| Objective: The protein of Sohlh1(Spermatogenesis and Oogenesis Specific Basic Helix-loop-helix 1)gene contains helix loop helix domain,belongs to b HLH family,and is a transcription factor specifically expressed by germ cells.Sohlh1 was mainly expressed in spermatogonial cells at A1~A4 stage(during which spermatogonial cells undergo cell proliferation and differentiation),and gradually decreased from the stage of B spermatogonial cells.In the testes of Sohlh1 knockout mice,a large number of spermatogonial cells were apoptotic at 14.5 days after birth,and the adult testes were atrophied and infertile.Our previous study found that Sohlh1 gene knockout mice showed that spermatogonial differentiation was blocked,spermatogonial differentiation could not enter meiosis stage to form spermatogonial cells,resulting in male sterility.SOHLH1 protein directly regulates transcription of Neurogenin 3(Ngn3),SRY-box transcription factor 3(Sox3)and differentiation spermatogonial marker gene c-kit,thus promoting spermatogonial differentiation.However,it is not clear whether Sohlh1 regulates spermatogonial proliferation.In the early stage of this study,gene chip was used to find that Bmp7 expression was significantly down-regulated in Sohlh1 knockout testis at 7.5 days after birth,and two E-boxes of SOHLH1-binding sites were found in the range of-2000 ~ +100 bp of the mouse Bmp7 promoter.The differentially expressed genes in Sohlh1 knockout testis were searched in GEO database,and Bmp7 expression was also significantly down-regulated,which was consistent with our gene chip results.However,it is not clear whether SOHLH1 directly regulates Bmp7 transcription.Bone Morphogenetic proteins(BMPs)exert biological effects through Smad and PI3K/AKT signaling pathways.Bmp7 was expressed in both male and female embryonic gonads before 11.5 dpc(days post coitum,dpc),but only in XY gonads after 12.5 dpc.The activation of PI3K/AKT signaling pathway by Bmp7 has been widely confirmed in different species,tissues and cells.In mouse spermatogonial cells,activation of PI3K/AKT signaling pathway upregulated the expression of cyclin D3,promoted the cell cycle from G1 phase to S phase,and then played a role in promoting spermatogonial cell proliferation.However,whether Bmp7 activated PI3K/AKT signaling pathway in spermatogonial cells? whether Sohlh1 activates PI3K/AKT signaling pathway through Bmp7,thereby promoting spermatogonial proliferation?These questions are not clear.Based on the above,this project intends to use Sohlh1 gene knockout mice and mouse GC-1 spg spermatogonial cell lines to explore the transcriptional regulation of SOHLH1 protein on Bmp7,and its effect on PI3K/AKT signaling pathway in animal and cell experiments,and to clarify the molecular mechanism of Sohlh1 affecting spermatogonial cell proliferation and apoptosis.Methods:1 Individual level In this study,male Sohlh1 knockout mice and male C57BL/6J mice were selected as research objects.Immunohistochemistry,Real-time PCR and Western blot were used to analyze the relative quantification and localization of BMP7 and PI3K/AKT pathway proteins in testis at 7.5 days and 8 weeks after birth.2.Cell level After upregulating or down-regulating Sohlh1 and Bmp7 expression levels,the apoptosis of GC1 spg mouse spermatogonial cells was detected by flow cytometry.CCK8 assay and Ed U methods were used to detect cell proliferation rate.The activation of PI3K/AKT pathway by Sohlh1 and Bmp7 was investigated.3.Molecule level In the range of Bmp7 promoter-2000 ~ +100 bp,two SOHLH1 binding sites(E-box)were searched and obtained,and whether SOHLH1 directly bound to Bmp7 promoter was verified by Ch IP assay.Wild-type Bmp7 promoter luciferase expression vector and containing mutant E-box Bmp7 promoter were constructed,and the transcriptional regulation of SOHLH1 on Bmp7 promoter was studied by using dual luciferase reporter gene.Results:1.Testicular growth retardation in adult Sohlh1 knockout mice At PD 7.5 d,there was no significant difference in testicular appearance between Sohlh1 knockout(KO)males and wild-type(WT)C57BL/6J males.However,at PD 8w,Sohlh1 KO testis were significantly smaller than WT C57BL/6J testis.2.The expression of Bmp7 and PI3K/AKT signaling pathway genes was down-regulated in Sohlh1 knockout testis At PD 7.5 d,comparing with WT testicle,the results of testicular immunohistochemical showed that the expression of BMP7 protein in