The cultured rodent follicle as a model for investigations of gonadotrophin surge-attenuating factor (GnSAF) production

Abstract

Gonadotrophin surge-attenuating factor (GnSAF) bioactivity (the suppression of GnRH-induced but not basal LH and FSH secretion from pituitary gonadotrophs) is produced by granulosa cells in vitro. Previous studies to investigate this bioactivity used dispersed granulosa cells which lack some cell types and the structural components of the follicle in vivo. The aim of this study, therefore, was to investigate whether intact rodent follicle culture was a suitable model for the study of the production of GnSAF bioactivity, allowing GnSAF to be investigated in a more physiologically realistic environment while still retaining culture conditions from which, as with granulosa cell cultures, extraneous factors can be excluded. Follicles from 16-day-old rats and 21-day-old mice were cultured for 3–6 days in the presence or absence of FSH and/or LH. The follicle-conditioned medium, and matching samples of unconditioned culture medium were added to our established rat pituitary monolayer GnSAF bioassay. Both mouse and rat intact follicles produced GnSAF bioactivity, reducing GnRH-induced LH secretion significantly. GnSAF output from the mouse follicles was highest during days 1–3 of culture, when follicles were at an early antral stage of development, and fell on days 4–6 as the follicles grew to the mid antral stage. While the stimulatory effects of FSH on rat follicle GnSAF secretion was dose-dependent, LH alone did not increase GnSAF production. An antibody against human GnSAF blocked GnSAF bioactivity produced by rat follicles, and recognised proteins within the expected pI and molecular weight range for GnSAF in two-dimensional gels of rat follicle-conditioned medium, showing a good homology between rodent and human GnSAF proteins. In conclusion, the release of GnSAF bioactivity is principally from small follicles stimulated by FSH. Therefore, intact rodent follicle culture systems offer an excellent model for the investigation of factors controlling GnSAF production under relatively physiological conditions.