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Search for other papers by S. L. JEFFCOATE in
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SUMMARY
Rat stalk median eminence (SME) extract was incubated with various quantities of an antiserum raised against synthetic luteinizing hormone releasing hormone (LH-RH) and the resulting material was used at two or more dose levels to stimulate release of LH and FSH from anterior pituitary tissue, obtained from male rats, incubated in vitro. The resulting dose–response lines were used to obtain the LH- and FSH-releasing potency of the material, expressed as a percentage of SME extract pre-incubated with normal rabbit serum. Treatment with various doses of antiserum reduced LH-releasing and FSH-releasing potencies to a similar extent.
Stalk median eminence extract and synthetic LH-RH were incubated with antiserum and directly compared in the same experiment in vitro. The changes in LH and FSH release caused by pre-treatment with antibody were similar for SME extract and synthetic LH-RH.
In a final experiment, SME extract was passed through a column of Sepharose 2B particles to which was coupled anti-LH-RH antibody. The resulting material, when mixed with synthetic LH-RH and used to stimulate rat pituitary tissue in vitro caused a similar increase in the LH- and FSH-releasing potencies of the synthetic decapeptide.
It is concluded that rat SME extract contains a single releasing factor for LH and FSH immunologically indistinguishable from synthetic LH-RH.
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SUMMARY
Hypothalamic extracts from three mammalian species (rat, rabbit and sheep) were found to contain several ng of immunoreactive thyrotrophin releasing hormone (TRH)-like activity. This substance chromatographed on ion exchange chromatography (carboxymethyl cellulose) as a single peak that was indistinguishable from synthetic TRH. Hypothalamic TRH was also inactivated by normal human plasma at a rate (1·21–1·46%/μl plasma/h and 1·59–1·77%/50 μl plasma/min) similar to that of synthetic TRH (1·42%/μl plasma/h and 1·73%/50 μl plasma/min). This combination of chromatographic and enzymic techniques can be applied to the identification of immunoreactive TRH in body fluids.
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SUMMARY
Serum samples from rabbits, sheep and rats containing immunoreactive luteinizing hormone releasing hormone (LH-RH) have been extracted and fractionated by ion exchange chromatography on carboxymethylcellulose followed by radioimmunoassay of the fractions. Control experiments showed that the extraction and chromatographic procedures did not alter the mobility of synthetic LH-RH. Four immunoreactive components of circulating LH-RH in blood samples from various species at various times were identified on CM-cellulose columns. One of these had a mobility identical with that of synthetic LH-RH; of the others, two were eluted before and one after synthetic LH-RH. The nature, site of formation and possible significance of the extra components are discussed.
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ABSTRACT
Three ampouled preparations of purified human prolactin were assessed by 20 laboratories in eight countries for their suitability to serve as International Standards for the estimation of human prolactin in serum. Bioassays (pigeon crop sac assays and NB2 cell assays) were carried out in two laboratories, radioreceptor assays by one laboratory and radioimmunoassays by 17 laboratories.
By physicochemical analysis the preparations appeared similar. Each preparation contained small amounts of contaminants and/or prolactin variants. No major differences among the three preparations were detected by immunoassay although, in one radioreceptor assay system, one of the preparations was found to differ from the other two.
On the basis of all the available information, the Expert Committee on Biological Standardization of the World Health Organization (ECBS) in 1986 established the preparation in ampoules coded 83/562 as the Second International Standard for Prolactin and in October 1988 established the preparation in ampoules coded 84/500 as the Third International Standard for Prolactin. A value of 0·053 IU (53 mIU) prolactin activity/ampoule was assigned to both the Second and Third IS on the basis that this unitage would, insofar as possible, maintain continuity of the IU defined by the First International Reference Preparation of Prolactin, human, for Immunoassay (coded 75/504).
Journal of Endocrinology (1989) 121, 157–166
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ABSTRACT
Oestrogen-2/4-hydroxylase activity was measured in whole brain, thalamus, amygdala, hypothalamus and pituitary gland of lactating rats and in whole brain of rats on different days of the oestrous cycle. Enzyme activity was increased in whole brain and in each of the brain regions examined (with the exception of the amygdala) in lactating rats. This increase in enzyme activity was associated with an increase in serum prolactin levels. During the oestrous cycle, enzyme activity in whole brain was higher on metoestrus and dioestrus than on pro-oestrus and oestrus. The decrease in enzyme on pro-oestrus was associated with an increase in both serum oestradiol and prolactin levels. These results are consistent with the hypothesis that changes in oestrogen-2/4-hydroxylase activity are associated with changes in prolactin and oestradiol secretion and may play a regulatory role in reproduction.
J. Endocr. (1985) 107, 191–196
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The ability to assay small amounts of the peptide releasing hormones in biological fluids would aid greatly in the assessment of hypothalamic function. We have recently described a specific radioimmunoassay for luteinizing hormone releasing hormone (LH-RH) (Jeffcoate, Fraser, Gunn & Holland, 1973a, b) and in this study we report a radioimmunoassay for the tripeptide, thyrotrophin releasing hormone (TRH).
