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Development of a Homologous Radioimmunoassay for Ovine Follicle Stimulating Hormone: Studies after Estrus, Ovariectomy, Estradiol and Releasing Hormone^1,2,

US Department of Agriculture, Beltsville, MD 20705

Abstract

The development of a specific, sensitive homologous ovine follicle stimulating hormone (FSH) radioimmunoassay (RIA) is described. The key procedural modifications of basic RIA methods were immunoabsorption of the anti-ovine FSH serum with lutenizing hormone (LH) to improve specificity and adsorption chromatography of the radioiodinated ovine FSH on hydroxyapatite (HTP) to improve immunoreactivity. Cross-reactivity of the immunoabsorbed FSH antisera (R5-4IA) was less than .5% with NIH-prolactin-SlO, NIH-GH-Sll, NIH-TSH-S5 and LER-LH-1374a. Sensitivity (5.5 ng of NIH-FSH-S10) of the assay was adequate for quantification of plasma FSH in all ewes studied. Accuracy of the assay was high. The recovery of FSH from ovine plasma was nearly quantitative (y = –5.5 + 1.1X, r² .999), and the mean ratio of biological potency by bioassay to immunological potency by RIA of several FSH preparations was 1 ± .2. Intraassay and interassay coefficients of variation were both less than 15%. Four studies with Rambouillet ewes were conducted. Study 1 was with ovariectomized ewes. FSH and LH both increased at a similar rate after surgery. Values for both hormones were four- to sixfold greater 15 days after surgery than before surgery. Study 2 was with ewes at estrous. Two peaks of similar magnitude were observed in plasma FSH. The first occurred about 10 hr after the onset of estrus and coincided with the large peak in plasma LH. The second occurred about 24 hr later, when plasma LH was low. Study 3 was with ovariectomized ewes that were injected with estradiol. Plasma concentration of both FSH and LH tended to decline for the first 6 hr postinjection. The large peak in plasma LH, which occurred about 14 hr postinjection, was similar in both height and duration to that observed in estrous ewes. The small increase in plasma FSH, which coincided with the LH peak, was not significant. Study 4 was conducted with ovariectomized ewes given single or multiple injections of LH-RH/FSH-RH. Both FSH and LH increased significantly, but the response was considerably greater for LH. These results confirm that the anti-ovine FSH serum after immunoabsorption and radioiodinated ovine FSH after absorption chromatography on HTP were reliable reagents for the development of a specific, sensitive homologous ovine FSH radioimmunoassay.

¹ The author gratefully acknowledges L. Reichert, Jr., H. Papkoff and the National Pituitary Agency for providing purified pituitary hormones. Appreciation is also expressed to Dr. C. Kragt for supplying a partly specific ovine follicle stimulating hormone antiserum (J. Anim. Sci. 36:908) that was used in preliminary testing.

² Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.

³ Reproduction Laboratory, Animal Science Institute, Beltsville Agricultural Research Center, SEA.