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Notes: Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 μmol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l–1 μmol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 μmol/l; P〈0.05). PACAP also stimulated FSH secretion in cells from some animals although this effect was small. The GH response to PACAP was inhibited by PACAP(6–38), a putative PACAP antagonist, but not by (N-Ac-Tyr1, D-Arg2)-GHRH(1–29)-NH2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1–24 h, VIP (1 μmol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l–1 μmol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l–1 μmol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 μmol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.

Notes: This study was undertaken to investigate the roles of PACAP and VIP in the control of pituitary hormone secretion in the ewe. The first experiment was designed to identify any direct effects at the level of the pituitary and was conducted during the luteal phase of a prostaglandin-synchronized oestrous cycle. PACAP (0.008, 0.04, 0.2 and 1.0 nmol/min) or VIP (0.06, 0.2, 0.6 and 1.8 nmol/min) was infused into the carotid artery over a 10 min period. Blood samples were taken before and after the infusions so that plasma PRL, LH and GH concentrations could be measured. Blood pressure was also monitored to determine if the doses used were biologically active. In no case was an effect on hormone secretion observed. In contrast, the highest dose of each peptide induced an increase in heart rate to almost three-fold the resting value. Although both peptides are active in vivo, this result suggests that neither peptide has a direct effect on hormone release from the pituitary of prostaglandin-synchronized ewes. In a second experiment, we investigated whether the peptides had central effects on hormone secretion. Intracerebroventricular (ICV) injection of PACAP or VIP at the dose 10nmol was tested in ovariectomized ewes. After injection, PACAP suppressed PRL and GH secretion so that plasma hormone concentrations from 1–3 h after injection were significantly different from the control (P〈0.05 for PRL, P〈0.01 for GH). In addition, PACAP significantly reduced mean LH concentration (P〈0.05) and LH pulse frequency (P〈0.01). A similar suppressive effect on LH secretion was also observed after ICV injection of VIP (P〈0.05 for both parameters), although PRL and GH release were not affected. These results suggest a possible role for PACAP in the neuroendocrine control of PRL, GH and LH secretion in sheep. In addition, VIP may be involved in the control of LH secretion. In contrast, there is no evidence to suggest that either peptide is a hypophysiotropic factor for PRL, LH or GH in prostaglandin-synchronized ewes.

Notes: The present study was undertaken to determine the effects of the endogenous opioid ligandβ-endorphin on pulsatile luteinizing hormone (LH) secretion and plasma prolactin concentrations during the follicular phase of the ewe. Oestrous cycles were synchronized by injection of prostaglandin analogue and, commencing 13 h later, saline or β-endorphin (2, 10 or 50 μg) was injected intracerebroventricularly at hourly intervals for 3 h. Treatment with β-endorphin was followed by a significant reduction in LH pulse frequency at all doses due to almost complete cessation of pulses. There were no significant changes in LH pulse amplitude or mean LH concentrations. At the lowest dose ofβ-endorphin, LH pulses recommenced within 3 h of the last injection in all animals and pulse frequency was not significantly different from the saline-injected controls during the 3 h post-treatment period. Following treatment with 10 or 50 μg β-endorphin, LH pulse frequency remained suppressed during the 3 h post-treatment period but was not different from saline-treated controls on the following day. The time to the onset of the LH surge was not affected by intracerebroventricularβ-endorphin. Plasma prolactin concentrations were significantly increased following intracereb-roventricular injection of 10 or 50 μg β-endorphin, declining to control values soon after treatments stopped. Intravenous administration of 50 μg β-endorphin had no effect on LH but was accompanied by a small increase in prolactin concentrations.While these results indicate that hypothalamicβ-endorphin may be involved in the central control of LH and prolactin secretion, they provide no evidence for subtle modulation of LH pulse frequency by this neuropeptide.

Notes: This study was undertaken to determine whether dopaminergic suppression of pulsatile luteinizing hormone (LH) secretion during seasonal anoestrus in the ewe is mediated via the dopamine D1 or D2 receptor. This was tested by 1) assessing the response to dopamine D1 and D2 antagonists during seasonal anoestrus, and 2) determining the ability of D1 and D2 agonists to suppress pulsatile LH secretion during the breeding season. In seasonally anoestrous ewes the D2 antagonist pimozide increased LH pulse frequency although this effect did not reach significance (P = 0.07). The D1 antagonist SCH 23390 had no effect on LH pulse frequency. LH pulse amplitude and mean LH were not affected by either treatment. During the breeding season, ovariectomized oestradiol-implanted ewes were injected intracerebroventricularly with vehicle, LY 171555 (dopamine D2 agonist) and SKF 38393 (D1 agonist) with each drug tested at 50 μg and 200 μg. At the higher dose, LY 171555 significantly (P〈0.05) reduced LH pulse frequency in the 2 h period immediately after treatment. Mean LH declined at both doses but only in the first hour after treatment. SKF 38393 did not affect LH pulse frequency, pulse amplitude or mean LH. These results suggest that the D1 receptor is not involved in the suppression of pulsatile LH secretion during seasonal anoestrus. Dopaminergic suppression of pulsatile LH secretion is mediated via the D2 receptor but the significance of this neurotransmitter in the seasonal suppression of LH remains to be elucidated.

