THYROID-HORMONE AND FOLLICLE-STIMULATING-HORMONE REGULATE MULLERIAN-INHIBITING SUBSTANCE MESSENGER-RIBONUCLEIC-ACID EXPRESSION IN CULTURED NEONATAL RAT SERTOLI CELLS

Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T-3 in Mullerian-inh ibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertol i cell secretory protein that induces Mullerian duct regression and al so may be critical for germ and Leydig cell development, is maximal pe rinatally, then decreases as Sertoli cells mature. The fall in MIS mRN A expression is delayed by hypothyroidism in vivo, indicating that T-3 could regulate MIS mRNA. However, understanding of the hormonal regul ation of MIS has been limited due partly to the lack of a primary Sert oli cell culture system in which sustained expression of MIS or its mR NA can be obtained. We have developed a Sertoli cell culture system fo r examining hormonal regulation of MIS mRNA. We then tested the effect s of T-3 and/or FSH treatment on MIS mRNA levels in this new system. I nitial studies indicated that MIS mRNA production by Ei-day-old rat Se rtoli cells was minimal in vitro. Therefore, Sertoli cells from a-day- old rats were cultured for 2 or 4 days. After 2 days in vitro, steady state MIS mRNA levels were decreased to 36% of the levels seen in fres hly isolated Sertoli cells from a-day-old rats. However, by day 4 of c ulture, steady state MIS mRNA production had recovered to 67% of that seen in freshly isolated a-day-old Sertoli cells, which closely parall eled the decrease seen in MIS production in vivo from days 2-6. MIS mR NA levels were decreased 53%, 64%, and 86% in cultures treated with 0. 01, 0.1, and 1.0 nM T-3 (P < 0.05), respectively. This decrease in Ser toli cell MIS mRNA did not reflect a nonspecific effect on cell viabil ity and/or activity, as shown by a dose-responsive increase in inhibin -alpha mRNA in these same cultures. FSH (2.5-100 ng/ml) also produced a dose-responsive decrease in MIS mRNA levels, and FSH and T-3 togethe r had an additive inhibitory effect on MIS mRNA levels, indicating tha t these hormones may act through distinct mechanisms. In summary, this is the first primary culture system in which sustained MIS mRNA produ ction can be demonstrated, and it should prove useful for understandin g the regulation of MIS in developing Sertoli cells. In addition, T-3 and FSH are major regulators of the postnatal decrease in MIS producti on by the rat Sertoli cell, and these hormones may act through separat e pathways.