Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells

Abstract

Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T3) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T3 on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N6,2′-O-dibutyryladenosine-3′:5′-cyclic monophosphate ((Bu)2cAMP) that was clearly down-regulated by T3. The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [3H]H2O released after incubation with [1β-3H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)2cAMP and markedly down-regulated by T3. In contrast, the strong increase in aromatase mRNA upon (Bu)2cAMP stimulation was apparently unaffected by T3 administration. This paper shows how the identification of an altered transcript induced by T3 coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T3-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T3 on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T3 in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.