Distinct cAMP Dynamics Mediated by PDE4 isoforms Regulate Granulosa Cell Function

Minnu Jayapal¹, Tasman Daish¹, Darryl Russell¹

1. Robinson Research Institute, The University of Adelaide, Adelaide, SA, Australia

Abstract Text:

Ovulation is the timely release of a functional oocyte from the ovary.  The LH surge activates the key cAMP/PKA pathway, by activating Adenyl Cyclase (AC), which converts ATP to cAMP.  Increased cAMP activates protein kinase A (PKA), which upon translocation into the nucleus phosphorylates CREB (pCREB), triggering ovulatory gene expression.   Phosphodiesterase (PDEs) metabolise cAMP, and Pde4d and Pde4b are shown to be expressed in ovary. PDE4D knockout mice has shown to have an infertile phenotype with reduced ovulation rate.  Our hypothesis is that Pde4d and b and splice variant isoforms regulate granulosa cell function via unique cAMP dynamics.   

Gene expression studies showed that LH-surge induces both Pde4d and Pde4b isoforms, more specifically, the short splice variant isoforms of these 2 genes, Pde4d1 and Pde4b2 were significantly higher 2 hours post hCG, while the long forms were elevated at 8 hours post hCG in mouse granulosa cells (mGCs), in-vivo.   Immunohistochemistry analysis revealed that Pde4d and Pde4b were localized in granulosa cells of follicles of all stages.  We used live fluorescent imaging with cAMP sensor ‘Pink Flamindo’ to demonstrate that a Pde4d-specific inhibitor D159687 at 30, 10 and 3 uM doses transiently increased cAMP levels in mGCs, but Pde4b specific inhibitor was less effective.  Immunofluorescent analysis of pCREB showed D159687 and A33 dose-dependently induced pCREB within a dose range of 0. 01uM to 3 uM.  pCREB was also induced in mGCs after knockdown of total Pde4d, Pde4d7 (long,) or Pde4d1(short), as well as total Pde4b, Pde4b1(long), or Pde4b2(short) isoforms.  Live monitoring and time course analysis of mGCs treated with PDE4 inhibitors demonstrated that D159687 and A33 at 10uM dose cause morphological changes such as increase in circularity, in GCs.  In addition, immunofluorescent analysis, revealed that D159687 at 10 uM disrupted F-actin and Vimentin organization in mGCs in comparison to A33 at 10uM, which had a lesser effect. Progesterone ELISA showed that PDE4B inhibitor A33 at 10uM and 3uM caused a significant increase in progesterone levels compared to control, in contrary to slight reduction in P4 production with D159687 treatment at both the dose, although it did not reach significance.  Gene expression analysis of mGCs treated with D159687 at 10 uM for 2 hours showed a significant increase in Star gene expression and a significant decrease in cyp19a1 expression, however A33 did not show such an effect within 2 hours of treatment.

Taken together, these results suggest that PDE4 isoforms differentially modulate cAMP dynamics to distinctly regulate crucial granulosa cell functions such as steroidogenesis, by altering morphology, cytoskeletal organization and progesterone production. A deeper understanding of PDE4 mediated mechanism of ovarian steroidogenesis is important for developing treatments for reproductive and hormone dependent disorders and also in drug development for female infertility and contraception.