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T V Novoselova, D Jackson, D C Campbell, A J L Clark and L F Chan

The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R–MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis. In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.

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C. G. Tsonis, D. T. Baird, B. K. Campbell, J. A. Downing and R. J. Scaramuzzi

ABSTRACT

The secretion rates of bioactive inhibin, oestradiol and progesterone were measured during the mid-luteal phase and at various times during the follicular phase of the cycle by a sensitive bioassay using sheep pituitary cells in culture in 12 Booroola ewes with and without copies of the Fecundity (F) gene in which the left ovary had been auto-transplanted to the neck.

Inhibin secretion was high during the luteal phase and fell in the early follicular phase in all genotypes (P < 0.01). In Booroola ewes with a F/- genotype, inhibin secretion then increased again, towards luteal rates, in the mid and late follicular phases. In Booroola ewes without a copy of the F gene (+/+) inhibin secretion remained low at all three sampling times in the follicular phase. The secretion rate of inhibin at 36 h (P < 0.1) and 48 h (P < 0.01) were significantly lower in ewes from the +/+ (no copy of the gene) ewes than in F/(one copy of the gene) ewes. Oestradiol secretion was low during the luteal phase and increased steadily during the early (24 h) to a plateau in the mid (36 h; P < 0.01) and late (48 h; P < 0.05) follicular phase. Progesterone secretion was high during the luteal phase, and decreased to a very low rate by 24 h after prostaglandin (PG) treatment (P < 0.001) and remained low. At 24 h after PG the concentration of FSH was significantly lower (P < 0.01) than that during the luteal phase and remained suppressed until the onset of the LH surge. There were no significant differences in LH concentrations. We conclude that (1) the secretion of inhibin by the ovary is highest in the luteal phase and (2) inhibin secretion is significantly raised during the mid to late follicular phase in Booroola ewes with a copy of the Fecundity gene compared with those without.

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R. K. Iles, N. C. Wathen, D. J. Campbell and T. Chard

ABSTRACT

Sixteen matched samples of first trimester amniotic fluid (AF), extraembryonic coelomic fluid (EECF) and maternal serum (MS) were assayed for intact human chorionic gonadotrophin (hCG) and free subunits. Total β-hCG (free β-subunit and intact hCG) levels in the EECF (median 410 kIU/l) were 61 times greater than levels in AF (median 6·73 kIU/l) and 2·8 times greater than in MS (median 141·5 kIU/l). Levels of intact hCG in the EECF (median 245 kIU/l) were 142 times greater than in AF (median 1·73 kIU/l) and 1·6 times greater than in MS (median 157 kIU/l). Free α-subunit levels in EECF (median 17·3 mg/l) were 66 times greater than in AF (median 0·262 mg/l) and 12 times greater than in MS (median 1·3 mg/l). Virtually all of the total β-hCG immunoreactivity in MS can be attributed to intact hCG, but only 60% of total β-hCG in the EECF and 20% of that in AF can be accounted for by the intact hormone. In both EECF and AF the free α-subunit was a major constituent; on a molar basis the ratio of free α:free β:intact hCG was 1:1·2:0·3 in AF, 1:0·6:0·5 in EECF and 1:0:5 in MS. Chromatography of MS, EECF and AF on Sephadex G-100 confirmed the hCG and subunit composition of the fluids. On the basis of these findings it seems likely that previous studies showing very high levels of hCG in AF during the first trimester may have incorrectly sampled the EECF. In reality, the levels of total hCG (and free subunits) are low in the AF, and only 20% is intact hCG. In both AF and EECF the free subunits may have been derived by dissociation of intact hormone, or possibly by independent synthesis. These and other findings suggest that either the amnion acts as a barrier to the transfer of proteins or that there may be dynamic removal from this compartment. By contrast, the EECF might act as a relatively stable reservoir for these proteins.

Journal of Endocrinology (1992) 135, 563–569

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C G Gutiérrez, B K Campbell, D G Armstrong and R Webb

Abstract

Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.

As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.

It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

Journal of Endocrinology (1997) 153, 231–240

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N.C. Wathen, S. Egembah, D.J. Campbell, A. Farkas and T. Chard

ABSTRACT

Concentrations of IGF-binding protein-1 (IGFBP-1) were measured by radioimmunassay in 176 amniotic fluid samples from 9 to 20 and 36 to 42 weeks of pregnancy. Low levels of IGFBP-1 were present at 9 and 10 weeks (median values 35.0 and 45.0 μg/l respectively). After 10 weeks, the levels increased by four orders of magnitude to reach a peak at 16 weeks (median 145.2 mg/l). After 16 weeks levels fell. Near term, the levels (median 27.1 mg/l) were lower than in the second trimester. The rapid increase in IGFBP-1 shown by radioimmunoassay was confirmed by Western ligand blotting. The findings suggest that the regulatory role of IGFBP-1 in the growth or differentiation of the fetus or of its surrounding membranes may change as pregnancy advances.

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T Chard, W F Blum, J Brunjes, D J Campbell and N C Wathen

Abstract

Insulin-like growth factor-II (IGF-II) and IGF-binding protein-2 (IGFBP-2) were measured in amniotic fluid, extraembryonic fluid and maternal serum from 20 women with apparently normal first trimester pregnancies prior to termination. A further 111 specimens of amniotic fluid were collected from women at 10–20 weeks of pregnancy. Levels of IGFBP-2 were similar in coelomic fluid and maternal serum. Levels in amniotic fluid were lower than those in serum and coelomic fluid (Mann–Whitney test; P=0·0002 and P<0·0001 respectively). The levels of IGF-II were much higher in maternal serum than in coelomic fluid, and higher in the latter than in amniotic fluid (Mann–Whitney test; P<0·0001 for both situations). The levels of IGFBP-2 were relatively low at 10–11 weeks (medians 19·8 and 61·1 μg/l) but thereafter increased to 20 weeks (median 1400 μg/l). The levels of IGF-II showed a similar pattern. The findings suggest that the role of IGF-II and IGFBP-2 in the regulation of growth or differentiation of the fetus or of its surrounding membranes may change with advancing pregnancy.

Journal of Endocrinology (1994) 142, 379–383