Regulation Mechanism Of FSH On Autophagy And Against Oxidative Stress Of Testicular Sertoli Cells In Dairy Goats

The reproductive capacity of male mammals depends on continuous and stable spermatogenesis in the seminiferous tubules of the testis.Sertoli cells,as the only somatic cells in the seminiferous tubules,are directly involved in regulating the development of germ cells during spermatogenesis.Recent studies have found that Sertoli cell-mediated autophagy plays important roles in maintaining Sertoli cell function and ensuring semen quality.Sertoli cells are the target cells of oxidative injury in oxidative stress-induced spermatogenesis disorder.Follicle-stimulating hormone（FSH）plays a key role in spermatogenesis and Sertoli cell function,but the regulatory mechanisms of FSH on the autophagy and against oxidative stress of Sertoli cells remain unclear.Moreover,mammalian spermatogenesis is regulated by reproductive hormones.Dairy goat breeding has significant seasonal changes,and semen quality and hormone levels show a dynamic pattern with seasonal changes.However,changes in spermatogenesis,testicular histology and Sertoli cell in dairy goat,during breeding and non-breeding seasons,have never been investigated.In this study,male dairy goats were used.Hormone levels,Sertoli cell autophagy,testicular histology,and differences in signaling pathways related to spermatogenesis were analyzed using ELISA,immunohistochemical staining,and transmission electron micrographs.At the cellular level,FSH was added to the medium of dairy goat Sertoli cells to explore the regulatory effect of FSH on the autophagy and against oxidative stress of Sertoli cells.To further clarify the protective effect of FSH in oxidative injury of Sertoli cells in vivo,we used an in vivo oxidative stress model targeting mouse testes.After pretreatment with FSH,intraperitoneal injection H₂O₂ stimulates oxidative stress in mice.Then testicular histology and Sertoli cell ultrastructural were analyzed.And the expression levels of apoptosis,oxidative stress,autophagy,and Sertoli cell-associated proteins were detected.The main results were as follows:1.Compared with breeding season（mid-October）,semen quality of dairy goats during non-breeding season（mid-April）significantly decreased（P<0.01）,and serum FSH and testosterone were reduced by 49.63%and 36.63%（P<0.01）,respectively.The number of apoptotic germ cells significantly increased（P<0.05）and proliferation activity of germ cells reduced（P<0.05）during the non-breeding season.However,the testis size did not change seasonally（P>0.05）,indicating spermatogenesis in dairy goats is seasonally regulated and is not completely arrested.Compared with breeding season,Sertoli cell autophagy was more active during the non-breeding season.And we found significant changes in the ultrastructure of Sertoli cells during the non-breeding season.During the breeding season,the expression levels of FSHR,WT1,ABP,GDNF,and SCF were significantly higher（P<0.05）than those in non-breeding season goats.Transcriptome sequencing analysis showed that the number of significantly up-regulated genes enriched in the PI3K/Akt signaling pathway was the largest（a total of 10）in the testis during the breeding season,suggesting that the PI3K/Akt signaling pathway plays an important role in the seasonal spermatogenesis of dairy goats.2.After treatment with 2 IU/m L FSH for 48 h,Sertoli cell viability was significantly increased（128.30%±7.82%,P<0.01）.FSH activates PI3K/Akt/m TOR signaling pathway,thereby increase LC3-II/I ratio and reduce p62 protein degradation,suggesting that the downstream of autophagic flux was inhibited.Ch IP-PCR results indicated that transcription factor EB（TFEB）can bind directly to the LAMP2 promoter.Moreover,FSH decreased the expression levels of LAMP2 protein（P<0.01）and lysosomal gene m RNA（P<0.05）by regulating the nuclear translocation of TFEB,and reduced lysosomal biogenesis,thereby inhibiting autophagy in dairy goat Sertoli cells.Meanwhile,FSH-mediated inhibition of autophagy extends the biological half-lives of ABP,GDNF,and SCF,and promotes the secretion of ABP and SCF.3.H₂O₂ was used to stimulate dairy goat Sertoli cells to construct an in vitro model of oxidative stress.After H₂O₂treatment,the viability of Sertoli cells was significantly decreased（P<0.001）,and ROS level was significantly increased（P<0.05）.The cell viability of Sertoli cells was reduced to 47.25%±5.16%（P<0.001）following treatment with 0.6 mmol/L H₂O₂.Additionally,pretreatment with 2 IU/m L FSH for 48 h increased antioxidant enzyme activity in Sertoli cells,and attenuated the H₂O₂-induced apoptosis of Sertoli cells,indicating that FSH can prevent H₂O₂-induced oxidative injury of Sertoli cells.FSH-inhibited Sertoli cell autophagy increased p62 accumulation,which maintains persistent activation of Nrf2.FSH pretreatment significantly increased the expression level and nuclear translocation（P<0.01）of Nrf2 in Sertoli cells,which enhanced the antioxidant gene expression levels.Additionally,interference with Nrf2 or p62 abolished the protective effect of FSH,indicating that both Nrf2 and p62 are required for FSH-mediated protective role in oxidative stress-induced Sertoli cell injury.These results suggesting that FSH can increase antioxidant enzyme activity through p62/Nrf2 signaling pathway to protect dairy goat Sertoli cells against H₂O₂-induced oxidative injury.4.In an experimental mouse oxidative stress model,H₂O₂ treatment caused more vacuolar spaces in testicular seminiferous tubules and damaged seminiferous epithelium.FSH injection significantly increased SOD activity and total GSH content（P<0.05）,and decreased the MDA content（P<0.05）.Moreover,FSH pretreatment reversed the apoptosis and loss of germ cells caused by H₂O₂,and attenuated the structural damage of seminiferous epithelium,suggesting that FSH reduced testicular oxidative injury.And mice pretreated with FSH showed a lower number of autophagosome-like structures in Sertoli cells（P<0.01）.FSH pretreatment effectively prevented oxidative stress-induced ultrastructure injury of Sertoli cells,and promoted the expression levels of FSHR,ABP,GDNF,and SCF proteins.Taken together,this study showed that spermatogenesis in dairy goats is seasonally regulated,and PI3K/Akt signaling pathway may be involved in the regulation of seasonal spermatogenesis in dairy goats.In vitro experiments showed that FSH reduced lysosomal biogenesis through the PI3K/Akt/m TOR signaling pathway,inhibited the autophagy level of dairy goat Sertoli cells,resulted in the accumulation of p62 protein,and maintained the continuous activation of Nrf2,thereby defending against oxidative stress of dairy goat Sertoli cells.This study provides a theoretical basis for exploring the mechanisms of FSH-mediated autophagy of Sertoli cells to regulate spermatogenesis in dairy goats.It also provided theoretical references for researches on spermatogenesis disorders and male infertility in mammals.