Expression Of Cell-age-related Molecules In The Testis And Epididymis Of Different Months Rat

No matter people or animals, there are the time differences of characteristic changes and time of tissue/cell between somatic cell and epididymis in aging process, but aging itself is a common rule. Our previous studies showed that the expression level of tau protein increased and microtubule associated proteins (MAPs) decreased in Leydig cells of aged rats [1]. This indicates that the performance of testicular aging is aging substance tau protein accumulated in the microtubule. The decreased expression of MAPs leads to the abnormal physiological effects of Leydig cells function. There are inconsistent expression of androgen receptor (AR) and heat shock protein 90α(HSP90α) in the aged testis (castrated testis of prostate cancer) [2], which can be partly explained that reduction of HSP90αexpression lead to the marginalization of AR expression thereby affecting the biological function of androgen. Although there are many aging signs such as angiosclerosis, tissue ischemia and degeneration of Leydig cell in the aged testis, what lead to up-regulation of HSP90αexpression? There were many characteristic morphology structure changes in all segments of epydidymis in aging process [3]; meanwhile, the expression level of aging associated genes increased in the epididymis epithelial cell nuclei of aged rats [4]. The expression of tau protein showed a obvious aging rule in cerebral ganglion and hippocampal of rat, which was positive correlation to the expression level of Gas7 protein (unpublished data). From these, the aging mechanism of organism is far obscure. Therefore, it is necessary that the expression of cell-age-related molecules of different stages should be carried out in-depth study in the rats of testis and epididymis. We hope to explain some mechanism of the two male organs from maturity to senescence and then provide a theoretical basis for the male aging.The cell-age-related molecules c-Kit, HIWI, Vimentin and Gas7 were used to examine the differences in the expression of them in the testis and epididymis of different months rat. c-Kit and HIWI can be represented by the molecules of cell proliferative stage, and Gas7 can be represented by the molecules of cell quiescent state. Meanwhile, Vimentin could be represented by the molecules exiting cell cycle. The work was divided two parts, and the immunohistochemical SP method with the antibody against c-Kit, HIWI and Vimentin was performed in the testis and epididymis of the rats in PartⅠ. In partⅡ, at first, the glutathione S-transferase (GST) fusion protein with bioengineering technology was prepared for polyclonal antibody as immunogen. Then, the immunohistochemical (IHC) examination and In situ hybridization (ISH) technology were performed in the testis and epididymis of the rats. We are trying to find some methods for investigating the mechanisms of tests and epididy aging.PartⅠExpression of c-Kit,HIWI and Vimentin in the Testis and Epididymis of Different Months RatObjective: To examine the differences in the expression of c-Kit, HIWI and Vimentin in the testis and epididymis of different months rat, investigate the expression of characteristic molecules of regularity change in the ripening and senescence process of testis and epididymis, and to provide a theoretical basis for anti-aging male reproductive system. Methods: Sprague-Dwaley male rats were divided into a young group (6-month-old, n=10), an adult group (12-month-old, n=10) and an aged group (24-month-old, n=10). The immunohistochemical S-P method with the antibody against c-Kit, HIWI and Vimentin was performed in the testis and epididymis of the rats. Results: The positive immunohistochemical reaction to c-Kit was observed mainly in spermatogonia but weakerly in others cells. There was also expression in the epithelium and lumens of epididymis. The abundant expression of HIWI was detected in all stages of spermatogenic cells in testis and epididymis of 6-month-old (6M) and 12-month-old (12M) rats. Moreover, spermatogonia and primary spermatocyte displayed intense expression. Sertoli cells, Leydig cells, myoid cells and vascular endothelial cells (VEC) were also positive expression cells. But the positive expression decreased with age in the testis and epididymis of 24-month-old(24M)rats (P>0.05). Vimentin expression pattern of the testis and epididymis demonstrated that 6M expression was very weak and 24M displayed a significant increase (P >0.01). Conclusion: The expression of c-Kit and HIWI in the testis and epididymis of 24M rat is lower, while Vimentin expression increases with age and markedly enhances. The results suggest the testis and epididymis aging are closely related to the abnormal cell signal transduction.PartⅡExpression of Gas7 in the Testis and Epididymis of Different Months RatExperimentⅠExpression of Gas7-GST protein in prokaryotic cell and preparation of polyclonal antibody against Gas7Objective: The aim of this study was to prepare anti-Gas7 polyclonal antibody and make it widely useful in Gas7 detection of testis and epididiymis relationships of the structure and function. Methods: The 456bp DNA fragment encoding Gas7 C-terminal was obtained by PCR and was cloned into pGEX-3X plasmid expressing glutathione S-transferase (GST). The GST-Gas7 fusion protein was expressed by E. coli BL21 (DE3) after IPTG induction and purified through a Glutathione Sepharose 4B affinity columella. The rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum. The antiserum was identified by Western Blot. Results: The prokaryotic expression plasmid pGEX-3X-Gas7 was successfully constructed, and the Mr 43 kD fusion protein GST-Gas7 was expressed efficiently. The polyclonal antibody was also successfully prepared after immunizing the rabbit. Western Blotting analysis showed that the titer of the antiserum could achieve 1:100 000; and Gas7 expression of brain and testis accordance with the foreign literature, meanwhile, Gas7 expression was found in the rat epididymis. Conclusion: The Gas7- GST fusion protein has the good antigenicity and cheap cost. The Gas7 polyclonal antibody with high titer and specificity could satisfy Western Blot and immunohisochemisty experiment requirement.ExperimentⅡExpression of Gas7 protein and its mRNA in the testis and epididymis of different months ratObjective: To examine the differences in the expression of Gas7 protein and its mRNA in the testis and epididymis of different month rats, to investigate the regularity change in the ripening and senescence process of testis and epididymis, and to research the pathomechanism of male reproductive system. Methods: Sprague-Dwaley male rats were divided into a young group (6-month-old, n=10), an adult group (12-month-old, n=10) and an aged group (24-month-old, n=10). The immunohistochemical (IHC) examination and In situ hybridization (ISH) technology were performed in the testis and epididymis of the rats. Results: In both the 2 examination, the expression pattern of the testis and epididymis demonstrated that 6M and 12M expression was very weak and 24M displayed a significant increase. Clear brown staining was observed in round spermatids, in the tails of elongating spermatids, in Leydig cells, in myoid cells, in the tails of mature spermatozoon and in the duct wall of epididymis. Conclusion: The expression level of Gas7 protein and its mRNA increases with age and markedly enhances in the testis and epididymis. The results suggest that the testis and epididymis aging are closely related to the abnormal pathway of the cell signal transduction. The accumulation of aging molecule leads to abnormal cell behavior.