DISCRIMINATIVE ANALYSIS OF RAT SERTOLI AND PERITUBULAR CELLS AND THEIR PROLIFERATION IN-VITRO - EVIDENCE FOR FOLLICLE-STIMULATING HORMONE-MEDIATED CONTACT INHIBITION OF SERTOLI-CELL MITOSIS

A new methodological approach using immunohistochemical markers for Se rtoli cells (alpha inhibin), peritubular cells (alpha smooth muscle ac tin), and S-phase cells (bromodeoxyuridine; BrdU) is presented that al lows an accurate and simultaneous analysis of morphogenetic and mitoge nic changes occurring in vitro. Sertoli cells and peritubular cells we re isolated by sequential enzymatic digestion from 7-day-old rats. Lam inin, as a major component of the extracellular matrix of the seminife rous tubule, and FSH, as a hormone stimulating Sertoli cell proliferat ion, were tested for their ability to influence the morphology or mito tic activity of the cultured cells. After fixation, the coverslips wer e stained for these antigens with use of specific primary antibodies a nd horseradish peroxidase- or alkaline phosphatase-labeled secondary a ntibodies for visualization of the respective antigens with different- colored precipitates. This approach allowed us to distinguish the two cell populations, which could not be done unequivocally without the an tibody staining. We scored striking changes in cell densities and cell ratios during the culture period. Peritubular cells showed a consiste ntly higher BrdU-labeling index than Sertoli cells. While Sertoli cell s were not labeled until Day 7, peritubular cells proliferated as soon as on Day 3, and their density doubled from Day 3 to Day 7. A linear negative correlation was established for Sertoli cell proliferation in response to their local density on the coverslip, indicating contact inhibition as a signal for cessation of mitosis. At high cell densitie s, this inhibition was partially overcome in the presence of FSH. The presence of laminin had striking effects on the morphogenetic response but only a minor influence on mitogenesis.