ABSTRACT

Melatonin-binding sites have previously been identified in the suprachiasmatic nucleus (SCN) and median eminence (ME) of the rat. We have further investigated the localization of melatonin-binding sites in the rat hypothalamus and pituitary using the ligand [¹²⁵I] iodomelatonin and in-vitro autoradiography. The presence of specific melatonin-binding sites in the SCN is confirmed; however the second area of melatonin binding is identified as the pars tuberalis of the pituitary and not the ME as previously described. No other areas which bound melatonin were found in either the pituitary or the hypothalamus.

Abstract

The pineal hormone, melatonin, is important in the timing of seasonal reproduction in the sheep. Melatonin of maternal origin readily crosses the placenta; its function in the fetal sheep is, however, unclear. To gain an insight into the role of melatonin in ovine development we have identified specific melatonin receptors throughout gestation using 2-[¹²⁵I]iodomelatonin and quantitative in vitro autoradiography. Specific binding was found at the earliest time studied at 30 days of gestation, over the developing thyroid (term=145 days). At 31 days of gestation specific labelling was found over the thyroid and pituitary glands, the spinal nerves, nasal cavity and developing bronchi. This binding was diminished by over 50% in the presence of 10⁻⁴ m GTPγS (an analogue of guanosine triphosphate) indicating that the 2-[¹²⁵I]iodomelatonin binding at this early stage of gestation represents a receptor coupled to a regulatory G-protein. By 40 days of gestation specific binding was found over the nasal epithelium, cochlear epithelium, regions of the brain, especially the hind brain and the vestibulocochlear and glossopharyngeal nerves, and both the pars distalis and pars tuberalis of the pituitary. As gestation proceeded, labelling over the pars distalis appeared to become more scattered in nature while that on the pars tuberalis remained consistent. Saturation studies of both the neuronal and pituitary binding sites at 121 days of gestation and in the newborn lamb revealed a single class of high-affinity binding sites with K _d values in the picomolar range. Also at 121 days of gestation, binding over the fetal pars tuberalis was diminished in a dose-dependent manner by GTPγS, again confirming that specific binding is indicative of a receptor coupled to a regulatory G-protein. These data demonstrate a potential for sensitivity to melatonin from early in gestation, as well as the developmentally specific expression of the melatonin receptor in certain tissues, and suggest a wider role for melatonin in ovine fetal development than previously considered.

Journal of Endocrinology (1994) 142, 475–484

Abstract

Cortisol inhibits growth hormone (GH) release in short-term culture and is stimulatory in long-term cultures of rat and human pituitary cells. This study sought to determine the in vitro effects of cortisol on GH release and the signal transduction pathways mediating the effects of cortisol on GH release from cultured ovine somatotrophs. Pituitary cells were dispersed with collagenase and placed in culture medium for 4 days. The data indicate that cortisol inhibited growth hormone-releasing hormone (GHRH)-stimulated GH release by at least 2 h. In short-term culture GHRH-, forskolin- and dibutyryl cyclic AMP-stimulated GH release were inhibited by cortisol, suggesting an effect distal to the membrane and involving a protein kinase A (PKA)-dependent pathway. GH release initiated by KCl was inhibited by cortisol, but GH release caused by the calcium ionophore A23187 was unaffected. This suggests a possible action of cortisol on the calcium channels. The inhibition by cortisol of the calcium-dependent secretion of GH release appeared to play a smaller role in mediating cortisol inhibition of GH release than that seen with PKA. Attempts to overcome cortisol inhibition of GH release using puromycin, arachidonic acid or pertussis toxin were unsuccessful. Since cortisol inhibition of GH release does not occur via the mechanisms found in other cell types, cortisol inhibition of pituitary cell secretions appears to be cell-specific rather than utilizing a single inhibitory mechanism. The majority of cortisol actions on the somatotroph appear to act at a site distal to the production of cyclic AMP. In contrast to man and the rat, the sheep somatotroph does not appear to increase GH release when treated with cortisol for 24 h, perhaps related to the lack of effect of cortisol on somatotroph content of GH.

