Cellular basis for follicle-stimulating hormone-stimulated calcium signaling in single rat sertoli cells: Possible dissociation from effects of adenosine 3′,5′-monophosphate

To study the cellular basis for FSH-stimulated dose-dependent graded increases in intracellular Ca²⁺ concentrations in populations of Sertoli cells, we investigated the effects of FSH on free Ca²⁺ ion concentrations ([Ca²⁺]_i) in individual rat Sertoli cells using the Ca²⁺-sensitive dye fura-2/AM and digital fluorescent videomicroscopy. Ovine or rat FSH elicited a hormone-specific rise in [Ca²⁺]_i within 20-140 sec, with a peak level 2.7 ± 0.9-fold greater than the basal value (mean ± SEM; n = 8) lasting for 4-16 min. The amplitude and kinetics of the FSH-induced [Ca²⁺]_i signal were not dose dependent. Instead, increasing doses of FSH recruited a higher percentage of responding cells. Chelation of extracellular Ca²⁺ or cotreatment with verapamil or cobalt abolished FSH-induced [Ca²⁺]_i increases. Furthermore, in the presence of extracellular Mn²⁺, direct evidence for FSH-mediated Ca²⁺ influx was obtained from the quench of fura-2 fluorescence. Induced Ca²⁺ increases were mimicked by forskolin or protein kinase-A type I activators [8-(6-amino-hexyl)amino-cAMP and N⁶-benzoyl-cAMP (N⁶B)]. However, the cAMP analogs, 8-bromo-cAMP, N⁶,2′-O-dibutyryl cAMP, or protein kinase-A type II activators (8-thiomethyl-cAMP and N⁶B), induced [Ca²⁺]_i increases even in the absence of extracellular Ca²⁺, and the time course of the [Ca²⁺]_i rise induced by cAMP analogs was more rapid than that induced by FSH. Similarly, the uninhibited rise in [Ca²⁺]_i induced by FSH in pertussis toxin-pretreated Sertoli cells suggests that PT-sensitive G-proteins are not involved in the action of FSH on [Ca²⁺]_i. In summary, we demonstrate that FSH evokes sustained [Ca²⁺]_i increases in single Sertoli cells in a nongraded fashion and recruits increasing numbers of responding cells in a dose-dependent fashion. We also provide explicit evidence that FSH induces Ca²⁺ influx. Mimicry of the FSH-induced [Ca²⁺]_i rise by certain cAMP analogs [8-(6-amino-hexyl)amino-cAMP and N⁶B; protein kinase-A type I activator] or forskolin suggests that Ca²⁺ may be part of a dual pathway of cAMP-initiated intracellular signaling.