Evaluation of Vitamin B12 effect on the mitotic and meiotic phases of spermatogenesis and in the sperm DNA of cimetidine-treated rats

Abstract

Cimetidine has been used for the treatment of digestive cancer due to its antagonist effect on the histamine H2 receptors since histamine acts as growth factor for different tumoral cell types via H2 receptors. Moreover, the tumoral growth inhibition seems to be related to the antiangiogenic effect of cimetidine. In the male reproductive system, cimetidine has caused harmful effects mainly in the testes and vas deferens. In the testes, this drug has caused seminiferous tubule alterations, including epithelial area reduction due to germ cell sloughing, Sertoli and peritubular myoid cells death. Additionally, Leydig cells and testicular microvasculature has also been injured by cimetidine treatment. Recent studies have demonstrated that epithelial alterations, caused by cimetidine, are softened by a concomitant treatment with vitamin B12. Although this beneficial effect has not been clarified, it has been suggested that vitamin stimulates mitotic and/or meiotic activities in spermatogonia and/or spermatocytes, respectively. Thus, based on the harmful effect of cimetidine on spermatogenesis and the beneficial effect of vitamin B12, we purpose to evaluate: 1) which germ cells (spermatogonia and/ou spermatocytes) are stimulated by vitamin B12 either in the treated animals only with vitamin or in the animals treated with cimetidine+vitamin; 2) whether spermogram and spermatic DNA are damaged by cimetidine treatment and 3) whether this spermatic injures can be softened or inhibited by vitamin B12 supplement. Adult male rats will be treated with: 100mg of cimetidine/Kg b.w. (CMTG), 3µg of vitamina B12 (B12G), cimetidine+vitamin B12 (CMT/B12G) and saline (CG). After treatment for 50 days, the left caudal portion of epididymis will be collected and the morphology, concentration and motility of sperm will be analyzed. From the right caudal portion of epididymis, sperm will be collected for the analysis of spermatic DNA integrity (Comet Assay) and mitochondrial activity. The testes will be fixed in formaldehyde and embedded in paraffin (right testes) and historesin (left testes). Historesin sections stained by H.E. will be used for the quantification of undifferentiated A spermatogonia and spermatocytes; the results will be statistically evaluated. With the aim of evaluating the deleterious effect of cimetidine and the mitogenic effect of vitamin B12 in the spermatogonial cells, immunofluorescence for detection of GFR±1 or E-cadherin (undifferentiated A spermatogonia markers), PCNA or Ki67 (cellular proliferating markers) and caspase-3 (apoptosis marker) will be performed. Double immunofluorescent reactions will also be performed to confirm these effects in these cells. (AU)