Journal of Endocrinology (1994) 141, 517–525

Abstract

To define the hormonal influences that are directly involved in the hair follicle cycles of animals with differing patterns of fibre growth and moulting, we have investigated the possible presence of IGF-I and melatonin receptors on the dermis and hair follicles of cashmere and Angora goats, sampled in February, March and June, using quantitative in vitro autoradiography. The presence of IGF-I receptors in the dermis of both breeds of goat was determined using cryostat sections incubated with 50 pm ¹²⁵I-labelled IGF-I in the presence or absence of 50 nm IGF-I. Sections of the growing tip of deer antlers containing the cartilaginous zone, a tissue known to contain high concentrations of specific IGF-I receptors, were used as a positive control. As the production of antler velvet uniquely involves the generation of hair follicles de novo, the presence of IGF-I receptors in the velvet-producing region was also investigated. In both breeds of goat, specific ¹²⁵I-IGF-I binding was localised over the inner and outer root sheath, the matrix, the germinal matrix, the dermal papilla and the sebaceous glands and satisfied the basic kinetic criteria considered to be representative of a specific IGF-I receptor. Analysis of saturation isotherms using a one-site binding model revealed dissociation constants (K _d) in the range 0·1–0·9 nm and theoretical maximal numbers of binding sites (B _max) between 21·4 and 45·6 fmol/mg tissue. K _d and B _max values derived from cashmere and Angora goats sampled at different times of the year did not differ significantly between breeds or sampling times. Specific ¹²⁵I-IGF-I binding was also localised to the developing follicles on the deer antler dermis. The presence of melatonin receptors within the goat dermis was also investigated. Sections were incubated with 100 pm 2-[¹²⁵I]iodomelatonin with or without 0·1 μm melatonin, along with sections of sheep pars tuberalis which are known to contain high levels of high-affinity melatonin receptors. No displaceable 2-[¹²⁵I]iodomelatonin binding was found on any sections of the cashmere or Angora skin analysed. It is therefore concluded that melatonin receptors are not present on the hair follicles or associated structures. IGF-I receptors are present on the hair follicle and sebaceous gland and may be involved in the growth of both seasonally and non-seasonally produced fibre and in the development of antler velvet.

Journal of Endocrinology (1996) 151, 55–63

ABSTRACT

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N⁶,O²′-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3·5 mmol/l), significantly (P < 0·01) increased aldosterone output by at least 1·5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P < 0·01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 ± 14 vs 105 ± 19 pmol/10⁶ cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion.

Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P < 0·01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine.

These observations suggest that: (1) the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; (2) with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and (3) activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

J. Endocr. (1986) 110, 315–325

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.

Naturally occurring diabetogenic substance (NDS), isolated from clinical grade human growth hormone (hGH), induces insulin release from isolated pancreatic islets of hypo-physectomized rats in the presence of Krebs–Ringer bicarbonate solution (KRB) containing 2·8 mmol glucose/l. To determine the role of extracellular glucose in insulin release mediated by NDS, islets were perifused with glucose-free KRB containing 200 μg NDS/ml. Under these conditions NDS induced prompt insulin release (284 ± 34 (s.e.m.)% over basal insulin secretion) (P < 0·0005). Islets perifused with 16·7 mmol glucose/l before and during exposure to 200 μg NDS/ml released additional insulin with exposure to NDS (199 ± 28% over basal insulin secretion) (P< 0·0005). Purified intact hGH (200 μg/ml) did not induce insulin release in the presence of 16·7 mmol glucose/l. Mannoheptulose (5 mmol/l) did not inhibit insulin release mediated by NDS but did inhibit insulin release stimulated by 16·7 mmol glucose/l (P< 0·0005). Islets were pre-incubated for 90 min with 200 μg NDS/ml or 200 μg intact hGH/ml KRB and 2·8 mmol glucose/l to determine what effect either protein might have on subsequent glucose-stimulated (16·7 mmol/l) insulin release. Islets pre-incubated with NDS responded with no less insulin release than islets pre-incubated with intact hGH. Naturally occurring diabetogenic substance initiated insulin release in the absence of extracellular glucose, stimulated additional secretion in the presence of stimulatory glucose concentrations and did not inhibit subsequent islet response to glucose. Naturally occurring diabetogenic substance did not depend on glucose phosphorylation to initiate insulin release.

