Abstract
Infertility affects 10–15% of couples worldwide, and male factors account for 50%. Spermatogenesis is precisely regulated by genetic factors, and the mutations of genes result in abnormal spermatogenesis and eventual male infertility. The aim of this study was to explore the role and transcriptional regulation of P63 in the apoptosis and mouse spermatogenesis. P63 protein was decreased in male germ cells of P63(+/−) mice compared with wild-type mice. There was no obvious difference in testis weight, sperm motility, and fecundity between P63(+/−) and wild-type mice. However, abnormal germ cells were frequently observed in P63(+/−) mice at 2 months old. Notably, apoptotic male germ cells and the percentage of abnormal sperm were significantly enhanced in P63(+/−) mice compared to wild-type mice. Spermatogonia, pachytene spermatocytes and round spermatids were isolated from P63(+/−) and wild-type mice using STA-PUT velocity sedimentation, and they were identified phenotypically with high purities. RNA sequencing demonstrated distinct transcription profiles in spermatogonia, pachytene spermatocytes, and round spermatids between P63(+/−) mice and wild-type mice. In total, there were 645 differentially expressed genes (DEGs) in spermatogonia, 106 DEGs in pachytene spermatocytes, and 1152 in round spermatids between P63(+/−) mice and wild-type mice. Real time PCR verified a number of DEGs identified by RNA sequencing. Gene ontology annotation and pathway analyzes further indicated that certain key genes, e.g., Ccnd2, Tgfa, Hes5, Insl3, Kit, Lef1, and Jun were involved in apoptosis, while Dazl, Kit, Pld6, Cdkn2d, Stra8, and Ubr2 were associated with regulating spermatogenesis. Collectively, these results implicate that P63 mediates the apoptosis of male germ cells and regulates three stages of spermatogenesis transcriptionally. This study could provide novel targets for the diagnosis and treatment of male infertility.
Highlights
Introduction been reported thatP63 is expressed in various kinds ofThe P63 gene, known as Trp[63] gene, encodes two tissues, including heart, epidermis, urothelium, cervix, isoforms, namely TAp63 and ΔNp63, and alternative spli- testis, kidney, thymus, and prostate in humans and mice[1].cing at the C-terminus forms three different proteins, TAp63 mainly induces cell cycle arrest and/or apoptosis, including α, β and γ for both TAp63 and ΔNp63
Immunohistochemistry revealed that P63 protein was expressed in the nuclei of spermatogonia, spermatocytes, and round spermatids in wild-type male mice (Fig. 1d) and the P63(+/−) male mice (Fig. 1e)
We found that the transcripts of Gpr[125], Gfra[1], Thy[1], and Zbtb[16] were expressed in isolated spermatogonia (Fig. 5b), and Scp[3], Crest, and Mlh[1] mRNA was detected in isolated pachytene spermatocytes (Fig. 5c)
Summary
Cing at the C-terminus forms three different proteins, TAp63 mainly induces cell cycle arrest and/or apoptosis, including α, β and γ for both TAp63 and ΔNp63. It has whereas ΔNp63 usually has an opposite effect on TAp631,2. The α isoform of TAp63 and ΔNp63 is the longest one, Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao. Tong University, Shanghai 200127, China 2Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai. Jiao Tong University, 145 Shangdong Road, Shanghai 200001, China which contains the Sterile A Motif (SAM).
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