Thyrotrophin releasing hormone (2 mg) was conjugated to 10 mg bovine serum albumin in borate buffer pH 9·0 by the bis-diazotized benzidene method (Bassiri & Utiger, 1972 a). After 2 h at 5 °C the mixture was dialysed against distilled water for 48 h and against 0·15 m-NaCl for 24 h. This technique conjugates TRH by the imidazole ring of histidine to the protein. A sample of conjugate (2·5 mg) in saline was homogenized with Freund's complete adjuvant and injected into 20 intradermal sites in a White New Zealand rabbit.
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SUMMARY
The clearance of synthetic luteinizing hormone releasing hormone (LH-RH) after intravenous injection has been investigated in man. Thirteen tests were performed in ten subjects, four of whom were normal, the other six had proven disease of the hypothalamo-pituitary region. The rate of disappearance of LH-RH from the circulation could be represented as a double exponential with half-times of the two components of 5·3 and 27·4 min respectively. The initial volume of distribution was 11·1 ± 1·6 (s.d.) 1 and the metabolic clearance rate 1480 ± 170 (s.d.) 1/day. There was no difference in any of these parameters between normal and abnormal subjects. Between 0·75 and 2·8% of the injected dose was excreted in the urine within 8 h, of which 48% was excreted in the first hour. The daily production rates of LH-RH were calculated from the urinary excretion rates; these gave higher results than production rates calculated from the blood metabolic clearance rate. Possible reasons for this are discussed.
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ABSTRACT
Catecholoestrogens are naturally occurring metabolites of oestrogens which are found in brain tissue and for which a neuroendocrine role has been postulated. However, reports of their effects on prolactin secretion are ambiguous and as yet no defined function has been attributed to them.
The effects of 2-hydroxyoestradiol (2-OHE2) and dopamine on the release of prolactin in vitro by perfused pituitary glands from normal adult female rats at different stages of the oestrous cycle have been investigated. The purity and stability of the 2-OHE2 preparation before and after exposure to pituitary tissue was confirmed by radioenzymatic assay and subsequent thin-layer chromatography. Dopamine (500 nmol/l, 100 nmol/l) was found consistently to suppress release by 60%; this effect was immediate and reversible upon removal of the dopamine. In contrast, the effects of 2-OHE2 (10 nmol/l, 100 nmol/l) were found to vary during the cycle. No effect on prolactin release was evident during either dioestrus or pro-oestrus, but during oestrus a similar, though less potent, suppression of prolactin secretion to that of dopamine was observed (35% suppression compared with controls).
The cyclical variation in the suppressive effect of 2-OHE2 on prolactin secretion in the female rat is compatible with a postulated neuroendocrine role for this catecholoestrogen.
J. Endocr. (1986) 111, 199–204
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Search for other papers by S. L. JEFFCOATE in
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The occurrence of a peak of luteinizing hormone (LH) in the peripheral blood of the sheep within the first 16 h of oestrus is well established (Geschwind & Dewey, 1968; Niswender, Roche, Foster & Midgley, 1968; Goding, Catt, Brown, Kaltenbach, Cumming & Mole, 1969). Changes in the LH releasing hormone (LH-RH) content of the hypothalamus have been correlated with the occurrence of the plasma LH peak and the accompanying decline in pituitary LH content (Crighton, Hartley & Lamming, 1973). Using a radioimmunoassay for LH-RH, Kerdelhué & Jutisz (1972) detected increases in plasma LH-RH content in one ewe 2 days before the LH peak and again from 1 h before the start of the LH peak to 8 h after its end. The present report describes the simultaneous determination of LH and LH-RH in samples taken at frequent intervals from onset of oestrus in the sheep.
Serial blood samples (2·5 ml)
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The purpose of this study was to determine if luteinizing hormone releasing hormone (LH-RH) could be detected in the peripheral blood of the rat on the afternoon of pro-oestrus by the radioimmunoassay described elsewhere (Jeffcoate, Fraser, Gunn & Holland, 1973a; Jeffcoate, Fraser, Holland & Gunn, 1973b).
Female Sprague—Dawley rats, weighing 220–270 g, were maintained in a room illuminated from 05.00 to 19.00 h. The animals were observed for at least two consecutive 4-day oestrous cycles as judged by daily vaginal smears. At various times on the afternoon of pro-oestrus the animals were lightly anaesthetized with ether and rapidly desanguinated by cardiac puncture. Plasma luteinizing hormone (LH) was determined using the radioimmunoassay described by Daane & Parlow (1971) with minor modifications. This utilized purified rat LH for radio-iodination (NIAMD-Rat LH-1–3), NIAMD-Anti-Rat LH serum-1 and a rat LH preparation (NIAMD-Rat LH-RP-1) was used as standard. Serum (1–2 ml) samples were
