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

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.