Study On The Effects And Mechanisms Of Palmitic Acid And Octanoic Acid On Sertoli Cells

The prevalence of dyslipidemia resulting from an unhealthy diet has been on the rise in recent years,adversely affecting male fertility.A comprehensive understanding of the key lipid components involved in this process and their potential mechanisms of regulation of spermatogenesis could significantly advance our knowledge and treatment of male infertility.In this study,we employed mass spectrometry to analyze the changes in serum fatty acids in patients with spermatogenic dysfunction,identifying palmitic acid and octanoic acid as potential fatty acids with effects on spermatogenic function.Our findings offer insights into possible intervention targets and treatment measures,providing a theoretical foundation for the protection of male fertility.Part 1:Palmitoylation of Calnexin is involved in palmitic acid-induced Sertoli cells barrier disruptionObjective:The relationship between dyslipidemia and male infertility has garnered increasing attention in recent years.Dyslipidemia is often accompanied by abnormal levels of palmitic acid(PA)in serum,which has been shown to have lipotoxic effects on Sertoli cells.Our previous research has demonstrated that PA induces palmitoylation of proteins in Sertoli cells,leading to activation of the endoplasmic reticulum(ER),damage to the blood-testis barrier(BTB),and impaired sperm production.However,the key palmitoylated proteins involved in this process remain unknown.Additionally,studies have suggested that ω-3 polyunsaturated fatty acids(PUFAs)can mitigate the lipotoxicity of PA to Sertoli cells in unsaturated fat.Nevertheless,further investigation is required to determine the effects of ω-3 PUFAs on palmitoylation and ER stress.Materials and Methods:To identify the palmitoylated proteins with the most significant effect of PA,mass spectrometry was employed.In vitro experiments were conducted to verify the effect of palmitoylation on endoplasmic reticulum stress and cell barrier by constructing a plasmid with a mutation of the palmitoylation site.Mice were randomly assigned to three groups:control,PA exposure,and PA exposure withω-3 PUFAs intervention.Sperm quality was assessed using a hemocytometer,while the integrity of the blood-testis barrier was examined using FITC-I test and electron microscopy.In vitro experiments were conducted on the mouse Sertoli cell line TM4 cells,which were treated with PA and ω-3 PUFAs.The protein level was detected using.Western blotting,and the cell barrier function was assessed using a transmembrane resistance(TER)test.Results:The results of mass spectrometry revealed that Calnexin is the most significantly affected palmitoyl protein by PA.In vitro experiments demonstrated that the mutation of the palmitoylation site led to a significant decrease in the palmitoylation and ER stress levels of Calnexin,as well as an improvement in the barrier function of the TM4 cells.In mouse models,ω-3 PUFAs intervention was found to enhance sperm quality,restore BTB integrity,and reduce the palmitoylation levels of proteins in the testis compared to the exposed group.In vitro experiments showed that ω-3 PUFAs treatment reduced the ER stress and palmitoylation levels of Calnexin in TM4 cells,leading to the restoration of tight junction protein expression and cell barrier function.Conclusion:Our study has revealed the critical role of Calnexin in the damage induced by PA to Sertoli cells.Furthermore,we have demonstrated that ω-3 PUFAs can mitigate ER stress by modulating the palmitoylation of Calnexin,thereby reducing the reproductive toxicity of PA.These findings offer a promising therapeutic direction for the treatment of spermatogenic disorders resulting from lipid metabolic disorders.Part 2:Octanoic acid ameliorates busulfan-induced Sertoli cells barrier damageObjective:In recent years,dyslipidemia has emerged as a crucial factor in the management of male infertility.Therefore,it is imperative to investigate its pathogenesis and identify relevant intervention measures to safeguard male fertility.Octanoic acid(OCA)has been shown to possess protective effects on lipid metabolism,anti-inflammation,and anti-oxidation,and may hold potential as a treatment for male infertility.The present study aims to elucidate the role and mechanism of OCA in busulfan-induced spermatogenesis disorders.Materials and Methods:The mice in this study were randomly allocated into a control group,a Busulfan group,and four treatment groups,each receiving a different dose of OCA(32mg/kg,64mg/kg,128mg/kg,256mg/kg).Sperm quality was assessed using a hemocytometer,while the integrity of the blood-testis barrier was examined using a biotin tracer test.The morphology of the testicular seminiferous tubule and changes in epididymal sperm were observed using HE staining.Gene expression was evaluated using qPCR,and protein expression was assessed using Western blotting.In vitro experiments were conducted on TM4 cells treated with OCA and Busulfan to investigate the underlying mechanism of action.Results:In vivo experiments revealed that OCA intervention at a dose of 32 mg/kg significantly improved sperm quality,the morphology of testicular seminiferous tubules,the integrity of the blood-testis barrier,and the expression of tight junctionrelated proteins,as compared to the Busulfan group.Furthermore,OCA intervention was found to reduce busulfan-induced oxidative stress and autophagy in the testis.In vitro experiments demonstrated that OCA pretreatment significantly mitigated the damage to the cell barrier induced by autophagy agonist(Rapamycin)or Busulfan.Conclusion:The present study has demonstrated that OCA can protect Sertoli cells by mitigating busulfan-induced oxidative stress and autophagy.These findings provide a deeper understanding of the toxicology of Busulfan and offer a new theoretical basis for the treatment of male infertility.