A New Sensitive Bio-ass ay for Follicle-Stimulating Hormone (FSH) M. IGARASHP AND S. M. McCANN Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia,

response when administered in doses up to 200 ng. Contamination of FSH with over 10 ng NIH-LH sometimes reduced the uterine response caused by FSH; however, contamination with less than 6 /xg NIH-LH did not interfere with the response. Other anterior and posterior hypophysial hormones did not give any significant response when administered with HCG. The minimal effective dose of NIH-FSH-S-1 ovine standard and International Reference Preparation for Human Menopausal Gonadotrophin was 2 and 10 jug1, respectively. (Endocrinology 74: 440, 1964)

ABSTRACT. A specific and very sensitive assay method for FSH using intact immature female mice was devised. This method is based on the synergism between FSH and a small amount of human chorionic gonadotrophin (HCG). The test material mixed together with 0.25 IU or 0.1 IU HCG is injected subcutaneously daily for 3 days into intact immature female mice. On the fourth day, necropsy is performed and uterine weight is measured after evacuation of any fluid. Using NIH-FSH and IRP-HMG, linear log-dose response curves were obtained. By contrast, the ovine NIH-LH standard evoked no significant

S

EVERAL methods for the assay of follicle-stimulating hormone (FSH) have been devised and used; however, most of them are complicated and relatively insensitive. Steelman-Pohley's (1) and P. S. Brown's (2) methods are being applied widely since they are easy to carry out and are specific for FSH, but these two methods suffer from a lack of sensitivity. Among the various bio-assay methods for gonadotrophins, the mouse uterine weight method has been recognized as the most sensitive assay by several workers (3-7), but it is not only nonspecific for FSH, but also still too insensitive to assay small amounts of gonadotrophin. In order to obtain a Received August 28,1963. This work was supported by NIH Grant No. A-1236, C-6, and by a grant from the Population Council for Dr. Igarashi's laboratory expenses. 1 Fellow of the Population Council, on leave from the Department of Obstetrics and Gynecology, Gunma University Medical School, Maebashi, Japan.

Pennsylvania

more sensitive FSH assay, a new modification of the mouse uterine weight method which takes advantage of the synergism between FSH and HCG has been devised. The initial experiments were performed at Gunma University in Japan, and the work was completed at the University of Pennsylvania. The results of the basic experiments on this improved assay method are reported here. Materials and Methods Immature female mice of the Swiss albino strain, weighing 7.5-10 g, were used in all experiments. Groups of 7-10 animals were injected daily subcutaneously (sc) for 3 days with gonadotrophins such as FSH,2 luteinizing hormone (LH),3 International Reference Preparation for Human Menopausal Gonadotrophin (IRP-HMG),4 and HCG,6 either 2 3 4

NIH-FSH-S-1 ovine standard. NIH-LH-S-3 ovine standard. Kindly provided by National Institute for Medical Research, London. 6 Kindly provided by Dr. John B. Jewell of Ay erst Laboratories.

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March 1964

BIO-ASSAY OF FSH

alone or in various combinations which are described in the results. In all cases the mice received 0.7 ml of the solution to be injected on each of the first 2 days and 0.6 ml on the third day. Care was exerted to avoid intraperitoneal injection, which often resulted in death of the mouse or a reduction in uterine response, and to avoid leakage from the injection site, which reduced precision. The mice were sacrificed on the fourth day; the uteri were cut at the oviductal and vaginal junctions, freed of fat, squeezed between about 4 layers of filter paper to remove fluid and weighed immediately. Body weight of all the mice was measured each day just prior to injection and at sacrifice. Any mice showing a reduction of body weight were discarded.

50

40 O)

30

Results

E

Synergism between FSH and HCG—the sensitivity of the method. FSH, alone, had

D

no effect on uterine weight until a minimal effective dose (MED) of 50 jug was reached (p