Effects of cyclic AMP and phorbol ester on transepithelial electrical resistance of Sertoli cell monolayers in two-compartment culture

Abstract

The effects of dibutyryl cyclic AMP ((Bu) 2cAMP) and phorbol ester (TPA), in the absence or presence of follicle-stimulating hormone (FSH) and/or testosterone, on the development of tight junctions by immature rat Sertoli cells (Sc) were investigated in vitro using the two-compartment culture system. The tight junction status was evaluated by repeated measurements of transepithelial electrical resistance (TER). Untreated cell monolayers developed stable TER of approximately 120 Ωcm 2 during 3 days of culture. Continuous presence of FSH (200 ng/ml) from day 1 onward significantly increased the TER up to approximately 300 Ωcm 2 after a transient (24–36 h) delay. The initial delay was prolonged to 3–4 days by the addition of 1-methyl-3-isobutylxanthine (MIX) (0.2 mM), whereas the subsequent increase of TER was significantly potentiated by the concomitant presence of testosterone (10 μM). Cholera toxin (CHT; 10 ng/ml) and forskolin (FR; 50 μM) mimicked these FSH effects. (Bu) 2cAMP, at concentrations which maximally stimulated immunoactive inhibin secretion (100–500 μM), inhibited the initial TER increase and significantly decreased the TER level when added on days 1 and 5 of culture, respectively. In contrast, low concentrations of (Bu) 2cAMP (4–20 μM) consistently stimulated the TER development, mimicking the stimulatory phase of FSH action. TPA (100 nM) alone had no effect on TER development, but potentiated the stimulatory effect of testosterone in a manner similar to FSH, CHT, FR or low concentrations of (Bu) 2cAMP. These results demonstrate, for the first time, a concentration-dependent, dual effect of exogenous cAMP on the Sc function. Moreover, our data suggest that, at least in vitro, FSH regulates the formation of inter-Sc tight junctions via a cAMP-mediated mechanism and that protein kinase C may modulate the testosterone-, but not FSH-dependent development of tight junctions by immature Sc.