Sertoli cell production of müllerian inhibiting substance in vitro.

Sertoli cell cultures were monitored for müllerian inhibiting substance with a solid phase radioimmunoassay. Sertoli cells from newborn calf testes were placed in defined media free of serum in monolayer culture after treatment with trypsin-collagenase followed by gravity separation. Immunoreactive müllerian inhibiting substance was detected in the culture media of Sertoli cells but not National Institutes of Health 3T3 cells. To verify that the Sertoli cells were intact, cyclic adenosine monophosphate levels were determined after follicle-stimulating hormone stimulation. Cyclic adenosine monophosphate levels were elevated in Sertoli cells but not National Institutes of Health 3T3 cells. The newborn calf Sertoli cell culture provides a useful system in which to study factors affecting müllerian inhibiting substance production and release, and documents this substance as another reliable marker for the Sertoli cell. Müllerian inhibiting substance levels also could be measured in media beneath testicular fragments in organ culture, and were increased and prolonged in comparison to müllerian inhibiting substance release from Sertoli cell monolayer cultures. Modulation of müllerian inhibiting substance release from monolayer and organ cultures then was attempted. Neither gonadotropin nor steroid additions affected the release of müllerian inhibiting substance during 24 to 72 hours in organ or tissue culture. We are using the müllerian inhibiting substance radioimmunoassay to monitor attempts to immortalize a Sertoli cell line capable of continuous müllerian inhibiting substance production using viral deoxyribonucleic acid transfection techniques.