Abstract
Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.
Highlights
Sertoli cells are hormonally regulated by follicle- FSH’ while fulfilling numerous functions geared toward supstimulating hormone (FSH) acting upon a G-protein- porting the development and maturation of germinal cells
Using freshly iso- Previous studies have suggested a role for cyclic AMP as a lated rat Sertoli cells we measured cytosolic free ion- second messenger mediating FSH action in Sertoli cells [2]
The present findings provide the first direct measurement of cytosolic-free calcium in rat Sertoli cells and demonstrate that physiological doses of FSH, the major trophic hormone for Sertoli cells, stimulate a prompt rise in cytosolic calcium
Summary
From the DerJartments of Obstetrics and Gynecology and Medicine, University of Sydney, Sydney, ~~. Measurement ofCytosolic Calcium in Sertoli Cells-Preliminary conditions before and after administration of either FSH (1 experiments using fluorescencemicroscopy indicated that theoptimal ng/liter) or saline vehicle control. Emission fluorescence for calculation of cytosolic calcium was ob 60 routinely recorded for 60 s prior to andfor 180 s after administration of ovine FSH and/or other drugs (or saline vehicle) in 30 p1 of cell. Free calcium (nM) = K d X ( R - Rmin/R,,, - R) X Sf2/Sb, prolactin (1 ng/liter; 85 f 13 nM, n = 4) andrat growth hormone (1ng/liter; 89 k 25nM, n = 4)] had no effect on with Kd being the Fura dissociation constant at 37"C (224 nM); R cytosolic calcium compared with saline-treated Sertoli cells defined as the ratio of fluorescence measured at 340- and 380-nm excitations, respectively; and Sf2/Sb2 the ratio of fluorescence at 380 nm in low calcium to thatof high calcium. Bay K8644 analysis was performed by analysis of variance for repeated measures (1000 nM), a selective dihydropyridine agonist, induced and unpaired t test as appropriate
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