Steroid receptors are key transcriptional regulators of mammary growth, development and lactation. Expression of estrogen receptors alpha (ERα) and beta (ERβ), progesterone receptor (PR), and estrogen-related receptor alpha-1 (ERRβ) have been evaluated in bovine mammary gland. The ERRα is an orphan receptor that, in other species and tissues, appears to function in the regulation of estrogen-response genes including lactoferrin and medium chain acyl-CoA dehydrogenase and in mitochondrial biogenesis. Expression of ERα, ERβ, PR and ERRα was characterized in mammary tissue obtained from multiple stages of bovine mammary gland development using quantitative real-time RT-PCR. Expression was evaluated in prepubertal heifers, primigravid cows, lactating non-pregnant cows, lactating pregnant cows and non-lactating pregnant cows (n=4 to 9 animals/stage). In addition, ERα, ERβ, PR and ERRα were mapped to chromosomes 9, 10, 15 and 29 respectively, by linkage and radiation hybrid mapping. Results indicated that expression of ERα, PR and ERRα was largely coordinately regulated and they were present in significant quantity during all physiological stages evaluated. In contrast, ERβ transcripts were present at a very low concentration during all stages. Furthermore, no ERβ protein could be detected in bovine mammary tissue by immunohistochemistry. The ERα and PR proteins were detected during all physiological states, including lactation. Our results demonstrate the presence of ERα, PR and ERRα during all physiological stages, and suggest a functional role for ERRα and a relative lack of a role for ERβ in bovine mammary gland development and lactation.

ABSTRACT

The induction of ovulation in early post-partum ewes is associated with a high incidence of premature luteal regression which is independent of the suckling stimulus but dependent on the stage post partum. The aim of the present study was to determine whether oxytocin receptors are present on uterine endometrium early in the luteal phase and hence ascertain whether oxytocin-induced uterine prostaglandin F_2α release is a possible mechanism involved in the premature regression of these post-partum corpora lutea. Ovarian and uterine tissues were collected on day 4 of the cycle in ewes induced to ovulate at either 21 or 35 days post partum (n = 4 per group). A further four cyclic ewes were similarly synchronized to ovulate and acted as controls. Corpora lutea from the 21-day post-partum group were significantly (P < 0·01) smaller, had a lower progesterone content and a reduced capacity to secrete progesterone in vitro than corpora lutea from 35-day post-partum or control ewes.

A highly specific oxytocin receptor ligand ¹²⁵I-labelled d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH₂ ⁹]-vasotocin was used to localize and characterize high affinity oxytocin receptors in uterine endometrium (dissociation constant 145 pmol/l). Oxytocin receptor concentrations in endometrium from ewes induced to ovulate at 21 days post partum were on average five-fold higher (P < 0·05) than in 35-day post-partum and control groups.

Journal of Endocrinology (1991) 128, 253–260

Endogenous purines including inosine have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for protection is very high because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic-resistant purine analogue, INO-2002, exerts anti-inflammatory effects in two animal models of type I diabetes. Type I diabetes was induced chemically with streptozotocin or genetically using the non-obese diabetic (NOD) female mouse model. Mice were treated with INO-2002 or inosine as required at 30, 100, or 200 mg/kg per day, while blood glucose and diabetes incidence were monitored. The effect of INO-2002 on the pancreatic cytokine profile was also determined. INO-2002 reduced both the hyperglycaemia and incidence of diabetes in both streptozotocin-induced and spontaneous diabetes in NOD mice. INO-2002 proved to be more effective in protecting against diabetes than the naturally occurring purine, inosine, when administered at the same dose. INO-2002 treatment decreased pancreatic levels of interleukin (IL)-12 and tumour necrosis factor-α, while increasing levels of IL-4 and IL-10. INO-2002 also reduced pancreatic levels of the chemokine MIP-1α. The inosine analogue, INO-2002, was protected more effectively than the naturally occurring purine, inosine, against development of diabetes in two separate animal models. INO-2002 exerts protective effects by changing the pancreatic cytokine expression from a destructive Th1 to a protective Th2 profile. The use of analogues of inosine such as INO-2002 should be considered as a potential preventative therapy in individuals susceptible to developing type I diabetes.