| Mammalian spermatogenesis is an intricate cell differentiation process to generate spermatid from spermatogonia stem cell,there are three main phases during spermiogenesis: the self-renew or differentiation of spermatogonia stem cells;spermatocytes undergoing meiosis to generate haploid round spermatids;round spermatids completing a series of morphological changes to mature spermatozoa.Errors at any stage can result in abnormal sperm shape or stunted development.Spermatogenesis disorder is one of the major causes of male infertility,but there are still many unknown researches on the regulation and pathogenesis of spermatogenesis.In this study,yeast two-hybrid screening was used to explore the interaction molecules of corresponding proteins during the meiosis of spermatocytes and the deformation of round spermatocytes,to elucidate the molecular mechanism of their participation in the regulation of spermatogenesis,and to provide a new scientific explanation and basis for clinical male infertility cases.Therefore,the function of BBOF1 and MZIP2 in male reproduction was studied in this study,which enriches the protein regulatory network in spermatogenesis.It is known that Bbof1 is a ciliary matrix component,which can locate on the motile cilia of Xenopus laevis and regulate its swing direction.BBOF1 is highly conserved among vertebrates and is widely expressed in various tissues and organs of mice,with the highest abundance in adult testes.However,the specific biological function of the protein is still unknown.We observed male sterility,decreased sperm motility and incomplete axoneme structure of flagellum in BBOF1 knockout mice.To investigate the role of BBOF1 in spermatogenesis,yeast two-hybrid screening was carried out in mouse testis c DNA library using full-length BBOF1 as bait protein.A total of 51 candidate proteins were identified,9 of which were related to flagellar development or protein transport within flagellum,and they all had a stable interaction relationship with BBOF1.Among these proteins,both ODF2 and MNS1 are structural components of flagellum,and their deletion can lead to male sterility in mice.Secondly,we divided BBOF1 into two truncated proteins with amino terminus and carboxyl terminus,explored the specific domain of protein interaction,and determined that both ODF2 and MNS1 were bound to the carboxyl terminus of BBOF1.These results suggest that BBOF1 maintains the structural integrity of sperm by binding to flagellar structural proteins at the carboxyl terminal.MZIP2 is a protein specifically expressed during meiosis of mouse germ cells,it can be located to the chromosomes during leptotene to zygotene of first meiosis during germ cell genesis.During meiosis,MZIP2 can recruit SPO16,TEX11 and other proteins to regulate the synaptonemal complex synthesis and crossover assurance.Firstly,we found that MZIP2 can interact with SPO16 in mouse germ cells to format MZIP2-SPO16 complex,besides,the specific interacting domain can be restricted to the XPF domain,which is the carboxyl terminus of MZIP2.Since MZIP2 is a macromolecular protein which is encoded by 1481 amino acids,we speculated that it can work as a scaffold protein during meiosis regulation.On the one hand,MZIP2 can recognize and bind on branched DNA structure,on the other hand,its deletion causes many proteins to be unable to locate on chromosomes.In order to explore other unknown proteins that can interact with MZIP2 and participate in regulating meiosis,we used MZIP2 aa601-1481 as bait to conduct yeast two-hybrid screening in mouse testis c DNA library.Thirty-six candidate proteins were obtained,among which SERTAD2 had the highest number of repeats.The interaction between the carboxyl terminus of MZIP2 and SERTAD2 was verified in yeast.In conclusion,these results provide new genetic targets for the diagnosis and treatment of male infertility diseases and improve the success rate of in vitro assisted reproductive technology. |
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