Function Of HnRNPK In Mouse Spermatogenesis

Spermatogenesis is a continuous and complex dynamic process of highly coordinated cell proliferation and differentiation,which mainly includes three stages:spermatogonia mitosis,spermatocyte meiosis and sperm cell deformation.Each stage of spermatogenesis is precisely regulated by gene expression,which can ensure that spermatogonia differentiate into spermatocytes and finally form haploid long sperm.At present,the biological mechanism of spermatogenesis has not been fully revealed.To explore the genes,molecules and their mechanisms related to spermatogenesis is still the focus of reproductive biology research in the future.Heterogeneous nuclear ribonucleoprotein K（hnRNPK）is a multifunctional protein and an important transcription regulator,which plays a key regulatory role in animal embryonic development and cell proliferation and differentiation.Previous studies have shown that hnRNPK is highly expressed in animal testis and may play an important role in spermatogenesis,but there is no systematic report.In this study,we constructed a male germ cell specific knockout mouse model to study the role of hnRNPK in mouse spermatogenesis and its molecular mechanism.The main results are as follows:（1）It was confirmed by q RT-PCR and Western blot that hnRNPK was highly expressed in mouse testis;the results of immunohistochemistry showed that hnRNPK was highly expressed in spermatogonia,pachytene spermatocytes and round spermatids.（2）Using Cre-lox P system,we hybridized Stra8-Cre^+/-mice with hnRNPK^flox/flox mice to obtain hnRNPK^flox/-/Stra8-Cre^+/-heterozygous mice,and then hybridized female mice with male hnRNPK^flox/flox mice to obtain hnRNPK gene knockout mice(hnRNPK^flox/flox/Stra8-Cre^+/-,referred to as KO)The efficiency of gene knockout in testicular tissue of knockout mice was verified by Western blot and immunofluorescence,which can be used for further study.（3）The birth rate of KO mice conformed to Mendelian genetic law,and the growth and development of KO mice were normal.There was no significant difference in appearance between KO mice and wild-type mice,but the testicular volume became smaller,and the ratio of testicular weight to body weight from 14 days old was significantly smaller than that of wild-type mice（p<0.05）;The results of HE staining showed that the diameter of seminiferous tubules was significantly smaller than that of wild type（p<0.05）from 14 days old,and vacuoles and absence of germ cell layer appeared in most areas,and the number of lumen cells decreased;HE staining of epididymal tissue showed that most of the epididymal lumens in KO mice were empty, and there was no cluster spermatogenesis.Fertility test showed that KO mice were malesterile.These results suggest that specific hnRNPK knockout in pre-meiotic germ cells affects spermatogenesis and testicular development.（4）The results showed that compared with wild-type mice,the number ofγH2AX positive cells in KO mice was significantly decreased,especially in pachytene or diplotene spermatocytes stained withγH2AX dotted（p<0.05）,while there was no significant difference in the number of PLZF and SOX9 positive cells（p>0.05）The number of Ki67 positive staining cells was also significantly reduced（p<0.05）,and most of the decreased cells were in meiotic phase;TUNEL detection showed that the number of apoptotic cells in the KO mice was more than that in the wild type（p<0.05）.These results suggest that hnRNPK knockout inhibits germ cell proliferation,promotes apoptosis,and then leads to a significant reduction of meiotic cells in the lumen,suggesting that hnRNPK knockout may block spermatogenesis by affecting spermatocyte meiosis.（5）The expression of DNA damage associated proteinsγH2AX and hnRNPK,acrosome marker proteins PNA and hnRNPK were detected by Immunohistochemistry.It was found that hnRNPK negative pachytene spermatocytes could be observed in KO mice,but not hnRNPK negative diplotene spermatocytes.Meanwhile,hnRNPK negative round spermatozoa could not be observed in KO mice.Furthermore,the expression of synaptonemal complex proteins SYCP3 and hnRNPK were detected by chromosome spreading and Immunofluorescence.It was found that pachytene spermatocytes were observed in KO mice,while the number of diplotene spermatocytes was very small.These results suggest that hnRNPK knockout may affect the transition process of pachytene spermatocyte to diplotene spermatocyte,and then lead to the arrest of spermatogenesis meiosis,so round spermatozoa can not be formed.（6）We performed high-throughput RNA-seq on testicular tissues of 4-week-old KO and WT mice,q RT-PCR was used to further verify that the expression pattern of differentially expressed genes was consistent with that of RNA-seq.Compared with wild type,4W had 6227 up-regulated genes and 3841 down regulated genes（q<0.05,fold change≥1.5）.Further GO functional cluster analysis of differentially expressed genes showed that the up-regulated genes were mainly concentrated in the process of apoptosis and negative regulation of cell proliferation,while the down regulated genes were mainly concentrated in the process of spermatogenesis and sperm motility,which revealed that hnRNPK was closely related to spermatogenesis.Among the down regulated genes in KO mouse testis,the genes with high expression and large down- regulation were mainly related to sperm development,testis specific histone variants and round sperm formation.KEGG enrichment analysis showed that PI3K-Akt signaling pathway and ECM-receptor interaction pathway were up-regulated gene enrichment pathways,and c AMP signaling pathway was down regulated gene enrichment pathway,which suggested that hnRNPK might participate in these signaling pathways to regulate spermatogenesis.In conclusion,we successfully constructed pre-meiotic germ cell specific hnRNPK knockout mice by Cre-lox P system for the first time,and systematically studied the effect of hnRNPK knockout on spermatogenesis in mice at cell,tissue and individual levels.It was found that hnRNPK knockout could decrease the proliferation and increase the apoptosis of meiotic cells.Spermatogenesis was blocked earlier than round spermatozoa,and round spermatozoa could not be formed,leading to male infertility;hnRNPK negative pachytene spermatocytes were observed in the lumen of seminiferous tubules of KO mice,but no hnRNPK negative diplotene spermatocytes were observed,which indicated that there were some defects in the prophase of meiosis;RNA-seq showed that hnRNPK was involved in the PI3K-Akt signaling pathway and affected sperm development,testis specific histone variants and the expression of genes related to round spermatogenesis.These results suggest that hnRNPK,as a key transcription factor,may be involved in the regulation of gene expression from pachytene to post meiosis,thus affecting sperm development.