Human chorionic gonadotropin induces an inverse regulation of steroidogenic acute regulatory protein messenger ribonucleic acid in theca interna and granulosa cells of equine preovulatory follicles

The time- and gonadotropin-dependent regulation of steroidogenic acute regu latory protein (StAR) has not been characterized in vivo in preovulatory fo llicles of large monoovulatory species or sexually mature animals. The obje ctives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementar y DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equ ine StAR that differ only in the length of their 3'-untranslated region (3' -UTR); a long form of 2918 bp and a short form of 1599 bp. The StAR long fo rm cDNA contains a 5'-UTR of 117 bp, an open reading frame (ORF) of 855 bp, and a 3'-UTR of 1946 bp. Primer extension analysis showed that the cDNA cl one lacked the first 10 bp of the primary transcript, giving a total of 127 bp for the complete StAR 5'-UTR. The ORF encodes a 285-amino acid protein that is 86-90% identical to StAR of other species characterized to date. Th e regulation of StAR mRNA in vivo was studied in equine preovulatory follic les isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 foll icles/time point) after an ovulatory dose of hCG. Results from Northern blo ts showed no significant changes in StAR mRNA levels after hCG treatment wh en analyses were performed on intact follicle wall (theca interna with atta ched granulosa cells). However, Northern blots performed on isolated follic le cells revealed an unexpected regulation of StAR mRNA. In granulosa cells , StAR transcripts were undetectable at 0 h but were significantly increase d at 30 h post-hCG, and this induction was associated with a rise in follic ular fluid concentrations of progesterone (P < 0.05). In contrast, StAR mRN A levels were high in theca interna at 0 h, remained unchanged until 33 h p ost-hCG, and dropped dramatically thereafter (P < 0.05). Thus, this study d escribes the primary structure of equine StAR, documents the regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory specie s, and identifies a novel inverse regulation of StAR transcripts in theca i nterna and granulosa cells of equine follicles before ovulation.