A highly specific and sensitive radioreceptor assay for FSH has been developed, using partially purified plasma membranes from bovine testes. Highly purifed hFSH was radioiodinated by the lactoperoxidase method. The limit of detection of purified hFSH was 1 ng/ml on the basis of 2 times standard deviation of the zero value. The assay was applicable for measurements of FSH in serum; however, the sensitivity decreased slightly to 2.5 ng/ml. The precision of the assay was less than +/- 10% within-assays and +/- 15% between-assays as expressed by the coefficient of variation. The assay was highly specific for FSH of various species. Slight inhibition of uptake of 125I-hFSH was observed with LH and TSH; but no competition was seen with 10,000 ng/ml of insulin, prolactin, hGH, hCG, and subunits of LH. Purified hFSH (LER-1575C) was measured to be 200 times the potency of the reference FSH/LH (LER-907); and purified ovine FSH (LER-1491) and rat FSH (FSH-I-1) were estimated to be 35 times and 71 times that of oFSH-S-1 (NIH), respectively. The content of FSH in human pituitary was estimated to be 226.8 +/- 118.8 mIU/mg, and the index of discrimination (radioimmunoassay/radioreceptor assay) for pituitary FSH was demonstrated to be 1.71 +/- 0.49. For measurements of serum hFSH, the index of discrimination (radioimmunoassay/radioreceptor assay) was demonstrated to be 1.08 +/- 0.20.
A radioimmunoassay for bovine FSH has been developed using a rabbit antiserum against highly purified bovine FSH (160 X NIH-FSH-S1) and a 125I-labelled tracer of the same preparation. The sensitivity was 0.25 ng/ml for purified bovine FSH or 25 ng/ml with reference to a crude bovine FSH (NIH-FSH-B1) standard. Purified bovine LH and TSH showed cross-reactivities of less than 1%, while their subunits and other purified protein hormones showed no significant inhibition at concentrations up to 100 ng/ml. Ovine FSH cross-reacted significantly but in a non-parallel manner; human and rat FSH cross-reacted only slightly. The concentrations of FSH (means +/- S.E.M.) in the sera of cows (n = 12), cows at oestrus (4), bulls (7) and steers (9) were measured as 25.4 +/- 4.7, 58.4 +/- 12.6, 45.7 +/- 9.5 and 120.2 +/- 36.0 ng/ml respectively. The FSH content of bovine pituitary glands varied greatly between individuals, from 2 to 25 microgram/mg tissue. The inhibition curves of pituitary and serum FSH were parallel to that of the bovine FSH standard.
The changes in the binding of FSH during follicular maturation were examined in the hen using 125I-labelled bovine FSH (bFSH) and unlabelled bFSH. The binding of 125I-labelled bFSH was not inhibited by bovine LH or chicken LH but was inhibited by extracts of chicken pituitary glands. The ovarian stroma, which contained both interstitial tissue and small follicles, bound the greatest amount of FSH. As the follicles progressed through the yolk-filled hierarchy of maturation, they bound decreasing amounts of FSH. In the two largest follicles of the hierarchy, there was a significant increase in the binding of FSH 12-16h before ovulation. There were two peaks in the concentrations of LH; a preovulatory peak occurred 4-6h before ovulation and a second peak occurred 14-16h before ovulation. Plasma concentrations of testosterone, oestradiol and progesterone began to rise 9, 8 and 6h, respectively, before ovulation. These data are consistent with the hypothesis that changes in the gonadotrophin concentration and binding regulate the order of the follicular hierarchy and the onset of preovulatory steroidogenesis in the hen.
Acetone dried human pituitary powder was preextracted for gonadotropins according to the method of Hartree (1). Two components of human neurophysin, designated H-Np I and H-Np II, were purified from the 75% ethanol supernatant which remains after precipitation of the gonadotropins. The neurophysin in the supernatant was precipitated by the addition of two volumes of acetone. The precipitate was redissolved and fractionated by gel nitration on Sephadex G-100. The two components of neurophysin were resolved by DEAE-cellulose chromatography and purified further by gel filtration on Sephadex G-75. H-Np
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