Granulosa cell aromatase bioassay for follicle-stimulating hormone: validation and application of the method.
Stimulation of rat granulosa cell aromatase activity by FSH has recently been used as a sensitive biological end point to develop an in vitro FSH bioassay. The present report provides a detailed validation and application of this assay. In the presence of androstenedione and diethylstilbestrol, FSH stimulated estrogen production in a dose-dependent manner. Although addition of high doses of a phosphodiesterase inhibitor [1-methyl-3-isobutyl xanthine (MIX)] decreased maximal estrogen production, treatment with 0.125 mM MIX increased the sensitivity of granulosa cells to FSH, presumably by minimizing endogenous cAMP breakdown. Addition of insulin and human CG (hCG) further synergistically enhanced granulosa cell sensitivity to FSH. Although inclusion of gonadotropin-free serum obtained from hypophysectomized male rats decreased the assay sensitivity, pretreatment of serum with polyethylene glycol [(PEG) 10-14%] resulted in a dose-dependent decrease in the serum-interfering effect. Studies using exogenous [125I]iodo-rat FSH or RIA measurement indicated recovery of 94-98% FSH after pretreatment of serum with 12% PEG. In the presence of the PEG-pretreated gonadotropin-free serum (4%), ovine, rat, and human FSH preparations induced parallel dose-response curves for estrogen production with minimal detectable doses of 0.12 ng, 0.12 ng, and 0.12 mIU/culture, respectively. In contrast, treatment with GH, PRL, TSH, and ACTH did not affect estrogen production. The apparent stimulatory effect of high doses (greater than 60 ng/culture) of LH and hCG could be attributed to FSH contamination or intrinsic FSH activity in these preparations. Changes in serum bioactive FSH levels were studied in adult male rats after GnRH administration. GnRH (5 micrograms/rat) treatment significantly elevated FSH levels within 30 min after injection. Maximal increases (approximately 2.8-fold) in serum bioactive FSH were observed between 60-120 min. At 8 h after treatment, FSH levels decreased to control levels. Comparison between granulosa cell aromatase bioassay and RIA results indicated no apparent changes in the bio- to immuno- ratio of FSH after GnRH treatment. In conclusion, extreme sensitivity of the bioassay allows the measurement of circulating levels of bioactive FSH. Since rat granulosa cells respond to FSH preparations from different species, the in vitro assay should also provide valuable information on FSH levels in many animal species including those lacking a specific RIA. Measurement of serum levels of bioactive FSH should provide insight regarding the role of FSH in various physiological and pathological conditions.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000735:Androstenedione / Q000494:pharmacology; D000818:Animals; D001141:Aromatase / Q000378:metabolism*; D001681:Biological Assay; D001769:Blood; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004054:Diethylstilbestrol / Q000494:pharmacology; D004357:Drug Synergism; D004967:Estrogens / Q000096:biosynthesis; D005260:Female; D005640:Follicle Stimulating Hormone / Q000097:blood* / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D007328:Insulin / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity
D006063:Chorionic Gonadotropin; D004967:Estrogens; D007328:Insulin; D011092:Polyethylene Glycols; D000735:Androstenedione; D004054:Diethylstilbestrol; D005640:Follicle Stimulating Hormone; D001141:Aromatase; D015056:1-Methyl-3-isobutylxanthine