Study On The Role And Mechanism Of Stra8 In Spermatogenesis

Retinoic acid(RA)is an important signaling molecule that regulates germ cell development,initiates meiosis,and facilitates spermiation.Stimulated by retinoic acid gene 8(Stra8),a RA-induced response gene,is a marker of spermatogonial differentiation and meiotic initiation.In humans,aberrant expression of Stra8 associates with human oligozoospermia and azoospermia,and Stra8 knockout male mice are infertile.Specifically,spermatogenesis is halted at the leptotene stage,with seminiferous tubules lacking post-meiotic germ cells and testes reducing in size.In older Stra8 knockout mice,the testes are enlarged,the seminiferous tubule structure is destroyed,and the number of undifferentiated spermatogonia is high.As such,the production of sperm depends on the timely expression of key genes such as Stra8;however,its exact role in spermatogenesis is still unclear.To understand the role of Stra8 in spermatogenesis,we investigated three aspects of its function using in vitro and in vivo modelsPart I:Study on the interaction between Stra8 and Setd8 in spermatogenesisObjective:To explore the interaction between Stra8 and SET domain-containing protein 8(Setd8)in regulating meiosis during spermatogenesisMethods:The transcriptional regulation of Stra8 and Setd8 was analyzed by dual-luciferase reporter gene assays,and the key promoter binding region was verified by chromatin immunoprecipitation(CHIP)assays.The main interacting region of Stra8 and Setd8 was examined using different fragments in immunoprecipitation experiments,and the interaction between proliferating cell nuclear antigen(Pcna)and Stra8 was also analyzed.Western blotting experiments were performed to quantify Stra8 and Setd8 levels in P19 cells.A transient Stra8 expression vitamin-A model(vitamin A deficiency followed by recovery of vitamin A deficiency)was established in which the levels of Setd8,histone H4 lysine 20(H4K20me1),and Pcna were examined by quantitative real time polymerase chain reaction(qRT-PCR),western blotting,and immunofluorescence staining experiments.The levels of cell cycle regulators(cyclin A2[Ccna2],cyclin E2[Ccne2])and ubiquitination-related factors(cullin-4A[Cul4A],DNA damage-binding protein[Ddb1],chromatin licensing and DNA replication factor 1[Cdtl],denticleless E3 ubiquitin protein ligase homology[Dtl])were analyzed in the vitamin-A model.The differentially expressed testicular genes in 11-day postpartum(dpp)knockout mice were studied by RNA sequencing,and qRT-PCR was used to quantify the levels of cell cycle regulators and ubiquitination-related factorsResults:The results of dual-luciferase reporter gene assays and CHIP assays revealed that Setd8 could bind to the Stra8 proximal promoter region(-1364 to-1 bp),which contained E-boxl,E-box2,E-box3,DMRT1bs,RARE(DR2),and RARE(DR4)motifs,and negatively regulate the transcriptional activity of this promoter.Furthermore,Stra8,as a transcription factor,increased the activity of the Setd8 promoter dose-dependently.The results of immunoprecipitation experiments showed interactions among Stra8,Setd8,and Pcna.A fragment,designated as Stra8 F1R2(1-193 aa),which contained a glutamic acid(GA)-rich region(143-193 aa)with no self-activating activity,could interact with Setd8 F1R2(1-213 aa)and F2R1(214-350 aa).The treatment of P19 cells with RA increased Stra8 and Setd8 expression and decreased H4K20me1 expression.Compared to control cells,there was no difference in the Stra8 level in P19 cells overexpressing Setd8;however,Stra8 and H4K20mel levels were down-and up-regulated in RA-stimulated P19 cells overexpressing Setd8,respectively.In the transient Stra8 expression vitamin-A mouse model,testicular Stra8 expression decreased to an undetectable level in the absence of dietary vitamin A and returned to a normal level in its presence.Compared to control testes,there were differences in the levels of Setd8,H4K20me1,Pcna,Ccna2,Ccne2,Ddb1,CUL4A,Cdtl,and Dtl.The changes in differentially expressed genes(Setd8,Pcna,Ccne2,Ddb1)in the transient Stra8 expression vitamin-A model were similar to those in the Stra8 knockout testis modelConclusions:Setd8 binds to the proximal promoter region of Stra8 to