SUMMARY

Merino ewes whose foetuses had surgically-implanted, indwelling vascular cannulae were used to determine the influence of maternal nutritional status on foetal plasma hormone levels during the last month of gestation. Observations were made during feeding of the ewes on a lucerne chaff diet ad libitum, during restricted feeding on the same diet and during fasting. Foetuses survived for an average of 31 days after the operation and seven out of the ten ewes lambed normally. In both ewes and foetuses, plasma concentrations of insulin were significantly higher 3–12 h after feeding than in prefeeding samples. Similar changes were seen with restricted and ad-libitum feeding. Fasting for 48 h caused significant decreases in insulin levels of both ewes and foetuses, but the decrease in foetal plasma insulin concentration was less than that in ewes. In agreement with earlier studies, foetal and maternal glucose concentrations were closely correlated and foetal fructose concentrations were closely related to foetal glucose concentrations. Foetal plasma insulin concentrations were closely correlated with glucose and fructose concentrations. Plasma growth hormone (GH) levels increased significantly in both ewes and foetuses during fasting. There were also significant increases during fasting in the plasma corticosteroid levels of the ewes, but not in those of their foetuses. Feeding did not cause significant changes in foetal plasma GH or corticosteroid concentrations although maternal GH concentrations were significantly increased and corticosteroid concentrations decreased 3–12 h after feeding on the restricted diet.

The results suggest that alterations in foetal plasma hormone concentrations could play an important role in foetal adaptation to fluctuations in maternal nutrient supply during the last month of gestation.

Early life diet influences metabolic programming, increasing the risk for long-lasting metabolic ill health. Neonatally overfed rats have an early increase in leptin that is maintained long term and is associated with a corresponding elevation in body weight. However, the immediate and long-term effects of neonatal overfeeding on hypothalamic anorexigenic pro-opiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP)/neuropeptide Y (NPY) circuitry, and if these are directly mediated by leptin, have not yet been examined. Here, we examined the effects of neonatal overfeeding on leptin-mediated development of hypothalamic POMC and AgRP/NPY neurons and whether these effects can be normalised by neonatal leptin antagonism in male Wistar rats. Neonatal overfeeding led to an acute (neonatal) resistance of hypothalamic neurons to exogenous leptin, but this leptin resistance was resolved by adulthood. While there were no effects of neonatal overfeeding on POMC immunoreactivity in neonates or adults, the neonatal overfeeding-induced early increase in arcuate nucleus (ARC) AgRP/NPY fibres was reversed by adulthood so that neonatally overfed adults had reduced NPY immunoreactivity in the ARC compared with controls, with no further differences in AgRP immunoreactivity. Short-term neonatal leptin antagonism did not reverse the excess body weight or hyperleptinaemia in the neonatally overfed, suggesting factors other than leptin may also contribute to the phenotype. Our findings show that changes in the availability of leptin during early life period influence the development of hypothalamic connectivity short term, but this is partly resolved by adulthood indicating an adaptation to the metabolic mal-programming effects of neonatal overfeeding.

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na⁺/Cl⁻ transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na⁺/Cl⁻ cotransporter (Ncc), the number of Ncc ionocytes, Na⁺/Cl⁻ influxes of ionocytes, and whole-body Na⁺/Cl⁻ contents. In FW with low Na⁺ and low Cl⁻, the expression levels of mRNA for Na⁺/H⁺ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na⁺/Cl⁻ contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na⁺/Cl⁻ uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

We explored the involvement of oxytocin receptor (Oxtr)/transient-receptor-potential-vanilloid-1 (TRPV1) genes and oxytocin (Oxt) on the adaptation of skeletal muscle to cold stress challenge in mice. Oxtr expression in hypothalamic paraventricular (PVN), supraoptic nuclei (SON), and hippocampus (HIPP) were evaluated by immunohistochemistry in parallel with the measurement of circulating Oxt. The Oxtr and TRPV1 gene expressions in soleus (SOL) and tibialis anterior (TA) muscles were investigated by RT-PCR. Histological studies of the cardiac muscle after cold stress were also performed. Male mice (n = 15) were divided into controls maintained at room temperature (RT = 24°C), exposed to cold stress (CS) at T = 4°C for 6 h , and 5 days. Immunohistochemical studies showed that Oxtr protein expression increased by two-fold (P = 0.01) in PVN and by 1.5-fold (P = 0.0001) in HIPP after 6 h- and 5 days of CS but decreased by 2-fold (P = 0.026) in SON in 5 days. Both Oxtr and TRPV1 gene expression increased after 6 h and 5 days of CS in SOL and TA muscles. Oxtr vs TRPV1 gene expression in SOL and TA muscles evaluated by regression analysis was linearly correlated following CS at 6 h and 5 days but not at control temperature of 24 ± 1°C, supporting the hypothesis of coupling between these genes. The circulating levels of Oxt are unaffected after 6 h of CS but decreased by 0.2-fold (P = 0.0141) after 5 days-CS. This is the first report that Oxtr and TRPV1 expressions are upregulated in response to cold acclimation in skeletal muscle. The up-regulation of Oxtr in PVN and HIPP balances the decrease of circulating Oxt.

