Expression And Functional Analysis Of Asymmetric Factors In Mouse Oocyte Maturation

Asymmetric cell division,in which a cell divides into two cells of different developmental potentials,is a fundamental means of generating cell diversity.An asymmetric division produces two cell types that differ in size,morphology,and/or the developmental determinants that they inherit.In the latter case,determinants are polarized within a cell and the division plane partitions these determinants unequally between the daughter cells,producing two cells with different development in many organisms.In all organisms,oocyte maturation is an extreme example of asymmetric division.Many asymmetric divisions require the establishment of asymmetries in the cell prior to division,which implies the existence of polarity-estbalishing molecules. Some of these asymmetries are set up already in the oocyte,before fertilization.In the mouse,the female germ cells undergo two consecutive meiotic divisions with unequal cytokinesis thus forming two polar bodies at the animal pole of the egg.The polarity of meiotic divisions develops during the first meiotic metaphase spindle and the polar location of the first meiotic metaphase spindle and the polar location of the first polar body,which is followed by the polar localization of the second meiotic spindle and of the second polar body.As a result,the mouse secondary oocyte,arrested in the metaphase of the second meiotic division,is already a polarized cell.The polarity of the oocyte is reflected in the localization of meiotic spindle and polar body and the asymmetrical distribution of cytoplasm.It is believed that the structural and molecular polarity of the oocyte cytoplasm,and its oolemma are caused by the polar positioning of the meiotic spindle during maturation.The mechanism that in mammals regulates asymmetrical positioning of the spindle during meiotic division of the oocyte remains elusive.But the mechanism between different organisms is largely evolutionarily conserved.The cellular distribution of maternal factors and their functions are usually studied in single cell organisms and multi-cell epithelium.Accordingly,this study was designed with the purpose to disclose the role of two conserved genes or pathway in oocyte maturation and early preimplantation embryos, which is divided into the following two parts:1.Expression and functional analysis of the cAMP/Epac/Rap1 pathway at oocytes and early perimplantation embryos.2.Expression and functional analysis of Dlg1 during oocyte maturation. PART ONE:EXPRESSION AND FUNCTIONAL ANALYSIS OF DLG1 IN MOUSE OOCYTESⅠ:MEIOTIC MATURATION INDUCES ASYMMETRIC DISTRIBUTION OF DLG1 IN MOUSE OOCYTES.Background and Purpose:Discs large homolog 1(Dlg1) proteins had been demonstrated to play an important role in cell polarity in Drosophila neuroblasts, where they induced the cortical localization of two conserved proteins(Pins and Gαi). These two cortical proteins play central role in regulating asymmetric cell division and spindle-cortex interactions from worms to mammal.But Dlg1's role in mammal oocyte has never been investigated.Here we aim to reveal the distribution of Dlg1 in different stages of oocytes and find its relationship with cytoskeleton.Materials and Methods:Firstly,we used Western-blot and RT-PCR to identify Dlg1's existence at different stages of mouse oocytes.Next we used immunohistochemistry to disclose its distribution in ovary.Oocytes collected in vitro or matured in vivo were fixed for immunocytochemistry to demonstrate the localization of Dlg1.Finally,we disclosed the relationship between the distribution of Dlg1 and cytoskeleton by adding specific drugs to the maturation medium at different time.Results:Western-blot and RT-PCR analysis showed that Dlg1 was present in GV and MII-stage oocyte,and its quantity seemed doubled during meiotic maturation.For the first time,we showed that Dlg1 was expressed in both germ and follicle cell through different stages of oocyte development from primordial follicle(＜80μm) to Graafian follicle(＞380μm).For the oocytes,Dlg1 staining distributed throughout the cytoplasm and various stages of oocytes.It appeared that the intensity of Dlg1 staining