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Serum and urinary gonadotrophin assays have been performed in three pregnant mares. The assays were conducted by the mouse uterus and mouse ovarian weight tests and the results expressed in terms of the international standard for Pregnant Mare's Serum Gonadotrophin as i.u. per litre.Gonadotrophic activity was detected in the urine as well as in the serum of all three animals but the values in urine were much lower than those in serum.Derivatives of dithiocarbamoylhydrazine, administered orally early in pregnancy, had no pronounced effect on the gonadotrophin levels in either serum or urine.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (National Institutes of Health—NIH have been incubated with varying concentrations of urea at different temperatures and for different periods of time.The luteinizing activity of NIH-LH was destroyed to a greater extent than the follicle-stimulating activity of NIH-FSH at all concentrations of urea employed.Higher temperatures tended to increase the degree of inactivation of both FSH and LH, while the time of incubation did not affect the results.It is suggested that at concentrations of urea below 6 m inactivation of gonadotrophic activity is produced by ammonium cyanate formed from urea during incubation.

Separation of the subunits of human pituitary luteinizing hormone (LH) has been accomplished by dissociation of the hormone with 8 m-urea followed by chromatography on CM-cellulose at pH 5·0 or by counter-current distribution. Each procedure resulted in isolation of one subunit, the other subunit being contaminated with reassociated LH. The isolated subunits (α and β) are each of low biological activity, but when recombined in a 1:1 ratio at pH 7, reassociation occurs with restoration of full activity. The amino acid compositions of the two subunits are significantly different from each other, but the composition of the α subunit is very similar to published data on the corresponding subunit of human chorionic gonadotrophin (HCG) and human thyroid-stimulating hormone. Significant differences were observed when comparing the compositions of the β subunits of the hormones. By immunoradiometric assay using antibodies to HCG, the α subunit was as active as native LH on a weight basis, but the β subunit was significantly lower in immunological activity.

The luteinizing hormone (LH), follicle-stimulating hormone (FSH) and 'total gonadotrophic' activities of various hormones have been studied following incubation with 6 murea at 40° c for 24 hr. LH activity was estimated by the ovarian ascorbic acid depletion test in rats, FSH activity by the augmentation test in mice, and 'total gonadotrophic' activity by the mouse uterus test.Following incubation with 6 murea the LH activities of NIH—LH, NIH—FSH, human chorionic gonadotrophin and Pergonal were almost completely destroyed, while the LH activity of pregnant mares' serum gonadotrophin (PMSG) was reduced to a smaller extent. The FSH activity of NIH—FSH was little affected by this form of treatment, but in the case of Pergonal a considerable reduction of FSH activity occurred. The 'total gonadotrophic' activity of NIH—FSH, PMSG and Pergonal was reduced after incubation with 6 murea, the degree of inactivation being greatest in the case of Pergonal.After control incubations with water no appreciable loss of biological activity was observed with any hormone other than Pergonal.

A modification of the ovarian ascorbic acid depletion (OAAD) method for luteinizing hormone (LH) is described in which the standard and test materials are administered by the intraperitoneal rather than by the intravenous route.The potency of NIH-LH when administered i.p. and i.v. was the same and the slopes of the log dose-response curves for the two routes of administration were very similar.Studies on the effect of seasonal factors on the OAAD method showed that both the slopes of the log dose-response curves and the absolute levels of ascorbic acid varied from one assay to another. The necessity for using a standard preparation in all assays of LH activity by the OAAD method is emphasized.

A new assay method for luteinizing hormone (LH) activity is described and the criteria for its reliability are assessed. The technique depends on ovarian cholesterol depletion in intact immature rats pretreated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG).The main advantage of the ovarian cholesterol depletion (OCD) test is its high sensitivity, the assay being approximately five million times more sensitive than the ovarian ascorbic acid depletion (OAAD) method. The chief disadvantage of the technique is its relatively low degree of precision.The LH activity of various gonadotrophin preparations has been determined by the new OCD test. The results obtained agree well with those found by the OAAD method.The extreme sensitivity of the OCD test suggests that it will be eminently suitable for clinical use.

A double antibody radioimmunoassay for canine luteinizing hormone (LH) was devised. The assay can measure immunoreactive LH in the peripheral plasma of the normal male and female dog. An increase in hormone concentration was found on the 2nd day of oestrus in the female. Ovariectomy raised plasma concentrations of the hormone to levels greater than those observed during oestrus. The administration of LH-releasing factor induced a rapid increase in the plasma concentrations of LH.

