The Role Of Rab Proteins In Oocyte Meiosis

The oocyte is a central component of female fertility. Growth and maturation of the oocyte are highly coordinated processes involving both nuclear and cytoplasmic events. Accurate control of spindle assembly and chromosome organization is required for orderly meiosis during oocyte maturation, where errors in this process can lead to the generation of aneuploid eggs. Fertilization of aneuploid eggs in humans is a major cause of pregnancy loss, and if there is survival to term, it will result in developmental disabilities. Therefore, it is important to maintain the integrity of the genome and the stability of genetic material of the oocyte. Although various molecules have been proposed to contribute to spindle/chromosome defects in oocyte meiosis, the pathways and mechanisms that modulate the meiotic apparatus remain to be discovered. In this study, we used mouse oocyte as a model, through PMO and RNAi technology toknock down Rab5a and Rab6a. We also used immunofluorescence and confocal laser technology to study the role of Rab5a and Rab6a in oocyte meiosis and to explore its mechanism. In addition, we used in-vitro aged porcine oocyte as a model to study the effects of Sirtl on oocyte aging. This study contains the following 6 parts.Experiment 1. Rab5a affects meiotic progression in oocytesThe aim of this experiment was to examine Rab5a localization and effects on spindle, chromosomes and kinetochore during mouse oocyte meiosis, through specifically reducing the expression of Rab5a protein in oocytes with morpholino microinjection. The results showed the presence of Rab5a-containing vesicles in oocytes at different developmental stages, these vesicles reside in the entire immature oocytes, and many of them appear to be accumulated in the GV. As the oocytes enter into metaphase, the vesicles became uniformly localized in the cytoplasm. Of note, during anaphase and telophase, numerous vesicles became concentrated around the spindle/chromosome region. Such a dynamic distribution pattern suggested that vesicular Rab5a may have a function in the formation or stability of meiotic spindle/chromosomes or in regulation of meiotic progression. Next, to investigate the function of Rab5a in oocyte meiosis, we microinjected the Rab5a-targeting morpholino (Rab5a-MO) into fully grown oocytes. As a result, Pbl extrusion was decreased in Rab5a-MO oocytes compared with controls, Confocal microscopy revealed that Rab5a-MO oocytes displayed a high frequency of chromosome misalignment and obvious spindle morphology defects, remarkably including spindle elongation. We immunostained kinetochores with CREST and microtubules with anti-tubulin antibody, quantitative analysis demonstrated that the loss/merotelic attachment in Rab5a-MO oocytes was greatly increased in frequency relative to control cells, which could contribute to the chromosome alignment failure observed in Rab5a-depleted oocytes. And loss of Rab5a resulted in 3-fold increase in incidence of aneuploidy eggs compared with controls. These findings suggested Rab5a play an important role during meiosis of mouse oocytes.Experiment 2. The study on signal pathways of Rab5a during meiosisThis experiment was to study the effects of Rab5a on Lamin, NuMA, CENPF by immunofluorescence. In order to find a potential mechanism that would explain the requirement of Rab5a for maintaining spindle morphology and chromosome alignment in oocytes. Immunostaining with anti-lamin A/C antibody showed the presence of intact nuclear lamina in control GV oocytes. With the resumption of meiosis, nuclear lamina was completely dispersed in most of prometaphase/metaphase of control oocytes, In contrast, persistent lamin around the region of meiotic chromosomes, was observed in Rab5a-MO metaphase oocytes more than control oocytes (P<0.05). Double staining further revealed colocalization of NuMA and Rab5a vesicles inside the GV. However, demonstrated that Rab5a depletion in oocytes had no significant effects on NuMA localization or levels, suggesting that NuMA is unlikely to be an effector protein for Rab5a in oocytes. CENPF puncta were uniformly distributed in GV oocytes, double staining was also performed in metaphase oocytes and confirmed that CENPF puncta colocalize with kinetochores. Notably, following Rab5a knockdown, localization of CENPF to kinetochores was severely reduced and total CENPF was significantly decreased, based on immuno staining and Western blotting respectively. These data suggested that Rab5a