got higher in follicle cells as the progression of oogenesis.And we investigated thoroughly its spatial-temporal subcellular location during oocyte maturation.Firstly,Dlg1 was evident around the GV membrane and uniformly distributed in the cytoplasm.Dlg1 was restructured to colocalize with spindle at the time of GVBD.GVBD was essential for the initiation of Dlg1 reorganization,since Dlg1 structure did not change in GV-arrested oocyte.Dlg1 reorganization was also prevented by the microtubule inhibitor nocodazole.Dlg1's redistribution at GVBD was therefore MTs(microtubules)-dependent and cell cycle-dependent.Then the colocaliztion of Dlg1 with spindle began to migrate to the oocyte cortex,companied by emerging network of Dlg1 at the vegetal cortical.Distinctive cresent vegetal cortical clusters of Dlg1 were formed close to the time of Pb1(polar body 1) extrusion and were ever evident in MII oocytes.Formation of the characteristic Dlg1 clusters was prevented by the deploymerisation of microfilaments.Conclusions:These experiments revealed that Dlg1 exists at different stages of oocytes and follicle cells,Dlg1 may involve in the regulation of tight junction of follicle cells.Mouse oocyte meiotic maturation induced the polarization of Dlg1 and this polarized action is highly related with cell cytoskeleton.Dlg1 may involve in the regulation and establishment of oocyte polarity.Ⅱ:MATERNAL DEFICIENCY OF DLG1 BLOCKS MEIOTIC MATURATION IN MOUSE OOCYTES.Background and Purpose:RNA interference(RNAi) is an effective tool for studying gene function in oocytes.Short hairpin RNAs(shRNA) has the advantage of reducing the potential for off-target interference and increase RNAi specificity in oocytes.Materials and Methods:Two couple(ID 160540 and ID 160541) of predesinged annealed 21-nt shRNA for Dlg1 was chemically synthesized by Ambion company. shRNA in a final concentration of 50μM and 100μM was microinjected into the cytoplasm of GV stage oocytes.Each oocyte was injected about 10 pL of siRNA.The control group was injected with same amount of positive(GAPDH) control and negative control.After microinjection,the oocytes were incubated in G1 medium containing 100μg/ml cAMP for 30min and then washed thoroughly with IVM medium.The oocytes were then cultured in IVM medium for the evaluation of GVBD and PB1 extrusion after 4 hr,8 hr and 18 hr respectively.The oocytes were collected for real-time RT-PCR to measure the mRNA levels and immunocytochemistry and confocal analysis at the same time.Results:In the first place,a concentration of 50μM was used for microinjection,both of the two couples of shRNA microinjected have no effect on cell cycle progression of oocyte maturation.So we increased the concentration of shRNA to 100μM.The two siRNA sequences all have silencing effect on Dlg1,but ID 160540 was better and its inhibitory rate was 70%.The most efficient time was 18 hr post microinjection.In 4 hr,8 hr and 18 hr,the rate of GVBD and Pb1 emission of Dlg1 siRNA injected were significantly lower than the negative control group.There was no difference between each group in GAPDH gene.Conclusions:The effect of Dlg1 siRNA in mouse oocyte was time-and concentration-dependent.The most efficient siRNA sequence was ID 160540 and the time was 18 hr.Maternal deficiency of Dlg1 blocked the oocyte maturation progress. PART TWO:EXPRESSION AND FUNCTIONAL ANALYSIS OF EPAC AND RAP1 IN OOCYTES AND PREIMPLANTATION EMBRYOS.Ⅰ:DYNAMIC EXPRESSION OF EPAC AND RAP1 IN MOUSE OOCYTES AND PREIMPLANTATION EMBRYOSBackground and Purpose:cAMP is an important second messenger that has long been recognized to control the initiation of meiosis through activation of Protein Kinase A(PKA) in oocytes.However,PKA is not the only target for cAMP.Recent work in cAMP-dependent and PKA-independent pathways suggests that Rap1 (Ras-related protein-1) is activated through its Epac proteins(exchange proteins directly activated by cAMP),cAMP responsive guanine exchange factors(GEFs) involved in various cellular processes.Evidence is accumulating that Rap1 is clearly linked to actin dynamics and functions in the spatial and temporal control of cell polarity.The aim of this study was to reveal the existence of an