negatively regulate its transcriptional activity.Stra8 and Setd8 expression patterns are cell cycle-dependent in germ cells.Stra8 plays an important role in the transition from mitosis to meiosis.Furthermore,it interacts with Setd8 and Pcna,and co-localizes with Setd8,Pcna,and H4K20me1 in spermatogonia.In the vitamin-A model,the levels of cell cycle regulators and ubiquitination-related factors were abnormal-regulated compared to the control.We speculate that Stra8 and Setd8 regulate spermatogenesis via the Cdl4-Clu4A-Ddb1 degradation axis in germ cells at S phase in a Pcna-dependent manner.Part II:The role of Stra8 in the prophase meiosis I of male miceObjective:To determine the stage of spermatogenesis arrested in Stra8 knockout mice and the chromosomal events at prophase I of meiosis,to construct the regulatory network of Stra8 during spermatogenesis,and to identify the possible downstream target genes.Methods:The morphology of testes and chromosomal spread of spermatocytes of Stra8 knockout mice during the first wave of spermatogenesis was analyzed by hematoxylin and eosin staining and immunostaining,respectively.RNA sequencing was performed to identify differentially expressed genes in 11 dpp knockout testes.The expression patterns and signaling pathways of differentially expressed genes were analyzed,and the genes directly affected by Stra8 were further examined.Interactions between genes were verified by dual-luciferase reporter gene assays,as well as immunoprecipitation and dual-immunofluorescence experiments.Results:Abnormal spermatogenesis was first observed in Stra8 knockout testes at 12 dpp Subsequently,a large number of cells with concentrated nuclei were detected in the seminiferous tubules of knockout testes.Spermatogenesis was halted at an early meiotic stage.The results of chromosomal spreading showed that spermatogenesis was arrested at the leptotene stage in knockout mice,and mid-and late-zygotene spermatocytes and pachytene spermatocytes were absent,although a few germ cells could develop into leptotene and pro-leptotene stages.In knockout testes,zygotene spermatocytes showed abnormal synapsis,gamma H2AX signals on autosomes degraded slower than that in wild-type spermatocytes,and pachytene spermatocytes exhibited incomplete synapsis.Furthermore,there was evidence of abnormal synapsis and pairing,abnormal formation of sex chromosomes,and abnormal transcriptional silencing of autosomes.Fifteen differentially expressed genes involved in spermatogenesis were studied by RNA sequencing and literature screening,among which reproductive homeobox 10(Rhox10)was involved in the maintainence of the spermatogonial stem cell pool;Utfl,Pou5F1,Upp1,and Inca1 in the proliferation and self-renewal of spermatogonia;Nanos3 and early growth response protein 4(Egr4)in the maintainence of the undifferentiated state of spermatogonia;spermatogenesis and oogenesis specific basic helix-loop-helix(Sohlh1)and Rhox10 in spermatogonial differentiation;and PR/SET domain 9(Prdm9),MutS protein homo log 5(Msh5),Egr4,Mtl5,Ccdc155,Dmc1,Pxt1,and Asb9 in meiosis.We constructed a Stra8 regulatory network and further analyzed the expression profiles of the differentially expressed genes in Stra8 knockout testes.We found that Stra8 interacted directly with Rhox10,Prdm9,Msh5,and Egr4.The analysis of transcriptional events revealed that Stra8 could increase Rhox10 promoter activity,whereas Rhox10 could increase Stra8 promoter activity dose-dependently.The results of dual-immunofluorescence staining experiments showed that Rhox10 was strongly expressed in spermatogonia and spermatocytes,but weekly in preleptotene spermatocytes of adult testes,whereas Stra8 was expressed in spermatogonia and preleptotene spermatocytes.Both proteins mainly co-localized in spermatogonia.The results of immunoprecipitation experiments demonStrated that Stra8 interacted with Rhox10.Conclusions:Stra8 knockout mice were infertile due to incomplete synapsis,abnormal synapsis and pairing,abnormal formation of sex chromosomes,and abnormal transcriptional silencing of autosomes.Spermatogenesis was halted at the leptotene stage in Stra8 knockout mice,and mid-and late zygotene spermatocytes and pachytene spermatocytes