Role of RNA and Protein Synthesis in Rabbit Ovarian Follicular Testosterone Production

Abstract

<p>The role of RNA and protein synthesis in follicular testeterone production has been investigated in the rabbit ovarian follicle in vitro.</p> <p>lsolated follicles were incubated (usually 2 hours) with labeled amino acids or uridine plus LH, cyclic AMP or cyclic GMP alone, or together with various metabolic inhibitors.</p> <p>In dose response studies, testosterone production was stimulated at all concentrations of LH (0.1 to 10 μg/ml) tested (p < .05), with optimal stimulation occurring at 5 μg LH/ml (p < .01). However, a significant uptake of ³H-leucine into follicular protein occurred only at > 2.5 μg LH/ml (p < .01), with optimal stimulation occurring at 5 and 10 μg LH/ml. Cyclic AMP (5 and 10 mM) enhanced both testosterone production and the uptake of ³H-leucine into follicular protein (p < .01). Lower cyclic AMP concentrations were ineffective. Neither LH nor cyclic AMP had any effect on the incorporation of labeled uridine into follicular RNA.</p> <p>In time course studies, testosterone was stimulated within 15 minutes (p < .01), in the presence of LH (5 μg/ml) or cyclic AMP (5 mM). The incorporation of ³H-leucine also increased with time in both LH and cyclic AMP treated follicles, compared to controls, but a significant difference was observed only after 90 and 60 minutes, respectively (p < .01). However, electrophoretic fractionation and radio autographic examination of total follicular proteins after exposure, of LH and ³⁵S-methionine for 15, 60 and 120 minutes showed no apparent difference in the distribution of protein bands when compared to controls.</p> <p>Actinomycin D (20, 80 and 160 μg/ml) together with LH (5 μg/ml) inhibited the incorporation of ³H-uridine into follicular RNA by 79, 85 and 86%, respectively (p < .01). At these concentrations, no inhibitory effect on LH-induced testosterone production was observed. Paradoxically, Actinomycin D (1 μg/ml) enhanced LH-induced testosterone production above that elicited by LH alone.</p> <p>Cycloheximide (20 and 10 μg/ml) inhibited LH-induced testosterone production by 64 and 57% (p < .01), as well as the uptake of ³H-leucine into follicular protein by 94 and 93%, respectively. However, cycloheximide (1 μg/ml) did rot inhibit LH-induced testosterone production, yet inhibited ³H-leucine incorporation by 81.7%. Similarly, puromycin (40 μg/ml) inhibited LH-induced testosterone production by 66%, and the uptake of ³H-leucine into protein by 74% (p < .01). However, puromycin (10, 1 or 0.1 μg/ml) did not inhibit LH-induced testosterone production, yet ³H-leucine incorporation was inhibited by 58, 37 and 31%, respectively (p < .01).</p> <p>The methylxanthines, theophylline (25, 10 and 1 mM) and MIX (5 and 0.5 mM) had no synergistic effects with cyclic AMP on follicular testosterone production. However, these methylxanthines inhibited the incorporation of ³H-uridine (35 to 68%) and ³H-amino acids (45 to 69%) into follicular RNA and protein.</p> <p>Cyclic GMP (25, 10 and 1 mM) had no stimulatory effect on follicular testosterone production or the uptake of protein. However, cyclic GMP (25 and 10 mM) significantly enhanced the uptake of ³H-uridine into follicular RNA (p < .01).</p> <p>These data collectively suggest that de novo RNA and protein synthesis are not required for acute LH-induced testosterone production in the rabbit follicle.</p>