Epac and Rap1 in mouse oocytes and embryos.Materials and Methods:Five-week-old B6D2F1 mouse ovaries were fixed for immunohistochemistry.Different stages of oocytes and early preimplantation embryos were used for RT-PCR and immunocytochemistry.Results:Results obtained indicate that Epac and Rap1 have different dynamic subcellular localizations and expression patterns in oocytes and embryos.For Epac,at germinal vesicle stage,Epac was first seen as small accumulations scattered in the cytoplasm;after germinal vesicle breakdown,a network of small clusters,distributed uniformly throughout the cytoplasm,then became evident;but at the meiosisⅡstage, the accumulations appeared to become reduced and spread out in the ovoplasm;at one-cell stage,the clusters disappeared and Epac became uniformly distributed throughout the embryos.But Rap1 dispersed uniformly as small punctuations throughout the cytoplasm during the progress of oocyte maturation.When developed into one-cell stage embryos,the small dots aggregated into visible clusters of particles that mainly occupied the cortical region of the blastomeres.This phenomenon was evident even in two-cell and four-cell stage embryos and persistent in subsequent early preimplantation embryos.Conclusions:These results indicate for the first time that the Eapc and Rap1 do exist and have differential dynamic subcellular distribution in mouse oocytes and embryos, which in turn suggests multiple and specific functions during oocyte maturation and embryo development.Ⅱ:FUNCTIONAL ANALYSIS OF EPAC AND RAP1 IN MOUSE OOCYTES AND EMBRYOSBackground and Purpose:In a former experiment,we demonstated that Epac and Rap1 have differential dynamic expression pattern in oocytes and embryos.According to the earlier experiment,Epac had a unique distribution during oocyte maturation but this characteristic expression was lost when entering MII stage.And Rap1 got its typical expression only beginning from pronuclear-stage embryos.So in the present study,we investigated the functional role of Epac and Rap1 in oocytes and embryos based on their unique subcellular locations.Materials and Methods:GV oocytes and pronuclear stage embryos were recovered from the ovaries and oviducts of primed 5-week-old B6D2F1 female mice.Epac and Rap1 antibodies(200 mg/ml in 1ml PBS containing 0.1%NaN3 and 0.2%gelatin) were microinjected into the cytoplasm of GV oocytes and 1cell stage embryos respectively.Each experiment was repeated three times,and 30-40 oocytes per group per experiment were used.A microinjection volume of about 10 pl per oocytes or embryos was used in all the experiments.The same amount of rabbit IgG(200 mg/ml in 1ml PBS containing 0.1%NaN3 and 0.2%gelatin) was injected as control.The oocytes and embryos were then cultured in IVM and G1 medium respectively for the evaluation of cell cycle progression and immunocytochemistry and confocal analysis at the same time.Results:We investigated the effects of Epac on oocyte maturation by antibody microinjection.The GVBD rate of oocytes 3hr after antibody injection(71.6%, 155/217) was significantly lower than that of the IgG-injected control group(85.3%, 91/107).The rate of the first polar body extrusion 13hr after antibody injection (12.9%,28/217) was significantly lower than that of the IgG-injected control group (67.2%,71/107).To determine importance of Rap1 in early embryo mitotic progression,anti-Rap1 antibody was microinjected into the cytoplasm of pronuclear-stage embryos and cleavage events were assessed in 24-hr intervals. Embryo development(99.3%,136/137) had not significant difference past the two-cell stage by 48 hr of culture compared to controls(100%,83/83).But the antibody microinjection did significantly compromise the outcome of blastula formation(42.3%,58/137) for a prolonged culture compared with control(80.7%, 67/83).Conclusions:Epac and Rap1 do have differential effect on cell cycle progression during oocyte maturation and preimplantation embryo development.Epac present in GV oocytes plays an important role in the process of GVBD and first polar body extrusion.Rap1 may have no effect on the development of early stages of embryos but is responsible for outcome of blastula formation.