Study Of Correlation Between The Activity And Expression Of HSL And The Spermatogenesis Of Guinea Pigs

Background and objectiveWorld Health Organization(WHO)predicted that the male infertility,cancer and cardiovascular disorder would be the three most severe diseases which would harm the human heath in 21th.During 50 years lately,both the quality and the reproductive activity of global human sperm have degraded consumedly.The statistic result of clinic cases showed that sterility and barrenness occupied 10-15%in the reproductive couple.One couple of ten reproductive couples was infertility.Some paper showed the statistic result that the visit number of people in later 5 years was 2-3 times than former 5 years in ten thousand cases of male infertility during 1980-1990.In Nanhai city,the number of visit during 1991-1995 was two times than the number of visit during 1981-1985.The data above-mentioned showed that the number of male infertility had been increasing gradually in scale of global.And the disorder of spermatogenesis is one of the most important factors which can induce the male infertility.So the study of spermatogenesis is urgent.Spermatogenesis is a developmental process depended on externalization and regulative,which characteristic of undifferentiated amphiploid stem cell transforming to high differentiated monoploid spermatozoa.The process occurs in contorted seminiferous tubules of testis and depends on contiguity between Sertoli cell and spermatogenic cell.Leydig cell excretes testosterone which functioning on Sertoli cell, so that it stimulates genetic transcription and germ cell differentiating on the function of LH and FSH. It is thus clear that T,FSH and LH are essential in spermatogenesis.And especially T is one of the significant factors.FSH is the important factor in starting the spermatogenesis.It regulates directly Sertoli cell via c-AMP or Ca²⁺signal channel,and which possibly depends on changing the structure and function of Sertoli cell so as to produce the condition in favor of spermatogenesis.LH functioned on Leydig cell.Every Leydig cell has 6000 LH acceptor which can combine whit LH tightly.Then the level of cAMP in Leydig cells increased obviously.This phenomenon indicates that LH can stimulate the activity of cAMP which can activate protein kinase and promote protein phosphorylation so as to activate cholesterol transforming to pregnenolone,which enhancing the testosterone biosynthesis. Testosterone is one of the most important factors which regulate spermatogenesis and its target cell is the peritubular contractile cells and Sertoli.Testosterone effects on peritubular contractile cells which produced Sertoli regulatory protein.In the end the double regulation was implemented through testosterone and Sertoli cell regulatory protein.On the level of molecular,spermatogenesis and sperm maturing is a process of a series of definite gene expression,of which contain testicular gene(cAMP SCF HSP70 TATA EDDY).A series of gene expression and control make that germ don't express form character,cell-behavior and special function in body cell.But germ growth and development require very strict environment.Now the correlated genes were studied by RNA interference,immunization technique,construction of cell line, transgene or knock out mice etc methods so that molecular basis congnited more.Our study is based on that HSL can regulate fatty acid and cholestoral metabolism,and cholestoral can be atabolismed to pregnenolone which can be atabolismed to testosterone which is the most important factor in spermatogenesis.Further more HSL expressed especially in testis.So we constructed animal model.The goal is that concerning theory of the correlation between spermatogenesis and HSL should be formed.Materials and MethodMaterials Napental,0.9%Sodium Chloride Injection,Trizol agent,Dahui RT-PCR reaction system,Spanish agarose,Da'an experimental gene companion,isopropanol,Xiangyi hydroextractor,Eppendof refrigerated centrifuge,Liuyi electrophoresis apparatus, Jieda gel-image analytical system,ABI9700 amplification equipment, ethylenediamine tetraacetic acid(EDTA),buffer,hormone(T,FSH and LH)kit,fat acid kit(triglyceride and cholesteryl ester)et al.Methods1)Guinea pigs in different developmental stages were grouped 3 according to the result of preliminary experimental result(6-day-old neonatal,16-day-old pubertal,and 70-day-old adult guinea pigs).These animals were put to death after anesthetized, testis and epididymis tissues and caval vein blood were collected.2)The left testis and epididymis tissues were homogenated(trisect).The testicular HSL mRNA was assessed respectively by semiquantitative RT-PCR and enzymatic activity was detected at 37℃via colorimetric method.The concentrationof triglyceride and cholesteryl ester were measured via enzymic method.3)The caval vein blood was centrifugated.The level of serum testosterone,FSH, LH was measured by radioimmunity.4)The fight testis and epididymis tissues section was stained in HE. Histomorphology alleosis and spermatogenesis or aspermatogenesis was assessed.5)Data handling and statistical methods were performed in SPSS software version 10.0.One-way ANOVA were analyzed among the group means in HSLmRNA, serum gonadal hormone.The comparation between two groups was used LSD-t with aqual variances assumed while Tambane's T2 Test with equal variances not assumed. Pearson correlation analysis was analyzed between HSL mRNA,enzymatic activity, serum gonadal hormone.Result1)Testicular HSL expression increased gradually with the ages of guinea pig increasing.The distinguished statistics difference existed between 70-day-old adult guinea pigs and 6-day-old neonatal or 16-day-old pubertal(P＜0.01).But no difference existed between 6-day-old neonatal or 16-day-old pubertal. 2)Meanwhile,the HSL activity increased gradually and the distinguished difference existed among each group by ONE-way ANOVA(F=145.418,P＜0.05). The apparent difference existed between 6d group and 16d group or 70d group,and also between 16d group and 70d group.The statistic positive correlation existed between the expression of HSLmRNA and HSL activity.3)The statistics difference existed among level of cholesteryl ester in testis of each group(F=13.666,P=0.000).But no statistic difference existed between 16d group and 70d group.The concentration of cholesteryl ester in testis decreased gradually.But,no statistic difference existed among the level of triglyceride in each group(F=2.887,P=0.087).Pearson analysis showed that the apparent inverse correlation existed between the activity of HSL and the concentration of cholesteryl ester,but no correlation existed between the activity of HSL and the concentration of triglyceride.4)And the apparently difference existed on serum testosterone levels (F=197.282,P＜0.01)or FSH(F=5.772,P=0.014)among each group.But no statistic difference existed between 70-day-old adult and 16-day-old pubertal in level of LH, but among other two groups.The statistic positive correlation existed between the activity of HSL and T,FSH or LH.5)Histopathotogy showed that spermatogonium was the main cell in the section of 6-day-old neonatal and no spermatozoa appeared.Spermatogonium of every period could be seen in the testis and epididymis tissues section of 16-day-old pubertal and a small quantity of sperm appeared.Large number of mature sperm could be seen in 70-day-old adult guinea pigs.Conclusion1)The expression and activity of HSL in testis or epididymis of ped-guinea pigs is very low.During the whole developmental period,the increasing tendency between HSL mRNA and the HSL activity was parallel and the possibility existed that the HSL expression determined the activity of HSL.2)HSL_teswas the substrate of hydrolyzing cholesteryl ester in guinea pig testis, and could be the only.The hormone-sensitive lipase could contribute to degrade cholesteryl ester to testosterone.3)By statistics analysis,we concluded that HSL was one of the important factors during the developmental period of guinea pig testis or epididymis.And HSL must participate in form and maintenance of testosterone.HSL was a probably important factor in spermatogenesis via regulating the level of T,FSH and LH.And it could maintain the reproductive activity of guinea pig.