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Keywords:

inhibin, igf-i, gonadotropin, granulosa, chicken, subunit gene-expression, complementary deoxyribonucleic-acid, follicle-stimulating-hormone, growth-factors, preovulatory follicles, immunoreactive inhibin, molecular-cloning, ovulatory cycle, domestic hen, luteal cells, Science & Technology, Life Sciences & Biomedicine, Endocrinology & Metabolism, Zoology, IGF-I, COMPLEMENTARY DEOXYRIBONUCLEIC-ACID, GROWTH-FACTORS, GENE-EXPRESSION, IMMUNOREACTIVE INHIBIN, PREOVULATORY FOLLICLES, INHIBIN/ACTIVIN ALPHA, MOLECULAR-CLONING, RAT INHIBIN, ACTIVIN-A, RNA, Animals, Cells, Cultured, Chickens, Female, Follicle Stimulating Hormone, Gene Expression Regulation, Developmental, Granulosa Cells, Inhibin-beta Subunits, Inhibins, Insulin-Like Growth Factor I, Luteinizing Hormone, Ovarian Follicle, Protein Isoforms, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, 0606 Physiology, 0608 Zoology, 0707 Veterinary Sciences, 3109 Zoology, 3202 Clinical sciences

Abstract:

A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin alpha- and beta(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 (1-100ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin alpha- and beta(A)-subunit was determined on cultured granulosa cells of F, 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 a-subunit mRNA levels in a dose dependent manner in both F, and the combined F4 + F5 whereas inhibin beta(A)-subunit was not affected. The effects of FSH, LH were more expressed in F, follicles compared to F4 + F5 on the alpha-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 alpha-subunit in F, follicles but had no significant effect on the inhibin beta(A)-subunit. The results suggest that the changing expression levels of inhibin alpha-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 beta(A)-subunit. (C) 2003 Elsevier Science (USA). All rights reserved.