nuclear Mitotic Apparatus (NuMA) protein is a multifunctional protein that is localized to the nucleus in interphase and to the poles of the mitotic apparatus during mitosis. In unfertilized porcine oocytes, NuMA is localized to the meiotic spindle. NuMA is removed along with the meiotic spindle during the enucleation process before reconstructing the egg by introducing the donor cell nucleus to produce cloned embryos. Questions have been raised regarding the source for NuMA in cloned embryos, as the enucleated oocyte does not contain detectable NuMA in the cytoplasm. To determine the source of NuMA in porcine nuclear transfer (NT) embryos, we conducted an immunofluorescence microscopy study with antibodies against NuMA to investigate the appearance and distribution of NuMA before and after reconstructing NT embryos with porcine skin fibroblasts. We used donor cells from a confluent culture with all cells in interphase. For comparative studies, we also determined the immunofluorescence pattern of NuMA, gamma-tubulin, and alpha-tubulin in porcine fibroblasts, parthenogenetic embryos and in vitro fertilized (IVF) embryos. Results show that NuMA was localized in nuclei of 33.5% (163/456) of the serum-deprived fibroblasts used as donor cells. No NuMA staining was detected in enucleated pig oocytes. Immediately after nuclear transfer, NuMA staining was absent in all donor cell fibroblast nuclei (0 h) but staining was detected by 6 h within the reconstructed eggs, at which time the transferred somatic cell nucleus swelled in most cells (19/27) and became a pronucleus-like structure. NuMA was localized exclusively within the pronucleus-like structures (15/27). At 25 h, NuMA was detected inside the nucleus (16/25) either in one-cell or in 2-cell stage embryos. Interestingly, in parthenogenetic embryos, NuMA staining was not detected in all 42 eggs examined at 1 h, and evident NuMA staining was only detected inside a few (4/51 at 6 h; 6/48 at 25 h) of the nuclei. In IVF embryos, NuMA was detected within the nucleus at 6 h (5/20) and 25 h (13/16). These results show that the donor cell nucleus contains NuMA that is contributed to the reconstructed embryo and possibly activated by mechanisms in the oocyte's cytoplast.

Cyclic adenosine 3',5'-monophosphate-dependent activation of mitogen-activated protein kinase in cumulus cells is essential for germinal vesicle breakdown of porcine cumulus-enclosed oocytes.

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation.

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

MEK1/2 regulates microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation.

It is well known that MAPK plays pivotal roles in oocyte maturation, but the function of MEK (MAPK kinase) remains unknown. We have studied the expression, subcellular localization and functional roles of MEK during meiotic maturation of mouse oocytes. Firstly, we found that MEK1/2 phoshorylation (p-MEK1/2, indicative of MEK activation) was low in GV (germinal vesicle) stage, increased 2h after GVBD (germinal vesicle breakdown), and reached the maximum at metaphase II. Secondly, we found that P-MEK1/2 was restricted in the GV prior to GVBD. In prometaphase I and metaphase I, P-MEK1/2 was mainly associated with the spindle, especially with the spindle poles. At anaphase I and telophase I, p-MEK1/2 became diffusely distributed in the region between the separating chromosomes, and then became associated with the midbody. The association of p-MEK1/2 with spindle poles was further confirmed by its colocalization with the centrosomal proteins, gamma-tubulin and NuMA. Thirdly, we have investigated the possible functional role of MEK1/2 activation by intravenous administration and intrabursal injection of a specific MEK inhibitor, U0126, and by microinjection of MEK siRNA into oocytes. All these manipulations cause disorganized spindle poles and spindle structure, misaligned chromosomes and larger than normal polar bodies. Our results suggest that MEK1/2 may function as a centrosomal protein and may have roles in microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte maturation.

Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage.

