Establishment Of Specific KDM2A Knockout Mouse Model And Its Effect On The Reproductive Of Male

Spermatogenesis and maturation are one of the influence factors of mammalian reproduction efficiency.In testicular seminiferous tubules,male germ cells undergo a series of differentiation and morphological changes to form mature male gametes,which are usually accompanied by epigenetic modifications.Lysine-specific histone demethylase is a regulatory factor of epigenetic modifications,which is involved in gamete formation and embryo development.The former researches indicated that several KDMs family members were involved in spermatogenesis and embryo development.KDM2A,as a member of the KDMs family,has regulated the cell proliferation,differentiation,apoptosis,aging by regulating the methylation level of H3K36 and the expression of related signaling pathways,it had proceeded to influence development and tumor formation.However,the effect of KDM2A on late spermatogenesis has not been reported.In this study,the expression of KDM2A in testis and sperm development was analyzed by real-time quantification PCR(qRT-PCR),immunohistochemistry and immunofluorescence.The mouse model of KDM2A specific knockout in sperm cell was established by Cre-loxP system.HE staining,in vitro fertilization,mating experiments and statistical analysis were used to compare the phenotypic of testicular development,sperm maturation and fertility after KDM2A knockout.The results are as follows:1.The tissues expression profile showed that the mRNA of KDM2A was highly expressed in testis and it was distributed dynamically during testis development.The expression of KDM2A increased rapidly in the prophase of testis development,and then remained stable;2.The results of immunohistochemistry showed that KDM2A was expressed in sertoli cells and spermatogenic cells except elongated sperm cells.Immunofluorescence results showed that KDM2A was mainly expressed in the tail of sperm.And its expression trend in testis development was consistent with qRT-PCR;3.The results showed that KDM2A specific knockout in sperm cell(KDM2A cKO)mice were successfully obtained.The knockout efficiency was high and the difference between the knockout and the control group was significant by qRT-PCR and immunohistochemistry(P<0.01);4.The results of testicular morphology and tissue structure comparison showed that no significant difference of the body weight,testicular weight,testes coefficient,testicular morphology,tissue structure,spermatogenesis process and the number of sperm cell between KDM2A cKO and the control group(P>0.05);5.Sperm count showed that the number of sperm in the epididymis of KDM2A cKO mice was slightly lower than KDM2A~fl/fll/fl mice,but there was no significant difference(P>0.05),and no obvious differences in the morphology of single sperm between KDM2A cKO and the control group.The results of the IVF experiment showed that no significant different in the cleavage and blastocyst rate(P>0.05),which revealed that no significant effect on the number,quality and in vitro reception of sperm after KDM2A specific knockout.6.The results of reproductive experiment showed that the average litter size and nest number of KDM2A cKO male mice decreased,but there was no statistical difference(P>0.05).The results indicated KDM2A knockout has no significant effect on the male fertility.In conclusion,the expression and localization of KDM2A in mouse testis development and mature spermatozoa were detected.The model of KDM2A specific knockout in sperm cell was successfully established to obtain KDM2A cKO mouse.There were no significant difference in testis,sperm and fertility between KDM2A cKO and KDM2A~fl/fll/fl mice.It indicated that the reproductive function of male mice had no significant change after KDM2A knockout.This may be because KDM2A only plays an important role in the prophase of spermatogenesis,but is not essential in the late spermatogenesis.In addition,the H3K36me1/2 demethylation function of KDM2A may be replaced by KDM2B.However,it is a better way to explore the biological function of KDM2B,another member of its subfamily,and its effect on spermatogenesis and fertility of male mice.