Rnf138 Is Essential For The Spermatogenesis In Juvenile Mice

E3 ligase can be classified by the domain into three major groups,including the HECT(homologous to E6-AP carboxy terminus)domain,the RING(really interesting new gene)finger domain,and the U-box.E3 ligase normally functions by ubiquitylating substrates,participating in the destruction of protein by proteasome,protein kinase activation,membrane trafficking,DNA repair,and chromatin dynamics.The RING domain family is characterized by the conserved sequence motif:C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-C/H-X2-C-X(4-48)-C-X2-C,which can be divided into the single subunit type E3 and the multi-subunit type E3.The single subunit type E3 proteins consist of the RING finger domain and the substrate recognition site on the same protein,While multi-subunit type E3 ligases are composed of at least a small RING finger protein,a substrate recognition subunit and a frame member of the cullin family of proteins.RNF138 is a new member of the RING family isolated by our lab from the human testis cDNA.Up to date,scientific reports about the function of RNF138 mainly focus on the regulation of Wnt pathway by ubiquitylating TCF/LEF,and the promotion of homologous recombination in DNA damage by ubiquitylating Ku and CtIP.The function of RNF138 in the testis development and spermatogenesis is rarely studied.Since RNF13 8 was firstly discovered in the human testis,this study tried to explore the role of RNF138 in the testis development and spermatogenesis.Firstly,we confirmed the expression of Rnfl38 at the levels of both RNA and protein in mouse that Rnfl38 was widely expressed in multi-tissues,but highly in testis.Secondly,there were several variants of Rnf128 transcribed in the testis,the levels of which changed according to the age.The variant 1 was highly expressed in the first three weeks,then decreased comparatively in the following weeks.But the variant 2 increased gradually from the first week.The localization of Rnf138 in the spermatogenic cells was then observed by using our antibody specific to Rnfl38 in immunohistochemistry.Rnf138 was expressed mainly in spermatogonia and spermatocytes before round spermatid.In consideration of this expression pattern of Rnfl38,we generated the knockout mouse of Rnf138 to further explore the function of Rnf138 in testis.After the knockout of Rnf138,the testes we harvested from the knockout group were much smaller than that from the control group at the same age.Compared to the fl/fl group,the testis weight/body weight ratio in Rnf13 8-/-increased much slower from 1 week to 7 week.Sperm counts showed that knockout Rnfl38 could cause the decrease of sperm number,even no spermatozoa at young age.The male fertility was slight impaired by the function loss of Rnf138.The diameters of the seminiferous were getting smaller,and the thickness of the seminiferous epithelium was thinner in the knockout group.Then we checked the development of spermatogenic cells at different ages,and found that the spermatogenesis were slightly impeded in the Rnf138-/-testes.The staining of markers,PLZF,STRA8,SYCP3 and SOX9,at 4 and 7 weeks all showed that the number of Sertoli cells and undifferentiated spermatogonia were unchanged,but the number of STRA8-positive cells,as well as SYCP3-positive meiotic spermatocytes,was significantly decreased in the Rnf138-/-testes.these implied that Rnfl38 may not function at the maintenance of Sertoli cells and undifferentiated spermatogonia,but had a role in the process of meiosis or differentiating.It is well know that the differentiation of spermatogonia is still a process of proliferation.To ascertain the function of Rnf138 in the differentiating,we examined the proliferation of spermatogonia.The Cyclin D1 positive cells in Rnfl38-/-were significantly fewer than that in control,which indicated that the knockout of Rnf138 impaired the proliferation of differentiating spermatogonia.Since recent report about the function of Rnfl38 in DNA repair,we examined the meiosis prophase I to find out if knockout of Rnfl38 might influent the homologous recombination.VWhereas the assembly of synaptonemal complex,the formation and repairing of double strain break,and the homologous recombination were not affected by the loss of Rnfl38.To find the proteins interacting with Rnfl 38,we performed yeast two hybrid system and human proteome microarrays,and found a group of potential interaction proteins.Two E2 ubiquitin conjugating enzyme were found interacting with Rnf138,one of which was reported before.In conclusion,we firstly reported that Rnfl 38 localized in the spermatogonia and the primary spermatocytes.It mainly functioned in the differentiating of spermatogonia.