Porcine Granulosa Cells Produce Gonadotropin Surge Inhibiting Factor (GnSIF) Activity In Vitro

Seo, Soo Hyung

Abstract

OBJECTIVES
It has been identified a novel protein in porcine and bovine follicular fluid termed Gonadotropin Surge Inhibiting Factor (GnSIF) which inhibits GnRH-stimulated LH secretion by pituitary cells in culture. The purpose of this study was to investigate the production of GnSIF by porcine granulosa cell in vitro.
MATERIALS AND METHODS
Granulosa cells were aspirated from different size of porcine follicles (1-3mm, 3-5mm, >6mm in diameter) and cultured in 24-well plates in Ham's F-12/DMEM at 500,000 cells/well for 24h. After plating, cells were washed and incubated in triplicate for 72h in DMEM/F-12 containing 2% FBS in the presence of 100ng/ml FSH and the media were collected. Granulosa cell-conditioned media were chromatographed on heparin-Sepharose to remove inhibin and follistatin, and assayed for GnSIF activity using a rat pituitary cell bioassay. Briefly, dispersed pituitary cells from adult female Sprague-Dawley rats were plated (500,000 cells/well) for 72h, washed, and incubated with granulosa cell-conditioned media in the presence of 10nM GnRH for 4h. The media were collected and assayed for LH by RIA. GnSIF activity is defined as the suppression of GnRH-stimulated LH secretion.
RESULTS
Granulosa cell-conditioned media significantly inhibited GnRH-stimulated LH secretion by rat pituitary cells in vitro (69% of control, P<0.001, n=2 experiments). Preliminary data suggest that granulosa cells from small follicles (1-3mm) produce greater amounts of GnSIF than those obtained from larger follicles (>6mm).
CONCLUSIONS
GnSIF activity is produced by porcine granulosa cells in vitro. Granulosa cell production of GnSIF, along with other gonadal proteins such as inhibin, follistatin, and activin may be an important component of the ovarian control of pituitary gonadotropin secretion.