Real-time RT-PCR quantification of pregnancy-associated plasma protein-A mRNA abundance in bovine granulosa and theca cells: Effects of hormones in vitro

Abstract

Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5 mm) and large (LG) (8–22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3–500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected ( P > 0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect ( P > 0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased ( P < 0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.