RAT SERTOLI-CELL CALCIUM RESPONSE TO BASEMENT-MEMBRANE AND FOLLICLE-STIMULATING-HORMONE

Sertoli cells cultured on basement membrane substrates differentiate m orphologically into polarized cells and exhibit an enhanced responsive ness to FSH. The signal transduction mechanisms by which the extracell ular matrix induces changes in the morphology and function of Sertoli cells are not known. Since calcium has been implicated in mediating ch anges in cytoskeletal assembly and organization, we investigated to se e if basement membrane can modulate cytosolic free calcium concentrati ons during the process of adhesion and spreading of Sertoli cells. A d irect quantification of the intracellular free cytosolic calcium conce ntration [Ca2+](i) in freshly isolated immature rat Sertoli cells plat ed on laminin was performed by digital imaging microscopy using the fl uorescent probe Fura-2 AM. [Ca2+](i) levels rose by 1.5-2-fold within 1 h after plating on laminin, suggesting that calcium may be involved in adhesion and spreading of the cells on basement membrane. Furthermo re, the possibility that matrix influences [Ca2+](i) levels upon stimu lation with FSH was examined by adding FSH directly to the cells sprea ding on laminin. A dramatic decrease in [Ca2+](i) was observed compare d to the level in untreated cells. Similarly, a significant decrease i n [Ca2+](i) in response to FSH was observed in cells already spread on laminin or Matrigel, Addition of dibutyryl cAMP did not significantly alter the basal calcium levels. Long-term exposure of Sertoli cells c ultured on either laminin or Matrigel to FSH was studied by incubating the cells with (CaCl2)-Ca-45 in the presence or absence of FSH for 24 h. FSH induced a decrease or no change in Ca-45 concentration in cell s cultured on basement membrane. Addition of dibutyryl cAMP, instead o f FSH, did not alter the basal Ca-45 concentrations. In cells cultured on the peptides derived from laminin (RGD and SIKVAV), FSH increased the uptake of Ca-45 significantly, whereas on YIGSR, also a laminin-de rived peptide, it did nor have any effect. Thus, basement membrane ind uces an early increase in [Ca2+](i) in cultured Sertoli cells during s preading, and FSH appears to significantly decrease [Ca2+](i) levels.