KO testicle was decreased in spermatogonial cells and had no difference in mesenchymal cells.SOHLH1 was not expressed in spermatogonial cells and mesenchymal cells.The expression of PI3 K protein was decreased in spermatogonial cells,but had no difference in mesenchymal cells.The expression of AKT protein was decreased in spermatogonial cells,but had no difference in mesenchymal cells.The expression of pho-PI3 K protein was decreased in spermatogonial cells,but had no difference in mesenchymal cells.pho-AKT protein expression was decreased in spermatogonial cells,but had no difference in mesenchymal cells.At PD 8 w,comparing with WT testicle,the results of immunohistochemical showed that the expression of BMP7 protein in KO testicle was significantly decreased in spermatogonial cells,and had no difference in mesenchymal cells.SOHLH1 was not expressed in spermatogonial cells and mesenchymal cells.The expression of PI3 K protein was significantly decreased in spermatogonial cells,but had no difference in mesenchymal cells.The expression of AKT protein was significantly decreased in spermatogonial cells,but had no difference in mesenchymal cells.The expression of pho-PI3 K protein was significantly decreased in spermatogonial cells,but had no difference in mesenchymal cells.Pho-AKT protein expression was significantly decreased in spermatogonial cells,but had no difference in mesenchymal cells.Compared with WT testis,the results of real-time PCR showed that the expression levels of Sohlh1 and Akt in KO testis were downregulated at PD 7.5 d,and the difference was extremely significant(P < 0.01);The expression levels of Bmp7 and PI3 K were significantly downregulated(P < 0.05).Compared with WT testis,the expression levels of Bmp7,Sohlh1,PI3 K and Akt in KO testis were down-regulated at PD 8 w,and the difference was extremely significant(P < 0.01).Compared with WT testis,the results of Western blot results showed that the expression levels of SOHLH1,AKT and pho-AKT protein in KO testis were downregulated at PD 7.5 d,and the difference was extremely significant(P < 0.01).The expression levels of BMP7,PI3 K and pho-PI3 K were downregulated,and the difference was significant(P < 0.05).The protein expression levels of BMP7,SOHLH1,pho-PI3 K,AKT and pho-AKT in KO testis were downregulated at PD 8 w,and the differences were extremely significant(P < 0.01).The expression of PI3 K protein was downregulated with significant difference(P < 0.05).3.Sohlh1 si RNA knockdown induced decreased proliferation rate and increased apoptosis rate in GC1-spg cells The proliferation rate of Sohlh1 si RNA knockdown group was significantly lower than those of control group at 24,48 and 72 h after transfection(P < 0.05).Ed U staining showed that 48 hours after transfection,the cell proliferation rate of sohlh1 interference group was significantly lower than that of sohlh1 overexpression group and control group(P < 0.05).48 h after Sohlh1 si RNA knockdown,cell apoptosis rate was significantly higher than that of control group(P < 0.01).4.The expression levels of BMP7 and PI3K/AKT signaling pathway related proteins were down-regulated after Sohlh1 si RNA knockdown The results of Western blot showed that the expression levels of BMP7,PI3 K,AKT,pho-PI3 K and pho-AKT were significantly downregulated after Sohlh1 si RNA knockdown in GC1-spg cells,which was consistent with testicular Western blot results.However,the downregulation of PI3K/AKT signaling pathway proteins inducing by Sohlh1 si RNA knockdown can be partially recovered by Bmp7 overexpression.regulatory effect on Bmp7 Ch IP results confirmed that the transcription factor SOHLH1 binds to the E-box at-97 ~-92 bp site of Bmp7 promoter.Dual luciferase reporter gene confirmed that SOHLH1 protein had transcriptional promotion effect on Bmp7 promoter.5.The transcription factor SOHLH1 binds to Bmp7 promoter,and has a positiveConclusion:1.In Sohlh1 KO testis,the expressions level of BMP7 protein in spermatogonial cells were downregulated and PI3K-AKT signaling pathway was blocked.2.In mouse GC1-spg spermatogonial cells,Sohlh1 si RNA knockdown inhibited spermatogonial proliferation,promoted apoptosis,inhibited BMP7 protein expression and PI3K-AKT signaling pathway was blocked.3.Transcription factor SOHLH1 can directly bind to Bmp7 promoter and have a transcriptional activation effect on Bmp7. |
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