Functional Roles Of Ddb1 In Testis Cord Development And Spermatogenesis Of Mice

Spermatogenesis is a complicated and sophisticated process,which is regulated by various factors.It starts with spermatogonial stem cells self-renewal and differentiation,then spermatocyte meiosis,terminating into spermiogenesis.Any unusual step will result in reduction of sperm production and even extermination of sperm formation.This complex process requires precisely temporal and spatial expressions of enzyme and structural proteins,which are controlled by gene transcription,translation,and post-translational regulations,including protein ubiquitination and degradation.Cullin-RING finger ligases(CRL),one of the largest ubiquitin ligases of eukaryotic cells,are involved in a variety of cellular processes.Among the CRL family members,CRL4 participates in a series of biological activities by using DNA Damge-binding protein(DDB1)as an adapter,such as DNA damage repair,cell cycle regulation and transcription processes,etc..Ddb1 knockout in oocytes leads to massive oocyte loss and premature ovarian failure(POF).However,the role of Ddbl in spermatogenesis has not been reported yet.In this research,we abrogated Ddb1 in mouse testis to study the role of Ddb1 in spermatogenesis.We found that:?.Amh-Cre driven Ddb1 specific knockout in Sertoli cells resulted in:(1)an increase of testis cord diameter and a decrease of testis cord number per unit area in neonatal mouse testes;(2)a reduction of the proliferation rate of Sertoli cells and a significant decrease of Sertoli cell number per unit area in neonatal mouse testes;(3)gather of germ cells in single testis cord and apoptosis of some germ cells in neonatal mouse testes;(4)a significant change of SET8 protein(increased)and phosphorylated-SMAD2 protein(decreased)in both 18.5 day post-coitum(dpc)and 0 day post-partum(dpp)testes;(5)smaller testes,shorter seminiferous tubules and some vacuole-like structures in adult testes.?.Stra8-Cre driven Ddb1 specific knockout in spermatgonia resulted in:(1)smaller testes,appearance of apoptosis-like cells and vacuole-like structures in seminiferous tubules and sub-fertility in the adult knockout mice;(2)smaller testes,thinner seminiferous epithelium in 14 dpp mouse testes;(3)a decreased number of spermatocyte while unaffected prophase ? process;(4)a reduction of the proliferation rate of spermatogonia and a decrease of spermatogonia number in 6 dpp mouse testes.These results proved that:(1)Ddb1 specific knockout in Sertoli cells,SET8 ubiquitination and degradation are blocked,which suppresses SMAD2 activation and Sertoli cell proliferation,further delay the testis cords convolution and expansion,finally causes shorter seminiferous tubules and smaller testes in adult mice;(2)Ddb1 specific knockout in spermatogonia,the spermatogonia number is significantly reduced,which produce decreased spermatocytes,finally causes thinner seminiferous epithelium,smaller testes and sub-fertility.In summary,our study provides two different animal models of small testis,one is shorter seminiferous tubules,and the other is thinner seminiferous tubules.This research disclosed the important role of Ddb1 in spermatogenesis via regulation of Sertoli cell and spermatogonia proliferation.Furthermore,our findings will provide a new theoretical basis for human small testis biogenesis and point out a novel direction for diagnosis and treatment of the related human disease.