Establishment Of Mouse Plcz1 Gene Knockout Model Based On CRISPR-Cas9 Technology And Its Reproductive Ability Evaluation

With the development of industrial production,the incidence of reproductive disorders is increasing year by year,which not only causes economic losses to animal husbandry,but also poses a serious threat to human health.It has become the focus of current reproductive research to explore the mechanism of birth defects via constructing male animal models of birth defects.In recent years,many regulatory factors that play an important role in animal reproduction have been found.During fertilization,the sperm-specific phospholipase C zeta（PLCζ）enters the oocyte cytoplasm through the fusion pore,initiates the Ca2+oscillation in the oocyte,and then activates oocyte.It has been found that the deficiency of Plcz1 gene expression in male mice can lead to subfertile but not sterile,affect the early embryo development and decrease the blastocyst rate.However,the role and regulatory mechanism of PLCζ in spermatogenesis and early embryo development of offspring have not been fully clarified.There is also a lack of effective animal models of reproductive disorders in the study.In this study,Plcz1 gene knockout（Plcz1-/-）mouse model was generated via CRISPR-Cas9 system.The reproductive ability of Plcz1-/-mice was evaluated by counting the number of offspring,analyzing sperm motility and observing tissue pathological changes.The mechanism of the effect of Plcz1 gene deletion on the mice reproductive ability was preliminarily explored by RNA sequencing.（1）In order to generate Plcz1 gene knockout mouse model,this study selected exon 6 and 7 of Plcz1 gene as target sites,designed two pairs of sgRNAs and inserted them into pX330 plasmid to construct pX330-sgRNA recombinant plasmid by Golden Gate method.The plasmid was introduced into mouse fertilized eggs by microinjection for embryo transfer.After delivery,DNA was extracted from mouse tail,and the target gene fragment sequence was analyzed by DNA sequencing.The expression of PLCζ protein was detected by Western blot.The results showed that the pX330-sgRNA recombinant plasmid was successfully constructed;three Plcz1-/-mice were obtained through breeding and sequencing identification:Plcz1m3（3078 bp deletion in exon 6,7）,Plcz1m4（7 bp deletion in exon 7,the mouse died in the course of feeding）and Plcz1m5（1 bp deletion in exon 6）;Western blot showed that PLCζ protein of Plcz1m3 mice was deleted.VI（2）In order to explore the effect of Plcz1 gene knockout on the fertility of male mice,sexually mature Plcz1m3 and Plcz1m5 were mated with wild-type（WT）mice respectively to get F1 generation Plcz1+/-mice,and F2 generation mice were obtained by selfing.Plcz1-/male mice were identified and their fertility was evaluated.The early embryos were cultured in vitro to analyze the embryo development rate of Plcz1-/-male mice.The oocytes after fertilization were collected and the phenomenon of polyspermy into eggs was observed by immunofluorescence.The sperm suspension was prepared,the sperm motility and kinematics parameters were measured by Computer-aided sperm analysis（CASA）automatic detection system.The growth indexes of reproductive organs were measured.The pathological changes of male reproductive organs were observed by HE staining.The results showed that compared with WT mice,the fertility of Plczlm3 and Plcz1m5 mice decreased significantly（P<0.05）;the blastocyst rate of Plcz1m3 and Plcz1m5 mice decreased significantly（P<0.05）,and the ratio of abnormal fertilized eggs increased significantly（P<0.05）;Plcz1m3 and Plcz1m5 mice showed polyspermy and abnormal activation of oocytes;the sperm motility and kinematics parameters of Plcz1m3 mice decreased significantly,but there was no significant change in Plcz1m5 mice;the relative weight of testis and epididymis in Plcz1m3 mice decreased significantly and the relative weight of testis in Plczlm5 mice increased significantly;the observation results of epididymal and testicular tissue sections showed spermatogenesis disorder in Plcz1m3 mice,but there was no significant change in Plcz1m5 mice.（3）In order to explore the mechanism of Plcz1 gene knockout on spermatogenesis,the RNA of epididymis in Plcz1m3 mice was sequenced by RNA-Seq method.The expression of related genes was verified by qPCR.And the distribution of cytoskeleton proteins in early embryos was observed by immunofluorescence.The results showed that compared with WT mice,the expression of Tub α 3a in epididymis of Plcz1m3 and Plcz1m5 mice decreased significantly（P<0.05）;Plcz1m3 and Plcz1m5 mice had effects on sperm production and regulation;the fluorescence distribution of α-Tubulin in Plcz1m3 and Plcz1m5 mouse embryos was uneven,and the fluorescence distribution of α-Tubulin in Plcz1m5 mouse embryos showed marginalization.In conclusion,this study successfully established Plcz1 gene knockout mouse models:Plcz1m3 and Plcz1m5,and their reproductive ability decreased significantly;the decreased reproductive capacity of Plczlm3 mice was related to spermatogenesis disorders,and the decreased reproductive capacity of Plcz1m5 mice was only related to polyspermy during fertilization;the spermatogenesis disorder of Plcz1m3 mice was closely related to the functional defects of cytoskeleton protein in epididymis.This study could provide theoretical basis and effective research tools for further revealing the pathogenesis of reproductive disorders.