INSULIN-LIKE GROWTH-FACTOR-I GENE-EXPRESSION IN HUMAN GRANULOSA-LUTEIN CELLS

IGF-I is an important local regulator of ovarian function, stimulating follicular growth and steroidogenesis in human granulosa cells. However, it is not known whether ovarian IGF-I is derived from the circulating serum pool or from local production. IGF-I peptide has only been detected in human thecal cells and not in granulosa cells. This study has used the sensitive technique of reverse transcription of mRNA followed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells. Granulosa-lutein cell (GLC) samples were obtained by follicular puncture of seven women enrolled in an ovulation induction programme. Treatment had included buserelin acetate, human menopausal gonadotrophin to stimulate follicular growth and human chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), extracted from the GLCs, was amplified by RT/PCR, using combinations of leader and 3' IGF-I exon-specific primers, to yield four IGF-I gene products: IGF-IA (exons 1, 3, 4, 6), IGF-IB (exons 1, 3, 4, 5), IGF-IA' (exons 2, 3, 4, 6) and IGF-IB' (exons 2, 3, 4, 5). As controls from other tissues, an identical procedure was undertaken on TRNA from peripheral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression differed between the tissues; in liver and GLCs, the IGF-IA transcript was dominant, but in monocytes the IGF-IA' species was the most prominent. Quantitative RT/PCR using standardization to the house-keeping gene for glyceraldehyde-3'-phosphate dehydrogenase revealed that IGF-IA mRNA was 300-fold more abundant in liver than GLCs. This study has indicated that: i) RT/PCR can be used to detect multiple IGF-I mRNA species in small amounts of TRNA from human tissue, ii) the IGF-I gene is expressed at low level in human preovulatory GLCs and iii) there is preferential use of leader exon 1 in human GLCs and leader exon 2 in monocytes. Further studies of IGF-I gene expression by this method in other ovarian cell types and granulosa cells from the developing follicle may help to clarify the local role of IGF-I in the human ovary.