Early pregnancy is susceptible to oxidative stress, and thus characterisation of antioxidant systems and pro- and anti-apoptotic pathways would improve understanding of placental development and function. We aimed, therefore, to determine the activities of the antioxidant enzymes, copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), catalase (CAT), glutathione (GSH) peroxidase (GPX) and GSH reductase (GSR); and to quantify the expression of BAX and MCL1 proteins in relation to the developmental changes in antioxidant defences in sheep placentomes sampled on days 35, 55 and 80 of pregnancy. Placentome progesterone content was analyzed to determine steroidogenic capacity. Malondialdehyde (MDA) and protein carbonyl were quantified in placentomes as biomarkers of lipid peroxidation and protein damage respectively. Placentome tissues demonstrated significantly increased content of progesterone and MDA at day 80 of pregnancy and protein carbonyl as early as day 50 of pregnancy. Progesterone and MDA contents were not different between days 35 and 55 of pregnancy. While SOD1 and CAT activities did not alter significantly, SOD2 activity decreased from days 35 to 55. GPX activity increased from days 35 to 55 and increased further to day 80 of pregnancy. GSR activity increased from days 35 to 55 of pregnancy. BAX protein expression decreased, while MCL1 increased from days 35 to 55 and 80 of pregnancy. The increased GPX activity was associated with a decrease in the BAX/MCL1 protein expression ratio. Changes in the antioxidant enzymatic defences could be a part of placentome adaptation to reactive oxygen species-induced oxidative stress at specific early developmental stages of pregnancy.

Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.

At birth, the endocrine pancreas must assume a glucoregulatory role if the neonate is to survive the transition from parenteral to enteral nutrition. In species like the horse, neonatal hypoglycaemia is common, which suggests that the glucoregulatory mechanisms are not always fully competent at birth. Hence, this study examined pancreatic beta cell function in newborn foals during nutritional adaptation over the first 10 days post partum. Over a 48 h period at three time intervals after birth (days 1-2, 5-6 and 9-10 post partum), the beta cell responses to suckling and to intravenous administration of glucose, arginine and saline were measured in seven normal pony foals. Basal plasma concentrations of proinsulin, but not insulin or glucose, increased significantly between days 1 and 10. Suckling caused a gradual increase in plasma glucose, which was accompanied by a significant increase in plasma insulin concentrations 15 min after the onset of suckling on days 5 and 9, but not day 1. There was no significant change in plasma proinsulin concentrations in response to suckling at any age. At all ages studied, glucose and arginine administration stimulated an increase in the plasma concentrations of insulin and proinsulin; these beta cell responses did not change significantly with postnatal age. The insulin responses to glucose were significantly greater than those of arginine at each time period. Glucose clearance was significantly slower on day 1 than subsequently. Proinsulin and glucose, but not insulin, concentrations decreased significantly after saline administration at all three ages. At each time period, there was a significant positive relationship between the plasma insulin and proinsulin concentrations, the slope of which was significantly shallower on days 1-2 than subsequently. These results show that equine beta cells are responsive to glucose and arginine and release both insulin and proinsulin during the immediate postnatal period. They also suggest that newborn foals may be insulin resistant on the first day after birth.

A preparation for estimations of rates of secretion of parathyroid hormone (PTH) in conscious goats is described. The procedure involved surgical isolation of a thyroid with its indwelling parathyroid gland, leaving the blood supply intact. Permanent access to the arterial and venous supplies of the preparation was secured by exteriorization of the jugular vein and carotid artery in skin-covered loops. The remaining parathyroids were removed leaving the isolated parathyroid gland as the only source of the hormone. For the seven goats operated on, the preparation has worked satisfactorily from the start in five and also in the remaining two after a reoperation to cut an accessory venous drainage. The loops have been catheterized repeatedly for extended periods of time and the preparation has proved useful for periods of up to 3 years.