RhoA, a small GTPase, plays versatile roles in many aspects of cell function such as stress fiber formation, cytokinesis, and cell polarization. In this study, we investigated the subcellular localization of RhoA and its possible roles during oocyte maturation and fertilization. RhoA was localized in the cytoplasm of eggs from the germinal vesicle (GV) stage to 2-cell stage, especially concentrating in the midbody of telophase spindle when oocyte extruded PB1 and PB2. The RhoA kinases (ROCKs) specific inhibitor Y-27632 blocked GV breakdown (GVBD) and first polar body extrusion, but did not affect apparatus formation and anaphase/telophase I entry. Anti-RhoA antibody microinjection into the oocytes showed similar results. RhoA inhibitor caused abnormal organization of microfilaments, failure of spindle rotation, PB2 extrusion as well as cleavage furrow formation, while sister chromatid separation was not affected. Microinjection of RhoA antibody also blocked PB2 emission. Our findings indicate that RhoA, by regulating microfilament organization, regulates several important events including GVBD, polar body emission, spindle rotation, and cleavage.

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos.

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, gamma-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. gamma-tubulin translocated into the two poles of the transient spindles, while no accumulated gamma-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, gamma-tubulin was translocated to the spindle poles. The distribution of gamma-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. gamma-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse.

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.

Cell cycle-dependent localization and possible roles of the small GTPase Ran in mouse oocyte maturation, fertilization and early cleavage.

The small GTPase Ran controls numerous cellular processes of the mitotic cell cycle. In this experiment, we investigated the localization and possible roles of Ran during mouse oocyte meiotic maturation, fertilization and early cleavage by using confocal laser scanning microscopy, antibody microinjection and microtubule disturbance. The results showed that Ran was localized mainly in the nucleus (except for the nucleolus) in the oocyte, zygote and early embryo. At pro-metaphase of meiosis I, Ran distributed throughout the cell, but predominantly concentrated around the condensed chromosomes. During the completion of meiosis I and meiosis II, it concentrated to the meiotic spindle microtubules except for the midbody region. After sperm penetration, Ran dispersed with the extrusion of the second polar body and gradually concentrated in the male and female pronuclei thereafter. Ran was also observed to exist diffusely in the cytoplasm in prophase; it concentrated at the mitotic spindle, and migrated to the nucleus during early cleavage. Ran's concentration around the spindle disappeared when microtubule assembly was inhibited by colchicine, while it was concentrated around the chromosomes after microtubule stabilization with taxol treatment. Ran did not display any role in cytokinesis during division when pseudo-cleavage of germinal vesicle-intact oocytes was induced. Anti-Ran antibody microinjection decreased the germinal vesicle breakdown and the first polar body extrusion, and distorted spindle organization and chromosome alignment. Our results indicate that Ran has a cell cycle-dependent localization and may have regulatory roles in cell cycle progression and microtubule organization in mouse oocytes, fertilized eggs and early embryos.

Inducible nitric oxide synthase-derived nitric oxide regulates germinal vesicle breakdown and first polar body emission in the mouse oocyte.

The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I-telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase-telophase transition.

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos.

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.

[Expression of c-fos in various functional state of rat ovaries].

AIM AND METHODS: The method of labeled streptavidin biotin was used to study the expression of c-fos in various functional state of rat ovaries and its relationship with the levels of serum estradiol and progesterone. RESULTS: (1) In the mature rat, c-fos expression was found higher in interstitial gland and stroma of proestrous ovaries and lower in estrous ovaries, and was found higher in luteal cells and stroma of pregnant ovaries and lower in diestrous ovaries. There was a positive correlation between the area and optical density of c-fos expression and the levels of serum E2 and P. (2) In the immature rats, c-fos expression was not found in the ovaries containing preantral follicles from DES-treated rats,and was found both in interstitial gland and stroma of the ovaries containing preovulatory follicles from PMSG-treated rats ,and the expression was higher in day 4 luteinized ovaries and lower in day 9 luteinized ovaries from PMSG with hCG treated rats. There also was a positive correlation between the area and optical density of c-fos expression and the levels of serum E2 and P. CONCLUSION: The results suggested in rat ovaries and might play an important role in follicles development, ovulation, luteum formation and regression.

[Experimental study of c-erbB2 on the fertilization in mouse].

