Effects Of The Testis-specific Protein TEX33 On Spermatogenesis

Mammalian spermatogenesis involves three stages:(1)Spermatogonium cells self-renew,proliferate and differentiate to produce primary spermatocytes.(2)Primary spermatocytes produce spermatids(Round Spermatocytes)by meiosis.(3)Round spermatids finally transform into elongated spermatids(Spermiogenesis).There are about 2,000 genes taking participate into the procedure of spermatogenesis.Autosomal gene mutations mainly incur gonadal axis-related hypogonadism,sperm abnormalities,idiopathic asthenozoospermia and non-obstructive azoospermia,etc.Mutations in sex chromosomal genes lead to more severe spermatogenic disorders.So far,about 7% of men worldwide are suffering from infertility.Infertile men are accompanied with abnormal sperm morphology and semen parameters.Male infertility is caused by many factors,and genetic-related infertility is one of them.Many germ cell-specific transcripts are produced,and these transcripts include many testicular enriched and testicular-specific genes.Their expression is specific to the developmental and spermatogenic stages.Some testis-specific genes are essential to the testicular spermatogenesis.However,there are still many testicular-specific genes whose functions are unknown.In order to understand the role of these testis-specific transcripts in spermatogenesis,gene editing technology can be used to knock out one or more genes in model animals to study the role of target genes in spermatogenesis and further elucidate the mechanism of male infertility.Some genes are necessary for the formation sperm tails.The sperm flagella ultrastructure contains a "9 + 2" microtubule structure: the axial filament is surrounded by a pair of central microtubules by 9 duplex microtubules.The formation of sperm filaments is the basis for the function of sperm flagella.Proteins used to assemble flagella(such as SEPTIN,TEKTINS,AKAP,DRC,etc.)flow bidirectionally through the flagellar intracellular transporter(Intra Flagellar Transport,IFT).IFT has multiple subunits,and the absence of these subunits can cause severe flagellation disorders.The absence of sperm flagellin components(such as Tcte1)sometimes does not cause abnormal morphology of the sperm tail but the sperm's ability to move is often impaired.Tex33(Testis expressed 33)is a recently discovered testis-specific gene that is evolutionarily conserved in vertebrates.C57BL/6 male adult mice were subjected to Tex33 in situ hybridization with seminiferous tubules,and Tex33 expression was found in the circular sperm cytoplasm of adult male C57BL/6.However,no study reports the in vivo function of Tex33.In this study,we constructed a-62 bp in-frame deletion on the second exon of Tex33 in C57BL/6 mouse via CRISPR/Cas9 gene editing technology.Adult Tex33 knockout homozygous C57BL/6 male mice are fertile.In adult C57BL/6 homozygous mutant male mice after Tex33 knockout,there were no significantl alteration in testicular/weight ratio,testicular/epididymal histology,sperm count,and sperm motility.TUNEL analysis also showed that there was no change in the germ cell apoptosis rate in adult Tex33 knockout male C57BL/6 mice.These results show that Tex33 is not an essential gene in spermatogenesis of adult mice under physiological conditions.We found that Tex33 has two evolutionarily conserved amino acid sequence motifs,NIRH and SYT.We speculate that Tex33 may function similarly to the protein Cplane1,which also has the motif of the amino acid sequence of NIRH and SYT.