Human chorionic gonadotrophin (HCG), pregnant mare serum gonadotrophin (PMSG) and human menopausal gonadotrophin (HMG) were incubated with varying concentrations of urea at different temperatures for different times.The luteinizing hormone (LH) activity of HCG was progressively destroyed with increasing concentrations of urea. The degree of inactivation was greater at higher temperatures but the time of incubation did not affect the results.The follicle-stimulating activity of PMSG was reduced at high urea concentrations; the time of incubation was without effect.Under the experimental conditions used the LH activity of HMG was generally destroyed to a greater extent than its follicle-stimulating hormone activity.

Estimations of human pituitary gonadotrophins (HPG), total 17-hydroxycorticosteroids, total 17-oxosteroids and oestrogens have been performed in seven hospitalized postmenopausal subjects receiving treatment with the dithiocarbamoylhydrazine derivatives Compound 22365 and Compound 33828 (Imperial Chemical Industries Ltd.).Both compounds were shown to be inhibitors of pituitary gonadotrophic function as indicated by urinary HPG assays; Compound 33828 was more active in this respect than Compound 22365.When Compound 22365 was administered to individual subjects at two different dose levels the time taken for urinary HPG levels to return to pretreatment values was longer in the case of the higher dose.Neither of the compounds studied produced any effect on adrenocortical function as judged by urinary assays of 17-hydroxycorticosteroids, 17-oxosteroids and oestrogens.Side effects were noted in two of the seven patients studied. In one subject with pre-existing liver damage it was necessary to stop treatment with Compound 22365 because of the development of mild jaundice.

The effect of the administration of 25 i.u. human chorionic gonadotrophin (HCG) on the induction of ovulation in intact immature rats treated with 50 i.u. pregnant mare serum gonadotrophin (PMSG) has been studied.After the administration of HCG a marked increase in ovarian wet weight was observed. The maximum increase, which occurred 10 hr. after hormone treatment, was noted 2 hr. before ova were found in the oviducts.The alteration in ovarian wet weight was associated with a fall in percentage solids. However, it appears likely that an increase both in follicular fluid and in cell mass occurred before ovulation. Possible reasons for the absence of any marked effect on uterine wet weight or percentage solids are discussed.

The effect of the administration of 50 i.u. pregnant mare serum gonadotrophin (PMSG), followed 72 hr. later by 25 i.u. human chorionic gonadotrophin (HCG), on ovarian weight, ascorbic acid and cholesterol has been investigated in rats of the Wistar strain bred in a closed colony.In the ovarian ascorbic acid depletion (OAAD) test for luteinizing hormone (LH) the bioassay is generally carried out from 5 to 9 days after the administration of HCG. The present investigation has shown that at this time ovarian ascorbic acid levels in untreated animals are in the same range as in rats pretreated with PMSG and HCG.The reasons for conducting the ovarian cholesterol depletion (OCD) test for LH on the 11th day after treatment with HCG are discussed. It is concluded that in the strain of animal used assays conducted before this time are likely to be unsatisfactory.It is postulated that the main function of the pretreatment procedure in both assay methods is to produce a large amount of highly reactive ovarian tissue which is readily responsive to the administration of LH.

Several gonadotrophin preparations from different sources have been investigated for biological activity using a number of assay methods including highly specific tests for both follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In the great majority of assays the results obtained were expressed in terms of the International Reference Preparation (I.R.P.).The FSH activity of NIH-FSH was seventy times higher and its LH activity thirty times higher than that of the I.R.P. The specific activity of NIH-LH was high, the material being approx. 2000 times more potent than the I.R.P. Material prepared from human pituitary tissue contained the highest FSH activity of any preparation tested. Its LH activity was considerably lower than that of NIH-LH.Pergonal had ten times more FSH and five times more LH activity than the I.R.P. Pregnant mares' serum gonadotrophin contained, in addition to its FSH activity, a considerable amount of LH activity, the ratio FSH/LH being 2.10. The international standard for human chorionic gonadotrophin was shown to be a relatively weak LH preparation, being only slightly more active than the I.R.P.Results obtained by the mouse uterus test did not appear to bear any constant relationship to results obtained by specific methods for either FSH or LH.

The effect of clomiphene on hormone excretion in four normal males has been investigated.In all subjects there was a marked rise in the urinary excretion of oestrone and oestradiol both during and after administration of the drug. The rise in oestrone excretion preceded and was more marked than the increase in oestriol output.All subjects showed an increase in total 17-oxosteroid excretion during and after clomiphene administration. This was associated with a rise in the output of dehydroepiandrosterone in 2 of the 4 men studied.The effect of clomiphene on steroid output occurred without any consistent change in the pattern of urinary gonadotrophin excretion.