The Function Of Mitogen Activated Protein Kinase(MAPK) In Meiosis Reinitiation Of Zebrafish Oocytes

Mitogen Activated Protein kinase(MAPK) is a kind of Ser/Thr protein kinases that involve in many physiological course of various cells.The enzyme family is divided into three categories:the extracellular signal regulated kinase(ERK), the c-Jun NH terminal kinase( JNK)and p38 MAPK. The ERK family include a variety of protein kinases including ERK1 and ERK2 which have been widely researched. ERK1 and ERK2 are usually referred to as MAPK. Recent studies showed that MAPK is believed to play an essential role in meiosis. To study the function of MAPK in meiosis of zebrafish (Danio rerio) oocytes, we first cultured zebrafish oocyte in vitro induced by various concentrations of FSH and 17α,20β-DHP .Secondly, we identified the relationship between GVBD(germinal vesicle breakdown)and phosphorylated MAPK(p-MAPK) by the method of Western Blot. At last, the localization of MAPK is identified through immunohistochemistry.The first part: Comparison of the effects of different hormones and Ebstablishment of the zebrafish oocytes culture method in vitro. Zebrafish oocyte maturation were induced by various concentrations of FSH and 17α,20β-DHP. The results showed that:①The GVBD% of the oocytes treated with FSH at 1IU/mL was higher than that in other groups and control . Compared with the control group, the GVBD% of oocytes at 2 h, 4 h, 6 h, 8h increased by 11.98%, 16.67%, 6.31%, 14.25%, separately, Add 10μg/mLU0126, its rate of GVBD at 2h, 4h, 6h, 8h decreased by 26.11%, 29.2%, 23.12%, 28.73%.②The GVBD% of the oocytes treated with 17α,20β-DHP at 5ng/mL was the higher than that in other groups and control . Compared with the control group, the GVBD% of oocytes at 2h, 4h, 6h, 8h increased by 17.84%,44.67%,27.62%,23.03% , separately; Add 10μg/mLU0126, its rate of GVBD at 2h, 4h, 6h, 8h decreased by15.19%,46.82%,32.38%,27.4%. The results indicated that both of FSH and 17α,20β-DHP initiated germinal vesicle breakdown (GVBD). The 5 ng/mL of 17α,20β-DHP significantly increased GVBD of oocytes and activated MAPKThe second part: The function of Mitogen Activated Protein kinase(MAPK) in meiosis reinitiation of zebrafish(Danio rerio) oocytes. On the basis of experiments part one above, we detected the MAPK activity in zebrafish oocytes treated with 17α,20β-DHP at 5ng/mL by Western Blot. The results showed that MAPK was activated before GVBD occurred in mass of oocytes. The higher expression of MAPK, the higher ratio of GVBD.The activated MAPK in control groups was found at 4h and the expression was gradually increased from 4h to 8h. Compared with the control group, The activated MAPK in oocytes treated with 17α,20β-DHP was found at 2h and the expression was gradually increased from 2 h to 6 h. The expression of MAPK reached the maximum at 6h. At last, we inhibited the MAPK acticity with 10μg/mL U0126, then the ratio of GVBD and the MAPK activity were significantly reduced.The results indicated that MAPK played an important role in promoting the ovary development.The third part: The spacial and temporal distribution of MAPK during GVBD At first, on the basis of traditional paraffin sections, we improved the paraffin section protocol to make ovarian tissue and optimized the paraffin section process to obtain the high quality structures of oocyte tissues. The oocytes at different developmental stage could be distinguished clearly. The results could meet the test requirements. Next, the position of MAPK in oocytes was localized at different stages by means of immunohistochemistry. The histochemistry assay showed that MAPK was activated in the oocyte plasm at the oocyte development stages II and III while no MAPK activity was observed at the stage I. At the stage V, the acitvated MAPK could be observed both in the plasm and nucleus . The results indicated that both of FSH and 17α,20β-DHP initiated germinal vesicle breakdown (GVBD) of zebrafish oocytes.The secretion of 7α,20β-DHP was induced by FSH. 17α,20β-DHP significantly increased GVBD% of oocytes and activated MAPK. Activated MAPK was in the oocyte plasm firstly and then moved into nucleus. All results indicated that activated MAPK played an important role in promoting the ovary development.