| [Background and aims]:Obesity is a complex metabol ic disease that is determined by lifestyle factors,including environmental(food variety and intake,physical activity)and genetic factors.In recent decades,obesity has become a predominant health problem and has increased globally at an alarming rate.Worldwide obesity has nearly tripled since 1975.Most of the world s population live in countries where overweight and obesity kills more people than underweight.Raised BMI is a major risk factor for noncommunicable diseases such as:cardiovascular diseases(mainly heart disease and stroke),which were the leading cause of death in 2012;diabetes;musculoskeletal disorders(especially osteoarthritis-a highly disabling degenerative disease of the joints);some cancers(including endometrial,breast,ovarian,prostate,liver,gallbladder,kidney,and colon).The risk for these noncommunicable diseases increases,with increases in BMI.Apart from the above risks-related-to-obesity,the negative impact of obesity on male reproduction is gradually recognized.There is a universally accepted fact that the development of male infertility increased rapidly worldwide,which coinciding with the epidemic of obesity.Infertility has become a serious worldwide problem.Forty-eight point five million couples were infertile in 2010,and about 50%infertility is caused by male factors.Male infertility is a complex,multifactorial and polygenic disease.Plenty of studies have demonstrated that excess weight and obesity factors serve an important role in the development of male infertility.According to the clinical studies,men with overweight or obesity can decrease sperm quality including sperm concentration,sperm motility,ejaculate volume,acrosome reaction decline,increased sperm DNA damage and lower embryo implantation rates as well,comparing to those of normal BMI men.Some studies estimate that male sperm counts continue to decrease by as much as 1.5%per year in the USA;similar findings pertaining to other Western countries,where obesity is more prevalent,have also been reported.Chavarro et al.demonstrated that ejaculate volume decreased steadily with increasing BMI levels;men with BMI>35 kg/m2 had a lower sperm count than normal-weight men;considerably more sperm with high DNA damage were in obese men than in normal-weight men,when they studied male partners of subfertile couples.Hofny et al.found BMI correlated positively with abnormal sperm morphology.Hammoud et al.found prevalence of a low progressively motile sperm count increased with increasing BMI.Furthermore,plenty of nonhuman animal experiments have also shown that a high-fat diet can increase the male infertility rate and decrease sperm parameters.Carla DB Fernandez et al.found diet-induced obesity in rats leads to a decrease in sperm motility.Yong Fan et al.fed male C57BL/6 mice with a high-fat-diet,and found the percentage of sperm motility and sperm acrosome reaction significantly decreased,the proportion of teratozoospermia dramatically increased in HFD mice compared to those in normal diet fed controls.These alteration of sperm function parameters strongly indicated that the fertility of HFD mice/rats was indeed impaired,which was also validated by a low pregnancy rate in their mated normal femal.However,the underlying mechanism research still remains unclear.Currently the reasons for high-fat-diet-induced-damage on male fertility are mainly concentrated in hypothalamic-pituitary,aromatase/peripheral lipids,germ cells and Leydig cells,Sertoli cells(SCs)have not received enough attention.SCs nurse developing germ cells and form the BTB between opposing SCs and adjacent Sertoli-germ cells.The SC is considered to be a spermatogenic epithelial support structure,and extends into the entire spermatogenic epithelium,so that spermatogenic cells at all levels may complete all morphological and physiological changes from spermatogonia to mature sperm along the SCs.With the advent of puberty,the SCs lose their ability to proliferate,and the number is basically fixed,and meanwhile,the main role of SCs is transformed to support and nutrient the development of germ cells to ensure the orderly spermatogenesis.The number of germ cells that can be supported by a single SC is limited,therefore,the number of SCs determines the number of germ cells,which in turn determines the final sperm production and then affects fertility.The current study focuses on puberty and sexual maturity mice,whose number of SCs is fixed already,so the function of SC,and especially its derived structure-BTB was mainly studied target.The BTB is one of the tightest blood-tissue barriers and physically divides the seminiferous epithelium into the basal and apical compartments,in which different stages of germ cell development occur,and these compartments are crucial to male fertility.The BTB in the mammalian testis is constituted by coexisting TJ,basal ectoplasmic specialization[basal ES,a testis-specific atypical adherens junction type],gap junction,and desmosome.Functions of the BTB are mainly the 3 aspects:1."Fence" and"Gate-Keeper" Functions:a.Restricting paracel lular flow of biomolecules,b.Segregating cellular events during the epithelial cycle of spermatogenesis;2.Creating an immunological barrier to avoid the production of anti-sperm antibodies and autoimmune disease;3.Confering cell polarity in the seminiferous epithelium.An intact and functional blood-testis barrier is necessary for spermatogonial stem cell differentiation during spermatogenes.Several previous studies have demonstrated that various stimulators,such as high