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

Fluprostenol, an analogue of prostaglandin F_2α, administered s.c. to rats on day 18 of pregnancy increased cervical creep, or softness, by the following day. Doses of fluprostenol 100-fold larger were necessary to increase uterine contractions. Fluprostenol produced falls in serum progesterone concentrations, increases in 20α-dihydroprogesterone concentrations, no changes in oestradiol or relaxin concentrations and a reduction in the ovarian human chorionic gonadotrophin binding capacity in vitro. Fluprostenol was less potent in inducing cervical softness when administered per vaginam, and a dose which produced softening in pregnant rats was ineffective in ovariectomized steroid-maintained pregnant or pro-oestrous rats. The findings suggest that cervical softening by fluprostenol does not result from a simple direct action on the cervix or by increasing uterine contractions, but rather by an indirect hormonal action mediated by the ovaries. The results with the lowest dose of fluprostenol indicate that cervical softening could be produced without a sustained fall in serum progesterone concentrations. Fluprostenol is much more potent at increasing cervical softness in the pregnant rat than prostaglandin F_2α or prostaglandin E₂. With fluprostenol the ratio of dose to induce uterine contractility relative to that to produce cervical softness was greater than with these natural prostaglandins, indicating the greater selectivity of fluprostenol in the pregnant rat.

ABSTRACT

The influence of endogenous opioids on the posterior pituitary response to stress was investigated by measuring plasma hormone levels in immobilized male and female rats following either acute naloxone treatment or prolonged morphine administration. Naloxone significantly potentiated the oxytocin and arginine vasopressin (AVP) response to immobilization, but in female rats only. The responses of morphine-treated male rats showed differences compared with vehicle-treated controls, although chronic morphine treatment did not reliably alter the oxytocin or AVP responses to immobilization in males or females.

In a further experiment to investigate the role of gonadal hormones in determining the sex difference in responsiveness to naloxone, it was found that acute naloxone treatment significantly potentiated the posterior pituitary response to stress in castrated male rats.

These results extend previous studies showing a sex difference in stress-induced secretion of posterior pituitary hormones, providing evidence of a sexual dimorphism in the endogenous opioid regulation of this response which is partly determined by circulating gonadal hormones.

J. Endocr. (1986) 111, 239–244

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

Abstract

3,5,3′-Tri-iodothyronine (T₃), 1α,25(OH)₂-vitamin D₃ (D₃) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T₃R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T₃, D₃ and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T₃R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T₃, D₃, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T₃R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.

Journal of Endocrinology (1997) 154, 63–74

One of the methods most commonly used in the biological assay of gonadotrophic extracts is the quantitative measurement of the response elicited in the ovaries of the immature rat. This method, used on account of its convenience, suffers from the disadvantage that the animal's own pituitary gland is present and must be regarded as a potential source of complication in the assay results. Experience has shown that the complication may be quantitative or qualitative or both. Smith [1930] found that the difference in response of normal and hypophysectomized rats to injections of gonadotrophic extracts of the pituitary gland was quantitative rather than qualitative, whereas the difference in the response of the ovaries of normal and hypophysectomized rats to extracts of urine of pregnant women (luteinizing hormone) and of the urine of ovariectomized women (follicle stimulating hormone) has been found to be most definitely qualitative [Selye, Collip, and Thomson, 1933; Leonard

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

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.

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