HIGHER GONADOTROPIN SURGE-ATTENUATING FACTOR BIOACTIVITY IS FOUND IN SMALL FOLLICLES FROM SUPEROVULATED WOMEN

Ovine and rat pituitary bioassays for gonadotrophin surge-attenuating factor (GnSAF) were utilized to determine whether the level of GnSAF b ioactivity in pooled human follicular fluid (hFF) from superovulated w omen varied according to follicle diameter (less than or equal to 11 m m, 12-15 mm and 16-21 mm follicles examined using the ovine bioassay, or less than or equal to 10 mm, 11-13 mm, 14-17 mm, 18-20 mm, 21-24 mm and greater than or equal to 25 mm follicles examined using the rat b ioassay). When tested using dispersed ovine pituitary cells, GnSAF bio activity, expressed in terms of the reduction in gonadotrophin-releasi ng hormone (GnRH)-induced LH secretion, was inversely related to folli cle diameter (P < 0.01). In response to 5 mu l hFF/well from follicles of less than or equal to 11, 12-15 and 16-21 mm diameter, GnRH-induce d LH secretion was reduced to 40.5 +/- 6.9%, 65.2 +/- 6.6% and 83.7 +/ - 7.9% of control respectively. A similar inverse relationship was obs erved when a second batch of hFF samples from different sized follicle s was tested using rat pituitary cell monolayers. Expressing GnSAF bio activity in terms of the dose required to suppress GnRH-induced LH sec retion by rat pituitary cells to 50% of the maximal suppression observ ed (ED(50)), the three smallest follicle size pools contained the most GnSAF (EDS, values of 0.13, 2.79 and 5.36 yl hFF/well from follicles of less than or equal to 10, 11-13 and 14-17 mm respectively). The ED( 50) values for follicles of 18-20, 21-24 and greater than or equal to 25 mm were 8.81, 27.1 and 60.0 mu l hFF/well respectively. Thus hFF fr om follicles less than or equal to 11 mm was over 450 times more poten t than hFF from follicles greater than or equal to 25 mm in suppressin g GnRH-induced LH release. The ED(50) values for inhibin bioactivity ( measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity ( between 0.85 and 0.13 mu l hFF/well). Inhibin immunoreactivity, measur ed by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follic les (41.96 ng/ml) and highest concentrations in the three largest foll icle size groups (56.48-64.48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant hum an inhibin at doses of 0.025-25 ng/well. In both the sheep and rat pit uitary monolayers, basal FSH was suppressed (ED(50) = 0.02 and 0.16 ng /well respectively). However, while inhibin markedly stimulated GnRH-i nduced LH secretion from ovine pituitary monolayers (ED(50) = 0.04 ng/ well), it suppressed GnRH-induced LH secretion from rat pituitary mono layers (ED(50) = 0.31 ng/well) by 13%. The divergent effects of inhibi n on GnRH-induced LH secretion in the two culture systems, and the rel ative insensitivity of GnRH-induced LH secretion to recombinant human inhibin in the rat system, indicates that the inverse relationship bet ween GnSAF concentrations and follicular diameter cannot be an artefac t of inhibin bioactivity. In addition, when hFF was fractionated by hy drophobic interaction chromatography using phenyl Sepharose, fractions which contained the greatest amounts of GnSAF bioactivity differed fr om those which contained peak levels of bioactive or immunoreactive in hibin. These results support in vivo observations that small follicles are important regulators of gonadotrophin secretion in superovulated women. Concentrations of GnSAF fall as the follicles approach an ovula tory size which enables positive steroid feedback on pituitary respons es to hypothalamic GnRH, leading to the preovulatory LH surge.