Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.
There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.
Department of Obstetrics and Gynecology, St. Mary's Hospital Medical School, Paddington, London, United Kingdom.
H D
HD
Mason
S C
SC
Cwyfan-Hughes
G
G
Heinrich
S
S
Franks
J M
JM
Holly
D015153:Blotting, Western; D010450:Endopeptidases / Q000096:biosynthesis*; D005260:Female; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000032:analysis / Q000096:biosynthesis*; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis*; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis*; D010053:Ovary / Q000166:cytology / Q000378:metabolism*; D011085:Polycystic Ovary Syndrome / Q000378:metabolism*; D011863:Radioimmunoassay; D017154:Stromal Cells / Q000378:metabolism; D013799:Theca Cells / Q000378:metabolism
D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D010450:Endopeptidases; C080473:insulin-like growth factor binding protein-3 protease