The objective of this study is to explore the role of polyamines (putrescine, spermidine and spermine) in spermatogenesis and to test the hypothesis that transmission of the polyamine putrescine from Sertoli cells (SC) to differentiating germ cells (GC) is essential for normal spermatogenesis. Efforts will be made to determine cellular levels of free and nuclear conjugated polyamines in population of isolated rat interstitial cells, purified GC and cultured SC during testicular maturation by using high performance liquid chromatography after dansylation of biological samples. Attempts will be made to assess the activity and distribution of ornithine decarboxylase (ODC) in testicular cells, especially differentiating GC, by using L-[1-14C]-ornithine in an """"""""in situ"""""""" ODC assay and using [3H]-alpha-difluoromethyl ornithine to localize ODC by autoradiography. The [3H]-alpha-difluoromethylornithine-bound molecules will be extracted and analyzed by SDS-gel electrophoresis. The distribution of ODC has been investigated by immunochemical/enzymatic detection method using ODC antibody. The observed ODC-positive molecules in late spermatids and spermatozoa (located exclusively in the acrosome region) will be further identified, after extraction, by gel electrophoresis and Western Blot analysis. We also intend to study dynamics of polyamine synthesis, metabolism and release in SC using L-[14C-(U)]-ornithine, the uptake of exogenous [14C]-putrescine by GC during different stages of differentiation. If the transmission [14C]-putrescine from SC to GC requires direct contact of both cell types, the phenomenon will be studied in a co-culture system. Further efforts will be made to study the effect of hormonal (androgen/FSH) depletion and supplementation on the release of putrescine by SC and to correlate these data with GC polyamine profiles and degree of differentiation. Furthermore, steroid-suppressive agents (aminoglutethimide) and antiandrogens (e.g. flutamide and cyproterone acetate) are known to interfere with normal spermatogenesis at different stages of differentiation, and their effects on testicular polyamines are not clear. Therefore, these agents and polyamine inhibitors will be employed to probe the role of polyamines in spermatogenesis. The information gained will broaden our knowledge of spermatogenesis, elucidate the roles of polyamines in this process and may establish cellular cooperation in biochemical terms between Sertoli cells and differentiating germ cells.
| Shubhada, S; Tsai, Y H (1990) Differential effects of FSH on the activities of S-adenosyl-L-methionine decarboxylase and ornithine decarboxylase in Rat sertoli cells. J Androl 11:414-21 |
| Shubhada, S; Lin, S N; Qian, Z Y et al. (1989) Polyamine profiles in rat testis, germ cells and Sertoli cells during testicular maturation. J Androl 10:145-51 |
| Shubhada, S; Daver, R; Tsai, Y H (1989) The changing profiles of L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities in testicular cell types during sexual maturation of male rats. J Androl 10:152-8 |
| Madhubala, R; Shubhada, S; Steinberger, A et al. (1987) Inhibition of ornithine decarboxylase activity by follicle stimulating hormone in primary culture of rat Sertoli cells. J Androl 8:383-7 |
| Tsai, Y H; Lin, S N (1985) Differential release of polyamines by cultured rat Sertoli cells. J Androl 6:348-52 |