Secretion of PTH was measured in one goat at two consecutive parturitions. In late pregnancy, secretion rates of 0·7–0·9 ng/kg per min were observed when the dietary calcium intake was adequate. In the first weeks of lactation, secretion increased to 1·2–1·5 ng/kg per min when the intake of calcium was adequate and to 2·0–2·2 ng/kg per min when calcium intake was inadequate. Reductions in calcium intake led to increased rates of secretion of PTH both during pregnancy and during lactation. The results indicate that the preparation may be useful in the study of changes in PTH secretion both acutely and in connection with long-term studies of dietary adaptations of the calcium metabolism.

The influence of stress on the secretion of corticosterone has been investigated by radioimmunoassay in domestic ducks (Anas platyrhynchos) of 6–8 weeks of age. Intravenous and i.m. injections of synthetic ACTH (Synacthen, 25 i.u./bird and Synacthen Depot, 100 i.u./bird respectively) markedly increased (2·7- to 10-fold) basal concentrations of corticosterone in plasma (3–6 ng/ml) in comparison with those in birds injected with 0·9% saline (1 ml/bird). Maximum levels of 30–40 ng/ml plasma were reached after 30 min and 5 h respectively. Increased levels of corticosterone in plasma were also seen within 5 min of i.v. administration of saline and remained as high as those in birds treated with Synacthen for at least 20 min after injection. Blood sampling (from the brachial vein) was, by itself, sufficient to increase levels of corticosterone in plasma; a large (twofold) rise being observed as soon as 1 min after initial handling and bleeding, with concentrations of 30–40 ng/ml being found in birds bled 15 times during a period of 14 min. However, in a flock of birds, the alarm created in unhandled birds while others were being bled had no effect on concentrations of corticosterone in plasma. Deprivation of food or water for 24 h also enhanced levels of corticosterone in plasma, as did 24 h of adaptation to 0·2 m-NaCl drinking water. These results have demonstrated the lability of the secretion of corticosterone in response to stress and demonstrated its usefulness as a physiological indicator of stress in ducks.

Abstract

The reindeer in its natural habitat is subject to great annual variations in ambient temperature, illumination and nutrition. To ascertain the effect of these environmental factors on thyroid function, serum thyroid-stimulating hormone (TSH), thyroxine (T₄), tri-iodothyronine (T₃) and reverse T₃ (rT₃) concentrations were measured four times a year (2 June, 8 October, 21 November, and 24 February) in 14 animals housed outdoors at latitude 69°10′N. They all showed statistically significant (P<0·05) seasonal changes. Serum TSH and T₄ were highest in February (623 ± 30 ng/ml and 287 ± 19 nmol/l respectively). TSH was lowest in October (318 ± 47 ng/ml) and T₄ in November (199 ± 19 nmol/l). The T₃ concentration was highest in November (3·0 ± 0·3 nmol/l) and lowest in June (1·8 ± 0·2 nmol/l). In contrast, rT₃ was highest in June (3·6 ± 1·2 nmol/l) and lowest in November (1·9 ± 0·6 nmol/l). Thus, there was an inverse relationship between T₃ and rT₃ (linear regression r= −0·406, P<0·01).

TSH, T₄, T₃ and rT₃ responses to exogenous thyrotrophin-releasing hormone (synthetic TRH; 500 μg i.m.) were determined in ten animals. The magnitude of their response to TRH was significantly (P<0·05) dependent on the time of year. When compared with the control level all the parameters rose significantly (P<0·05). The greatest rise in serum TSH occurred in October (219 ± 151% and the smallest in February (66 ± 53%). The corresponding figures for T₄ were 56 ± 60% in June and 11 ± 18% in February, for T₃ 143 ± 45% in October and 73 ± 46% in June, and for rT₃ 25 ± 13% in October and 12 ± 10% in February.

In conclusion, significant annual variations occur in the reindeer serum concentrations of TSH and thyroid hormones as well as in their responses to exogenous TRH. Environmental factors greatly affect the pituitary-thyroid axis in this mammalian species living far in the North. The observed variations probably reflect an adaptation of the species to the annual environmental cycle.

Journal of Endocrinology (1994) 141, 527–533