Forskolin, a reversible stimulator of the catalytic subunit of adenylate cyclase, has been used to determine: 1) whether an increase in hamster cumulus cell cyclic adenosine monophosphate (CAMP) results in an elevation of intraoocyte cAMP and an accompanying increase in the maintenance of meiotic arrest (%GV where GV is germinal vesicle) when heterologous coupling is maintained, 2) whether the hamster oolemma possesses the catalytic subunit of adenylate cyclase in an amount adequate to stimulate sufficient cAMP synthesis to maintain arrest, and 3) whether release from meiotic arrest is accompanied by a decrease in the content of intraoocyte CAMP. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically.
While the %GV of both cumulus-enclosed (intact) and cumulus-free (denuded) oocytes was dose-dependent upon forskolin, that of intact oocytes was much more sensitive to the drug (intact: ID50 3.4 pM; denuded: ID50 65.0 pM, where ID50 is the dose of forskolin that inhibits the maturation of 50% of cultured oocytes). Forskolin stimulated a significant, dose-dependent increase in the amount of cAMP within the cumulus mass [(r) = 0.789, P < 0.001)], the intact oocyte [(r) = 0.715, P < O.OOl], and the denuded oocyte [(r) = 0.673, P < 0.01)].
The cAMP content of intact oocytes was significantly greater than that of denuded oocytes above 6.25 pM forskolin (25 pM forskolin: 9.28 k 1.01 vs. 3.98 f 0.15 fmol CAMP, intact and denuded oocytes, respectively; P < 0.001, paired t test). A highly significant positive correlation was established between the amount of cAMP in groups of cumulus masses and that in the corresponding enclosed oocytes [(r) = 0.635, P < O.OOl]. The enhanced sensitivity of meiotic arrest in intact, as compared to denuded, oocytes was due to the presence of adherent cumulus cells but was not attributable to a significant increase in the cAMP content of intact oocytes (at 6.25 pM forskolin; %GV intact = 73.0 & 10.7, denuded = 20.3 7.4; fmol cAMP intact = 5.02 & 1.50; denuded = 4.63 k 0.81). The arresting action of forskolin on intact oocytes was transient and fully reversible, but release from arrest was not accompanied by a decrease in either intraoocyte cAMP or heterologous metabolic coupling. These data show that 1) the hamster oocyte can synthesize cAMP 2) when heterologous metabolic coupling is maintained cumulus cell cAMP may be transferred to the hamster oocyte, and 3) that release from forskolin-maintained meiotic arrest is not correlated with a decline in intraoocyte cAMP or in heterologous coupling. The findings indicate that forskolin stimulates hamster cumulus cells to produce a "factor," which may not be CAMP, to maintain meiotic arrest.