were absent In summary,Stra8 is involved in several aspects of spermatogenesis,including the maintenance of the spermatogonial stem cell pool,spermatogonial proliferation,differentiation,and meiosis.It may directly regulate spermatogenesis by interacting with Rhox10,Prdm9,Msh5,and Egr4.Part?:Study on the roles of Morinda officinalis polysaccharide and Icariin in the differentiation towords germ cells induced by retinoic acidObjective:To investigate the effects of Morinda officinalis polysaccharide and icariin(ICA)on the differentiation of P19 cells into spermatogonia induced by retinoic acid(RA).Methods:P19 cells were pretreated with RA at concentrations of 1,2,3,4,6,and 8 ?M for 24,48,and 72 h.The effects of different concentrations and exposure times on the Stra8 expression level were examined.Subsequently,RA-stimulated P19 cells were treated with M officinalis polysaccharide at concentrations of 0.0625,0.125,0.25,0.5,1,and 2 mg/L and ICA at concentrations of 2.5,5,7.5,10,and 20?M for 24 h.Cell proliferation was examined with the MTT assay.Subsequently,RA-stimulated cells were treated with the optimal concentration of M.officinalis polysaccharide or ICA,and the optimal exposure time was determined.Flow cytometry was used to determine the apoptotic rate in treated and untreated cells.qRT-PCR and western blotting experiments were performed to quantify the levels of octamer-binding transcription factor 4(Oct4,an embryonic stem cell marker),promyelocytic leukemia zinc finger(Plzf,an undifferentiated spermatogonial marker),and Stra8(a meiotic initiation marker)in M.officinalis polysaccharide-and ICA-treated P19 cells previously exposed to RA.Results:Stra8 expression increased significantly in P19 cells stimulated with 4 ?M RA for 24 h,and these conditions were selected for further experiments.M.officinalis polysaccharide could further promote the proliferation of RA-stimulated cells at all concentrations,except 0.0625 mg/L,and the effect was most significant at 0.5 mg/L.At concentrations of 7.5 and 10 ?M,ICA could further promote the proliferation of RA-treated cells,and the effect was most significant at 7.5 ?M.A final M.officinalis polysaccharide concentration of 0.5 mg/L and a final ICA concentration of 7.5 ?M were used to treat RA-stimulated P19 cells for 24-72 h.Compared to control cells,M.officinalis polysaccharide and ICA could further promote the proliferation of RA-treated cells for 24-48 h,and the effect of ICA was most significant at 24 h.The early apoptotic rate of P19 cells treated with RA was 2.31%± 0.79,and that of cells treated with M.officinalis polysaccharide at 0.5 mg/L was 2.27%± 0.62.The early apoptotic rate of P19 cells treated with RA was 2.32%±0.40,and that of cells treated with ICA at 7.5?M was 2.15%±0.53.There was no significant difference between the groups.Compared to control cells,the levels of Plzf and Stra8 increased significantly,whereas that Oct decreased with prolonged RA treatment time,indicating that RA induced the differentiation of P19 cells into spermatogonia.Compared to RA-treated cells,the Oct4 level decreased,whereas those of Plzf and Stra8 increased in P19 cells treated with either M.officinalis polysaccharide or ICA.Conclusions:RA could induce the differentiation of P19 cells into spermatogonia that expressed mouse Vasa homolog(Mvh,a germ cell marker),c-Kit(a differentiated spermatogonial marker),and Stra8.Both M.officinalis polysaccharide and ICA could further promote RA-induced cell proliferation at optimal concentrations of 0.5 mg/L and 7.5 ?M,respectively,after incubation for 24 h.The results of flow cytometry revealed no significant difference in the apoptotic rate between RA-treated and untreated cells.In P19 cells,the Stra8 level was highest at 24 h after treatment with RA.Furthermore,M.officinalis polysaccharide and ICA promoted the differentiation of P19 cells into spermatogonia,while decreasing the expression of Oct4 and increasing the expression of Plzf and Stra8.In summary,M.officinalis polysaccharide and ICA are traditional Chinese medicines that can activate the RA-Stra8 pathway,indicating that both compounds promote the proliferation and differentiation of P19 cells into spermatogonia through this pathway.