Study On The Roles Of Protein Kinases CK2 And ERK5 During In Vitro Maturation Of Mammalian Oocytes

Oocyte meiotic maturation is a precise and complicated process of development and is one of the most important determinants of female animal fertility,which directly affects the efficiency of livestock production.This process involves the faithful regulation of many functional proteins,thereby ensuring the normal operation of a series of organelles and the orderly occurrence of various biological events in the oocytes.Casein kinase 2(CK2)and extracellular signal regulated kinase 5(ERK5),a class of serine/threonine protein kinases that are widely present in diverse types of cells,have been found to participate in the regulation of the cell cycle,such as cell proliferation,cell differentiation,apoptosis and DNA damage repair.However,the exact roles of CK2 and ERK5 during oocyte meiotic maturation have not been fully determined.We thus proposed that CK2 and ERK5 take essential parts in oocyte development.The present study investigated the functions and potential mechanisms of CK2 and ERK5 during in vitro maturation in porcine and mouse oocytes using their specific inhibitors,providing the theoretical basis for improvement of oocyte in vitro maturation and livestock fertility.The specific research content and results are as follows:Experiment 1:CK2 modulates the spindle assembly checkpoint to orchestrate porcine oocyte meiotic progressionIt has been shown that CK2 is involved in multiple processes related to cell cycle regulation,including cell proliferation,cell differentiation,apoptosis,DNA repair and circadian regulation.Taking advantage of porcine oocyte in vitro maturation system,immunofluorescence and intensity measurement analysis,immunoblotting analysis and inhibitor treatment,we demonstrated that CK2 is essential for meiotic maturation of porcine oocytes by regulating the spindle assembly checkpoint(SAC).The results of Immunofluorescence and immunoblotting analysis showed that CK2 was phosphorylated and constantly present on the chromosome during the oocyte meiotic maturation.Inhibition of CK2 activity by its specific inhibitor CX-4945 on the activity of CK2 hindered the meiotic progression by showing the dramatical reduction of polar body extrusion and metaphase I(M I)arrest,which was caused by the aberrant spindle assembly-induced SAC activation.In addtion,we observed that inhibition of CK2 led to the decreased level of acetylated tubulin and weakened microtubule stability,thereby impairing the spindle/chromosome structure in oocytes.Last,we found that the level of DNA damage was remarkably increased in CK2-inhibited oocytes as assessed by yH2A.X staining,indicating that DNA damage might be another critical factor resulting in the meiotic arrest of oocyte maturation.In sum,our findings document that CK2 orchestrates the porcine oocyte maturation by ensuring normal spindle assembly and DNA damage repair.Experiment 2:ERK5 modulates the spindle assembly to coordinate the oocyte meiotic maturationERKs are a member of the mitogen activated protein kinase(MAPK)family and contain five subfamily proteins ERK1-ERK5.Among of them,ERK1 and ERK2 signalling pathways have been well studied.They are widely expressed and involved in the regulation of mitosis and meiosis in a variety of cells.However,the research of ERK5 is lacking,and particularly the exact function of ERK5 during oocyte meiosis is unknown.The immunostaining result in the present study showed that ERK5 was uniquely localized in the spindle pole region at M I and M II stages in oocytes,indicating that ERK5 might be implicated in the spindle assembly event.By contrast,ERK5 is localized predominantly in the cytoplasm and secondarily in the nucleus in the mitotic cells.We further employed ERK5 specific inhibitor XMD8-92 to investigate the role of ERK5 during oocyte maturation and observed that inhibition of ERK5 caused the substantially decreased rate of first polar body extrusion and arrest of oocytes at M I stage.In addition,inhibition of ERK5 enhanced the level of acetylated tubulin and over-strengthened microtubule stability,which compromised the microtubule dynamics and spindle assembly,thereby provoking SAC and preventing the metaphase to anaphase transition in oocytes.Together,our study demonstrates that ERK5 has a unique regulatory function in spindle assembly during oocyte meiotic maturation.