Cloning and characterization of the cyclic guanosine monophosphate-inhibited phosphodiesterase PDE3A expressed in mouse oocyte

In the preovulatory follicle, oocyte meiotic resumption occurs soon after t he LM surge and is associated with a decrease in cAMP, Inhibition of cAMP d egradation blocks germinal vesicle breakdown as well as activation of meiot ic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacalogical analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compa rtment, Here we have investigated the structure and properties of the PDE f orm expressed in mouse oocytes. Polymerase chain reactions using a mouse oo cyte cDNA library as a template, and primers based on the conserved sequenc e of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent l igation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this P DE, the cDNAs encoding the full-length PDE3A and NH2-truncation forms Delta 1 (Delta 346aa) and Delta 2 (Delta 608aa) were expressed in mouse Leydig t umor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta I and Delta 2 truncated PDE3As were re covered mostly in the soluble fraction. The Michaelis constant values for h ydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2-0.5 muM) More importantly, t here was good correlation between the rank of potency of selective and nons elective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble for m of PDE3A and that activity of this enzyme is involved in the control of r esumption of meiosis.