BASIC FIBROBLAST GROWTH-FACTOR AND N-CADHERIN MAINTAIN RAT GRANULOSA-CELL AND OVARIAN SURFACE EPITHELIAL-CELL VIABILITY BY STIMULATING THE TYROSINE PHOSPHORYLATION OF THE FIBROBLAST GROWTH-FACTOR RECEPTORS

Both granulosa cells (GCs) and ovarian surface epithelial cells underg o apoptosis in vivo. Although basic fibroblast growth factor (bFGF) an d N-cadherin-mediated cell contact inhibit GC apoptosis, little is kno wn about the factors that influence rat ovarian surface epithelial (RO SE) cell apoptosis. The present studies were designed to determine whe ther bFGF and N-cadherin maintain the viability of both GC and ROSE ce lls by stimulating separate signaling pathways. For the GC studies, la rge GCs were collected from immature rat ovaries after Percoll gradien t centrifugation and placed in serum-free culture for 24 h. These stud ies confirmed that about 10% of the aggregated GCs and more than 50% o f single GCs were apoptotic after culture. bFGF reduced the percentage of apoptotic single GCs, but did not influence aggregated GCs. A neut ralizing antibody to bFGF blocked bFGF's antiapoptotic action, but did not alter the percentage of apoptotic aggregated GCs. The antibody tp N-cadherin not only increased the percentage of aggregated apoptotic GCs, but also blocked bFGF's ability to maintain the viability of sing le GCs. The effect of the FGF receptor antibody was similar to that of the N-cadherin antibody. Like GCs, ROSE cells also undergo apoptosis in serum-free medium. Exposure to either the N-cadherin or FGF recepto r antibody, even in the presence of serum, increased the percentage of apoptotic aggregated ROSE cells. As tyrosine kinase activity is invol ved in maintaining cell viability, the pattern of tyrosine-phosphoryla ted proteins was examined after culture in control(ascites) or N-cadhe rin antibody-supplemented medium. Exposure to the N-cadherin antibody altered the pattern of tyrosine-phosphorylated proteins, decreasing th e tyrosine phosphorylation of proteins in the 130- to 180-kDa range an d increasing the tyrosine phosphorylation of one or more proteins of a bout 50 kDa. The identity of the 50-kDa protein is unknown. However, i mmunoprecipitation studies demonstrated that the N-cadherin antibody r educed the amount of tyrosine-phosphorylated FGF receptor in both GCs and ROSE cells by 50%. This decrease corresponds to an increase in apo ptosis among aggregated cells. Taken together, these data suggest that hemophilic N-cadherin binding and bFGF-FGF receptor binding activate signal transduction pathways that converge at the level of the FGF rec eptor and subsequently promote the viability of both GC and ROSE cells .