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. Howev er, it is not known whether ovarian IGF-I is derived from the circulat ing serum pool or from local production. IGF-I peptide has only been d etected in human thecal cells and not in granulosa cells. This study h as used the sensitive technique of reverse transcription of mRNA follo wed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells. Granulosa-lutein cell (GLC) sample s were obtained by follicular puncture of seven women enrolled in an o vulation induction programme. Treatment had included buserelin acetate , human menopausal gonadotrophin to stimulate follicular growth and hu man chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), ext racted from the GLCs, was amplified by RT/PCR, using combinations of l eader and 3' IGF-I exon-specific primers, to yield four IGF-I gene pro ducts: 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 oth er tissues, an identical procedure was undertaken on TRNA from periphe ral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression diff ered 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 mRN A was 300-fold more abundant in liver than GLCs. This study has indica ted 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 expr essed at low level in human preovulatory GLCs and iii) there is prefer ential use of leader exon 1 in human GLCs and leader exon 2 in monocyt es. Further studies of IGF-I gene expression by this method in other o varian cell types and granulosa cells from the developing follicle may help to clarify the local role of IGF-I in the human ovary.