Cellular Heterogeneity of the Luteinizing Hormone Receptor and Its Significance for Cyclic GMP Signaling in Mouse Preovulatory Follicles

Abstract

Meiotic arrest and resumption in mammalian oocytes are regulated by 2 opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic guanosine 5′-monophosphate (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 incompletely understood, due in part to imprecise information about the cellular localization of these 2 proteins. To investigate their localization, we generated mouse lines in which hemagglutinin 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 results showed that the LHR protein is absent from the cumulus cells and inner mural granulosa cells, and is present in only 13% to 48% 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. An inhibitor of gap junction permeability attenuates the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with this mechanism contributing to how NPR2 is inactivated in cells that do not express the LHR.

Transfer of mitochondria and endosomes between cells by gap junction internalization

“…While the fate of the mitochondria within internalized gap junctions of ovarian follicles is not clear, one could speculate that like gap junctions connecting two cells, 26 at some point, internalized gap junctions may be permeable to metabolites such as ATP, allowing for the controlled release of small molecules generated by mitochondria into the receiving cell (see Figure 4). In the ovarian follicle, only the outermost layer of granulosa cells has proximity to vasculature, and only some of these cells express the receptor for luteinizing hormone 27 . Gap junction internalization may be a means for increasing the delivery of metabolites to cells in more interior regions of the follicle, or those that do not express the luteinizing hormone receptor.…”

“…In the ovarian follicle, only the outermost layer of granulosa cells has proximity to vasculature, and only some of these cells express the receptor for luteinizing hormone. 27 Gap junction internalization may be a means for increasing the delivery of metabolites to cells in more interior regions of the follicle, or those that do not express the luteinizing hormone receptor. Thus, the process of gap junction internalization may facilitate not only the transfer of mitochondria, but also the release of small molecules from the enclosed mitochondria into the receiving cell cytosol.…”

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth

“…To allow specific labeling of the NPR2 protein, the mice were genetically modified to insert a 9–amino acid hemagglutinin (HA) tag on the N-terminus of NPR2 (HA- Npr2 ; ref. 43 ) ( Supplemental Figure 3 ). We compared the phosphorylation state of NPR2 in chondrocytes with and without LB-100 preincubation — and with and without subsequent exposure to FGF.…”

Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

“…Mouse ovarian antral follicles were high pressure frozen 30 minutes after exposure to luteinizing hormone (LH). This tissue was used because it has large numbers of Cx43 gap junctions (Okuma et al, 1996;Norris et al, 2008Norris et al, , 2017Baena et al, 2020), which are caused to internalize in response to hormone (Larsen et al, 1987). As in our previous study, the follicles were embedded in lowicryl, serial sectioned, then labeled with primary antibody to Cx43 and gold-labeled secondary antibody (Norris et al, 2017).…”