Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins

Abstract

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1–5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased ( P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased ( P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased ( P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased ( P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect ( P>0.10). Estradiol (3 and 300 ng/ml) decreased ( P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased ( P<0.05) IGFBP-4 expression, but had no effect ( P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased ( P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.