| The semen quality of dairy goat is one of the important factors affecting the pregnancy rate and reproduction performance.The special immune tolerance microenvironment of testis provides a strong guarantee for spermatogenesis and testosterone synthesis.As one of the specific cells in testis,Leydige cells ability of testosterone synthesis can affect directly the reproductive performance of animals.Furthermore,LCs can release a variety of immunomodulatory factors,which are essential for maintaining the local immune tolerance microenvironment.Nevertheless,when the testis is invaded by various pathogenic microorganisms in the blood and downstream reproductive tract,it is easy to cause inflammatory damage,which ultimately affects testosterone synthesis and spermatogenesis and leads to animal infertility.Chinese herbal medicine icariin(ICA)has a variety of pharmacological effects such as anti-inflammatory,anti-oxidative stress,promoting immune regulation,and promoting reproductive endocrine.Most of the studies on ICA focus on human medicine,and there is a relative lack of research data in the reproductive endocrine of animal.Therefore,in this study,dairy goat LCs were used as research materials,and lipopolysaccharide(LPS)was used to cause inflammatory damage to dairy goat LCs.On this basis,ICA was added in vivo and in vitro for inflammatory stimulation,and the anti-inflammatory and reproductive endocrine regulation effects of ICA were analyzed.The main experimental and research results are as follows.(1)The morphological and ultrastructural characteristics of LCs in dairy goat at different developmental stages were observed by light microscopy and transmission electron microscopy.The results of H&E staining showed that the lumen of ST in young dairy goat was small,and the seminiferous epithelium was only composed of spermatogonia and Sertoli cells.The ST lumen of the testes of puberty and adult Dairy goat increased significantly.The seminiferous epithelium was composed of spermatogenic cells and supporting cells at all levels,and the lumen contained a large amount of sperm.Dairy goat LCs are often distributed in groups between STs.LCs are large,round or oval,and the nucleus is round,often located in the center of the cell.The expression and localization of LCs-specific marker protein 3β-hydroxysteroid dehydrogenase(3β-HSD)were detected by immunohistochemistry(IHC).The results showed that 3β-HSD was mainly distributed in the cytoplasm of LCs,and the expression level was weakly positive in young age,while the expression level gradually increased in adolescence and adulthood,and showed strong positive expression in adulthood.ELISA results showed that the contents of luteinizing hormone(LH)and testosterone in serum of dairy goats were significantly higher in adolescence and adulthood than in infancy.RT-q PCR further detected that the gene levels of key enzymes in testosterone synthesis : Steroidogenic Acute Regulatory Protein(St AR),Cytochrome P450 Cholesterol Side Chain Lyase(P450scc,also known as CYP11A1),3β-HSD and 17β-hydroxysteroid dehydrogenase(17β-HSD)were significantly increased with sexual maturity.The results of transmission electron microscopy showed that the nucleus of LCs in young dairy goats was round,the nucleolus was obvious,and the organelles such as smooth endoplasmic reticulum and mitochondria were scattered in the cytoplasm around the nucleus.In adolescence and adulthood,the nucleus is oval,the membrane is irregular,and a large number of smooth endoplasmic reticulum,mitochondria and lipid droplets are distributed in the cytoplasm.The above results showed that the testosterone synthesis ability of dairy goat LCs increased significantly with sexual maturity,and a large number of steroid synthesis-related organelles in the cytoplasm laid a material foundation for the rapid synthesis of testosterone.(2)Bacterial lipopolysaccharide(LPS)can cause testicular inflammatory injury and affect spermatogenesis and androgen testosterone synthesis.In this experiment,0.5 mg LPS(Escherichia coli 055 : B5)was injected into the testis to induce inflammatory injury in the testis of dairy goats,and the control group was injected with the same amount of normal saline.Anatomy showed that compared with the control group,the testis of dairy goats in LPS treatment group was enlarged and congested.Light microscopy showed that the number of ST spermatogenic cells in the LPS-treated group was significantly reduced,the arrangement of spermatogenic epithelial cells was disordered,and spermatogenic cells fell into the lumen.ELISA showed that the serum testosterone content of dairy goats in LPS treatment group was significantly lower than that in control group.The dairy goat LCs were further isolated and cultured,and the cultured LCs were stimulated with gradient concentrations of LPS.In vitro results showed that 1.25 μg / m L LPS could significantly inhibit the cell viability and testosterone synthesis of dairy goat LCs.RT-q PCR and Western blot analysis showed that LPS treatment significantly down-regulated the gene and protein levels of St AR,P450scc(CYP11A1),3β-HSD and 17β-HSD,which are key enzymes in testosterone synthesis.ELISA and RT-q PCR were used to further detect and analyze the expression changes of LCs-related immunomodulatory factors.The results showed that LPS treatment could significantly up-regulate the expression levels of IL-1β,IL-18,TNF-α,TGF-β,PROS1 and GAS6.(3)Traditional Chinese herbal medicine ICA has many pharmacological effects such as anti-inflammatory,anti-oxidative stress and promoting reproductive endocrine.In this experiment,dairy goat were fed with grass containing 0.1 % ICA for one spermatogenic cycle(about 60 days),and then LPS inflammatory stimulation was performed.ELSA detection showed that the content of testosterone in serum of ICA group was significantly higher than that of control group after 30 days of feeding.H & E staining results showed that ICA feeding group could significantly alleviate LPS-induced testicular inflammatory injury.In vitro cell experiments showed that LPS treatment group inhibited cell proliferation,while ICA pretreatment could alleviate the inhibitory effect of LPS.RT-q PC assay showed that ICA pretreatment of LCs could significantly inhibit the expression of inflammatory cytokines IL-1β,IL-18,TNF-α,TGF-β,PROS1 and GAS6.It was further found that ICA pretreatment could protect LPS-induced inflammatory injury by inhibiting TLR4 / NF-κB pathway and maintain the dynamic balance of testosterone synthesis and immunomodulatory factors in dairy goat LCs.In summary,this study showed that the testosterone synthesis ability of dairy goat LCs increased significantly with sexual maturity.Bacterial LPS can induce inflammatory damage of LCs,resulting in decreased testosterone synthesis and disordered secretion of immune cytokines.Traditional Chinese herbal medicine ICA can significantly alleviate LPS-induced inflammatory injury and maintain the normal physiological function of LCs.The results of this study will provide a reference for the clinical development and utilization of ICA as a new feed additive and the prevention of testicular inflammatory injury in livestock production. |
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