The presence of IGFs and their associated binding proteins (IGFBPs) in human follicular fluid is well documented. Furthermore, most of the constituents of the IGF system in follicular fluid have been found to vary, either in total amount or by proteolytic cleavage, depending on the health status of the follicle. In this study we have examined the acid-labile subunit (ALS) and found that levels in follicular fluid (mean 146 nmol/l) were almost 50% of those in the circulation. This amount of ALS was considerably greater than that found in other extracirculatory fluids (20.9 for synovial fluid and 31.4 nmol/l for skin blister fluid). As in the circulation, ALS levels were in molar excess and did not vary between atretic and dominant follicles. Although the source of ALS is probably from blood (conditioned medium from ovarian cell cultures had no measurable ALS) it would appear that this glycoprotein is not merely diffusing from the circulation as the capillary endothelium becomes more permeable in dominant follicles and this is not reflected in the level of ALS. Analysis of the distribution of IGF-I, IGF-II and IGFBP-3 in fluid from healthy and atretic follicles revealed that the majority of these growth factors (> 80% of total IGF-II) were in the 150KDa complex, indicating that the ALS present was functional, in that it formed the ternary complex with a molecule of IGFBP-3 and IGF. No free IGF-II was found in any of the follicular fluids analysed nor was there any increase in the amount of unsaturated IGFBP-3 in atretic follicles. In summary, we have shown that the majority of IGF measured in follicular fluid, whether from healthy or atretic follicles, is bound in the ternary complex.
SC Hughes, HD Mason, S Franks and JM Holly
J Bonser, J Walker, A Purohit, MJ Reed, BV Potter, DS Willis, S Franks and HD Mason
Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range 4-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and androstenedione production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for androstenedione and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.