SUMMARY At 75 days of age, female rats neonatally sterilized with oestradiol benzoate or testosterone propionate were compared with normal and ovariectomized rats with regard to their 6-hr. response to 0·2 μg. oestradiol 17β. The greatest increases in uterine weight, glucose and glycogen concentrations and per cent uterine water occurred in the ovariectomized animals. A marked oestrogen response also occurred in the animals neonatally sterilized with oestradiol benzoate. The response of the normal rats was slight, and the testosterone propionate-treated rats were the least affected. Adrenal, pituitary, and ovarian weights were found to be affected by the neonatal hormone treatments. Vaginal patency was completely inhibited in the rats injected with testosterone propionate. It is concluded that rats neonatally sterilized with steroids are much less suitable than ovariectomized animals for oestrogen assays.

A new micro-bio-assay method involving the increase of vaginal glycogen in response to local administration has been used to determine the oestrogenic content of urine and blood. In addition, various other steroid hormones (progesterone, cortisol, desoxycorticosterone, testosterone) were given to test the specificity of the assay. In no instance did the other hormones elicit a glycogen response when given alone, nor did they enhance the response when given in combination with oestrogen.In a previous report from this laboratory (Wrenn, Bitman & Wood, 1968), the rapid increase in vaginal glycogen caused by local administration of oestrogens to adolescent, ovariectomized rats has been characterized, and suggested as being suitable for the determination of the oestrogenic content of biological materials. The method was shown to be sensitive to small doses, with as little as 2-5 10"^g oestradiol being detected. This paper deals with the assay of biological fluids and the specificity of the reaction.The assay was conducted by instilling 0-01 ml of solution into the vaginae of 49 to 51-day-old adolescent rats which had been ovariectomized 10 to 12 days previously. The rats were killed 5 hr after the injection and the vaginae excised as described earlier (Wrenn et al., 1968). Glycogen determinations were made by the anthrone method of Seifter, Dayton, Novic & Muntwyler (1950).We have applied the assay to the determination of the oestrogenic content of a limited number of urine and blood samples. Table 1 shows the variety of materials assayed and the estimate of their potency. No activity was detected in human male and post-menopausal urines when they were instilled into the vagina in the undiluted condition. Samples of human pregnancy urine near term had the equivalent of about 2 µg oestradiol-17ß/ml of urine. It was necessary to dilute this urine 400 times with distilled water in order to get it within the limits of potency detectable by the assay. The oestrogenic potency of untreated and unconcentrated pregnant cow urine was low-only about 6°/0 of the usual urinary excretion values reported by Velie (1958). 301

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