Experimental Study On The Role And Mechanism Of Adiponectin In Testicular Tissue In The Effect Of High-Fat Induced Obesity On Male Reproduction Function

Objective: Obesity can adversely affect male reproductive function,but the mechanism of obesity affecting male reproductive development has not been elucidated,and some studies have found that adiponectin(ADPN)may play a role in it.ADPN is an adipokine,which is mainly secreted by adipose tissue.In recent years,the expression of ADPN and its receptors has also been found in the testis tissue,so the role of ADPN in reproductive development has also attracted attention.How is ADPN involved in the regulation of reproductive development? Some studies have found that high concentration of ADPN can inhibit the secretion of testosterone,and stable testosterone level plays an important role in maintaining male reproductive function.Therefore,we suggest that ADPN in testicular tissue may be involved in the regulation of male reproduction by affecting the level of testosterone.By what pathway does ADPN affect testosterone levels? Through literature review,we found that the change of ADPN level in testicular tissue may be through the AMP-activated protein kinase(AMPK)/phosphorylated extracellular regulated protein kinase(ERK)pathway or the c AMP/protein kinase A(PKA)/camp response element binding protein(CREB)pathway.The expression of kissing gene(KISS1)and gonadotropin-releasing hormone(Gn RH)in the testis decreases,thereby affecting the production of testosterone.ADPN may also directly affect testosterone-producing proteins or enzymes at the testicular level,such as: Steroid acute regulatory protein(St AR),cholesterol side-chain cleavage cytochrome P450(P450scc),cytochrome P450 family member 17A1(CYP17A1)and 3β-hydroxysteroid dehydrogenase(3β-HSD),which in turn affect testosterone production and lead to reduced male reproductive function.In this study,we established a high-fat induced obesity model and an in vitro model of high-fat exposure to investigate the role and possible mechanism of ADPN expression in the testis in the effect of obesity on male reproduction,and to provide scientific basis for the study of the effect of high-fat induced obesity on male reproductive function.Methods:24 male C57BL/6J mice aged 4-5 weeks were randomly divided into CON group and OB group,with 12 mice in each group.The CON group was fed with normal diet,and the OB group was fed with 60% high-fat diet for 16 weeks to establish a high-fat induced obesity model.After 16 weeks of the experiment,the body composition was measured,and the sperm motility and number were detected.ELISA was used to measure the serum levels of ADPN,testosterone and estradiol.The pathological changes of testis were observed under light microscope and transmission electron microscope.Western Blot was used to detect the expression of ADPN,ADPN receptor 1,Kisspeptin,GPR54,Gn RH,PAMPK,P-ERK,c AMP,PKA,P-CREB,St AR,P450 scc,3β-HSD,and CYP17A1 in the testis.RT-q PCR was used to detect the m RNA expression of ADPN,ADPN receptor 1,KISS1,GPR54,and Gn RH in the testis.The Leydig cells of TM3 mice were cultured in vitro and divided into 4 groups: control group,palmitic acid(PA)intervention group,ADPN gene silencing group(ADPN-si RNA)group,and PA+ ADPN-si RNA group.Cell viability was detected by CCK-8 assay.ADPN si RNA was transfected into TM3 cells to construct an ADPN silencing model.ELISA was used to measure the level of testosterone.Western Blot was used to detect the expression of ADPN,ADPN receptor 1 and testosterone production related proteins.Results: 1.The body weight of OB mice was significantly higher than that of CON mice at 16 weeks(P<0.01).2.Body fat and percentage of body fat in OB group were significantly higher than those in CON group(P<0.05).3.The perirenal fat weight,perirenal fat coefficient and perirenal fat weight,perirenal fat coefficient,perirenal fat coefficient,perirenal fat coefficient,testis weight,epididymis weight and epididymis coefficient of OB mice were significantly higher than those of CON mice(P<0.01);The testis coefficient of the mice was significantly lower than that of the CON mice(P<0.05).4.Sperm count and motility in OB group were significantly lower than those in CON group(P<0.05),and the rate of abnormal sperm was significantly increased(P<0.01).5.Under the light microscope,the seminiferous tubules in the testicular tissue of the OB group were loosely arranged,not tightly packed,the outline was not clear,and the number of sperm was low.6.The number of interstitial cells in the testis of OB group was decreased under electron microscope.There were a large number of lipid droplets around the nucleus of interstitial cells in OB group mice.7.The serum levels of ADPN and testosterone in OB group were significantly lower than those in CON group(P<0.05),and the serum estradiol level was significantly higher than that in the CON group(P<0.05).8.The expression levels of ADPN protein and m RNA in the testis of OB group were significantly higher than those of CON group(P<0.05).9.The expression level of Gn RH protein in OB group was significantly lower than that in CON group(P<0.05).10.The protein and m RNA expression levels of Kisspeptin and GPR54 in the testis of OB mice were significantly lower than those of CON mice(P<0.05).11.The protein expression levels of AMPK,pAMPK,ERK,p-ERK,c AMP,PKA,CREB and p-CREB in the testis of OB group were not significantly different from those of CON group.12.The protein expression levels of P450 scc,St AR,CYP17A1 and 3β-HSD in OB group were significantly lower than those in CON group(P<0.05).13.Cell viability was significantly decreased in 200μM PA and400μM PA groups(P<0.05);The levels of testosterone in the 200μM PA treatment group were significantly lower than those in the CON group(P<0.05).14.The protein expression levels of ADPN and ADPN receptor 1 in the PA intervention group(200μM)were significantly higher than those in the CON group(P<0.05).15.Compared with CON group,the expression level of testosterone in PA group was significantly decreased(P<0.01),while the expression of testosterone in PA+ADPN-si RNA group was higher than that in PA group(P<0.01).16.the protein expression levels of P450 scc,St AR,CYP17A1 and 3β-HSD in PA group were significantly lower than those in CON group(P<0.05).Compared with the PA group,the protein expression levels of P450 scc,St AR,CYP17A1 and 3β-HSD in the PA+ADPN-si RNA group were significantly increased(P<0.05).Conclusion: 1.High-fat induced obesity can cause a decrease in male reproductive function.2.The decrease of male reproductive function induced by high-fat obesity may be related to the increased expression of ADPN in the testis,which inhibits the expression of Kisspeptin and Gn RH.However,AMPK/ERK or c AMP/PKA/CREB pathways are not found to be involved in the regulation of ADPN on Kisspeptin secretion.3.When obesity is induced by high fat diet,the increased expression of ADPN in the testis tissue can reduce the level of androgen by inhibiting the protein related to androgen synthesis,leading to the decline of male reproductive function.