Abstract
BackgroundOLA1 is a member of the GTPase protein family; unlike other members, it possess both GTPase and ATPase activities, and can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response, protein synthesis and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown.MethodsIn this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity.ResultsImmunofluorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.
Highlights
Mammalian gametes are yielded through an event named meiosis where two consecutive divisions are conducted to halve the chromosomes without an intervening replicative process (Verlhac & Terret, 2016)
Once the correct connection of chromosome with microtubule is established, all chromosomes are correctly aligned at the metaphase I plate (MI stage) spindle assembly checkpoint (SAC) proteins will dissociate from kinetochore to trigger the segregation of chromosomes, which bring out the polar body extrusion and oocyte will arrest at metaphase II (MII)
After 6 h post-germinal vesicle breakdown (GVBD) in vitro culture, most of meiosis resumed-oocytes in control group have reached at MI (Fig. 4A), while we found some oocytes were extruding the polar bodies in OLA1-knockdown oocytes
Summary
Mammalian gametes are yielded through an event named meiosis where two consecutive divisions are conducted to halve the chromosomes without an intervening replicative process (Verlhac & Terret, 2016). Spindle assembly and chromosome segregation are both vital courses to keep genomic stability during oocyte meiosis. After germinal vesicle breakdown (GVBD), as absent of centrosome at the mitosis, the meiotic spindle is emanated and nucleated from microtubule organizing centers (MTOCs) (Schuh & Ellenberg, 2007; Clift & Schuh, 2015; Bennabi, Terret & Verlhac, 2016). At this time, chromosomes and spindle microtubules has begun to interact with each other. We concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis
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