| Backgrounds:Testosterone plays an essential role in spermatogenesis.At present,it generally believed that the level of testosterone in the peripheral blood of oligoasthenospermia patients would decrease.Still,there are also a few patients whose serum T level is not lower than that of the lower limit of the normal reference range.The pathogenesis of male idiopathic oligoasthenospermia in male infertility is unknown.In the clinical practice,it mainly depends on empirical drug treatment,and androgen or its derivatives supplementary therapy is one of the commonly used medicines.Besides,drug therapy is widely used and also relatively simple in the clinical treatment of male idiopathic infertility,because it is more consistent with the expectations of natural fertility,and more easily acceptability,which is often considered and accepted by both doctors and patients first.Objectives:To verify the efficacy and feasibility of testosterone supplementary therapy(TST)and explore the possible mechanism by model rats with the oligoasthenospermia established.Methods:First,we systematically reviewed the literature on androgen alone or in combination with other medications for the treatment of male oligoasthenospermia and provided the basis for the further exploration of androgen for the treatment of model rats with oligoasthenospermia.Secondly,based on the previous studies,we selected 6-month-old healthy male SD rats to observe the effects of four different doses of TST on their reproductive organs and endocrine metabolism,to further clarify the physiological dose range and explore the appropriate drug dose in the next treatment experiment for the model rats.Thirdly,according to the physiological dose of TST suitable for the model rats treatment experiment derived from treated normal rats,we addressed the 6-month-old model rats with oligoasthenospermia induced by the Glucosides of tripterygium wilfordii and evaluated the therapeutic effects from multiple aspects such as organ weights,sperm quality,serum hormone levels,testicular tissue antioxidant index,and pathological histomorphology.Fourthly,we observed the ultrastructural changes of seminiferous tubules in testis by electron microscopy and investigated the metabolomics of oligoasthenospermia rats treated with TST by HPLC-MS.Through the quantitative analysis of metabolites in model rats,we tried to discover the relative relationship between metabolites and pathological changes,as well as the related apoptosis pathway and specific cell markers to explore the mechanism and accurate target of TST for model rats with oligoasthenospermia and provide fundamentally theoretical basis for TST clinical applicationResults:First of all,in terms of improving the semen quality of the patients with oligoasthenospermia,the meta-analysis showed that the effect of combined treatment with TU was superior to that with TU alone,and the effect of TU alone was no less than that of other drugs.Therefore,in some way,TU directly or indirectly promoted and improved the semen quality of the patients with oligoasthenospermia to a certain extent It also provides the literature basis and theoretical basis for exploring the androgen treatment of oligoasthenospermia rat model.Secondly,by exploring the effects of different doses of TU on the levels of serum reproductive hormones and spermatogenic function,and combining with organ mass,sperm quality,and pathological morphology of the normal rats,we further screened out and identified two ’physiological doses’ of 7.56mg/kg and 15.12mg/kg,which have the least influence on the physiological function of normal rats.Thirdly,by establishing the oligoasthenospermia model rats,the results showed that TST could improve sperm concentration and motility(P<0.05),but had no effect on the parameters of sperm movement(p>0.05);Besides,H&E results showed that compared with normal control group,the testicular histomorphology indicated that the number of spermatogenic cells in the treatment group increased,interstitial cells proliferated,the number of Sertoli cells relatively reduced,the spermatogenic epithelium further restored,and the Johnsen scores and the apoptosis rate were also lower than that of model rats group(P<0.05).In the aspect of eproductive hormone,the levels of FSH,LH were significantly restored(P<0.05),while the levels of T and E2 were also significantly increased(P<0.05);the levels of MDA and GSH-Px in testicular tissue were significantly reduced(P<0.05),especially the level of testosterone in the rat testis significantly increased(P<0.05).Fourthly,the ultrastructural changes of seminiferous tubules in testis were observed by electron microscopy,which given as follows the space between spermatogenic cells was significantly improved,the arrangement of spermatogenic cells was relatively closed,the structure of mitochondria basically completed,and the swelling reduced considerably,the arrangement of mitochondria cristae was disordered,and the vacuoles were also significantly reduced.The HPLC-MS explored the metabolic pathway of TST in model rats with oligoasthenospermia.There were significant differences among the four metabolic pathways of arachidonic acid,arginine,proline,phenylalanine,and glycerophospholipids in metabolomics(P<0.05)Fifthly,based on the glycerophospholipids metabolic pathway,the protein expression of the pathways such as Bcl-2/Bax-Caspase3/9 and RIPK1-mlkl/pMLKL were verified;compared with the solvent control group,the protein expression of Bax and Caspase-3/9 decreased significantly(P<0.05),while Bcl-2 increased significantly(P<0.05)in the mitochondrial apoptosis pathway;TNF-α,RIPK1 and pMLKL was significantly increased significantly(P<0.05),while the expression of pMLKL was significantly decreased(P<0.05)in the necrotic apoptotic pathway.However,the cell-specific markers of SOX9 as function marker of the Sertoli cell,StAR as functional markers of the Leydig cell,PGP9.5 as functional markers of the peptidergic nerve fiber and Occludin as the testicular tight junction marker,all significantly recovered after treatment(P<0.05)Conclusions:TST has significant effects on the improvement of spermatogenesis and restoration of reproductive functions in the model rats of oligoasthenospermia with low serum testosterone levels.1.By exploring the effects of different doses of TST on the levels of reproductive hormones and spermatogenic function in normal rats,we screened out.We identified two TU ’physiological doses’ of 7.56mg/kg and 15.12mg/kg as the treatment dosage used for treatmnetexperiment of oligoasthenospermia model rats;2.Through the establishment model rats of oligoasthenospermia with low serum testosterone level,we found that the TST group at a dose of 15.12mg/kg has better effects on improving the level of reproductive hormones and semen quality,and reducing the damage of oxidative stress products on the levels of tissue and cell;3.The results of electron microscopy showed that the TST group at a dose of 1 5.12mg/kg could improve the space of spermatogenic cells and restoration of mitochondria more significantly.Besides,the metabolic pathway of phosphoglycerides was significantly different displayed by HPLC-MS metabolomics;4.TST could achieve therapeutic effects through pathways of mitochondria apoptosis and cell necrosis apoptosis,on regulating spermatogenesis by related cell markers in testicular tissue. |
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