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SUMMARY
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.
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In 1964 Bell, Mukerji & Loraine described a new bioassay method for the quantitative determination of luteinizing hormone (LH) depending on the depletion of ovarian cholesterol after i.p. administration of the hormone to intact immature rats pretreated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. The pretreatment used in this method was identical with that employed by Schmidt-Elmendorff & Loraine (1962) in the ovarian ascorbic acid depletion (OAAD) test of Parlow (1958). In view of the marked difference in sensitivity between these two bioassay procedures it seemed desirable to ascertain whether the dose-response curve of the OAAD method is affected by the very small doses of LH similar to those used in the ovarian cholesterol depletion (OCD) test.
Three OAAD assays on 135 rats obtained from the University of Edinburgh Small Animal Breeding Station were conducted. The procedure used was similar to that of Schmidt-Elmendorff & Loraine (1962) except
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Bell, Mukerji & Loraine (1964) have described a bioassay method for the quantitative determination of luteinizing hormone (LH) depending on ovarian cholesterol depletion (OCD test) after i.p. administration of the hormone. The assay is conducted in intact, immature rats pretreated with 50 i.u. pregnant mare serum gonadotrophin (PMSG) followed 72 hr. later by 25 i.u. human chorionic gonadotrophin (HCG). As described by Bell et al. (1964) the bioassay proper is performed on the 11th day after the administration of HCG at which time the ovarian cholesterol level generally varies from 1000 to 2000μg./100 mg. ovary (Bell & Mukerji, unpublished observations). The main practical difficulty associated with the method is the lack of uniformity of the ovaries in any given assay both in relation to their appearance and their cholesterol content. This difficulty was further emphasized by Mukerji, Bell & Loraine (1965) who showed that the concentration of cholesterol varied considerably
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Peripheral plasma samples were obtained during pro-oestrus and oestrus from two adult Beagle bitches and from one animal (bitch 124) during its first oestrous cycle. The animals were housed in a constant environment kennel and each showed a normal oestrous cycle as judged by clinical appearance, vaginal cytology and behavioural tests; in addition, a rise in plasma progesterone was noted during late oestrus and early metoestrus (Christie, Bell, Horth & Palmer 1971). Plasma oestrogens were measured by the radioimmunoassay technique of Abraham (1969), with modifications. No separation of oestradiol and oestrone was undertaken and the results are thus an estimate of total immunoreactive oestrogens. The water blank for the method was 16 ± 6·5 (s.d.) pg/ml while the solvent blank was 19 ± 4·7 pg/ml. The results were corrected for the solvent blank. The recovery of oestradiol added to water was 102 ± 12 (s.d.)%, and to plasma from a bilaterally ovariectomized, hemi-adrenalectomized
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SUMMARY
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.
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SUMMARY
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.
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The luteinizing hormone (LH), follicle-stimulating hormone (FSH) and 'total gonadotrophic' activities of various hormones have been studied following incubation with 6 m urea 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 m urea 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 m urea, 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.
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SUMMARY
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.
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SUMMARY
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.
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A fractionation procedure used for the purification of glycoprotein hormones from pituitary glands of the human being (Stockell Hartree, 1966), horse (Stockell Hartree, Mills, Welch & Thomas, 1968) and chicken (Stockell Hartree & Cunningham, 1969) was also applied to acetone-dried dog pituitaries. The pituitaries were removed from dogs generally within 60 h of death. The glands were washed in acetone and stored in 20 volumes of acetone at 4 °C for several weeks. An acetone powder was prepared by drying the pituitaries in air and grinding them in a glass mortar. The yield of powder from 980 pituitaries was 10·3 g. This material was extracted with 6% ammonium acetate at pH 5·1 in 40% ethanol and the soluble extract chromatographed on a column of CM-cellulose equilibrated with 4 mm-ammonium acetate buffer at pH 5·5 as described previously (Stockell Hartree, 1966; Stockell Hartree et al. 1968; Stockell Hartree &
