Cloning and Characterization of the Cyclic Guanosine Monophosphate-Inhibited Phosphodiesterase PDE3A Expressed in Mouse Oocyte1

Abstract

In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis 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 microM). More importantly, there was good correlation between the rank of potency of selective and nonselective 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 form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.

Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation

“…An attractive hypothesis is that LH stimulation leads to the activation of oocyte PDE, which hydrolyzes cAMP (Conti et al 2002). The PDE3A isoform is a prevalent PDE in the mouse oocyte (Tsafriri et al 1996, Shitsukawa et al 2001. PDE3A activity in cumulusenclosed mouse oocytes has been shown to increase following LH receptor stimulation ; however, PDE3A activity has not been examined in isolated oocytes following stimulation of the LH receptor.…”

Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development

“…Phosphodiesterase (PDE) subtypes are differentially expressed in the somatic and germ cell compartments of the follicle (Tsafriri et al 1996, Park et al 2003. The oocyte-specific PDE that controls cyclic nucleotide accumulation is PDE type 3A (PDE3A) (Tsafriri et al 1996, Shitsukawa et al 2001. PDE3A is involved in in vitro induced and spontaneous resumption of meiosis in rat cumulus-enclosed oocytes (CEO): PDE3A activity is increased after stimulation by human chorionic gonadotrophin (hCG) and 2 h after oocyte isolation.…”

“…In both situations, this increase in PDE3A activity precedes oocyte nuclear maturation . Soluble forms of PDE3A exist in oocytes (Shitsukawa et al 2001), but the mechanisms of activation of the PDE3A remain unknown. Studies aiming to develop a new contraceptive strategy have shown that inhibition of PDE3A blocks oocyte meiosis in several species in vivo and in vitro (Tsafriri et al 1996, Wiersma et al 1998, Jensen et al 2002, Mayes & Sirard 2002, Nogueira et al 2003a.…”

Female infertility in PDE3A-/-mice

“…11,12 PDE3A, relatively highly expressed in mammalian oocytes, is the predominant PDE responsible for hydrolysis of oocyte cAMP. 13,14 Current thinking suggests that resumption of meiosis is triggered by LH, which increases cAMP and reduces cGMP in cumulus cells, leading to closure of gap junctions and decrease in oocyte cGMP. This relieves cGMP-induced inhibition RepoRt RepoRt Xenopus and human Cdc25C.…”

“…1C) is most likely due to increased cAMP/PKA signaling which results from the absence of PDE3A, the predominant cAMP-hydrolyzing PDE in mouse oocytes. 13,14 We therefore compared PKA activities in WT and PDE3A -/-oocytes. As shown in Figure 2A, in the presence of excess cAMP (2 μM), total PKA activity is similar in lysates from fresh WT and PDE3A…”