is essential for the properly disassembly of nuclear lamina and CENPF localization/levels at kinetochores depend on Rab5a during oocyte meiosis.Experiment 3. CENPF mediated the action of Rab5a to oocyte meiosisGiven the drastic effects of Rab5a depletion on CENPF localization/levels, we reasoned that functional ablation of CENPF may recapitulate at least some of the meiotic defects observed in Rab5a-MO oocytes. To investigate this possibility, fully grown oocytes were microinjected with CENPF-targeting morpholino (CENPF-MO) and then stained with anti-tubulin antibody to evaluate spindle and counterstained with PI for chromosomes. Endogenous levels of CENPF were partially reduced as evidenced by Western blot. Consistent with the hypothesis, CENPF-MO oocytes displayed similar phenotypes. Chromosome misalignment was readily observed in oocytes depleted of CENPF. CENPF knockdown elevated the proportion of K-MT misattachments in oocytes. In addition, to test whether Rab5a and CENPF might function in a parallel or in a linear pathway, simultaneous depletion of Rab5a and CENPF in oocytes was conducted. We found that the aberrant meiotic phenotypes of the single knockdowns were not exacerbated in the double knockdown oocytes, consistent with Rab5a and CENPF operating in the same pathway. Together, these observations suggested that CENPF is a major, if not unique, target mediating the action of Rab5a on meiotic apparatus during oocyte maturation.Experiment 4. The role of Rab6a in mouse oocyte meiosisThis experiment was designed to examine the effects of Rab6a on oocyte meiosis by depleting Rab6a with siRNA microinjection. The spindle and chromosome in control and RNAi oocytes were stained and observed under confocal laser scanning microscope. The results showed that, after RNAi, the expression level of Rab6a protein decreased significantly. Pb1 extrusion was decreased in Rab6a-KD oocytes compared with controls, P<0.05, laser confocal scanning revealed the Rab6a effects on chromosomes alignment (P<0.05). In this chapter, the results indicate the involvement of Rab6a in meiotic process.Experiment 5. The effect of Rab6a on cell organelles during meiosisRab6a, as a small GTPases, closely associated with vesicular transport. In this experiment, we knocked down Rab6a by microinjection siRNA, and investigated the influence of Rab6a on the organelles which are related to vesicle transport during oocyte meiosis. Cortical granule distribution, endoplasmic reticulum and Golgi were labeled by immunofluorescence in control group and Rab6-KD group, and then the effects of Rab6a on cortical granules distribution, endoplasmic reticulum and Golgi apparatus were evaluated. The results showed that the distribution of cortical granules were affected significantly and the percentage of failed CGFD in MII stage oocytes increased (P<0.05). The distribution and fluorescence intensity of Golgi did not change obviously. But the distribution and fluorescence intensity of endoplasmic reticulum in the GV stage and MII stage oocytes were disrupted (P< 0.05). These results showed that, Rab6a has significant effects on oocyte maturation by disturbing the distribution of cortical granules and endoplasmic reticulum.Experiment 6. The preliminary study of the molecular regulation of oocyte agingOocyte quality is a very important factor in fertilization and subsequent embryo development. Therefore, it is necessary to investigate the molecular regulation of oocyte aging process. We uesd in vitro aged porcine oocytes as a model, and examine the effects of resveratrol (Sirtl activator) on oocyte aging. Sirtuins have been widely reported to be involved in multiple biological processes. However, their function during pig oocyte aging has not been reported yet. Here, we first identify that Sirtl expression is dramatically reduced in pig in vitro-aged oocytes. Furthermore, by confocal scanning and quantitative analysis, we found the increased frequency of spindle defects and chromosome misalignment, disturbed redistribution of cortical granules and mitochondria during oocyte in vitro-aging. Importantly, these aging-associated defective phenotypes could be ameliorated through resveratrol (Sirtl activator) treatment during pig oocyte maturation, providing the evidence for the hypothesis that decreased Sirtl is one of a number of factors contributing to oocyte in vitro-aging. In summary, our data indicated a role for Sirtl in pig oocytes and uncover a striking beneficial effect of Sirtl expression on aged oocytes.