glucose,perfluorooctanesulfonate,cadmium,amodiaquine and bisphenol A,adversely affect male fertility via impairment of the BTB;however,whether obesity that is induced by a high-fat diet can provoke deleterious effects on the BTB has not been elucidated.The BTB in the mammalian testis comprises tight junctions(TJs),basal ectoplasmic specializations(basal ESs),gap junctions,and desmosomes.TJs,which coexist with basal ESs between basal ESs and which are reinforced by basal ESs,make the BTB one of the tightest blood-tissue barriers in the mammalian body.The expression levels of TJ-related proteins(occludin and ZO-1)and basal ES-related proteins(N-cadherin and beta-catenin)are downregulated by exogenous stimulators,such as cadmium,perfluorooctanesulfonate,amodiaquine and bisphenol A.These studies indicate that TJs and basal ESs might be the molecular targets of exogenous stimulators.Obesity can increase oxidative stress in the whole body.Overconsumption of food leads to a dysmetabolism state where energy intake exceeds energy expenditure,and cellular oxidative stress follows.The increase of oxidative stress leads to lots of downstream effects including the induction of inflammatory cascades.This process starts from mitochondrial overload of free fatty acids(FFA)and glucose,which leads to an increase in the production of acetyl coenzyme A(acetyl CoA),an important enzyme in cellular metabolism.Higher levels of acetyl CoA result in an increase in nicotinamide adenine dinucleotide(NADH)generation from the tricarboxylic acid(TCA)cycle.Increased NADH increases electron generation by complex I of the mitochondrial electron transport chain and elevates membrane potential to the extent that complex III is stagnated resulting in a longer half-life for coenzyme Q.Increased coenzyme Q leads to an increased production/reduction of oxygen to superoxide(O2·-).Thus the main impact of overconsumption of free fatty acids and glucose leads to higher levels of superoxide in the mitochondria.Superoxide is a relatively unstable intermediate and in large part is converted to hydrogen peroxide in the mitochondria by superoxide dismutase(SOD).The newly formed hydrogen peroxide can then undergo a Haber-Weiss or Fenton reaction,yielding a highly reactive hydroxyl radical(·HO),which can oxidize mitochondrial proteins,DNA,and lipids,and amplify the effects of the superoxide-initiated oxidative stress.The generation of highly reactive oxygen radicals can activate redox-sensitive transcription factors and result in numerous downstream effects,including triggering inflammatory cascades and increasing reactive oxygen species(ROS)production.Depending on the cell types,the impact of oxidative stress-induced inflammation can result in various forms of dysfunction.Many events associated with male infertility are inducers of oxidative stress.For example,physical conditions of varicocele and cryptorchidism,X-irradiation and environmental toxicants have been demonstrated to increase testicular oxidative stress,which leads to an increase in germ cell apoptosis and subsequent hypospermatogenesis.Testicular oxidative stress appears to be a common phenomenon in much of what underlies male infertility.However,whether oxidative stress is involved in lipotoxicity-induced injury to SCs and the BTB is unclear.Metformin is an oral biguanide hypoglycaemic agent that is widely used for the treatment of type 2 diabetes mellitus.Therapeutic effects of metformin have been attributed to a combination of improving peripheral uptake and utilization of glucose,decreasing hepatic glucose output,decreasing rate of intestinal absorption of carbohydrates,and enhancing insulin sensitivity.Beyond its glucose lowering effects,metformin exhibits antioxidant properties in numerous tissues,an effect that is independent of its effect on insulin sensitivity,and acts to decrease lipid peroxidation.In 2003,Bonnefont-Rousselot,D,et al reported metformin could directly scavenge ROS or indirectly act by modulating the intracellular production of superoxide anion.In 2006,M.Mahrouf,N et al.reported metformin reduces angiotensin-mediated intracellular production of ROS in endothelial cells through the inhibition of protein kinase C.In 2007,Gonul Kanigur-Sultuybek et al.demonstrated short-term effect of metformin can protect against prooxidant stimulus-induced DNA damage in lymphocytes from elderly subjects,and the antioxidant activity of metformin might result from a direct effect on ROS or could have an indirect action on the superoxide anions produced by hyperglycemia.In 2012,Carolyn Algire et al.reported metformin reduces endogenous ROS and associated DNA damage;In the same year,Abdelkader E.Ashour et al.demonstrated metformin successfully prevents doxorubicin-induced cardiotoxicity in vivo by inhibiting doxorubicin-induced oxidative stress,energy starvation,and depletion of intramitochondrial coenzyme A-SH.In 2014,Ran-ran Zhao et al.found metformin protects against seizures,learning and memory impairments and oxidative damage induced by pentylenetetrazole-induced kindling in mice;Ahmed A and Abd-Elsameea demonstrated metformin improved the oxidative stress induced by ischemia and ischemia/reperfusion injuries in the cerebrum of rats.A recent clinical study has demonstrated for the first time that metformin improves semen quality in men with hyperinsulinaemia,but the underlying molecular mechanism is not clear.Moreover,a study in patients with PCOS(polycystic ovarian syndrome)has reported that metformin reduces angiogenesis through the NF-?B pathway.However,whether metformin can improve fertility in obese males and whether metformin can alleviate the damage to the BTB in the testis via the inhibition of oxidative stress remain unknown.In the present study,to determine the effect of metformin on fertility in obese males,an obese mouse model was induced with a high-fat diet,and metformin was administered.First,the effect of metformin on the reproductive ability of male mice was investigated.Alterations to the ultrastructure,BTB integrity,the expression of TJ proteins and the expression of basal ES proteins were further assessed.In addition,the levels of oxidative stress and NF-K B activity were measured to characterize the intracellular mechanism.This study demonstrates a crucial role for metformin in obesity-induced male infertility.