The Correlation Of P21 And DNA Double-stranded Breaks In Bovine Oocytes

P21 inhibit CDK1 kinase phosphorylation,that is mainlyresearch about P21 in mammalian meiosis.the correlation of P21 and DNA Double-stranded breaks in bovine oocytes has not been reported.Recent studies have reported that P21 can promotes DNA double-stranded breaks repair in somatic cell. In oocyte P21 whether has this effect is our focuse.P21 was determined the importance of maturation by over-expression and interference in bovine oocytes.Pvenus – P21 eukaryotic expression vector is stored by lab.Using DNA double-stranded breaks manufacturing reagent Zeocin, designed a concentration gradient test on Hela cells,and detected Hela cells’ proliferation rate.The purpose is to confirm the Zeocin concentration’ reference range for use on oocytes.Then use the reagent on bovin oocytes. Gamma H2 AX immunofluorescence appraisal the breaks are formed.Statistic analysis the relationship between DNA Double-stranded breaks and oocyte maturation rate.Analysis the gene P21、P53、about homologous and non homologous recombination expression changes.We find that oocytes and Cumulus-oocyte complexes’ s sensitivity to the reagent is not the same. We use ataxia telangiectasia mutated(ATM) specific inhibitor ku55933 processing oocytes, observing the oocytes maturation. At the same time detect whether P21 gene and oocyte quality evaluation gene’s expression are correlated. the purpose is to determine whether the expression of P21 quantity can be as a reference of oocyte quality evaluation.The results were as follows:1.first, I dentified the laboratory reserves p Venus- P21 gene eukaryotic expression vector of correctness. It can express in Hela cells and bovine oocytes. By over-expression and interferencein in bovine oocytes, determined the P21 gene on the important role of bovine oocyte maturation.2.Used reagent Zeocin the DNA double-strand breaks maker on Hela cell and detected cells’ proliferation rate.Determines the Zeocin’ concentration range on oocytes.Using the specificity of the double strand breaks marker gamma H2AX’ immunofluorescence to determine the formation of double strand breaks.Only DNA double-strand damage reaching a certain serious, can block the first polar body extrusion(PBE1).In DNA double-strand breaks cases the oocyte can’t PBE1’s P21 expression level higher with the increase of damage serious.First reaction is not sensitive, and then the expression increased(corresponding with the mature rate).In the case of DNA double-strand breaks, the relative expression of the ATM and the P21’s trend are the same in the immature oocytes(ATM is not dominant repair but the dominant P53- function of P21 cycle block). In the case of DNA double-strand breaks, KU70(participating in the homologous recombination)’s expression with the increase seriously of breaks increased at first and then decreased in mature oocytes.Not mature oocytes always declined. In the mature oocytes P21’ expression is always low;In immature oocytes P21,ATM, P53’s express trends are consistent.3.COCs on Zeocin damage is insensitive.4.ATM function and the completion of the first division has a very close relationship. when ATM is restrained, oocyte maturation rate reduce;Under ku55933 the regent of the specific inhibitor of ATM, GDF- 9, BMP- 15(oocytes evaluation factor) ‘ expression had a reduced trend. H1 foo contrary to their trend;In mature oocytes FSHR’expression raised significant.Under ku55933, immature oocytes’ p21 expressed reduced.Get the following conclusion: bovine oocytes are insensitive to DNA double-strand breaks during meiotic division for the first time. mature rate decreased when damage over a certain range, the cause of the decline that is applied to the ATM- P53- P21 affect.In the process of the first division suffered a DNA double-strand damage is obviously improved the homologous recombination KUN70 expression speculated that the homologous recombination dealing with DSBs plays a role.Cumulus-ooocyte complexes is insensitive to the regent of DNA double strend breaks. Granular cells play an important role in the first meiotic division.ATM activity is importent to the completion of the oocyte meiotic first division.when ATM is inhibited the mature rate decline. In the case of ATM are suppressed, immature oocytes’ H1 ffo trend and GDF- 9, BMP – 15 is contrary. Under the action of ATM inhibitors, mature oocytes FSHR’ expression increases significantly.