Biological determination of inhibin in rat ovarian-cell culture medium

Abstract

Rat ovarian-cell culture medium contains steroids and inhibin material that are able to influence basal and LHRH-stimulated secretion of FSH and LH from dispersed pituitary cells. Studies on procedures to remove steroids, such as ethyl acetate extraction and charcoal: dextran adsorption, showed that adsorption of the culture medium with 0.5 to 1.0% charcoal and 0.05 to 0.1% dextran, respectively, was the optimal method to eliminate most of the steroids in the medium without a detrimental effect on inhibin activity. The inclusion of serum or bovine albumin in the culture medium after the culture protected the inhibin from adsorption by the charcoal: dextran treatment. The inhibin present in the rat ovarian-cell culture medium treated with 1.0% charcoal: 0.1% dextran behaved like the inhibin reference standard (from rete testis fluid of the sheep: RTFS) in the basal secretion of FSH and the LHRH-stimulated secretion of FSH and LH in the in-vitro bioassay.

An in-vitro model for the study of inhibin production by rat ovarian cells

“…The population of cells harvested by this modified method of Hillier et al (1977) is probably heterogeneous, consisting of granulosa cells as well as theca and interstitial cells because no thecal layer remains after the collection and about 4 times more cells were obtained than in the original procedure. This was biochemically supported by the significant secretion of androgens as measured here and reported by Croze & Franchimont (1984). The influence of ovarian cell heterogeneity on the secretion of inhibin in long-term culture has not yet been investigated.…”

“…From the present study on the determination of the optimal culture conditions of rat ovarian cells to maintain inhibin production, we find that serum is an essential constituent of the culture medium and that the effect of serum is not only due to a protection of inhibin against charcoal : dextran adsorption (Croze & Franchimont, 1984). It is known that serum provides specific cellular nutrients in a physiologically balanced manner (May & Schomberg, 1981), enhances amino acid (Allen, Nilsen-Hamilton, Hamilton & Gospodarowicz, 1979) and glucose (Allen, Nilsen-Hamilton & Hamilton, 1981) uptake and can be mitogenic for ovarian cells (lohnson, Dawson, Turner & Wyche, 1980;Orly et al, 1980).…”

“…Serum-free DMEM was used to adjust the incubation volume to 1 ml. This bioassay was validated for the inhibin present in rat ovarian-cell culture medium by the method described by Croze & Franchimont (1984). The activity of inhibin present in the culture medium from rat ovarian cells was measured on one occasion in basal secretion and for all the experiments in the 6-h LHRH-induced secretion of FSH.…”

“…After the first 24 h of culture, medium was removed, the cells were rinsed with 3 1 ml Medium FIO and incubated with 2 ml Medium F10+ with 0, 2-5, 5-0, 7-5, 100 or 12-5% FCS. Before the adsorption by charcoal : dextran, the percentage of FCS in the various ovarian-cell culture media was adjusted to 12-5% to have the same protective effect against adsorption of inhibin by the charcoal : dextran treatment (Croze & Franchimont, 1984). Experiment 2.…”

“…The concentrations of FSH in the culture media from the pituitary cells were measured by an heterologous double-antibody radioimmunoassay as described by Croze & Franchimont (1984) using a first antibody raised against ovine FSH (Derivaux, Ectors, Hendrick & Franchimont, 1974) or the antibody raised against rat FSH (anti rFSH-S-11) supplied by the NIAMDD. The rat FSH used as standard, rFSH-RPl, and tracer, rFSH-I-5, was from the NIAMDD.…”