Study On The Abnormal Phenomenon Of Spermatocyte Meiosis And Its Mechanism In Sohlh1 Gene Knockout Mice

Objective:Spermatogenesis originated in spermatogenial stem cells?SSCs?,which have three different fates:?1?maintain stem cell character and remain dormant until the next spermatogenesis cycle;?2?apoptosis of abnormally developed stem cells to ensure the quality of germ cells;?3?development of spermatogonial progenitors-A_s,A_pr,A_al:A_al4,A_al8,A_al16.Spermatogonial progenitor cells undergo the process of gametophyte development——meiosis after undergoing mitotic development of type A?A₁,A₂,A₃,A₄?,intermediates and type B spermatogonia and completion of chromatin duplication.After the meiosis is the sperm formation period,during which sperm cells undergo a series of structural remodeling and morphological changes to form mature sperm.Spermatogenesis is influenced by a series of programmed expression of specific genes,and different transcription factor-regulated genes are expressed in space-time order at different stages of germ cell development.Spermatogenesis-specific and oogenesis-specific helix-loop-helix transcription factor 1?Sohlh1?is a germ cell-specific transcription factor belonging to the bHLH transcription factor family,which is located on mouse chromosome 2 and human chromosome 9.SOHLH1 is mainly expressed in A_al^B type spermatogonia in male mice and plays an important role in spermatogenesis.Through screening male patients with nonobstructive azoospermia?NOA?in different regions and ethnic groups,nonsynonymous mutation was found in different sites on SOHLH1 gene.Patients'testis biopsy showing that their spermatocytes reduced and there are no mature sperm,and pathological examination is"Sertoli-cell only syndrome".Testes of NOA patients have spermatogenic dysfunction and no mature sperm,resulting male infertility which seriously affecting the quality of personal life and the diversity of human genetic information.Sohlh1 knockout?KO?mice have a large number of apoptotic spermatocytes,resulting in failure of spermatogenesis and male infertility.Previously,the mechanisms by which SOHLH1 affects germ cell development mostly focus on mitosis,but the reasons for the large number of apoptotic spermatocytes in Sohlh1knockout males have not been reported yet,and whether SOHLH1 affects spermatogenesis by meiosis is still unknown.This study aimed to analyze the effect of Sohlh1 absence on mouse development and spermatogenesis by a series of morphological and histological observation methods.The differentially expressed genes of Sohlh1 knockout male mice were screened by microarray analysis.According to a series of experiments,we will discussion the role of SOHLH1 in the process of spermatocyte development,and whether the transcription factor can directly transcriptional regulate the SYCP1 and SYCP3.Finally,the effects of SOHLH1 on spermatocyte development in male mice and its mechanism were analyzed through comprehensive analysis.Methods:In this study,Sohlh1 gene knockout and wild-type C57BL/6J male mice were used as research objects.The appearance and testicular development of postnatal day 7?PD7.5?,PD10.5,PD14.5,PD23.5 and PD49.5 mice were observed.Differentially expressed genes in PD7.5 mice testis will be analyzed by microarray analysis.We will perform the co-localization of SOHLH1 and SYCP3 in PD7.5 wild male testis.We will detection the expression of SYCP1 and SYCP3 in PD14.5 testes.Transmission electron microscopy was used to observe the formation of synaptonemal complex in spermatocytes.By constructing the expression vectors of Sycp1 and Sycp3,using dual luciferase reporter gene to study the effect of SOHLH1 on Sycp1 and Sycp3 promoter and using ChIP assay to find the binding site of SOHLH1 on the Sycp1 and Sycp3 promoter sequences.Results:1.Compared with the wild type?WT?C57BL/6J mice,no abnormalities were observed in the appearance of Sohlh1 knockout?KO?male mice at each stage,whereas testicular development stagnated on PD14.5 and remained in the maintenance phase thereafter.We could observe the normal differentiated spermatogonia in WT mice on PD7.5,primary spermatocytes on PD10.5,and spermatogenic cells at all levels on PD23.5.Seminiferous tubules in Sohlh1 KO males are mainly spermatogonia and a small number of primary spermatocytes,and they did not contain sperm cells on PD23.5.Sohlh1 affects testicular development mainly by impeding the development of primary spermatocytes.2.In Sohlh1 knockout mice,there were 205 differentially expressed genes in PD7 testis,while 47.6%?66?were up-regulated and 67.8%?139?were down-regulated.Down-regulated genes include meiosis,signaling pathways and reproductive development-related factors.3.Quantitative real-time PCR results showed that Sycp1,Sycp3 and meiosis related factor Tex11 expression was significantly down-regulated in Sohlh1 KO mice.Immunofluorescence results showed there were correlations between SOHLH1 and SYCP3.The expressions of SYCP1 and SYCP3 were significantly down-regulated in Sohlh1 KO mice at PD14 and the result of transmission electron microscope showed that there were no SC in the nucleus of spermatocytes and the synapsis was abnormal,suggesting that Sohlh1 gene may affect the formation of SC during meiosis and this will cause the stagnation of spermatocytes.4.Luciferase assay was used to confirm that when SOHLH1 was increased,the activity of Sycp1 and Sycp3 was up-regulated,indicating that SOHLH1 can transactive the expression of Sycp1 and Sycp3.When a single E-box domain?-45bp,-94bp?or both E-box domains of the Sycp1-249bp⁸4bp region were mutated,the transcriptional activation of Sycp1 by SOHLH1 were disappeared.The transcriptional activation of Sycp3 by SOHLH1 did not disappear when the-43bp E-box domain of Sycp3-286bp⁶6bp was mutated,whereas the transcriptional activation of Sycp3 by SOHLH1 significantly decreased but did not disappear completely when the mutation of-64bp E-box domain of Sycp3-286bp⁶6bp or-43bp and-64bp E-box domains were mutated.The results showed that SOHLH1 has the possibility of transcriptional regulation of Sycp1 and Sycp3 through the E-boxes upstream of the transcription start site of Sycp1 and Sycp3.5.In mice testis,the results of ChIP showed that the transcription factor SOHLH1 can bind to the-1276bp,-708bp and-94bp E-box upstream of the transcriptional start site of Sycp1 and SOHLH1 can binds to-64bp and-43bp E-box domain upstream of the transcriptional start site of Sycp3.Conclusion:1.Sohlh1 affects testicular development mainly by impeding the development of primary spermatocytes.2.Differentially expression of meiosis related genes in testis is one of the causes of male sterility in Sohlh1 knockout mice.3.Sohlh1 can affect the synaptonemal complex formation during meiosis and thereby affecting spermatocyte development.4.Using luciferase assay we found that transcription factor SOHLH1 has transcriptional activation on Sycp1 and Sycp3.5.ChIP results showed that SOHLH1 can bind to the-1276bp,-708bp and-94bp E-box domains upstream of Sycp1 and play a transcriptional regulatory role on Sycp1.SOHLH1 binds to the-64bp and-43bp E-box domains upstream of Sycp3 and have transcriptional regulatory role on Sycp3.