Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa

Abstract

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of 3H-uridine into RNA. Penicillin-G (100 μg/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37°C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5–8 × 10 7 spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl 2 caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 μg/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of 3H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 μM) and of testosterone (15 and 30 μg/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.