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L. S. Young, S. I. Naik and R. N. Clayton

ABSTRACT

Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7–10 h routinely resulted in a 50–100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40–60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 μmol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 μmol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH.

Vinblastine (1 μmol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release. These results indicate that receptor stimulation by GnRH, K+ and dbcAMP is a calcium-dependent process requiring the integrity of the microtubule system and there is a different calcium requirement for the processes of GnRH receptor up-regulation and LH secretion.

J. Endocr. (1985) 107, 49–56

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J. L. YOUNG, P. HARSOULIS, S. F. KUKU and T. R. FRASER

SUMMARY

In human urinary concentrates and also in a urinary gonadotrophin standard (2nd IRP-HMG), gel-filtration analysis revealed three main peaks of immunoassayable luteinizing hormone (LH). A similar analysis of LH extracted from human pituitaries showed most of the activity in a peak of larger molecular weight, and only minor fractions in the positions of the urinary peaks. In an extract of normal human serum, analysis showed only one similar peak of large molecular weight, which also emerged before the urinary peaks.

During an i.v. infusion of pituitary LH into normal men, the urinary LH activity increased but was still found only in the same three peaks on gel filtration, and all were of a molecular weight smaller than that of the infused material; but a higher proportion of the urinary LH was found in the earliest of these peaks compared with that found before infusion. Conversely, 20–35 h after the i.v. infusion, there was a slightly higher proportion of LH activity in the third peak of smallest molecular weight.

These findings suggest that the urinary immunoassayable LH, which is found in three peaks of different molecular weights, is derived from the pituitary or serum LH of higher molecular weight. The changes in the proportions of larger or smaller molecular weight fractions in the urine during and after LH infusion suggest that the earliest peak may be disaggregated serum LH, while the last or smallest molecular weight peak may comprise metabolites of LH.

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L A Salamonsen, R J Young, S Garcia and J K Findlay

Abstract

Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 ± 820 c.p.m./well) and stromal (6368 ± 1350 c.p.m./well) cells and was displaced by ET-1 (1 μmol l−1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l−1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 ± 13% of control (untreated=100%) with dose-dependence between the range of 1 to 100 nmol l−1. Stimulation by fetal calf serum was to 377 ± 126% of control. The effects on proliferation by other growth factors (dose; % of control ± s.e.m., number of positives/total number of cell preparations) were: IGF-I (13 nmol l−1; 182 ± 14, 4/4), epidermal growth factor (EGF; 4·8 nmol l−1; 132 ± 5%, 7/7), platelet-derived growth factor-BB (0·4 nmol l−1; 146 ± 3, 2/2) and leukaemia inhibitory factor (0·4 nmol l−1; 110 ± 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 ± 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation.

Journal of Endocrinology (1997) 152, 283–290

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S. F. KUKU, P. HARSOULIS, J. L. YOUNG and T. R. FRASER

SUMMARY

The urinary excretion of thyroid-stimulating hormone (TSH) has been measured by double antibody radioimmunoassay after concentration by dialysis followed by lyophilization. Among 30 normal subjects, the excretion was 5·6 ± 0·31 (s.e.m.) μu./h. No diurnal variation nor differences between sexes were discerned. In 14 primary hypothyroid subjects the urinary excretion was raised (P < 0·001) to 25·1 ± 3·3 μu./h. In 14 hyperthyroid and 7 hypopituitary subjects subnormal levels of 2·6 ± 0·2 and 2·5 ± 0·22 μu./h (P < 0·001) respectively, were found. Serum and urinary TSH concentrations were measured before, during and after an infusion of human pituitary TSH (MRC 70/9) in two subjects and showed a correlation.

Urinary TSH measurement is thus a good discriminant between normal and hyperthyroid or hypopituitary patients.

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S. F. KUKU, P. HARSOULIS, J. L. YOUNG, N. D. QUE and T. R. FRASER

SUMMARY

To assess whether urinary immunoassayable thyroid-stimulating hormone (TSH) differed from pituitary and serum TSH, urinary concentrates from two hypothyroid subjects were analysed by Sephadex G-100 gel filtration.

The elution profiles, measured by radioimmunoassay, were then compared with those of neat sera from hypothyroid patients and human pituitary TSH preparations. The pituitary preparations and the hypothyroid serum were eluted as a comparable single symmetrical peak corresponding to that obtained from a highly purified radio-iodinated human TSH of pituitary origin; no evidence of 'big' TSH emerged. In contrast, however, the material eluted from the hypothyroid urine concentrates not only revealed an asymmetrical peak corresponding to that described above but several other minor peaks eluting later and probably corresponding to fragments of TSH.

When human pituitary TSH was infused into two normal subjects, gel filtration analysis of concentrates from urinary samples obtained during and at fixed periods after the infusion revealed a single peak during the infusion but more peaks appeared with the later samples.

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Julia M Young, Jennifer L Juengel, Kenneth G Dodds, Mhairi Laird, Peter K Dearden, Alan S McNeilly, Kenneth P McNatty and Theresa Wilson

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-β 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.