The Expression And Localization Of PA1 During Mouse Testis Development And The Function Of PA1 In Sertoli Cells In Mouse

BackgroundSpermatogensis is a precise process in mammals.As the only somatic cells in seminiferous tubules,sertoli cells play a critical role in structure,nutrition and regulation.The latest research indicates that,histone methylation play a critical role in gene activation and transcription.Histone H3K4 methylation is mediated by Histone Methyltransferases(HMTs).PA1 is the core component of H3K4 methyltransferases,which has been concerned since it was discovered in 2007.The researches on PA1 mainly focus on the expression of PA1 in different tissues and organs at present.Researches on the expression and function of PA1 in male reproductive system are almost blank.In order to explore the role of PA1 in male reproduction,we observed the expression and localization of PA1 during the development of mouse testis.Through gene knock out,we get the sertoli cell specific PA1 knock out mice.On the basis of above works,the reproductive phenotype of SC-PA1KO mice was studied.In the last,the mechanism of Reproductive deficiency caused by PA1 was studied,to further study the function and regulation of Pa1 in male reproductive system.Methods1.The expression and localization of PA1 in mouse testis were evaluated at various developmental stages up to 24 weeks after birth using q PCR,immunohistochemistry and immune fluorescent staining2.Sertoli cell specific Pa1 knock out mice was constructed,and Genomic DNA were extracted for identification of genotypes.Male SC-PA1KO mice and WT mice were respectively cohabitated with adult female mice in the ratio of 1:1 for 6 months,to obsearve the fertility of the mice.Sperm in the tail of epididymis was obtained to observe the motility and the total number of sperm,after Pa1 specific knocked out in sertoli cells.3.To investigate the function of PA1 in sertoli cells.The weight and volume of testis from 1-8 w mice were detected,and pictures of these testises were taken to study the development of SC-PA1KO mice testis.Through HE staining,we obsearved the changes about tissue structure of testis in SC-PA1KO mice.We count the number of sertoli cell and spermatogonia by IHC staining with sertoli cell specific marker sox-9 and histological characteristics of Spermatogonia.After that,TUNEL staining was used to detect apoptosis in seminiferous tubules of SC-PA1KO mice.In addition,FSH,LH,T levels were detected by radioimmunoassay.4.To explore the mechanism of the abnormalities in reproductive phenotype in SC-PA1KO mice.we injected evans blue through caudal vein to identify the integrity of the blood testis barrier after Pa1 was knocked out in sertoli cells.Transmission electron microscope was used to observe the integrity of tight junction between adjacent sertoli cells and the organelle in sertoli cells.q RT-PCR was conducted to examine the m RNA level of occludin?claudin-11?gelsolin?testin and laminin those were involved in cell junction in seminiferous tubule;cytokines such as transferrin?Bmp2?Fabp?Pdgf-A and Dhh;as well as molecules associated with serum hormone such as Ar?inhibin-B and activin-A.5.SC-PA1KO mice received intraperitoneal injection of human recombinant BMP2 for 21 days.after then,HE staining was used to identify whether human recombinant BMP2 is efficient for the deficiency of SC-PA1KO mice.Results1.PA1 expression in mouse testis was detectable one week after birth,peaked at week 2 and remained stable after 8 weeks.Immunohistochemical staining of 1 to 8 w mice testis showed PA1 mainly expressed in the nucleus of spermatogenic cells at all stages,Sertoli cells and Leydig cells.Immunofluorescent double staining further confrmed PA1 expression in Sertoli cells and Leydig cells.2.Mouse fertility detection showed that,no mouse was bred from 7 pairs of adult SC-PA1KO mice that cohabitated with female mice.Sperm counts in the tail of epididymis showed that,the SC-PA1KO mice showed decrease in sperm counts and motoricity.3.SC-PA1KO mice showed atrophy in testis volume and weight after 4 week old,comparing to the WT mice in the same age.HE staining showed that,SC-PA1KO mice showed no destinction between WT mice before 2 week;after that SC-PA1KO showed seminiferous tubule atrophy in 3 w;decrease in germ cell counts in 4 w;Testicular dysplasia,enlarged luman,further decrease in germ cell counts in 6 w,and vacuoloid changes could be observed in 8 w.IHC staining and cell counts in seminiferous tubules showed that,comparing to WT mice,SC-PA1KO mice showed no difference in sertoli cell counts before 24 w;The counts of Spermatogonia decreases in 8 w,few spermatogonia could be observed in semiferous tubule in 24 w;and no sertoli cell and spermatogoia could be observed in 72 w.TUNEL staining showed that,no apoptosis could be observed in semiferous tubules in both SC-PA1KO mice and WT mice.SC-PA1KO mice showed no difference in FSH level in serum,but the level of LH is upregulated,and T level reduced significantly,comparing to WT mice.4.Evans blue injection through caudal vein showed that,the integrity of blood testis barrier was destroyed.Transmission electron microscopy further identified that,the integrity of tight junction was destroyed between adjacent sertoli cells and the crest of mitochondrial fractured and the intimal of mitochondrial swelled in sertoli cells in SC-PA1KO mice.q RT-PCR showed abnormal expression of multiple proteins that,formed the cell junction.Amang them the level of claudin-11 m RNA reduced significantly,and high level of testin was detected;In addition,cytokines related to the development of spermatogoia detected by q RT-PCR showed aberrant expression in SC-PA1KO mice.Among them,Bmp2,Abp and Fabp showed low level of m RNA.5.After SC-PA1KO mice received intraperitoneal injection of human recombinant BMP2 for 21 days,HE staining showed significant improvement in the structural of spermatogonial epithelium,and a few sperms can be observed.Conclusion1.PA1 mainly expressed in the nucleus of spermatogenic cells at all stages,Sertoli cells and Leydig cells in all developmental stages.2.SC-PA1KO mice showed deficiency in testicular development,spermatogenesis,and serum gonadal hormone reversing that,PA1 play a critical role in male reproductive system.3.Pa1 knocked out leads to abnormal expression of multiple proteins associated with cell junction,destruction of tight junction,and the integrity of blood testis barrier was destroyed.Also the expression of cytokines related to development of spermatogia such as Bmp2,showed abnormal level in SC-PA1KO mice.Intraperitoneal injection of human recombinant BMP2 could improve the destruction in SC-PA1KO mouse testis.