Cellular heterogeneity of the LH receptor and its significance for cyclic GMP signaling in mouse preovulatory follicles

Meiotic arrest and resumption in mammalian oocytes are regulated by two opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase NPR2, and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic GMP (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains obscure, due in part to imprecise information about the cellular localization of these two proteins. To investigate their localization, we generated mouse lines in which HA epitope tags were added to the endogenous LHR and NPR2 proteins, and used immunofluorescence and immunogold microscopy to localize these proteins with high resolution. The LHR is absent from the cumulus cells and inner mural granulosa cells, and is present in less than 50% of the outer mural granulosa cells. In contrast, NPR2 is present throughout the follicle, and is more concentrated in the cumulus cells. Less than 20% of the NPR2 is in the same cells that express the LHR. These results suggest that to account for the LH-induced inactivation of NPR2, LHR-expressing cells send a signal that inactivates NPR2 in neighboring cells that do not express the LHR. Inhibitors of gap junctional permeability and of EGFR kinase activity both attenuate the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with both of these mechanisms contributing to how NPR2 is inactivated in cells that do not express the LHR.