Localization And Function Of MSpindly During Mouse Oocyte Meiotic Maturation.

As in mitosis, the spindle assembly checkpoint is ensuring correct segregation of chromosomes or sister chromatids by preventing the onset of anaphase. Even a single misaligned chromosome is sufficient to generate an anaphase waiting signal to maintain SAC activation and delay the anaphase onset. SAC signal pathway includes Mad1, 2, 3 (Mad for mitotic arrest deficient) and Bub1, 2, 3 (Bub for budding unperturbed by benzimidazole) as major components. Besides these proteins, Mps1 and Aurora B amplify the SAC signal and regulate mitotic checkpoint complex formation. In metazoan cells there are three additional proteins, Zw10, Rough Deal (Rod) and Zwilch, that play a crucial role in mitotic spindle checkpoint functions. Recently, it was determined that the proteins form a complex (the RZZ complex), which is required for recruitment of kinetochore components (dynein/dynactin complex, Mad1, and Mad2). Spindly is a newly found spindle assembly checkpoint protein which was first identified in S2 cells of Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all these species, Spindly and its homologues execute the function of recruiting dynein or dynein/dynactin complex to kinetochores and as a kinetochore-microtubules association regulator by cooperating with RZZ complex in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for recruiting dynein to unattached kinetochore and shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein/dynactin complex and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. To data, we have not seen any report on study of Spindly homologues in meiosis of any animal, and no study on mouse at all.The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockout of mSpindly. Firstly, by using Western Bolt and immunofluorescence we identified the expression and subcellular localization of mSpindly in the meiosis of mouse oocytes. And then, we used overexpression and RNAi techniques to study the function of mSpindly in the meiotic process of mouse oocytes.Female mice Kunming white,4- to 6-week-old, were used in our study. Before oocytes collection the mice were injected with 7.5～10 U PMSG for the superovulation.In the experiments, we collected 6 typical stage samples of mouse oocytes: GV, GVBD, pro-MⅠ, MⅠ, AⅠ-TⅠand MⅠstages. With Western blot, we found that mSpindly protein was expressed from GV to MⅠstages, without detectable change in expression level in the whole process. This implies that the Spindly homologue, mSpindly, was exist in mouse oocyte meiosis, and the level of its expression has been kept in a balanced level during the meiotic process. Next, we extracted the total RNA from mouse oocytes, and got the cDNA after RT-PCR. We used the cDNA as template to clone the full length mSpindly gene sequence and cloned this sequence to pCS2+MT vector which already has a Myc-tag in it. Then we got the Myc-mSpindly mRNA after transcription invitro. We microinjected the Myc-mSpindly mRNA into cytoplasm of GV stage mouse oocytes. After the exogenous mRNA was translated into Myc-mSpindly protein, we used FITC-anti-Myc antibody to incubate the oocytes, and then observed the subcellular localization of mSpindly in the mouse oocytes with confocal. We found that when the oocytes developed to GVBD stage, mSpindly was specifically localized to kinetochores. In MⅠand MⅠstage oocytes, mSpindly was typically localized to both the kinetochores and the spindle poles.As our result showed that mSpindly do express and take specific subcellular localization during meiosis of mouse oocytes; we then microinjected high concentration of Myc-mSpindly mRNA into mouse oocytes at GV stage to overexpress mSpindly, and observed the localization of the overexpressed Myc-mSpindly with confocal; we also calculated the time of the first polar body emission (PBE) and spread the chromosomes of the PBE oocytes to see that whether overexpression of mSpindly influenced exact segregation of the chromosomes. We found that overexpression of mSpindly factitiously did not change the subcellular localization of mSpindly in mouse oocytes meiosis, and did not promote the first polar extrude, too. In the chromosome spreading samples we observed no influence of overexpression of mSpindly on exact segregation of chromosomes. Next, we microinjected the mSpindly Morpholino into the GV stage mouse oocytes to depleted mSpindly, and then made observations on the spindle shape, chromosome alignment and the percentage of PBE. We found that depletion of mSpindly caused an arrest of MⅠstage. Most of the mSpindly-depleted oocytes couldn't enter anaphase and failed to extrude the first polar body, accompanied with abnormal spindle and misalignment of chromosomes.Our data suggested that mSpindly participates in SAC silencing and in spindle formation, the mechanism of its function might be as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but it would need to cooperate with other factors to fulfill its function.The present study found the Spindly homologues in meiosis for the first time, and proved that this Spindly homologue participates in meiotic SAC, and depletion of it caused constant SAC activation, pro-MⅠ/ MⅠstage arrest, abnormal spindle shape, unaligned chromosomes and failed extrude the first polar body. All the above phenomena suggested that the mSpindly-depleted mouse oocytes couldn't maturate normally, and it would affect the following fertilization and embryonic development. Our study also provides some basic materials for the therapy of mammal infertility.