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ME Guibbolini and M Avella

Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.

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AV Sirotkin, AV Makarevich, J Kotwica, PG Marnet, HB Kwon, and L Hetenyi

The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.

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Z Cheng, RS Robinson, PG Pushpakumara, RJ Mansbridge, and DC Wathes

Dietary polyunsaturated fatty acid (PUFA) intake in humans can affect the incidence of a variety of diseases including coronary heart disease. Feeding high PUFA diets to cows can alter the PUFA content of milk for human consumption. PUFAs supply the precursors for prostaglandin (PG) synthesis and PGs in turn influence many aspects of reproduction. This study examined the effects of a control (CONT), a high n-6 PUFA diet (derived from protected soya) and a high n-3 diet (derived from protected linseed) on uterine PG synthesis in the lactating dairy cow. Endometrial explants obtained on days 15-17 of the oestrous cycle were cultured for an initial 42 h in vitro in fully defined medium (basal production) and then challenged with control medium, oxytocin (OT; 20 or 200 nM) or calcium ionophore A23187 (CaI; 10 microM). PGF(2 alpha), PGE(2) and 6-keto-PGF(1 alpha) were measured in the spent medium. The experiments were repeated using tissue from two groups of cows, nine in Experiment 1 (three cows per diet) and seven in Experiment 2 (four CONT and three n-6). Results of the two experiments were consistent. The basal concentrations of all three PGs were significantly lower (>50% reduction) in the n-6-fed group in comparison with CONT and n-3 groups. The n-3 diet did not alter basal PGF(2 alpha) and PGE(2) but increased 6-keto-PGF(1 alpha). The n-6 diet also inhibited the ability of the tissue to respond to both OT and CaI, with significant reductions in the stimulated levels of all three PGs. In contrast, the n-3 diet only had minor effects; it did not alter the response to OT but did reduce the long-term response to CaI at 24 h post treatment. In conclusion, dietary PUFA intake can inhibit PG production in bovine endometrial explants, with a more pronounced effect following n-6 rather than n-3 supplementation. These data suggest that a high n-6 diet reduces the endometrial capacity to produce PGs and may therefore have implications for the control of luteolysis and other PG-mediated events such as ovulation.

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DM Sloboda, JP Newnham, and Challis JR

Synthetic glucocorticoids have become an important clinical tool with which to advance fetal lung maturation in women at risk of early preterm birth, and this has succeeded in reducing neonatal mortality and morbidity from respiratory distress syndrome. Although previous studies have shown that glucocorticoids have deleterious consequences on fetal development, there is little information regarding the effects of clinically relevant repeated maternal doses of glucocorticoids on fetal growth and hypothalamic-pituitary-adrenal (HPA) function. We hypothesised that repeated prenatal exposure to increased concentrations of glucocorticoids would alter fetal growth and HPA axis development. Pregnant ewes were injected with betamethasone (0.5 mg/kg) or vehicle at 104, 111 and 118 days of gestation (term 150 days). Animals were sacrificed at 125 and 146 days of gestation, at which time fetal weights were recorded. Maternal and fetal blood samples were gathered and fetal tissue collected. Maternal oestradiol concentrations were significantly greater than those in controls at 125 days of gestation, but were not different at 146 days. Maternal plasma progesterone concentrations were similar between groups at both 125 and 146 days of gestation. Weight at birth was significantly reduced by 23% at 125 days and 19% at 146 days of gestation (P<0.05) after exposure to glucocorticoid. Cord plasma ACTH concentrations were not significantly different between groups at day 125, but were significantly increased in day 146 fetuses of ewes that had received betamethasone (P<0.05). Cord plasma cortisol concentrations followed the same trend, although differences were not statistically significant. Cord plasma corticosteroid binding capacity (CBC) was significantly increased at 125 days of gestation in fetuses of betamethasone-treated animals (P<0.05), but not at 146 days of gestation. To examine the mechanisms regulating the increase in cord plasma ACTH of 146-day fetuses, we used in situ hybridisation to determine the distribution and levels of mRNA encoding key pituitary and hypothalamic neuropeptides of the HPA axis. In pituitaries of 146-day fetuses, there were no significant differences in the regional pattern of distribution or amounts of pro-opiomelanocortin (POMC) mRNA between betamethasone-treated animals and controls, in either the pars intermedia or the inferior and superior regions of the pars distalis. Neither prohormone convertase (PC)-1 nor PC-2 mRNA levels in pituitaries of 146-day fetuses were significantly different between treatment groups. After maternal betamethasone, immunoreactive ACTH peptide content in the fetal pars distalis was not different but glucocorticoid receptor (GR) mRNA levels in the pars distalis were increased significantly (P<0.05). No significant difference in distribution pattern or concentrations of corticotrophin-releasing hormone (CRH) mRNA, GR mRNA, oxytocin mRNA and pre-proenkephalin mRNA were found in hypothalami from fetuses at 146 days of gestation after betamethasone treatment. We conclude that antenatal betamethasone given to pregnant sheep in a manner similar to that used in human obstetric practice results in reduced weight at birth at 125 and 146 days, and altered basal cord levels of plasma ACTH and corticosteroid binding capacity, but these changes are not reflective of changes in steady state concentrations of POMC and CRH mRNA in the fetal pituitary or hypothalamus.

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Donald Pfaff

multiplicative set of gene inductions allows the female to participate in reproductive behaviour sequences. Anxiety reduction The oxytocin gene and the gene for its receptor are both expressed by hypothalamic neurons at

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Qinglei Li, Fermin Jimenez-Krassel, Anilkumar Bettegowda, James J Ireland, and George W Smith

of arachidonic acid to prostaglandins), the NAD+-dependent 15-hydroxy prostaglandin dehydrogenase (PGDH; the key enzyme that metabolizes PGE 2 and PGF 2α to biologically inactive 15-keto derivatives) and oxytocin, and follicular fluid progesterone

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Natasha Singh, Bronwen Herbert, Gavin R Sooranna, Nicolas M Orsi, Lydia Edey, Tathagata Dasgupta, Suren R Sooranna, Steven M Yellon, and Mark R Johnson

labour, when pregnancy is prolonged, are often unsuccessful and associated with significant morbidity and mortality. Certainly, prostaglandins and oxytocin are key players in the final common pathway, promoting myometrial contractility, meaning that their

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S P Sivarajasingam, N Imami, and M R Johnson

• This cytokine may play a role in labour by increasing the expression of oxytocin receptors on myometrial cells to increase their responsiveness to oxytocin • IL-6 can also increase oxytocin secretion by myometrial cells IL-8

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Vikki L Poole and Christopher J McCabe

expressed towards the end of gestation and throughout suckling; however, NIS levels are markedly reduced within 24 h of weaning ( Tazebay et al . 2000 ). With the hormones oxytocin and prolactin being heavily associated with lactation, investigations

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Roman A Romanov, Alán Alpár, Tomas Hökfelt, and Tibor Harkany

sites located outside the blood–brain barrier), neurons projecting to the brainstem and locally projecting hypothalamic neurons ( Daftary et al . 1998 , Krashes et al . 2014 ). Oxytocin and vasopressin ( Du Vigneaud 1954 ) are produced in