Notes: Dopamine receptors are pharmacologically grouped as D1 and D2 receptors. Previous research in the ewe has shown that central D1 receptors may have a role in facilitating prolactin release. The aims of this study were therefore to localize and characterize D1 binding sites in the hypothalamus of sheep. For comparison, a known D1 receptor-rich tissue (striatum) was also studied. The bioactivities of several D1 analogues were also assessed for their efficacy in sheep tissue. In vitro autoradiography with [125I]-SCH23982 was used to localize D1 binding sites. The ventromedial hypothalamic nucleus (VMH) displayed moderate levels of specific binding, localized to the medial portion of the nucleus. Low levels of specific binding were seen in the preoptic area, supraoptic nucleus and anterior hypothalamic area. The suprachiasmatic nucleus, median eminence and arcuate nucleus did not show specific binding. As expected the striatum displayed high levels of specific binding. The VMH, preoptic area, median eminence, striatum and anterior pituitary were examined with radioligand binding studies to quantify and characterize D1 binding sites. Scatchard analysis gave KD 1.04 nM and Bmax 127.4 fmol/mg protein for VMH and KD 1.99 nM and Bmax 454.6 fmol/mg protein for striatum. While specific binding occurred in the preoptic area and median eminence this binding did not show saturation characteristics. Specific binding was not observed in the anterior pituitary. Affinities determined by competitive binding studies showed that the binding sites in both VMH and striatum have the characteristics of a D1 receptor, that is, high affinity for the D1 agonists and antagonists, low affinity for dopamine and the serotonergic antagonist ketanserin and extremely low affinity for the D2 agonists and noradrenaline. Adenylate cyclase studies showed that in the striatum dopamine and the D1 agonists, fenoldopam and SKF38393, were able to cause significant dose-dependent increases in adenylate cyclase activity. In contrast the D1 agonist, SKF82958, was inactive in this system. The D1 antagonists SCH23390 and SCH39166, but not SKF83566, abolished the adenylate cyclase response to 50 μM dopamine. In the VMH the D1 agonist SKF38393, but not dopamine, stimulated adenylate cyclase activity. In conclusion, these results demonstrate that D1 binding sites exist within the hypothalamus in the VMH and that these binding sites have the characteristics of D1 receptors. These receptors are a potential site of action for dopamine in facilitating prolactin release. In addition, the results show that at least for some dopamine analogues, receptor binding affinity does not always correlate with biological activity.

Notes: We have examined the distribution of the pituitary adenylate cyclase activating polypeptide type I receptor (PAC1R) in the ewe hypothalamus by reverse transcription-polymerase chain reaction, in situ hybridization and immunohistochemistry. PAC1R mRNA was highly expressed in the mediobasal hypothalamus of the ewe, particularly in the arcuate nucleus and ventromedial hypothalamus, compared to other hypothalamic regions. Similar results were obtained from immunohistochemistry using a specific PAC1R antibody. Intense immunolabelling was observed in the arcuate nucleus, external zone of the median eminence and ventromedial hypothalamus. Only relatively weak immunolabelling was observed in other hypothalamic regions, including the paraventricular nucleus and supraoptic nucleus. In the ewe, PACAP acts via the arcuate nucleus to suppress prolactin secretion. Therefore we examined whether PAC1R was present on the tuberoinfundibular dopamine (TIDA) neurones in this nucleus. Dual immunofluorescence labelling for PAC1R and tyrosine hydroxylase revealed that 21.2 ± 1.7% of dopaminergic neurones in the arcuate nucleus (A12 cell group) also stained for PAC1R. By contrast, other hypothalamic dopaminergic cell groups (A11, A13, A14 and A15) exhibited little (〈 3%) or no colocalization. Overall, our results indicate that, in the ewe hypothalamus, PAC1R is most concentrated in the arcuate nucleus, where it is localized on a substantial proportion of dopaminergic neurones. These observations, together with previous in vivo studies, suggest that PACAP could act directly on TIDA neurones via PAC1R to increase dopamine release and consequently inhibit prolactin secretion in the sheep.

Notes: The Bennett's wallaby is a seasonal breeder in which photoperiod is an important proximate factor involved in regulating the timing of the breeding season. The present study was undertaken in order to describe the melatonin profiles of female wallabies under natural photoperiod and to examine the effects of abrupt changes in day length on the melatonin profile. Under artificial and natural photoperiods, plasma melatonin concentrations were low or nondetectable during the day but increased after the onset of darkness to values of up to 50 pg/ml. Under natural photoperiod, melatonin profiles were obtained from animals on December 17, April 3, June 26, and October 7. The duration of the nocturnal melatonin rise was positively correlated with the duration of the night, and there was no significant effect of time of year on the amplitude of the nocturnal melatonin peak. In a further study, 12 animals were placed in 9L: 15D and after 7 days, one-half were transferred to 18.5L: 5.5D for 6 weeks. Before and after each change of photoperiod, melatonin profiles were obtained from both groups. As demonstrated for animals on natural photoperiod, duration but not amplitude of the nocturnal melatonin rise was affected by day length. It is noteworthy that two of the 17 animals used in this study did not show evidence of a nocturnal rise in melatonin levels.