| Yaks are seasonally bred livestock living on plateaus at altitudes of more than 3,000 meters,with low natural reproduction rate and strong adaptability to cold and hypoxic environments.The tissue growth microenvironment and metabolic level of yaks of different ages vary greatly.As the most important reproductive organ of male animals,the testicular is regulated by a variety of factors during its development and reproductive function.In this study,yak testes of different ages: newborn(3 days),young(1 year),adult(3～4 years)and elderly(8～9 years)were the research objects.H&E was used to understand the healthy development of yak testes at different ages in the tissue level.The content and distribution characteristics of matrix metalloproteinase-2(MMP-2),tissue inhibitor of matrix metalloproteinase-2(TIMP-2),endothelin-1(ET-1)and endothelial nitric oxide synthase(eNOS)in yak testes were detected by RT-q PCR,Western blot,immunohistochemistry and immunohistofluorescence,providing a theoretical basis for exploring the function of yak testes in the natural growth process.The results of the study are as follows:1.Characteristics of yak testes tissue structure at different ages: from newborn to adult,the diameter of yak testes spermatogenic tubules gradually increases,the number of spermatogenic epithelial layers increases,and the connection between lumens is gradually tightened,but the yak enters the old age,the spermatogenic epithelium is irregularly arranged and the interstitial area increases,showing a fibrotic trend.2.Expression and distribution of MMP-2 and TIMP-2 in yak testes at different ages:RT-q PCR results showed that the expression of MMP-2 m RNA in young yak testes was significantly higher than that in newborn and adult yaks(P<0.01),and the expression of MMP-2 m RNA in adult yak testes was lower than that in old yaks(P<0.05).The expression of TIMP-2 m RNA in newborn and young yak testes was significantly higher than that in adult(P<0.01)and old yaks(P<0.05).Western blot results showed that MMP-2 and TIMP-2 protein expression decreased from newborn yaks to adult yaks,and increased in old yaks.The expression of MMP-2 and TIMP-2 proteins in adult yak testes was significantly lower than that in newborn yak testes(P<0.05).Immunohistochemistry and immunofluorescence showed that MMP-2 and TIMP-2 were localized to gonocytes of newborn yaks,Sertoli cells of young yaks,sperm of adult yaks,and Leydig cells of old yaks.3.Expression and distribution of ET-1 and eNOS in yak testes at different ages:RT-q PCR statistical results showed that the levels of ET-1 m RNA and eNOS m RNA in yak testes increased with age.ET-1 m RNA and eNOS m RNA in old yaks were significantly higher than other age groups(P<0.01).Western blot results showed that the protein expression levels of ET-1 and eNOS in adult and old yaks were significantly higher than those in newborn period(P<0.01),but ET-1 and eNOS in newborn yaks were lower than those in young period(P<0.05).Immunohistochemistry and immunofluorescence showed that ET-1 and eNOS were mainly localized to gonocytes of newborn yaks and Leydig cells of young yaks.However,in adult yak testes,ET-1 was localized to Leydig cells and eNOS to vascular endothelial cells;In old yaks,ET-1 was localized to Sertoli cells and eNOS to the population of Leydig cells in capillaries and their surroundings.In summary,the tissue structure of yak testes at different ages is obviously different.MMP-2 and TIMP-2 maintain homeostasis during the natural growth of yak testes,and the expression of spermatogenic epithelium of yak testes at different ages is crucial for testicular development and spermatogenesis.Strong expression of the interstitium in the testes of old yaks suggests that it is strongly associated with interstitial fiber degradation or remodeling.The expression of ET-1 and eNOS in Leydig cells suggests that they play an important role in androgen production.The expression of interstitial vessels showed that they were involved in the regulation of microcirculation in yak testes. |
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