Regulation of inhibin α- and β A-subunit messenger ribonucleic acid levels by gonadotropins and IGF-I in cultured chicken granulosa cells

Abstract

A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin α- and β A-subunits in chicken granulosa cells showed that these subunits are expressed in different amounts depending on follicular maturation. The present study determined the regulation of the expression of these subunits. The individual effect of different doses of IGF-I, LH or FSH (1–100 ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin α- and β A-subunit was determined on cultured granulosa cells of F 1 and the combined F 4+F 5 follicle. Cells were cultured for 48 h in 6-well plates with or without added hormones. Culture medium was discarded, cells were washed and total RNA was extracted from the cells. Five hundred nanograms of total RNA was reverse transcribed using specific primers and coamplified with an internal standard, as described previously, to determine expression level in the cells. IGF-I, LH, and FSH enhanced the inhibin α-subunit mRNA levels in a dose dependent manner in both F 1 and the combined F 4+F 5 whereas inhibin β A-subunit was not affected. The effects of FSH, LH were more expressed in F 1 follicles compared to F 4+F 5 on the α-subunit. The addition of IGF-I and either LH or FSH during the culture period significantly increased the stimulatory effects of both LH and FSH on the expression of inhibin α-subunit in F 1 follicles but had no significant effect on the inhibin β A-subunit. The results suggest that the changing expression levels of inhibin α-subunit during follicular development are the result of the regulatory effect of the interaction between IGF-I and the gonadotropins and that the regulation of this subunit may be the main factor for the regulation of the protein inhibin levels. Other factors may be also implicated in the changing expression levels of the β A-subunit.