[Mehods]:1.The construction of an obese male mouse model and method for adding metformin:The male C57BL/6 mice were randomly divided into two groups:the normal-diet group(N,n=20)was fed a standard diet,in which 10%of the calories were from fat,and the high-fat-diet group(H,n=30)was fed a high-fat diet,in which 60%of the kcal were from fat.Ten mice from each group were sacrificed at the end of the 8th week of feeding.The remaining mice in the N group were maintained on their standard diet(NN,n=10),whereas the mice fed a high-fat diet(n=20)were further subdivided into two subgroups.The first subgroup(HH,n=10)was maintained on the high-fat diet,and the second subgroup(HH+MET,n=10)was maintained on the high-fat diet with?200 mg/kg body weight/day metformin given in the drinking water.The body weights of the mice were monitored weekly during the feeding period.All mice were sacrificed at the end of the 16th week of feeding.2.Reproductive ability assay:At the 8th and 16th weeks after different feeding conditions,each male mouse was individually housed and mated with two female mice,which were randomly grouped by weight,for 5 consecutive days.On the second day,vaginal plugs were inspected to determine if coitus had occurred.Female mice with vaginal plugs were moved into another cage and observed until the pups were born.The number and weights of the pups were also statistically analysed.3.The assessment of lipid metabolism and sex hormone analysis:Serum levels of total cholesterol(TC),triglycerides(TG),high-density lipoprotein-cholesterol(HDL-c),low-density lipoprotein-cholesterol(LDL-c)and glucose(Glu)were determined using enzymatic methods with an Olympus AU5400 automatic biochemical analyser.To determine testicular ectopic lipid accumulation,frozen sections of the testis were stained with Oil red 0 for 10 min,and the degree of ectopic lipid deposition was determined according to the intensity of positive staining.The FSH level in serum was measured using an ELISA kit according to the manufacturer' s protocol for each assay.4.Morphological assessment of testis and blood testis barrier:Haematoxylin and eosin(H&E)staining was performed for morphological observation using a system incorporated in the Carl Zeiss microscope.Transmission electron microscopy(TEM)analysis,with a JEM-1200EX transmission electron microscope,was used to characterize the changes in testis and BTB at the ultrastructural level.5.BTB integrity and junction protein assay:A biotin tracer was used to assess the BTB integrity.The gaps below the testicular tunica albuginea were injected with 50 ?l of EZ-Link Sulfo-NHS-LC-Biotin,and after 30 min,the testes were removed and frozen in liquid nitrogen in preparation for cryosectioning(10 ?m).Alexa Fluor(?)568-conjugated streptavidin was used to integrate the biotin tracer.The sections were analysed by fluorescence microscopy.The related junction proteins,such as Z01,occludin,N-cadherin,beta-catenin,and nectin2,were analyed by immunoblotting analysis.The RNA level of the above proteins were also detected.6.Oxidative stress and NF-?B:The level of oxidative stress in testis was assessed through the measurements of malondialdehyde(MDA),superoxide dismutase(SOD)and reactive oxygen species(ROS).Assay kits for MDA,SOD and ROS were separately provided by Beyotime Biotechnology and Jiancheng Bioengineering Institute(China).The MDA content,SOD activity and ROS levels were measured using the kits according to the manufacturer ' s instructions,and these measurements were normalized to total protein.Immunofluorescence was used to detect the level of NF-?B in SC nuclei.The testicular tissues were fixed with MDF and embedded in paraffin blocks.The sections were deparaffinized,and antigen retrieval was performed with Tris-EDTA.NF-?B/p65(1:250)primary antibody was used to bind the NF-?B in the tissues.[Results]:1.High-fat-diet-fed mice presented evident lipid metabolic disturbances,disrupted BTB integrity,and decreased reproductive function.2.Metformin improved the decrease in male fertility,decreased ectopic lipid deposition in the testis and increased serum FSH(P<0.05).3.Further mechanistic analysis revealed that metformin ameliorated the high-fat-diet-induced injury to the BTB structure and permeability and restored the disordered BTB-related proteins,which might be associated with an improvement in oxidative stress and a recovery of NF-?B activity in SCs.[Conclusions]:Metformin improves obese male fertility,which may be related to alleviating oxidative stress-induced BTB damage.ROS-NF-?B pathway may be part of the inner mechanism of metformin in this process of regulating lipotoxic injury on Sertoli cell/blood-testis barrier.These findings provide new insights into the effect of metformin on various diseases and suggest future possibilities in the treatment of obese male infertility. |
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