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Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.

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Levels of LH and FSH released in vitro by rat anterior pituitary cells which were either co-cultured with isolated rat Sertoli cells or were grown with spent media recovered from the cultured Sertoli cells were measured by radioimmunoassay. The amounts of FSH released by pituitary cells grown for three days with Sertoli cells isolated from 31-36 day old rats or spent media from the cultured Sertoli cells, were significantly (P less than 0.01) lower compared to control pituitary cultures grown with fresh chemically defined medium. In contrast, the levels of LH were similar to the controls. The selective inhibition of FSH release was not observed when pituitary cells were co-cultured with rat spleen or kidney cells or with ruptured Sertoli cells. The FSH-inhibiting Sertoli-cell factor (SCF) was found to be a heat-labile macromolecule. It is suggested that the SCF may be secreted by the Sertoli cells in vivo, and regulate FSH secretion via negative feedback mechanism at the pituitary level.

Sertoli cells in vivo are highly polarized and interact with the inner (tubular) and outer (interstitial) fluids. To simulate this condition in vitro we developed a two-compartment culture system in which confluent Sertoli cell monolayers were grown on permeable supports (Millipore filters) separating the inner and outer fluid compartments. Monolayer permeability to (3H)-inulin decreased by 90% after 5 to 7 days of culture, presumably due to formation of tight junctions. This process was influenced by cell plating density. The cells were highly polarized morphologically, resembling their appearance in vivo, and secreted transferrin bidirectionally into both fluid compartments. The amount of transferrin secreted was 166% to 250% of that secreted by the same number of Sertoli cells cultured in plastic dishes. Testosterone (5 X 10(-8) M) doubled and testosterone + FSH (0.1 microgram/ml) increased transferrin secretion 3.6-fold. These results demonstrate that under suitable culture conditions the Sertoli cells remain both morphologically and functionally polarized, reflecting a more physiologic state.

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