Function And Regulation Mechanism Of Chk1 During Meiotic Maturation In Porcine Oocytes

Pig is a very important livestock for meat production in China.There are several problems to meet in our porcine breeding and production,including how to develop a good breed with high fertility,rapid growth,easy management,high meat-production rate and good meat quality.Traditional crossbreeding methods have long breeding cycle,however,the way of transgenosis and clone not only shortens the breeding cycle,but also maintains the characters which people need.The pig cloning needs larger of mature oocytes.It is difficult and expensive to to obtain mature oocytes in vivo.So these oocytes used for porcine cloning at present are got from ovaries and cultured in vitro.Compared to mouse and some other mammals,the rate of maturation in pig is still low.The maturation of porcine oocytes refers to the process that oocytes undergo the first meiotic division and reach the metaphase of the second meiotic division.The germinal vesicle stage and the onset of anaphase I(AI)are vital for maturation.In many kinds of cells,checkpoint 1 has impotent roles in the G2 stage and mitosis,which respectively are equal to the germinal vesicle stage and meiosis in oocytes.Therefore,it is important to study the functions and molecular mechanisms of checkpoint 1 during porcine oocytes maturation.Our main results are showed as follows:(1)cumulus oocyte complexes(COCs)obtained from porcine ovaries were respectively cultured for 0 h,24 h,28 h and 44 h.The mRNA and protein levels of Chk1 in porcine oocytes were detected respectively by quantitative Real-time Polymerase Chain Reaction(qRT-PCR)and western blot at each time point.We found that mRNA and protein levels of Chk1 appeared a peak at metaphase I.When we microinjected Chk1-eGFP mRNA into the oocytes,we found that Chk1 was located in the cytoplasm and at the spindle.(2)Porcine oocytes were microinjected Chk1 siRNA and respectively cultured to the time of 24 h and 44 h.We found that the oocytes underwent GVBD normally,but could not pass the metaphase I(MI).(3)We constructed the Chk1 overexpression vector and obtained Chk1 mRNA by in vitro transcription.When porcine oocytes were microinjected with Chk1 mRNA and respectively cultured to the time of 24 h and 44 h,we found that Chk1 overexpression delayed germinal vesicle breakdown in most of injected oocytes,which resulted in the decreasment of maturation rate.(4)When Chk1 knockdown porcine oocytes were cultured for 28 h,they were fixed in 4% paraformaldehyde and observed by fluorescence microscope.We found that the degradation of Cyclin B1 was blocked.(5)When Chk1 knockdown porcine oocytes were cultured for 28 h,we respectively detected the mRNA levels of Mad2L1,Mad2L2,Cdc20,Ndc80 and Cdh1 by RT-PCR.We found that only the mRNA levels of Mad2L1 and Cdh1 were declined.These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at AI and maintained transcriptional levels of Mad2L1 and Cdh1 to activate SAC and APC-Cdh1 and degrade MPF,which made sure that all chromosomes were accurately located in a line,then oocytes passed MI and AI and exited from the first meiotic division successfully.In addition,we found that the maturation of porcine oocytes did not need the DNA damage checkpoint,which was different from the mouse maturation.