AIM AND METHODS: The distribution of ErbB2 in mouse testis, epididymidis, ovaries, oocyte-cumulus cells-complexes in oviducts and sperms was investigated immunohistochemically. To study the effect of c-erbB2 on mouse fertilization in vitro, various concentrations of c-erbB2 antisense oligonucleotides (c-erbB2 ASODNs) were incubated with sperms and oocyte-cumulus cells-complexes during fertilization in vitro. To explore possible mechanisms involved in fertilization, the relationship between c-erbB2 ASODNs and GABA, or dbcAMP, or verapamil during fertilization in vitro was also observed. RESULTS: ErbB2 oncoprotein was observed in epithelial cells in epididymis, sperms and cumulus cells. C-erbB2 ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rate of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-erbB2 ASODNs group, and nonsense at oligonucleotides group (20 micromol/L) was 38.3%, 19.6%, 10.7%, 5.0%, and 33.8% respectively. Integral optical density immunostaining of ErbB2 in sperms was notably reduced. Medium and high concentration of c-erbB2 ASODNs notably inhibited cumulus cells adhering to inner wall of Petri dish. Treated alone with GABA or dbcAMP, the rate of fertilization was increased. Both GABA and dbcAMP partially inversed the ASODNs inhibition effect on fertilization rate, but neither of them showed significant effect on sperm integral optical density of ErbB2 immunostaining. In contrast, verapamil inhibited fertilization rate. Co-treated with c-erbB2 ASODN, verapamil showed synergic inhibiting effect on fertilization with c-erbB2 ASODN. Verapamil also inhibited the expression of c-erbB2 in sperms. CONCLUSION: It is suggested that c-erbB2 is closely correlated with fertilization. Ca2+ may inhibit fertilization in vitro through regulation the expression of c-erbB2 gene in sperm cells, while both of GABA and dbcAMP may affect the process of fertilization through the way other than c-erbB2 expression in sperm cells.

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos.

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization.

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

Localization of gamma-tubulin in mouse eggs during meiotic maturation, fertilization, and early embryonic development.

Gamma-tubulin, a member of the tubulin superfamily, is a peri-centriolar component which is considered to be essential for microtubule nucleation. The dynamics of gamma-tubulin during mouse oocyte meiotic maturation, fertilization, and early cleavage as well as the co-localization of gamma-tubulin and alpha-tubulin during the formation of the meiotic I spindle were studied by confocal microscopy. We found that gamma-tubulin was evenly distributed in the germinal vesicle (GV) stage oocyte. After germinal vesicle breakdown (GVBD) gamma-tubulin dots were localized in both the cytoplasm and the vicinity of the condensed chromosomes, and aligned at both poles of the meiotic spindle at prometaphase I and metaphase I. At anaphase I and telophase I, gamma-tubulin was detected between the separating chromosomes, while it was absent in the midbody. At the MII stage, gamma-tubulin was again accumulated at the spindle poles. Alpha-tubulin had a similar distribution pattern as gamma-tubulin in the cytoplasm and radiated from gamma-tubulin foci close to the chromosomes during the meiotic spindle formation. After fertilization, gamma-tubulin was translocated from spindle poles to the area between separating chromatids and distributed around the pronuclei. It aggregated into some dots during the interphase, but was distributed on the mitotic spindle poles in early embryos. Our results suggest that gamma-tubulin is essential for microtubule nucleation and spindle formation during mouse oocyte meiosis, fertilization, and early embryo cleavage.

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.

Somatic nucleus remodelling in immature and mature Rassir oocyte cytoplasm.

Successful production of cloned animals derived from somatic cells has been achieved in sheep, cattle, goats, mice, pigs, rabbits, etc. But the efficiency of nuclear transfer is very low in all species. The present study was conducted to examine somatic nucleus remodelling and developmental ability in vitro of rabbit embryos by transferring somatic cells into enucleated germinal vesicle (GV), metaphase I (MI) or metaphase II (MII) oocytes. Microtubules were organized around condensed chromosomes after the nucleus had been transferred into any of the three types of cytoplasm. A bipolar spindle was formed in enucleated MII cytoplasm. Most of the nuclei failed to form a normal spindle within GV and MI cytoplasm. Some chromosomes scattered throughout the cytoplasm and some formed a monopolar spindle. Pseudopronucleus formation was observed in all three types of cytoplasm. Reconstructed embryos with MI and MII cytoplasm could develop to blastcysts. Nuclei in GV cytoplasm could develop only to the 4-cell stage. These results suggest that (1) GV material is important for nucleus remodelling after nuclear transfer, and (2) oocyte cytoplasm has the capacity to dedifferentiate somatic cells during oocyte maturation.