Abstract
Summary
In order to examine a possible role of phosphodiesterase in mediation of the action of LH and FSH upon granulosa cell cAMP levels, porcine (GC) from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) follicles were incubated with human FSH (hFSH) and LH (hLH) for 30 and 60 min in the absence or presence of 3-isobutyl 1-methylxanthine (MIX), a potent phosphodiesterase inhibitor. Subsequently, the intracellular and incubation medium cAMP contents were determined by a protein binding assay. During a 30-min incubation, 10 μg of hFSH alone brought about an 11-fold, 5-fold, and 2-fold increase in intracellular cAMP accumulation and a 16-fold, approximately 4-fold, and 4-fold increase in incubation medium cAMP levels in GC from small, medium, and large follicles, respectively. Addition of 0.2 va.M MIX exerted a nonsignificant (P > 0.05) potentiating effect upon hFSH stimulation of intracellular cAMP accumulation in cells obtained from the three types of follicles. In the case of cells obtained from small and medium follicles, addition of 0.2 mM MIX in the presence of 10 μg/ml hFSH or 1.0 μg/ml hLH led to 41 to 69% potentiation (p < 0.001) of the effect of the FSH and LH upon cAMP accumulation in the incubation medium. This was evident after a 30-and 60-min incubation period. In the case of cells obtained from large follicles, addition of 0.2 mM MIX had a nonsignificant potentiating effect (p > 0.05) on either hFSH or hLH stimulation of cAMP accumulation in the incubation medium.
It may be concluded that probably there are low levels of intracellular phosphodiesterase in porcine granulosa cells and that gonadotropins act to stimulate the generation of cAMP rather than alter the rate of destruction of cAMP. The findings support the existence of an extracellular phosphodiesterase which may act to regulate or modulate the extracellular levels of cyclic AMP.
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