The Effect Of TRPV1 On Spermatogenic Cell Apoptosis

The process of spermatogenesis includes proliferation and differentiation of spermatogonia（SPG）,two meiotic divisions of spermatocyte（SPC）and deformation of spermatocytes,which are regulated by a variety of intrinsic and extrinsic factors.The normal temperature of the testes should normally be maintained at 2-6°C below the body’s core temperature.When testicular temperature is elevated or the body’s thermoregulation is impaired,this leads to reduced spermatogenesis,induces germ cell apoptosis and DNA damage,and affects sperm quality,resulting in reduced male fertility or infertility.Transient receptor potential vanilloid receptor-1（TRPV1）is a heat-sensitive receptor channel widely present in mammals that can be activated by stimuli such as harmful temperature（>43°C）,extracellular acidic environment（H⁺）and capsaicin,and these TRPV1 is a non-selective cation channel with high permeability to Na⁺and Ca²⁺,and can be involved in inflammation and pain transmission,apoptosis,proliferation and various physiological or pathological regulatory processes in vivo.Existing studies have found TRPV1 expression in human and rat prostate,testis,penis and bladder tissues,but its specific cellular localization in mouse testis has not been systematically studied.Meanwhile,after testes were treated with hot water bath at 42°C,the lumen of testicular spermatogenic tubules in Trpv1^-/-mice was deformed and apoptosis of spermatogenic cells increased,suggesting that TRPV1 may play a thermoprotective role on spermatogenic cells under high temperature;in addition,the expression of TRPV1 in testes of aged（32-month-old）mice was increased and apoptosis of spermatogenic cells in Trpv1^-/-aged mice was reduced,indicating that TRPV1 may play a pro-apoptotic role on spermatogenic cells in aged mice The results suggest that TRPV1 may have a pro-apoptotic effect on germ cells in aged mice.This suggests that TRPV1 is associated with germ cell apoptosis,but its specific role is controversial and needs further study.Objective:To investigate the relationship between TRPV1 protein and germ cell apoptosis,this study firstly clarified the expression pattern of TRPV1 in mouse testis,and then observed the change of TRPV1 expression through a cryptorchid model to determine whether TRPV1 was associated with germ cell apoptosis in mice after cryptorchidism;after that,we further observed the apoptosis of testicular germ cells in cryptorchid mice in Trpv1^-/-mice.The aim of this study was to explore the relationship between TRPV1 and germ cell apoptosis and to provide new ideas on the role of TRPV1in the male reproductive system.Methods:1.Quantitative PCR and protein immunoblotting were used to determine the amount of TRPV1 expression in the testis of postnatally growing mice.Immunohistochemical staining was used to detect TRPV1 expression in several cycles of adult mouse testicular germinal epithelium,and immunofluorescent double-labeling was used to determine TRPV1’s distribution in distinct kinds of germinal epithelial cells in adult mice tests.Fluorescence quantitative PCR and protein immunoblotting were used to determine the TRPV1 expression levels in all stages of germ cells in adult mouse testis.2.A mouse cryptorchid model was created,and the testis-to-body ratio,sperm count,and HE staining of the testis and epididymis at 3 d,6 d,9 d,12 d,and 15 d were used to evaluate the phenotypic alterations of spermatogenesis.By using TUNEL labeling,immunofluorescence staining of the apoptosis-related markers Bax and caspase3,and protein immunoblotting,researchers were able to examine the death of spermatogenic cells as well as the changes in TRPV1 expression following cryptorchidism.Protein immunoblotting and immunofluorescence labeling were used to detect changes in TRPV1 expression following cryptorchidism.3.The mean litter size,body weight and testis weight per cage,sperm count,testis and epididymis HE staining and testis TUNEL staining of the offspring mated with WT female mice were used to observe the fertility of Trpv1^-/-mice,to construct the Trpv1^-/-mouse cryptorchid model,and to observe Trpv1^-/-mice by comparing the testis-to-body ratio,sperm count,testis and epididymis HE staining of WT cryptorchid mice.Reproductive phenotypic changes were observed by comparing the testicular TUNEL staining of WT cryptorchid mice to observe the apoptosis of spermatogonia in Trpv1^-/-mice.Results:1.TRPV1 was expressed in the testis from 7 d after birth,and its expression level increased significantly at 21 d,peaking at 28 d.After that,its expression decreased gradually as mice developed into adults;TRPV1 was expressed in the spermatogenic epithelium of mouse testes at all stages and in the membranes of spermatocytes and supporting cells at all levels,and was mainly expressed in the membranes of spermatocytes.2.The volume and weight of testes as well as the number of spermatozoa in mice after cryptorchidism decreased with the increase of cryptorchidism time.The spermatogonia of testes after cryptorchidism started to shed from 6 d,the lumen of the duct was gradually deformed,the apoptosis of spermatogonia increased,the spermatogenesis of the caudal part of the epididymis decreased and the cavity increased.15 d after cryptorchidism,there were only a few spermatogonia and supporting cells in the lumen of the testes.3.Trpv1^-/-male mice showed no significant differences in body weight,testicular weight,sperm morphology,number,and mean sperm production from WT mice,and could have normal fertility.testicular morphology was better in Trpv1^-/-mice than WT mice after cryptorchidism,with rounded lumen of testicular spermatogenic tubules,reduced spermatogenic cell shedding,more spermatozoa in the caudal part of the epididymis,and reduced apoptosis of spermatogenic cells.Conclusion:1.TRPV1 is expressed in all stages of mouse testis development and its expression gradually increases as the testis develops.2.TRPV1 was expressed in spermatogenic cells at all stages of testicular spermatogenic epithelium and was mainly localized to the SPC membrane during prophase meiosis.3.The expression of TRPV1 increased with the duration of cryptorchidism,suggesting that TRPV1 is associated with apoptosis in testicular germ cells.4.Spermatogenesis was normal in Trpv1^-/-mice,and the reproductive phenotype was not different from that of WT mice.Apoptosis of germ cells was reduced in Trpv1^-/-mice after cryptorchidism,suggesting that TRPV1 promotes apoptosis of germ cells after cryptorchidism.