Role of protein synthesis in rabbit follicular testosterone production Academic Article uri icon

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abstract

  • The role of RNA and protein synthesis in follicular testeterone production has been investigated in the rabbit ovarian follicle in vitro.

    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.

    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.

    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.

    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.

    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).

    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.

    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).

    These data collectively suggest that de novo RNA and protein synthesis are not required for acute LH-induced testosterone production in the rabbit follicle.

publication date

  • March 1981