The Roles Of KIF11 During Porcine Oocytes Maturation

Compared with mitosis,meiosis is a specialized form of cell division.In meiosis,mammalian oocytes undergo one DNA replication and two meiotic cell divisions.During this process,chromosome segregation is a crucial event and particularly error-prone occurring in meiosis during cell division,which might elicit the generation of aneuploidy and further affect the developmental quality of embryos.Microtubules(MT)are polymers of tubulin that form part of the cytoskeleton and shoulder the responsibilities of structure and shape to the cytoplasm of cell.It can act as substrates for motor proteins that are involved in important cellular functions such as vesicle trafficking and cell division.Motor proteins utilize the energy from ATP hydrolysis to generate mechanical work that drives the protein along the MT.Kinesin superfamily proteins(KIFs)act as one of molecular motors and are involved in material transport along microtubules to maintain normal cellular functions.KIFs are classified into 14 subfamilies containing with over 100 proteins based on a homologous composition of amino acid sequences,which have been shown to be involved with transporting organelles,individual proteins,and mRNAs to appropriate places during the cell cycle and work by sliding along the microtubules while hydrolyzing ATP for energy.KIF11(also named kinesin-5,Eg5,and KSP)is a plus-end-directed homotetrameric kinesin that regulates spindle formation for actuate chromosomal separation during mitosis.Although several studies have reported KIF11 roles in mitosis using different models,its roles in meiosis have not been demonstrated.In present work,we investigated the roles of KIF11 in meiosis with a porcine oocyte model by a specific inhibitor SB743921.In order to conduct this experiment,some research methods are engaged,like immunofluorescence,immunoblotting,confocal microscopy etc.Experimental details include the following aspects.(1)detect the protein expression level of KIF11 at various developmental stages in porcine oocytes;(2)calculate the rate of first body extrusion after KIF11 inhibitor treatment;(3)detect cell cycle progress at different cultured time and the phosphorylation level of Cdc2 after inhibitor treatment;(4)observe spindle morphology,chromosomes alignment and the acetylation level of tubulin after inhibitor treatment;(5)detect KIF11 expression level in postovulatory aging oocytes.The major results are described below.Firstly,we found continuous KIF11 expression during porcine oocyte maturation and went up to the top level at first meiotic metaphase.As the inhibitor concentration rose,the rate of first body extrusion dramatically fell and 150 nM was chose as the work concentration.Meanwhile,results revealed that the majority of oocytes were arrested at the stage of germinal vesical breakdown after inhibitor treatment and this symptom did not recover with prolonging the cultured time,phosphorylated Cdc2 and acetylated α-tubulin were both detected and shown a decreased trend after inhibition treatment.Apart from that,we also found that increased expression of KIF11 in postovulatory aging oocytes as compared to young oocytes.In conclusion,our results showed that KIF11 regulated the cell cycle and tubulin acetylation related spindle formation in porcine oocyte meiosis.