Measurements of cAMP and cGMP in follicle-enclosed oocytes Proteins for injection were spin-dialyzed and concentrated in PBS. Follicles were kept in the injection slide for no more than 30 minutes between injections, they were returned to Millicell culture plates. Quantitative microinjection of 4 or 10 pl(equivalent to 2 or 5% of the 200 pl volume of the oocyte) was carried out as previously described, with the follicles held between two coverslips spaced 200 μm apart ( Jaffe and Terasaki,2004 Jaffe et al.,2009). Parlow, National Hormone and Peptide Program, Torrance, CA,USA) was used at 10 μg/ml. They were cultured for 24-32 hours on Millicell culture plates (PICMORG50,Millipore, Billerica, MA, USA) in media as previously described( Norris et al., 2008). These are crucial issues because, as noted above, cGMP is not the only regulator of PDE3A in addition, it is controversial whether relief of inhibitory signals from the somatic cells is the primary mode of LH action(see Eppig et al., 2004).Īntral follicles with their associated theca cells (see Norris et al., 2008) were dissected from 22- to 24-day-old B6SJLF1 mice (Jackson Laboratory, Bar Harbor,ME, USA), as approved by the University of Connecticut Animal Care Committee. LH-stimulated decreases in cGMP occur in oocytes and whole follicles from hamsters ( Hubbard,1986), but whether the magnitude of these changes is physiologically significant and whether these decreases occur in other species are unknown. Thus, it is unknown whether the decreases in cAMP that were observed in response to LH receptor stimulation were sufficient to affect the activity of PKA, which requires cAMP in the range of 70-500 nM for half-maximal activity ( Dostmann and Taylor,1991 Viste et al.,2005). However, the magnitude of these changes was difficult to interpret because the oocytes were removed from their follicles, which would decrease cAMP, and because phosphodiesterase inhibitors were present, which would increase cAMP. In mouse and rat oocytes isolated from follicles after LH receptor stimulation, immunoassays have shown that cAMP has decreased ( Schultz et al.,1983 Sela-Abramovich et al.,2006). LH causes a rapid and almost complete closure of the gap junctions between the somatic cells in the follicle( Norris et al., 2008), such that if cGMP from the somatic cells enters the oocyte prior to LH stimulation,the gap junction closure in response to LH should decrease cGMP, and indirectly cAMP, in the oocyte. Whether LH causes cAMP and cGMP in the oocyte to decrease to a level that allows meiotic resumption, and if so how this occurs, have also been uncertain. Inhibition of soluble guanylyl cyclase( Sela-Abramovich et al., 2008)or of inosine monophosphate dehydrogenase (needed for cGMP production)( Downs and Eppig, 1987 Eppig, 1991) causes meiotic resumption in follicle-enclosed oocytes, indicating that production of cGMP in the follicle is essential for the maintenance of meiotic arrest. Consistent with this hypothesis, cGMP in the oocyte decreases as a function of time after isolation of the oocyte from the follicle, and injection of cGMP into the oocyte delays meiotic resumption( Törnell et al., 1990a). Alternatively, because the primary cAMP phosphodiesterase in the oocyte is PDE3A ( Masciarelli et al.,2004), which is inhibited by cGMP( Hambleton et al., 2005), cGMP from the somatic cells could enter the oocyte through the gap junctions, thus inhibiting PDE3A and maintaining meiotic arrest( Törnell et al., 1991). One possibility is that the signal that maintains meiotic arrest is cAMP that diffuses from the somatic cells to the oocyte( Anderson and Albertini, 1976)(see Discussion). This causes a decrease in oocyte cAMP (from ∼700 nM to ∼140 nM), leading to the resumption of meiosis. The resulting decrease in oocyte cGMP (from ∼1 μM to ∼40 nM) relieves the inhibition of PDE3A, increasing its activity by ∼5-fold. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from ∼2 μM to ∼80 nM at 1 hour after LH stimulation)and by closing gap junctions between the somatic cells. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte,where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle.

Cyclic amp gmp