The Mechanism Of PA1 Of Mouse Sertoli Cells In Regulating Spermatogenesis

【Objective】The process of normal spermatogenesis could be divided into 3 stages including the mitosis of spermatogonia,the meiosis of spermatocytes,and the spermiogenesis.The seminiferous epithelium of the testis is the location for spermatogenesis,composed of the Sertoli cells and different germ cells.Sertoli cells,as the only somatic cells within the seminiferous epithelium,play important roles during the whole process of spermatogenesis.For example,Sertoli cells promote the proliferation of spermatogonia through the secretion of diverse growth factors including the GDNF and others,and Sertoli cells could affect the progress of meiosis in spermatocytes.Moreover,the structure of apical ectoplasmic specialization（a ES）formed between Sertoli cells and elongated spermatids is necessary for the spermiogenesis.The Blood-Testis Barrier（BTB）at the basal compartment is comprised of different junctional complexes formed between the basal adjacent Sertoli cells.The BTB is essential for maintaining the microenvironment inside the seminiferous tubules and the survival of different germ cells.Plenty of nuclear transcription factors,such as WT1 and AR,have been identified to be highly expressed in the nuclei of Sertoli cells and are indispensable for Sertoli cells to perform their normal functions,including regulating the integrity of the BTB.However,there are still many other specific factors in the Sertoli cells remained to be explored.PA1（PTIP Associated protein 1）is a unique component of the histone methylation transferase complexes,MLL3/4 complexes,and has been reported to be involved in many biological processes such as the embryonic development and adipose differentiation.However,the role of PA1 in the male reproductive system has not been reported.To investigate the function of PA1 protein in the male reproductive system,we firstly examined the expression pattern of PA1 protein in human and mouse testis,and then constructed a Sertoli cell Pa1 specific knockout mouse model with the cre-loxp system to demonstrate the mechanism of PA1 in regulating the spermatogenesis.【Methods】1.Immunohistochemistry（IHC）was used to examine the expression and localization of PA1 protein in normal human testis.The expression levels of PA1 in different organs of adult mice and in developmental testis were detected by Western blot and semi-quantitative PCR.The localization of PA1 in mouse testis was detected by immunofluorescence（IF）and IHC.2.The Sertoli cell Pa1 specific knockout mice(Amh-pa1^-/-)was constructed using the cre-loxp system.Western blot and immunofluorescence were performed to detect the efficiency and specificity of knockout method.3.The volume of testis,testis weight,body weight,the ratio of testis weight/body weight of Amh-pa1^-/-were analyzed.The fertility test was conducted.Hematoxylin and eosin staining（H&E）,PAS-H staining（Periodic Acid-Schiff and Hematoxylin staining）,TUNEL staining were performed to observe the seminiferous tubules of Amh-pa1^-/-mice testis.The immunofluorescence staining of F-actin was conducted to detect the structural defects of a ES.We testified the in-vivo BTB integrity in Amh-pa1^-/-mice testis and performed the transmission electron microscopy,immunofluorescence staining of BTB components and Western Blot to clarify the BTB defects.The ability of Pa1 knockout Sertoli cells to form cell junctions was further tested by isolating primary Sertoli cells in vitro with cell immunofluorescence.4.We performed RNA-seq as well as Cut-Tag experiments using the isolated primary Sertoli cells from 20-day-old Pa1^F/F and Amh-pa1^-/-testis and WT mice testis,respectively.The results were further analyzed through GO and KEGG pathway analysis.The targeted downstream genes were testified using q PCR.5.The motif analysis of Cut-Tag results and upstream analysis of RNA-seq results were performed to explore the potential binding motif and interacting transcription factors of PA1.The Co-immunoprecipitation（Co-IP）was conducted to testify the interaction between PA1and the transcription factor,JUN.We further use the IF,q PCR,WB and luciferase reporter assays to testify the alteration of downstream co-target of PA1 and JUN,Cx43.