Abstract
The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives 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 complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine 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 form 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 clone 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. The regulation of StAR mRNA in vivo was studied in equine preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h (n = 4–5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed no significant changes in StAR mRNA levels after hCG treatment when analyses were performed on intact follicle wall (theca interna with attached granulosa cells). However, Northern blots performed on isolated follicle cells revealed an unexpected regulation of StAR mRNA. In granulosa cells, StAR transcripts were undetectable at 0 h but were significantly increased at 30 h post-hCG, and this induction was associated with a rise in follicular fluid concentrations of progesterone (P < 0.05). In contrast, StAR mRNA levels were high in theca interna at 0 h, remained unchanged until 33 h post-hCG, and dropped dramatically thereafter (P < 0.05). Thus, this study describes the primary structure of equine StAR, documents the regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory species, and identifies a novel inverse regulation of StAR transcripts in theca interna and granulosa cells of equine follicles before ovulation.