Effect Of Meiotic Mitosis Inhibitor On Maturation And Development Of Procine Oocytes

In vitro maturation of pig oocyte had been studied for decades, but the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) remains limited. Asynchronous of cytoplasmic and nuclear maturation may be one of the main factors affecting the subsequent development competence of procine oocytes. This study aimed to improve the oocyte development capacity regulated by meiotic mitosis Inhibitor.In the first experiment, the optimum effective dose and culture period of times with cilostamide were determined. Oocytes were randomly cultured in the following4groups:medium without the inhibitor cilostamide (control)(i) and medium supplemented with cilostamide of20μM (ii);40μM (iii);60μM (iiii). Oocytes in groups (ii)(iii) and (iiii) were exposed to cilostamide for11h,22h and33h, respectively. GV arrest was assessed during the in vitro maturation (IVM) at11h、22h and33h. The cilostamide was subsequently removed by transfer of oocytes to a fresh IVM medium. Then the percentage of oocytes matured to metaphase II were calculated. The result showed that groups exposed to40μM and60μM cilostamide had a significantly higher proportion of oocytes arrested at GV stage (44.50%,48.60%vs34.67%, P<0.05), while group exposed to20μM cilostamide was increased, but not significant. All groups cultured with cilostamide for llh、22h and33h had a very significantly higher proportion of oocytes arrested at GV stage (65.78%,48.59%,11.29%, P<0.01). Oocytes treated with cilostamide were resumed meiosis and reached maturation after culture in IVM media without cilostamide for44h. The groups exposed to cilostamide had very significant increased than the control (61.80%,62.99%,57.69%vs53.43%, P<0.01).In the second experiment, the effect of cilostamide on microfilaments and microtubules were investigated. Microfilaments were observed in the cortex during the period of germinal vesicle and germinal vesicle breakdown and concentrated to the chromosomes during the period of pro-metaphase、metaphase I and anaphase I-telophase I. In the period of metaphase II, the microfilaments were around the first polar body. Microtubules were not detected during periods of germinal vesicle and germinal vesicle breakdown and then produced and encompassed the chromosome. Soon afterwards, microtubules were seen in the meiotic spindle during the period of metaphase I、anaphase I-telophase I and metaphase II, oriented perpendicularly or parallelly. There was no influence neither on ermorphology of microtubules and microfilamentsinthe nor on chromatin remodeling in the MⅡ stage of procine oocytes by prematuring with cilostamide.In the third experiment, investigation of embryonic development in vitro following treatment with cilostamide was carried out. The results revealed that oocytes exposed to20μM cilostamide were more likely to have higher embryonic development rate. Investigation of embryonic development via parthenogenetic activation revealed that the cleavage and blastocyst rates and blastocyst cell number of oocytes that were prematured with20μM cilostamide were higher from oocytes that underwent conventional IVM, but it was not significant (97.78%vs92.18%,40.72%vs35.09%,71.91vs64.20, P>0.05). While oocytes were fertilized in vitro, the cleavage and blastocyst rates prematured with20μM cilostamide were significantly higher comparing the control and the blastocyst cell number was lightly more than with the control, but not significant.These results from the present study indicate that cilostamide can reversibly arrest the meiosis resumption, and improve the ability of oocyte subsequent development, but have no negative effects on morphology of cytoskeleton and chromatin remodeling.