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1. A new method is described for labelling proteins to high specific radioactivities with (125)I. The protein is treated with a (125)I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the (125)I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55muCi/mug respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of (125)I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single (125)I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.

The concentrations of FSH, LH, prolactin, oestradiol and progesterone were measured in peripheral plasma and follicular fluid of women throughout the menstrual cycle. With the exception of prolactin, concentrations of pituitary and steroid hormones in follicular fluid correlated with those in peripheral plasma. Follicle-stimulating hormone was present in a greater number of small follicles (smaller than 8mm) during or just after the peaks of FSH in peripheral plasma. During the mid-follicular phase the concentration of both FSH and oestradiol in fluid from large follicles (larger than or equal to 8 mm) was high. During the late follicular phase the large follicles (larger than or equal to 8 mm) contained high amounts of progesterone in addition to oestradiol, low physiological levels of prolactin, and concentrations of LH and FSH about 30 and 60% respectively of those found in plasma. By contrast no large 'active' follicles (larger than or equal to 8 mm) were found during the luteal phase although many contained both LH and FSH. Luteinizing hormone was present in a proportion of small follicles (smaller than 8 mm) during the late follicular and early luteal but not at other stages of the menstrual cycle. It is suggested that a precise sequence of hormonal changes occur within the microenvironment of the developing Graafian follicle; the order in which they occur may be of considerable importance for the growth of that follicle and secretory activity of the granulosa cells both before and after ovulation.

The onset of production of spermatozoa (spermarche) is the basis for achievement of reproductive capacity in men. We collected 24-h urine samples every 3 months in a 7-yr longitudinal study of 40 normal boys initially aged 8.6-11.7 yr. After centrifugation, the urine was analyzed for the presence of spermatozoa by microscopic examination, and spermarche was estimated on the basis of age at first observed spermaturia. The results were corrected for the intermittent occurrence of spermatozoa in the urine after first observed spermaturia and the fact that the urine samples were collected quarterly. In addition, physical examination, including determination of testicular size by orchidometer measurement, pubic hair distribution (Tanner stage), and height, was carried out every 6 months. Spermarche occurred at a median age of 13.4 yr (range, 11.7-15.3 yr), at a time when testicular size was 4.7-19.6 ml (median, 11.5 ml), and pubic hair distribution was 1-5 (median, 2.5). In most boys, spermarche preceded the age of peak height velocity (median, 13.8 yr; range, 12.2-15.2 yr); at the time of spermarche, median peak height growth velocity was 9.9 cm/yr (range, 7.5-13.4 cm/yr), and median height was 160.4 cm (range, 151.7-175.9 cm). We conclude that spermarche is an early pubertal event and that a wide variation in testicular size and secondary sex characteristics is found at that time. In particular, spermarche may occur when little or no pubic hair has developed, and the testes have grown only slightly.

A number of attempts have been made to measure the amount of growth hormone in human plasma or in extracts thereof. By using the tibia-line biological assay Gemzell (1959) and Forsman & Gemzell (1959) detected growth hormone in extracts from subjects with acromegaly or diabetes but not in extracts of normal plasma. Owing to the lack of sensitivity and specificity of biological assays for growth hormone no further attempts with these methods have been reported. The sulphation factor in serum has been used as an indication of human growth hormone (Daughaday, Salmon & Alexander, 1959; ALnquist & Falkheden, 1961), but despite its usefulness the values obtained are not direct measurements of the hormone. An immunological approach to the problem was provided by the work of Hayashida & Li (1958) and of Read & Stone (1958), who produced antiserum to purified human growth hormone in the rabbit. The reaction between growth hormone and its antibody has formed the basis of a number of assays for the hormone. Read & Stone (1958) and Read & Bryan (1960) have used the haemagglutination-inhibition technique for this purpose. Subsequent experience (cf. Read, Eash & Najjar, 1962; Grumbach & Kaplan, 1962) showed that it was subject to gross interference by plasma. However, Ehrlich & Randle (1962) obtained reproducible results with the method and were able to use it for comparative purposes in studying diabetes mellitus. The removal of non-specific inhibition by an extraction procedure has been reported by Dominguez & Pearson (1962), and the concentrations of growth hormone in normal adults were found to be less than 20,umg./ml. of serum. Methods based on the quantitative precipitin reaction (

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