Author name:
وداد عبد جواد التميمي
Supervisor name:
جبار عباس احمد الساعدي | عدنان وحيد محمد البديري
Abstract:
The present study has been carried out at the department of biology, College of Education, Al - Qadisiya University, Iraq, to investigate the immunological localization of transforming growth factors beta (inhibins and activins) subunits during immature and mature periods and its involvement in male reproductive physiology of rats.At pre - pubertal stage, five male rats of 25, 30, 35, 40, and 45 days old, and at post - pubertal stage, five male rats of 55, 60, 65, 70, and 75 days old have been used in the present study. Experimental animals have been anesthetized and blood samples were obtained from abdominal vein for assesment of activin - A, inhibin - B, FSH, LH, testosterone, and estrogen, testis volumes were measured for each age period. Testes, epididymis, prostate and seminal vesicle, were obtained for molecular and immunohistochemical studyto investigate the expression levels of Inha, Inhba, and Inhbb genes using qRT - PCR and immunohistochemical technique.The present study demonstrated gradual increase of testis volume throughout the male rat life in parallel with the increase of serum inhibin - B and testosterone concentrations. Serum activin - A concentration increased significantly at 30 and 40 day periods. Throughout the post - pubertal stage, activin - A concentration gradually decreased. Serum inhibin - B concentrations gradually decreased at the pre - pubertal stage. Post - pubertal stage registered gradual increase. At 25, 30, and 35 day periods, serum FSH level registered no significant changes, whereas 40 day period recorded significant increase then decreased at 45 day period. Throughout the post - pubertal period, the level of FSH concentrations continued in gradual decrease. At 25, 30, 35, and 40 day periods, serum LH and testosterone levels showed no significant differences, whereas 45 day period recorded significant increase. Postpubertal period showed gradual significant increase. Serum estradiol concentration decreased gradually at the pre - pubertal stage and continued in decrement at the post - pubertal stage.The expression level of Inha gene in testis decreased as the age progress until 40 day period, and then slightly increased at 45 day period. At 55 day period, the expression significantly increased. At 60 and 65 periods, the levels recorded no increase, but 70 and 75 day periods recorded significant increase. The expression level of Inhba gene increased significantly as the age progress at the pre - pubertal stage, where the highest level was recorded at 45 day. At 55, 60, and 65 day periods, the highest expression level has been recorded, thenafter, the levels decreased at 70 and 75 day periods. The expression level of Inhbb gene increased significantly at 30, 35, 40, and 45 day periods of the pre - pubertal stage. At 55 and 60 day periods, no significant difference was recorded compared with 45 day period. At 65 day period, the highest level was recorded, thenafter, the levels decreased at 70 and 75 day periods. The expression level of Inha gene in epididymis recorded no significant difference at all periods of the pre - pubertal stage, but the post - pubertal stage showed gradual significant increase as age progressed. The expression level of Inhba gene recorded no difference at 25, 30, 35, and 40 day periods, but it was significantly higher at 45 day period. The levels at 55, 60, and 65 day periods recorded no significant difference when compared with each other or with 45 day period. Significant increase has been recorded at 70 and 75 day periods. The expression level of Inhbb gene increased significantly at 30 and 35 but it decreased at 40 day period, then it showed further increase at 45 day period. At 55, 60, 65, and 70 day periods, also increased but the highest expression level was recorded at 75 day period.The expression level of Inha, Inhba, Inhbb genes in prostate recorded no significant difference at all periods of the pre - pubertal stage, but the postpubertal stage showed significant increase at all periods.The expression level of Inha and Inhba genes in seminal vesicle recorded no significant difference at 25, 30, 35, and 40 day periods and significant increase at 45 day period. The 55, 60, and 65 day periods recorded no significant difference when compared with each other but significant increase has been recorded at 70 and 75 day periods. The expression level of Inhbb gene recorded no significant difference at 25, 30, and 35 day periods, but it increased as the age progressed, whereas the levels showed no significant difference at all periods of the post - pubertal stage, but they were significantly higher than that recorded at the pre - pubertal stage. The results of immunohistochemical study demonstrated positive immunostaining for inhibin - ? subunit in Sertoli cells and primary spermatocyte and no staining in Leydig cells of pre - pubertal rat testis, whereas post - pubertal testis showed positive immunostaining in Sertoli cells, Leydig cells and primary spermatocyte at 55 day period and positive immunostaining in Sertoli cells, Leydig cells, and spermatogonia at 60 day period. There was positive immunostaining in Sertoli cells, Leydig cells, spermatogonia, primary spermatocyte and spermatid at 65, 70, and 75 day periods. At 25 and 30 day periods of pre - pubertal stage, rat epididymis showed moderate positive immunostaining in epithelial cells, but strong positive staining in epithelial cells has been shown at 35, 40, and 45 day periods and at all periods of the post - pubertal stages. The result expressed positive immunostaining in the epithelial cells of the pre - pubertal rat prostate and strong positive immunostaining at the post - pubertal stage. on the otherhand both, pre - pubertal and post - pubertal rat seminal vesicle showed strong positive immunostaining in epithelial cells. Weak positive immunostaining of Inhba has been observed in the primary spermatocyte and no staining in Sertoli cells and Leydig cells in the prepubertal rat testis and at 55 day period of the post - pubertal rat testis, and no staining in Sertoli cells, Leydig cells and spermatogenic cells at 60, 65, 70, and 75 day periods. Moreover there was positive immunostaining in epithelial cells in both the pre - pubertal and post - pubertal rat epididymis and prostate. In the post - pubertal stage, rat prostate showed strong positive mmunostaining in epithelial cells. No staining was observed in epithelial cells in both the prepubertal and post - pubertal rat seminal vesicle. The pre - pubertal rat testis showed strong immunostaining for Inhbb subunit in Sertoli cells, Leydig cells and primary spermatocyte, in adittion to spermatid in post - pubertal rat testis. Strong positive immunostaining in epithelial cells of the pre - pubertal rat epididymis at 25 and 30 day periods was expressed, but moderate positive immunostaining in epithelial cells of prepubertalrat epididymis was observed at 35, 40, and 45 day periods and postpubertal rat epididymis. However Inhbb subunit in both pre - pubertal and postpubertal rat prostate showed positive immunostaining in epithelial cells. There was positive immunostaining in epithelial cells in the pre - pubertal rat seminal vesicle at 25, 30, 35, and 40 day periods, and strong positive immunostaining at 45 day period and post - pubertal stage.It could be concluded that serum inhibin - B has positive correlation with testis volum and testosterone concentration at pre - and post - pubertal stages, and positive correlation with FSH and LH concentrations at pre - pubertal but negative partial correlation at post - pubertal stage. There was relationship between serum inhibin and activin concentration and fold changes of Inha, Inhba, and Inhbb genes in testis, epididymis, prostate, and seminal vesicle tissues at all periods of the study. Positive immunostaining for inhibin ? - and ?B - subunits, but not for ?A - subunit has been shown in testis cells and epithelial cells of seminal vesicle, positive immunostaining for inhibin ?, ?A, and ?B subunits in the epididymis and prostate