【Results】1.PA1 protein is highly expressed in human testis and is mainly localized in the nuclei of Sertoli cells.Among the different organs of mouse,PA1 protein is abundant in mouse testis and its expression level was gradually increased with the development of testis.PA1 is expressed in the nuclei of testicular cells and is mainly localized in the nuclei of Sertoli cells.2.The expression level of PA1 in the testis of adult Amh-pa1^-/-mice was decreased significantly,and the positive signals of PA1 disappeared in the nucleus of the Sertoli cells in the Amh-pa1^-/-testis.Compared with the control mice,adult Amh-pa1^-/-mice are infertile and we could not find any mature sperm in the caudal epididymis of Amh-pa1^-/-mice.The Amh-pa1^-/-testis showed significantly reduced area and diameter of seminiferous tubules,increased apoptotic germ cells,and the significantly loss of different germ cells in the seminiferous tubule.3.The spermiogenesis of Amh-pa1^-/-testis were abnormal.We could find aberrant deformed sperm head since the Step9.The results of immunofluorescence staining of F-actin showed that the a ES structure of the sperm head was disordered.4.The BTB function of adult Amh-pa1^-/-mouse testis was destroyed.The transmission electron microscope showed the clefts between the Sertoli cells at the basal compartment of the seminiferous tubules in Amh-pa1^-/-mouse testis.The immunofluorescence staining of BTB components showed that the expression and localization of the components are abnormal.Western Blot found that the expression level of adaptor protein,ZO-1,was significantly downregulated.The ability to form cell junctions was compromised in isolated Amh-pa1^-/-Sertoli cells,compared with the control Sertoli cells.5.Transcriptomic analysis results found 1045 significantly down-regulated genes and 899significantly up-regulated genes in Pa1 knockout Sertoli cells,compared with the control group.The GO analysis of down-regulated genes showed that it was mainly related to cell junctions and the regulation of actin cytoskeleton organization.The Cut-Tag results of the PA1 protein in WT Sertoli cells showed that the PA1 protein was mainly located at the promoter region of 3553 genes.The GO analysis showed that downstream binding genes were also mainly associated with cell junctions and the actin cytoskeleton organization.6.The analysis of transcriptomics data and Cut-Tag results revealed that the downstream targeted genes of PA1 are also regulated by the transcription factor JUN.Co-IP results verified the interaction between PA1 and JUN.Immunofluorescence and Western Blot results showed that,Cx43,a key component of gap junction and potential target of JUN,was significantly downregulated in Amh-pa1^-/-mouse testes.【Conclusion】1.The high expression of PA1 protein in human and mouse testis suggest that PA1 plays an important role in normal spermatogenesis.The significant localization of PA1 protein in the Sertoli cell nucleus also indicates that PA1 may act as a nuclear transcription factor or cofactor in Sertoli cells to regulate the normal function of Sertoli cells.2.The Amh-pa1^-/-mouse knockout model was successfully constructed.PA1 plays an indispensable role in normal spermatogenesis by regulating the function of Sertoli cells.3.PA1 is involved in maintaining the normal transcription of Sertoli cells.Plenty of cell junctions and cytoskeleton-related genes are significantly downregulated in Pa1 deleted Sertoli cells.The Cut-Tag results of PA1 suggest that PA1 may bind to the promoter region of downstream target genes to regulate the transcription,to further affect the normal function of BTB and the actin cytoskeleton organization of Sertoli cells.4.The upstream transcription factor analysis of RNA-seq and the motif analysis of Cut-Tag results revealed that PA1 may cooperate with the transcription factor JUN to regulate the downstream gene expression.Co-IP experiments did confirm the interaction between PA1and JUN.And the IF results showed that PA1 and JUN were both located in the nucleus of the Sertoli cells in mouse testis.5.The results of RNA-seq,Cut-Tag combined with Ch IP-seq of JUN were co-analyzed and demonstrated that PA1 together with JUN regulated the expression of Cx43,a key component of gap junction.The results of Western Blot and IF both confirmed the downregulation of Cx43 in the testis of Amh-pa1^-/-mice.The Cx43 of Sertoli cell plays an important role in maintaining the normal structure of BTB and is indispensable for male fertility,suggesting that PA1 of Sertoli cell may contribute to the normal spermatogenesis via regulating the expression of Cx43 through interacting with JUN.