Endocrine Abstracts (2001) 2 P25


CM Perks2, RJ Wright1, JMP Holly2, M Grohmann2 & HD Mason1

1Dept. Ob/Gyn and Physiol, St. George's Hospital Medical School, London SW17, UK; 2Dept. Surgery, BRI, Bristol University, BS2, UK.

IGFs and IGFBPs have an important role in folliculogenesis, with the profile of IGFBPs being different in healthy and atretic follicles. In particular, IGFBP-4, which is a potent inhibitor of steroidogenesis, is preferentially proteolysed in healthy follicles. We therefore investigated the production and activation of IGFBP-4 protease in human follicular cells.

Follicles of 3-25mm were dissected from 13 pairs of ovaries collected with ethics committee approval. The androgen:oestrogen ratio in follicular fluid was measured to determine follicular health. Granulosa cells (GC) were also collected from IVF-flush. GC were plated at 0.1-1x106 per well in 200microlitres Medium 199 while theca (T) was incubated as 2-3mg explants in 1ml M199. GC and T from pooled small or separate large follicles were incubated with either medium alone, 10-7M testosterone, 5ng/ml IGF-II or gonadotrophin (Gn) at 1-5ng/ml. Conditioned medium (CM) was collected every 24-48h and stored at -80 degC. IGFBP-4 protease activity was assessed by the ability of CM to fragment 125I-IGFBP-4 in the presence or absence of 2mg/ml IGF-II (cell-free).

Protease activity was detected in GCCM from all sizes of follicle tested (3-25mm) and from both healthy and atretic follicles, but always required IGF-II for activation. In GC from large pre-ovulatory follicles, incubation with Gn caused an increase in the protease activity seen. Incubation of GC with IGF or testosterone did not change the level of protease activity detected. No IGFBP-4 protease activity was detected in any theca CM.

In conclusion, GC appear to be the source of IGFBP-4 protease in the follicle and production is regulated in larger follicles by Gn. Protease appears to be produced constitutively, even in small follicles, but activation of the protease by IGF-II is required and this may reflect an additional regulatory mechanism of IGFBP-4 proteolysis in healthy follicles.

This project is supported by the Wellcome Trust.

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