EVIDENCE THAT BASEMENT-MEMBRANE PREVENTS APOPTOSIS OF SERTOLI CELLS IN-VITRO IN THE ABSENCE OF KNOWN REGULATORS OF SERTOLI-CELL FUNCTION

Although the role of basement membrane in the morphological and functi onal differentiation of Sertoli cells has been well characterized, ver y little is known about its involvement in Sertoli cell survival and m aintenance throughout life. When cultured on laminin or Matrigel, 80-9 0% of Sertoli cells retained their viability. Sertoli cells prevented from attachment and basement membrane deposition by plating on plastic surfaces coated with polyhydroxyethylmethacrylate (poly-HEMA) exhibit ed a loss of viability by approximately 50% within 24 h. Addition of s oluble laminin did not prevent the loss of viability of Sertoli cells, whereas soluble Matrigel enhanced the survival significantly when add ed at a concentration of 100 mu g/ml or more. The addition of FSH, epi dermal growth factor, testosterone, retinoic acid, or a mixture of ins ulin, transferrin, and selenium had no significant effect on the viabi lity of Sertoli cells cultured on polyHEMA for up to 72 h. When all of these hormones and factors were added together, a significantly highe r percentage of cell survival was observed at 24, 48, and 72 h, but th e percent survival was significantly lower than that seen on either la minin or Matrigel. The nature of cell death occurring in the Sertoli c ells plated on polyHEMA was determined by agarose gel analysis that re vealed a ladder of similar to 200-base pair DNA multiple fragments. Fl ow cytometric analysis of propidium iodide-stained cells indicated tha t most of the cells were apoptotic. Freshly isolated Sertoli cells and adherent cells on basement membrane did not show internucleosomal DNA breakdown or an apoptotic peak in the flow cytometric analysis. These results suggest that basement membrane plays a crucial role in Sertol i cell survival in vitro when it is used as a solid substratum for cul ture, and in the absence of basement membrane, FSH and other regulator s of Sertoli cell function cannot prevent Sertoli cell apoptosis.