ABSTRACT

Dairy cows with a variety of clinical conditions were investigated in an attempt to identify the cause(s) of subfertility. Sequential or simultaneous injections of 20 μg gonadotrophin-releasing hormone (GnRH), 1 mg oestradiol benzoate and 0·06 mg ACTH(1–24) into five clinical cases of ovarian follicular cysts, two cases of poor body condition and one case of lameness and into control cows revealed a failure in the LH positive-feedback response to oestradiol in all eight clinical cases, but in only two out of twelve control cows. Two of the clinical cases and the two non-responding control cows had high or rising initial progesterone concentrations which would explain the absence of response. All cows studied responded similarly to GnRH and ACTH(1–24).

It is suggested that hypothalamus-pituitary control of LH release may involve a rate-limiting step (in the oestradiol positive-feedback system) and that lesions at this point result in subfertility in a variety of clinical situations.

J. Endocr. (1987) 113, 167–171

ABSTRACT

Stress is known to result in lowered female reproductive efficiency. The objective of this study was to examine how increased pituitary-adrenal activity may influence gonadotrophin release in anoestrous ewes.

Various doses (0·06–1·0 mg) of a synthetic adrenocorticotrophic hormone (ACTH(1–24)) preparation were injected into ewes 30 min or 3 h before an i.v. injection of 500 ng gonadotrophin-releasing hormone (GnRH). The LH response to GnRH given 30 min after ACTH(1–24) was similar to that after GnRH alone, whereas the response 3 h after ACTH(1–24) was significantly lower, irrespective of the dose of ACTH(1–24). At 30 min and 3 h after ACTH(1–24) the concentrations of cortisol exceeded 50 nmol/l compared with baseline values of < 10 nmol/l.

The effect of ACTH(1–24) on oestradiol-induced LH release was also examined. Those ewes receiving 0·8 mg ACTH(1–24) depot and 50 μg oestradiol benzoate simultaneously had a preovulatory-type increase in LH 14–20 h later, similar to when oestradiol benzoate was given alone. None of the ewes receiving an additional 0·8 mg ACTH(1–24) depot 10 h after oestradiol benzoate had increases in LH concentration. The cortisol concentrations in all ewes receiving either one or two injections of ACTH(1–24) were > 35 nmol/l at 10 h after the oestradiol injection. However, concentrations of progesterone increased from 0·9 ± 0·3 (s.e.m.) nmol/l at the time of the second ACTH(1–24) injection to 2·1 ±0·3 nmol/l after 2 h.

In summary, it would appear that the suppressive effect of ACTH(1–24) on LH secretion induced by GnRH or oestradiol in the anoestrous ewe is not dependent on increased plasma concentrations of cortisol.

J. Endocr. (1988) 118, 193–197

SUMMARY

Plasma levels of oestradiol-17β, progesterone and luteinizing hormone (LH) and pituitary levels of LH have been measured during the first 6 days of pregnancy, in normal rats and in rats receiving two doses of Tamoxifen (trans-1-(p-β dimethylamino-ethoxyphenyl)-1-2 diphenylbut-1-ene) on day 2 of pregnancy.

In normal rats oestradiol rose strongly from early on day 3 to reach a peak concentration between 22.00 h on day 3 and 08.00 h on day 4. Progesterone concentrations rose from day 2 to reach peak values on day 3–4. In animals in which implantation was delayed 20–24 h by administration of Tamoxifen (0·1 mg/kg) orally on day 2 the increased level of plasma oestrogen was also delayed by 20 h. A higher dose of Tamoxifen (0·2 mg/kg) on day 2, which prevented implantation, completely eliminated the increase in plasma oestradiol. Neither dose of Tamoxifen affected the levels of progesterone.

In both normal rats and rats treated with 0·1 mg Tamoxifen/kg, plasma LH levels declined by day 3 while pituitary levels rose steadily. There was no detectable change in either plasma or pituitary LH levels, accompanying the increase in plasma oestradiol in the normal rats. In animals receiving Tamoxifen (0·2 mg/kg), plasma LH increased to a maximum by day 4 while levels of pituitary LH decreased.

The results show that the oestrogen ' surge' of early pregnancy, occurs normally about midnight on day 3 and not late on day 4 as previously thought. It is considered that the plasma oestradiol peak in early pregnancy results from an increased release of FSH rather than an increased release of LH. Tamoxifen may owe part of its antifertility action to a capacity to inhibit the synthesis of oestradiol from progesterone.

