| Part?: Expression of P38?MAPK in the follicular development of female ratsObjectives: P38?MAPK signaling pathway plays a critical role in female reproduction, However, its expression and function in rat are still unclear. This study was carried out to explore the expression and function of P38?MAPK during the development of ovarian follicle in rats.Methods: SD male rats and adult female rats were randomly selected and Mating in accordance with the ratio of 1:2 to breed in neonatal rats and ovaries were collected from different ages of neonatal rat, including 2d, 4d, 8d, 12 d, 16 d, 20 d, 30 d respectively.Results: P38?MAPK was continuous expression in follicle cells and granulosa cells, In the different follicle cells the expression of P38?MAPK was relatively stable, however, the expression of p-P38?MAPK was gradually increased. In rat granulosa cells of different ages, P38?MAPK expression was also relatively stable, p-P38?MAPK expression showed dynamic changes trend,in 12 d, 30 d the expression of p-P38?MAPK was relatively less, but in 16 d, 20 d was more. Western-blot detected that the expression of P38?MAPK in ovary was relatively steady, p-P38?MAPK expression was increased at first and subsequently reduced, the expression was relatively more in 16 d and 20 d.Conclusion: P38?MAPK was expressed during the follicle development of rats and may play an important role in the development of ovarian follicles in rats.Part?: To explore the localization and function of P38?MAPK in oocytesObjectives: This part was designed to investigate the localization and function of MPKA alpha P38 in rat oocytes.Methods: Rat oocytes were cultured in vitro, the expression and localization of P38?MAPK, p-MK2, p-P38?MAPK during oocytes meiosis of different time periods were detected by immunofluorescence and the quantitative expression of p-P38?MAPK in oocytes were detected by Western-blot. P38?MAPK specific inhibitor SB20358 was used to co-cultured oocytes and oocyte germinal vesicle breakdown rate and maturity rate were detected, besides, the quantitative expression of p-P38?MAPK during SB203580 co-cultured oocytes in 3h and 12 h respectively was detected by Western-blot.Results: The study showed that at the GV phase P38?MAPK was distributed in whole cell, including the cytoplasm and nucleus, which was mainly concentrated in around nucleus; During M?, P38?MAPK mainly localized in the cytoplasm and in spindle regional gradually decrease. At the GV stage, p-mk2 localized in the cytoplasm and mainly around nucleus; by M?p-MK2 was gradually migrated to spindle and nucleus. p-P38?MAPK mainly localized in the germinal vesicle at the GV stage, there was no expression in the nucleus; After GVBD, p-P38?MAPK was distributed in the cytoplasm; by M?, p-P38?mapk was gradually migrated to spindle and nucleus;p-P38?MAPK tended gradually to the poles and distributed around the nucleus at the A?-T? stage; At the M?, p-P38?MAPK distributed around the spindle again. Western-blot assay showed that p-P38?MAPK expression began at the GV stage and the expression was the most at M?and M?. When the SB203580 was used to co-culture oocyte, there were obvious statistical differences between the experimental group and control group about germinal vesicle breakdown rate was 58.8±0.6% and 82.5±1.6% respectively(P<0.05) after 3h; When the oocyte was co-cultured for 12 h,the maturation rate in SB203580 treatment group(40.1±3.1%) was significantly lower than the control group(73.3±3.7%) and there was obvious statistical significance(P<0.05). Western-blot showed that the expression of p-P38?MAPK in SB203580 treatment group was obviously lower than the control group when oocyte was cultured at 3h or 12 h respectively and there was also obvious statistical significance(P<0.05).Conclusion: P38?MAPK may play a vital role in the process of rat oocyte meiosis and can promote oocyte maturation. |
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