We have applied the whole-cell configuration of the patch-clamp technique to investigate calcium currents in Sertoli cells from immature rat testis in primary culture. Cesium-filled pipettes were used to block potassium currents. In the presence of 10 mM extracellular calcium (Ca+), depolarizations elicited transient inward currents in the range -70 to +10 mV with a maximal amplitude of -1.3 pA/pF at -30 mV. This component activated in the range -70 to -20 mV (V0.5 = -42 mV) and inactivated in the range -75 to -45 mV (V0.5 = 61 mV). Currents were not modified when barium (Ba2+) was substituted for Ca2+. They were suppressed by Ca(2+)-free solutions, insensitive to Bay K 8644 (an L-type channel opener), and inhibited by 1 mM cobalt, 10 microM nickel, 10 microM isradipine, and 1-10 microM omega conotoxine GVIA (four calcium-channel blockers). We conclude that calcium channels with the properties of T-type calcium channels of excitable cells are located in the membrane of immature Sertoli cells in primary cultures. Because these channels do not appear to be directly sensitive to FSH, their involvement in calcium movements remains to be determined.

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The gap junction communication in Sertoli cells from immature rat testes, cultured either in absence or in presence of follicle-stimulating hormone (FSH), was studied by microinjection of a fluorescent dye and by Fluorescence Recovery After Photobleaching (gapFRAP). The cells cultured for 2-4 days in the absence of FSH showed a flattened "epithelial-like" appearance. They were poorly coupled, as judged by the low frequency of cell-to-cell spread of microinjected Lucifer Yellow, and by the value of the rate constant of dye transfer (k) estimated in gapFRAP experiments. However, when two different subpopulations of cells were separately analyzed, namely the cells forming small groups contacting over part of their circumference ("adjoining cells"), and the cells arranged in tight clusters, we found that the value of k in the latter group was much higher, reaching about 75% of that obtained in the presence of FSH. The cells cultured for two days in a medium containing ovine FSH underwent striking morphological changes and presented a rounded, "fibroblast-like" appearance. They were arranged in networks or in clusters. The frequency of cell-to-cell dye diffusion after microinjection and the rate constant of dye transfer were rapidly increased to the same final level by FSH, although they were initially different in these two groups. A concentration dependence of k, in the range 0.05 to 3 ng/ml, was observed in the cells in networks, contrasting with an all-or-none increase in the cells in clusters. Two days after FSH withdrawal, the dye transfer constant returned to prestimulation control values in the cells in clusters, but not in the cells in networks, which maintained a stable degree of coupling comparable to that of the unstimulated cells in clusters. This observation suggests (i) that an initial promoting effect of FSH already exists in the immature rat testis, which is preserved after enzymatic treatment in the cell clusters, but not in the more dispersed cells, and (ii) that the decreased junctional coupling is re-established in the dispersed cells by FSH, through a synthesis or a membrane insertion of connexin. The effects of FSH were mimicked by a brief exposure to 1 mM dibutyryl-cyclicAMP, but not to 10 nM human chorionic gonadotropin (hCG), indicating that the gap junction communication in Sertoli cells is upregulated by FSH through a specific membrane receptor, with cyclicAMP acting as a second messenger.

A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 microM and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state.