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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

[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.