Mechanism Of NAD~+De Novo Synthesis Pathway Regulating Spermatogenesis

BackgroundSperm needs to go through a series of complex and ordered processes,including spermatogonial self-renewal and differentiation,spermatocyte meiosis and spermiogenesis.Meiosis is an important biological process in spermatogenesis.The DNA of primary spermatocytes replicate once,and the cells divide consecutively twice,producing 4 haploid cells.Meiotic prophase I is the longest stage in the process of meiosis,which includes the programmed double-stranded DNA breaks(DSBs),the pairing of homologous chromosomes,synapsis,non-sister chromatids crossover,and in the process of meiosis,there are a lot of enzymatic reactions.What’s more important,whatever the key enzyme activity decreases,it will impede the meiosis process,leading to spermatogenesis obstacle.Nicotinamide adenine dinucleotide(NAD+)is an important metabolite in the body which is directly or indirectly involved in many important biological processes in vivo,including cellular energy metabolism,DNA repair,autophagy,stress response,cell senescence,genomic stabilization and intercellular signal transduction.NAD+is not only a coenzyme of redox reactions in the mitochondrial tricarboxylic acid cycle,but also an important cofactor of non-redox NAD+dependent enzymes in the body,including sirtuins,CD38 and poly(ADP-ribose)polymerase.There are three main pathways for NAD+synthesis in the body,including Preiss-Handler pathway,De novo pathway,and salvage pathway.The previous study of our research group indicated that with increasing age,the levels of NAD+in ovary of mice gradually decreased,the function of ovarian reserve decreased,the mitochondrial function of oocytes was incomplete,and the fertility of mice decreased.However,the effect of NAD+on male germ cell function has not been reported.ObjectiveOur aim is to explore whether indoleamine 2,3-dioxygenase 1(Idol)and quinoline phosphoribosyltransferase(Qprt),two key genes in the NAD+De novo synthesis pathway,have a functional effect on the reproductive system of male mice,as well as their influence on spermatogenesis and its regulatory mechanism,so as to provide more medical diagnosis for finding the causes of male infertility clinically.Methods1.Firstly,we applied CRISPR-Cas9 system to construct Idol and Qprt gene knockout mice,acquiring heterozygous F1 generation mice,then heterozygous F1 generations mated to obtain homozygous F2 generation mice.In order to obtain more experimental mice,homozygous F2 generation mice mated and were bred in the temperature of 18-22℃,humidity 40%-70%,12 hour alternating circadian cycles in an environment where adequate food and water are given.2.Immunofluorescence was used to detect the expression of Idol and Qprt in testis of wild-type mice.Western blotting was used to verify that the gene protein expression was lost in testicular tissue of Idol and Qprt knockout mice respectively.Testicular tissues of wild-type mice and Idol and Qprt gene knockout mice of the same age were taken to observe the changes in testicular volume and weigh the testicular mass.Spermatozoa from epididymis were collected and counted.NAD+levels in testis and pachytene spermatocytes were detected.HE staining of testicular tissue was used to observe the changes of cell types in the seminiferous tubules,and the diameter of seminiferous tubules was measured.Immunofluorescence VASA staining was used to observe the number of germ cells,and using the TUNEL staining was to observe cell apoptosis.The spread of spermatocytes in testis was detected by immunofluorescence,the proportion of spermatocytes in different stages of prophase meiosis I and the proportion of autosomal yH2AX positive spermatocytes in pachytene spermatocytes were analyzed and counted;Furthermore,the number of RAD51 and MLH1 in pachytene spermatocyte chromosomes was statistically analyzed.The distribution of silencing transcriptional activity related protein RNA pol Ⅱ in XY body and the expression of XY chromosomal related genes were further analyzed.Results1.Two key genes Idol and Qprt in the NAD+De novo synthesis pathway are mainly expressed in spermatocytes.Knockout of these two genes will lead to a decrease in NAD+levels in testis and pachytene spermatocytes,decreased testicular volume and weight,and reduced sperm count in epididymal fluid.2.The HE staining of testicular tissue showed that compared with wild-type mice of the same age,the cells in the convoluted seminiferous tubules and the tubule diameter after Idol and Qprt gene knockout were reduced.Further analysis of immunofluorescence VASA staining showed that the number of germ cells was reduced and the number of apoptotic cells was increased,and most of them were spermatocytes.3.Compared with wild-type mice,the proportion of pachytene spermatocytes increased but the proportion of diplotene spermatocytes decreased in Ido1-/-and Qprt-/-mice.The proportion of spermatocyte autosomal γH2AX positive cells increased in pachytene spermatocytes.Further analysis showed that the number of crossover MLH1 on the chromosomes of Ido1-/-and Qprt-/-spermatocytes at pachytene stage was significantly reduced,indicating that the repair mechanism of meiosis DNA was impaired and meiosis process was disturbed.4.Compared with wide type mice,in Ido1-/-and Qprt-/-XY body,the mean intensity of RNA pol II involved in transcription activity was higher,and XY chromosome related gene expression increased obviously,This suggested impaired gene silencing on sex chromosomes and maight further explain the increase in apoptotic cells.ConclusionTwo key genes of NAD+De novo synthesis pathway,Idol and Qprt,have a functional effect on the male reproductive system.Idol and Qprt gene knockout will lead to a decrease in NAD+levels in testis and pachytene spermatocytes,a decrease in testicular volume and weight,and a decrease in the number of germ cells and an increase in the number of apoptotic cells.In Idol and Qprt knockout mice,the DNA repair mechanism was damaged showing that spermatocytes were arrested in the pachytene stage of meiosis I.and further analysis of the causes of spermatocytes apoptosis found that the gene silencing function of sex chromosomes in pachytene spermatocytes was impaired which resulted in reduced sperm number.