Molecular Regulation Of Oocyte Polar Body Extrusion And Early Embryo Cleavage In Pig

Mammalian oocyte maturation and embryo development, which are defined as accurate and ordered a multi-stage regulation processes, is one of the important themes of life science research. The assembly and dynamics of cytoskeleton, especially actin and microtubule cytoskeleton play a crucial role on the regulation of oocytes maturation and early embryo development. Oocyte cortical reorganization and polarization is also essential for polar body extrusion during meiosis, in addition, accurate meiotic chromosome segregation is critical for successful oocyte meiosis and early embryonic development. This series of process is defined as molecular regulatory events, and during which, any defects are likely to result in abnormal of oocytes maturation or fertilized eggs development. But so far, the related molecular mechanism remains unclear during mammalian oocyte meiosis and early embryonic development. Therefore, it is important to clarify the molecular regulatory mechanisms of oocyte maturation and early embryonic development for better understanding the discipline and regulatory mechanism of mammalian reproduction, meanwhile, establishing the theoretical foundation for improving livestock reproductive efficiency and even possible give the treatments for human infertility diseases.In this experiment, pig was employed as the experimental animal model. Using in vitro culture, drug treatment, small interfering RNA (siRNA) injection, parthenogenetic activation, immunofluorescent staining and confocal microscopy, as well as western blot, we analysed the possible related molecular regulatory informations of oocyte maturation and early embryonic development. Firstly, using immunofluorescence staining, we examined the expression and localization of KIF20A, Dynamin2, RhoA, ROCK during pig oocyte maturation or parthenogenetic embryos development, respectively. Subsequently, we disrupted the protein function through protein-specific inhibitors or RNA interference (RNAi) so as to investigate their regulatory roles on oocyte maturation and early embryonic development, actin and spindle assembly, meiotic spindle migration and positioning, cortical granules (CG) reorganization and distribtion, as well as chromosome segregation. In addition, by western blot, we also examined expression of actin or microtubule-associated protein after disruption of Dynamin2 or RhoA, looking forward to put further insight in their molecular signal pathways of GTPase for the regulation of polarity during pig oocyte meiosis, and in order to establishing the theoretical foundation for furher understanding the molecular ragulatory mechanism during oocyte meiosis and embryonic development. This study is divided into six parts, the main results are as follows:Experiment 1. The research of KIF20A regulat pig oocyte meiotic cell cycleKIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. But until now, there is no evidence that KIF20A can able to participate in pig oocyte meiotic regulation. In the present study, we investigated whether KIF20A functions in pig oocyte meiotic maturation. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MⅡ). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that KIF20A inhibition effected on the cell cycle during meiosis, the percentage of oocytes that arrested at early metaphase I (MI) stage increased; however, the proportion of oocytes at anaphase/telophase I (ATI) and MⅡ stages decreased significantly. In addition, our study also proved that the inhibition of KIF20A activity had no effects on morphology of the meiotic spindle. Therefor, these results demonstrated that KIF20A is critical for meiotic cell cycle during pig oocyte maturation.Experiment 2. Distribution and regulation of KIF20A during early embryonic development in pigKinesin family member KIF20A, is one of the key regulatory elememts for ensure successful mitosis and benefit for reverse transport of Golgi vesicles to the nearby endoplasmic reticulum. Our previous studies have shown that KIF20A can regulate the meiotic cell cycle progression during pig oocyte maturation, whereas the role of KIF20A during early embryonic development is unclear. Our study was the first time to explore the localization and fuction of KIF20A during early embryonic development. The results of immunofluorescence staining showed KIF20A exhibited specific localization in the early embryos, mainly distributed in cortex and nucleus of 2-cell,4-cells and 8-cell embryos. Inhibition of KIF20A activity resulted in blocked early embryo cleavage. Statistical results reflected that the development rate of 2-cell and 4-cell significantly decreased. Our results show that, KIF20A expresses in pig early embryos, playing an pivotal regulatory role during early embryo development.Experiment 3. Large GTPase Dynamin2 regulates polar body extrusion during pig oocyte maturationMammalian oocyte meiotic maturation is distinguished by unique asymmetric division, but the regulatory mechanisms and signaling pathways involved are poorly understood. Dynamins are ubiquitous eukaryotic guanosine triphosphatease (GTPases) involved in membrane trafficking and actin related dynamics processes, whose roles in mammalian oocyte maturation have not been determined. In this study, we explore the localization, function and regulatory mechanisms of Dynamin2 during pig oocyte meiosis. By the immunofluorescence staining, the results showed that Dynamin2 localized at the cortex of GV oocytes, from GCBD to MⅡ stsge, Dynamin2 accumulated both at the meiotic spindle and in the cortex of oocytes, with a distribution similar to that of actin. In addition, the co-localization of Dynamin2 and α-tubulin showed the distribution of Dynamin2 at meiotic spindle. Inhibiting Dynamin2 activity in pig oocytes with the specific inhibitor dynasore resulted in failed polar-body extrusion. This phenotype may have been due to