N-cadherin-mediated human granulosa cell adhesion prevents apoptosis - A role in follicular atresia and luteolysis?

Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has bee n shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-c adherin regulates human GC apoptosis via a cAMP-dependent mechanism, N-cadh erin expression was evaluated in ovarian follicles and corpora lutea utiliz ing immunohistochemical techniques and in luteinized GCs in culture using i mmunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directe d against both the extracellular and cytoplasmic domains of the molecule. A poptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate o f GC apoptosis was found to be two- to three-fold lower among aggregated ce lls, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadh erin function disrupting antibodies or peptides exhibiting enhanced rates o f apoptosis, GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea, N-cad herin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expr ession in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through c leavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular d omain of N-cadherin and concomitantly inhibited the serum-deprivation-induc ed apoptosis of aggregated GCs, Collectively, these observations suggest th at down-regulation of N-cadherin or the absence of a functional extracellul ar domain of the molecule prevents cell aggregation and is associated with GC apoptosis, In addition, cAMP induces apoptosis in a dose-dependent manne r, and this process is dependent, at least in part, on regulation of the N- cadherin molecule at the surface of the cells, We conclude that N-cadherin- mediated GC signaling plays a central role in follicular and luteal cell su rvival.