Hormonal Regulation of 3′,5′-Adenosine Monophosphate Phosphodiesterases in Cultured Rat Granulosa Cells*

Abstract

The effect of gonadotropins on phosphodiesterase activity of rat granulosa cells was studied in an in vitro model. Granulosa cells were prepared from hypophysectomized or intact, estrogen-primed immature female rats and treated with FSH, hCG, or (Bu)2cAMP in vitro. Phosphodiesterase activity was determined in cell homogenates. FSH treatment for 2 days produced a marked increase in phosphodiesterase activity, while hCG was ineffective. FSH stimulation was potentiated by the addition of 1-methyl-3-isobutylxanthine, while treatment with the cAMP analog, (Bu)2cAMP by itself also markedly stimulated enzyme activity. FSH stimulated cAMP, but not cGMP, hydrolysis, suggesting that a phosphodiesterase specific for cAMP was stimulated by the gonadotropin. Time-course studies showed that an increase in phosphodiesterase activity was apparent after 1 h of incubation and was maximal at 48 h. FSH stimulation of phosphodiesterase was dose-dependent, with an ED50 of 30 ng/ml FSH and a maximal increase at 100-300 ng/ml. Treatment with cycloheximide (1 or 10 micrograms/ml) completely blocked the gonadotropin stimulation, suggesting that on-going protein synthesis is required for the FSH action. DEAE-cellulose chromatography of soluble extracts of control and FSH-treated cells indicated that two forms of phosphodiesterase were present in unstimulated granulosa cells. The first form, eluting at 0.17 M Na-acetate, hydrolyzed both cAMP and cGMP and was stimulated by Ca++ and calmodulin; the second form, eluting at 0.48 M Na-acetate, was insensitive to Ca++ or calmodulin and hydrolyzed mainly cAMP. FSH treatment markedly stimulated cAMP hydrolysis by the calmodulin-dependent first form as well as that by the second form. Double reciprocal analysis indicated that the FSH-stimulated enzymes are of high affinity for cAMP. In agreement with the data on total homogenate, the cGMP hydrolysis was not affected by the hormone treatment. These data demonstrate that FSH stimulates cAMP, but not cGMP, phosphodiesterase activity in rat granulosa cells in vitro. This stimulation might represent a mechanism for termination of the FSH primary stimulus and regulation of granulosa cell responsiveness to the gonadotropin.

Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice

“…Furthermore, follicle-stimulating hormone and luteinizing hormone regulate PDE4 activity in vitro (21), and in vivo studies have demonstrated that PDE4 activity changes in synchrony with the follicular cycle (27). These findings suggest that an impaired follicular function is the cause of the reduced fertility in PDE4D Ϫ/Ϫ females.…”

“…Aliquots of the homogenates were assayed for total PDE activity as well as rolipram-insensitive PDE activity by incubation in the absence or presence of 10 M rolipram. The PDE assay was performed according to the method of Thompson and Appleman (20) as detailed previously (21). The rolipram-sensitive rolipraminsensitive activity (PDE4 activity) was obtained by subtracting the RI activity from the total activity.…”

“…The cAMP response of granulosa cells to hCG was measured according to the procedure described previously (21). Briefly, adult mice were treated with PMSG for 44 h, and the granulosa cells were collected by puncturing the large antral follicles under a stereomicroscope.…”

Specific Role of Phosphodiesterase 4B in Lipopolysaccharide-Induced Signaling in Mouse Macrophages

“…After sonication with 20 bursts, the lysate was centrifuged at 4°C for 20 min at 16,000 ϫ g. The supernatant was assayed for both total PDE activity and rolipram-insensitive PDE activity in the presence of 10 M rolipram. The PDE assay was performed according to the method of Thompson and Appleman (12) as detailed previously (13). The rolipram-sensitive activity (i.e., PDE4 activity) was obtained by subtracting the rolipram-insensitive activity from the total activity.…”

Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways

“…Studies on endocrine and non-endocrine cells have shown that cAMP-phosphodiesterases 4 (cAMP-PDE4s) expression in granulosa and theca cells but not in oocytes (Tsafriri et al 1996), are part of a feedback control of cAMP levels stimulated by gonadotrophin (for review see Conti 2000). Stimulation of cultured rat granulosa cells with FSH causes a significant increase in PDE4s activity, which may in turn decrease the cAMP levels (Conti et al 1984). FSH, forskolin or db-cAMP significantly increases activin A expression while suppressing the expression of activin B, but no significant desensitization is noticed for forskolin and db-cAMP over the concentration ranges tested (Wang & Ge 2003).…”