ABSTRACT

It is now established that calcium is a second messenger mediating the action of calcitonin on the osteoclast. We have demonstrated that an increase in the concentration of intracellular free calcium ([Ca²⁺]_i) is associated with (and possibly mediates) the functional effects of calcitonin, including an acute reduction of cell spread area (the R effect) and, in the longer term, a reduction in enzyme release. The present study addresses questions relating to mechanisms of calcitonin action on osteoclast [Ca²⁺]_i. We have used asusuberic(1–7) eel and human calcitonin as agonists, and an indo-1-based dual-emission microspectrofluorimetric method for the measurement of [Ca²⁺]_i in single osteoclasts. Whilst asusuberic(1–7) eel calcitonin caused a biphasic increase in [Ca²⁺]_i, human calcitonin produced only a monophasic [Ca²⁺]_i response of a much lower magnitude. Each biphasic response consisted of a rapid initial transient increase, occurring within seconds of exposure, followed by a sustained increase in [Ca²⁺]_i. The magnitude of the latter response was more variable, but was consistently below the peak value of [Ca²⁺]_i. The sustained phase of the calcitonin effect was abolished in extracellular Ca²⁺-free medium. This phase is therefore dependent on extracellular [Ca²⁺] ([Ca²⁺]_e) whilst the rapid transient increase appeared to be dependent on Ca²⁺ _i redistribution. The effects of calcitonin on [Ca²⁺]_i were concentration-dependent, with neither latency nor oscillations. Repetitive 30-s exposures to calcitonin failed to produce subsequent responses. There was a marked concentration-dependent correlation between changes in osteoclast [Ca²⁺]_i and the magnitude of the R effect. Thus the likely components of the biphasic [Ca²⁺]_i response are a rapid redistribution followed by the transmembrane flux of Ca²⁺. We suggest that the increase in [Ca²⁺]_i may mediate, in part, the inhibitory effect of calcitonin.

Journal of Endocrinology (1992) 132, 241–249

ABSTRACT

Calcitonin is a circulating polypeptide that inhibits bone resorption by inducing both quiescence (Q effect) and retraction (R effect) in osteoclasts. Two structurally related members of the calcitonin gene peptide family, calcitonin gene-related peptide (CGRP) and amylin, inhibit osteoclastic bone resorption selectively via the Q effect. In the present study, we have made measurements of cell spread area in response to the application of amylin, CGRP and a peptide fragment of CGRP, CGRP-(Val⁸Phe³⁷). We found that, over a wide concentration range (50 pmol/l to 2·5 μmol/l), the selective Q effect agonists did not produce an R effect. Furthermore, the peptides, when used at a 50-fold higher molar concentration than calcitonin, did not antagonize calcitonin-induced cell retraction. Additionally, experiments designed to measure changes in the intracellular free calcium concentration ([Ca^{2 +} ]_i) in single osteoclasts revealed that, unlike calcitonin, the non-calcitonin Q effect agonists did not produce a rise in [Ca²⁺]_i. The peptides were also unable to attenuate the peak rise in [Ca²⁺]_i induced by calcitonin. The results support our hypothesis that the inhibitory activity of calcitonin on osteoclastic bone resorption is mediated by two sites which may or may not be part of the same receptor complex. One of these is the classical Q effect site coupled to adenylate cyclase via a cholera toxin-sensitive G_s. This site can be activated by nanomolar concentrations of calcitonin, amylin, CGRP or CGRP-(Val⁸Phe³⁷). A novel R effect site, possibly coupled via a pertussis toxin-sensitive G protein to a [Ca²⁺]_i elevating mechanism is predicted from this study. The site is highly specific for calcitonin and is not activated, even at micromolar concentrations, by amylin, CGRP or CGRP-(Val⁸Phe³⁷). Whether or not the two sites are part of the same receptor complex or are two receptor subtypes remains to be determined.

Journal of Endocrinology (1993) 136, 7–15

Mammals share common strategies for regulating reproduction, including a conserved hypothalamic–pituitary–gonadal axis; yet, individual species exhibit differences in reproductive performance. In this report, we describe the discovery of a species-restricted homeostatic control system programming testis growth and function. Prl3c1 is a member of the prolactin gene family and its protein product (PLP-J) was discovered as a uterine cytokine contributing to the establishment of pregnancy. We utilized mouse mutagenesis of Prl3c1 and revealed its involvement in the regulation of the male reproductive axis. The Prl3c1-null male reproductive phenotype was characterized by testiculomegaly and hyperandrogenism. The larger testes in the Prl3c1-null mice were associated with an expansion of the Leydig cell compartment. Prl3c1 locus is a template for two transcripts (Prl3c1 -v1 and Prl3c1-v2) expressed in a tissue-specific pattern. Prl3c1-v1 is expressed in uterine decidua, while Prl3c1-v2 is expressed in Leydig cells of the testis. 5′RACE, chromatin immunoprecipitation and DNA methylation analyses were used to define cell-specific promoter usage and alternative transcript expression. We examined the Prl3c1 locus in five murid rodents and showed that the testicular transcript and encoded protein are the result of a recent retrotransposition event at the Mus musculus Prl3c1 locus. Prl3c1-v1 encodes PLP-J V1 and Prl3c1-v2 encodes PLP-J V2. Each protein exhibits distinct intracellular targeting and actions. PLP-J V2 possesses Leydig cell-static actions consistent with the Prl3c1-null testicular phenotype. Analysis of the biology of the Prl3c1 gene has provided insight into a previously unappreciated homeostatic setpoint control system programming testicular growth and function.

The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E₂). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gα_o protein subunit potentiated ERα activity in the absence and presence of E₂. Transient transfection of the human breast cancer cell line MCF-7 showed that Gα_o augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gα_o revealed that Gα_o stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gα_o, through activation of the MAPK pathway, plays a role in the regulation of ERα activity.