Hydrogen Sulfide Improved Testicular Functions In Mice Fed With A High-cholesterol Diet

Background:As one of the most important organ in male reproductive and endocrine system,testis has the function of producing testosterone and sperms.Testosterone mainly comes from Leydig cells(LCs),and the formation of sperms mainly occurs in the germ cells(GCs)of testicular spermatogenic tubules,and Sertoli cells(SCs)are accepted to be imperative to provide adequate nutrients and protected environment for germ cells(GCs).Recent epidemiological studies have found that hypercholesterolemia becomes a crucial risk factor for testosterone deficiency,however,the effect of hypercholesterolemia on the overall testis function especially in fertility,still remains unclear,and the underlying mechanism is unknown.Therefore,currently there is no effective intervention or treatment.Hydrogen sulfide(H2S)is a gaseous signal molecule which is naturally produced in various cells and tissues.Studies have shown that H2S plays an important role in regulating the protein/enzyme functions in the body,especially in inhibiting inflammatory response and repairing damages.In addition,H2S protects tissues and cells from damages by reducing oxidative stress.H2S is a self-synthesizable biological reductant,but its effects on testicular dysfunction/hypogonadism which could probably be due to hypercholesterolemia and corresponding mechanisms have not been studied and determined.Objectives:In this study,high cholesterol diet was applied to induce hypercholesterolemia of mice.On this basis,exogenous H2S intervention was conducted to investigate the effect of H2S on mice testicular function,and the mechanism was explored.Methods:1.Construction of a HC mouse model:30 7-week-old male C57BL/6 mice weighed 19-21g were randomly divided into 3 groups of 10 mice:the normal diet group(NC group,n=10),the high-cholesterol diet group(HC group,n=10),and the GYY4137 intervention group(HC+GYY4137 group,n=10).The mice in NC group were fed with normal diet;the mice in HC group and in HC+GYY4137 group were fed with high-cholesterol diet.All mice were weighed weekly,and their blood samples were collected for the mice at 12th/18th week-feeding periods,then the levels of blood lipid,glucose and serum testosterone were detected.2.Exogenous H2S intervention:Since the end of the 18th week,the mice of HC+GYY4137 group,(n=10)began to pass through pharmacological intervention on the basis of the HC diet.Mice were intraperitoneally injected with GYY4137(a sustained release agent for H2S donor),while the mice of NC group and HC group were injected with the identical dose of PBS.After the 23rd week of feeding,male mice were mated with 8 weeks old C57BL/6 female mice.The female pregnancy rates,the number of offspring and body weight of offspring were estimated for the cause of assess male fertility.3.Male mice were sacrificed:(1)blood lipid,glucose and serum testosterone levels were analyzed by automatic biochemical analyzer;(2)sperm parameters of mice were analyzed by SSA-II system;(3)the cholesterols(TC and FC)accumulated in testicular tissues were detected qualitatively and quantitatively;(4)the morphology of mice testicular tissues was observed by H&E staining;(5)ELISA kits were used to detect the levels of sex hormone(T and LH)in testicular tissues;(6)Western Blotting and RT-PCR techniques were applied to detect the expressions of the key enzymes of testosterone synthesis and the specific marker of spermatogenic cells;(7)Western Blotting and RT-PCR techniques were used to detect endogenous H2S synthetase expressions in testicular tissue of mice.(8)Western Blotting were used to detect transcription factor NF-?B expressions in testicular tissue of mice.Results:1.After 24 weeks of feeding,there was no significant body weight difference among the NC group,the HC group and the HC+GYY4137 group.Compared with NC group,the TC and LDL-C levels in serum,the TC and FC levels in testicular tissues,and the lipid deposition significantly increased for the mice in HC group;also,the epididymis fat weight augmented remarkably for the mice in HC group.After GYY4137 intervention,the TC and LDL-C levels in serum,the TC in testicular tissues and lipid deposition were notably reduced;and the epididymis fat weight obviously decreased for the mice in HC+GYY4137 group.2.Compared with NC group,the serum and testicular tissue testosterone and LH levels were significantly reduced for the mice in HC group,and the expression of StAR and the key enzyme of testosterone synthesis decreased,also we found that the morphology and functions of testes were destroyed.After intervention,the testicular tissue testosterone level and the expression of the StAR increased for the mice of HC+GYY4137 group,and the damages of testicular morphology and function were restored.3.Compared with the NC group,the number of SPR,PR and the number of total sperm motility(PR+NP)for the mice of HC group were significantly reduced,and there was no significant difference in the expression of DAZL,the sperm concentrations and fertility.The parameters above for the mice of HC+GYY4137 group did not changesignificantly after intervention.4.Further analysis illustrated that the transcription levels of endogenous H2S synthase(CBS and CSE)were significantly reduced after 24 weeks of feeding with HC diet.GYY4137 intervention caused an obvious augmentation of CBS and CSE levels.However,the expression of NF-?B as well as the nuclear transcription factors had no significant distinction among the mice in three groups.Conclusions:1.High cholesterol diet led to lipid metabolism disorder and lipid ectopic deposition in testes of mice.2.High cholesterol diet led to the destruction of testicular structure in mice,the expression of StAR,a key enzyme in testosterone synthesis decreased,testosterone synthesis and sperm quality decreased.3.The intervention of exogenous H2S can improve the lipid metabolism disorder and testicular structure injury caused by high cholesterol diet;In particular,exogenous H2S improved testosterone production in testicular tissue.