aberrant actin distribution and/or spindle positioning as inhibitor treatment also disrupted the formation of the actin cap and cortical granule-free domain, which negatively impacted spindle migration and positioning. Moreover, the distribution of ARP2, a key actin-nucleation factor, was severely reduced in the cortex after dynasore treatment. Thus, our results suggest that Dynamin2 possibly regulates pig oocyte maturation through its effects on actin-mediated spindle positioning and cytokinesis, and that this may depend on regulating ARP2 localization.Experiment 4. The expression and functions of small GTPase RhoA during pig oocyte maturationMammalian oocyte meiosis includes a series of cellular events, finally, extrued first polar body after the establishment of oocyte polarity. Oocyte asymmetric division is regulated primarily by cytoskeleton, including microtubules and microfilaments. RhoA is a member of Rho guanosine triphosphatease (GTPases) superfamily of the Ras-related small GTP binding protein. Through binding its effector, the activation of RhoA can turn their downstream signaling pathways, including the regulation of cell polarity, migration and division as well as other important cellular processes. In this study, we investigated the roles of RhoA in mammalian oocyte meiosis by disruption the activation or expression through RhoA inhibition or RNAi. Immunofluorescence staining results showed that RhoA was expressed in all the stages of pig meiotic oocytes, from GVBD to MⅡ stage, RhoA not evenly distributed in the cortex of pig oocyte, but also in meiotic spindle microtubules. The co-localization of RhoA and a-tubulin analysis further confirmed the distribution of RhoA on the meiotic spindle. Disrupting RhoA activity or depleting the expression of RhoA caused the failure of COCs diffusion and polar body emission. This may have been due to decreased actin assembly and subsequent spindle migration defects. By the western blot analysis, we found that the involvement of RhoA in this process may have been though its regulation of actin nucleators ROCK, p-Cofilin, and ARP2 expression. In addition, spindle morphology was also disrupted and p-MAPK expression decreased in RhoA inhibited or RhoA KD oocytes, which indicated that RhoA also regulated MAPK phosphorylation for spindle formation. Thus, our results demonstrated indispensable roles for small GTPase RhoA in regulating cytoskeleton dynamics during pig oocyte meiosisExperiment 5. The expression and functions of RhoA in pig early embryoThe biological processes of emryos development begins from the fertilized eggs, small GTPase RhoA, is key regulatory factor for the assembly of the cytoskeleton, cell polarity, migration and mitosis and proliferation. Our previous study has demonstrated its indispensable roles during meiosis, in this study, we mainly explored the localization and functions of RhoA during erly embryo development. Our result showed that RhoA was specifically distributed in pig early embry, from 2-cell to morula stage, RhoA was stably localized at the cortex of pig early embryo, particularly cleavage furrow; in addition, RhoA was also enriched in the nucleus. In the zygote stage, inhibition of RhoA activity led to failure of early embryo cleavage, most embryos in 100 uM Rhosin groups even can not reach the 2-cell stage. Subsequently, by immunofluorescent staining of the nucleus, we observed the presence of two nucleus in only one blastomere of arrested embryo after suppression of RhoA activity, which indicated the failure of cytokinesis in pig embryo. Rhosin treatment also resulted in the decreased cortical actin fluorescence intensity, suggesting the reduced actin expression, which may be the main reason for embryonic cytokinesis defects. Therefore, our results indicate that RhoA regulates actin asssembly for cytokinesis, finally effects on the embryo cleavge.Experiment 6. RhoA effector ROCK regulats the pig oocyte maturationDuring oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex, subsequently, the oocyte extrudes the first polar body and forms a highly polarized egg; this process is primarily regulated by actin. ROCK, the downstream effector of Rho-GTPase, is involved in various cellular functions, such as stress fiber formation, cell migration, motility and tumor cell invasion, between which actin cytoskeleton assembly regulation is one of its most critical functions. In the present study, by inhibiton of endogenous ROCK via a specific selective inhibitor Y-27632, we investigated the effects on pig oocyte meiosis and possible regulatory mechanisms of ROCK. Firstly, we detect the localization of ROCK in pig oocyte, we found that ROCK accumulated not only at spindles, but also at the cortex in pig oocytes from GVBD to MⅡ stage. The co-labeled of ROCK with a-tubulin or actin further confirmed co-localization maner of ROCK with spindle or cortex. ROCK inhibition resulted in the failure of pig oocyte meiotic maturation and the significantly reduced rate of the polar body extrusion, which might have been due to the impairment of actin expression and actin-related spindle positioning. In addition, ROCK inhibition also disrupted the formation of actin cap and cortical granules free domain (CGFD), which further confirmed the disruption of oocyte polarity and spindle positioning defects. Thus, RhoA effector ROCK has significant effects on oocyte meiosis via its regulatory roles on actin-mediated spindle positioning and the subsequent polar body extrusion during meiosis in pig.In summary, we analyzed the subcellular localization, function and regulatory mechanisms of cell cycle molecular KIF20A, GTPase Dynamin2, RhoA as well as its effector ROCK during pig oocyte maturation and early embryonic development in our study. Our research can provide value for further improve the awareness of related molecular events and their potential regulatory mechanisms during mammalian oocytes meiosis and early embryonic development.