J. D. Curlewis

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe

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.

Effects of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) onHormone Secretion from Sheep Pituitary Cells in vitro

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.

Glucose transport in the equine hoof

REASONS FOR PERFORMING STUDY Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin-responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. OBJECTIVES To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. METHODS Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2-deoxy-D-[2,6-(3)H] glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin-induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushings syndrome. RESULTS Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin-independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. CONCLUSIONS The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1-mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. POTENTIAL RELEVANCE Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.

Successful Artificial Insemination in the Koala (Phascolarctos cinereus) Using Extended and Extended-Chilled Semen Collected by Electroejaculation

Abstract Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.

Dopaminergic Input to the Ventromedial Hypothalamus Facilitates the Oestrogen-Induced Luteinizing Hormone Surge in Ewes

In this study we examined the release of dopamine and noradrenaline in the ventromedial hypothalamus (VMH) of ovariectomized ewes during the oestrogen-induced luteinizing hormone (LH) surge by measuring their respective metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and methoxyhydroxyphenylglycol (MHPG) using microdialysis. Further we investigated whether inhibition of catecholamine synthesis in the VMH by bilateral reverse dialysis of α-methyl-p-tyrosine (α-MPT) would block the oestrogen-induced LH and/or prolactin surges. Oestradiol treatment (50 µg oestradiol benzoate) of ovariectomized ewes resulted in a biphasic LH response, significantly (p < 0.05) decreasing LH concentrations from 2.5 to 10.5 h after injection, followed by an LH surge beginning at 16 h. Prolactin concentrations were also significantly (p < 0.05) increased in oestradiol-treated ewes from 13 h. VMH DOPAC concentrations in oil-vehicle-treated animals were at the level of detection (0.02 ng/ml) in most samples over the 24-hour sampling period. In oestradiol-treated ewes, VMH DOPAC levels were initially low before and up to 8 h after oestradiol injection but then increased significantly (p < 0.05) at 10–12 h and remained elevated up to 20 h after injection. In contrast, oestradiol injection had no effect on MHPG concentrations in the VMH. Bilateral reverse microdialysis of α-MPT into the VMH significantly (p < 0.05) delayed the time from oestradiol injection to the onset of the LH surge, the time to peak LH concentration and attenuated the LH surge compared with reverse dialysis of Ringer solution alone. In contrast, α-MPT treatment had no effect upon the oestradiol-induced increase in prolactin concentrations. This study provides evidence that the VMH is an important hypothalamic site in the neuro-endocrine control of the LH surge in ewes. The results suggest that dopaminergic neurons with terminals in the VMH are part of a neuronal pathway mediating the positive feedback effects of oestradiol on gonadotropin-releasing hormone secretion and the LH surge.

PACAP stimulates dopamine neuronal activity in the medial basal hypothalamus and inhibits prolactin

In sheep intracerebroventricular injection of PACAP (10 nmol) significantly (P<0.01) stimulated the levels of the dopamine metabolite DOPAC within the medial basal hypothalamus (as measured by in vivo microdialysis) and this effect was temporally correlated with a significant (P<0.05) suppression in peripheral prolactin concentrations. This result is in accord with the hypothesis that PACAP suppresses prolactin secretion from the anterior pituitary gland by stimulating dopamine release from tuberoinfundibular dopaminergic neurons.

Hypothalamic dopamine D1 receptors are involved in the stimulation of prolactin secretion by high environmental temperature in the female sheep

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin‐stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol‐implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 °C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 μl/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390. GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress‐specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.

Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d(2×2×2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.

Control of the koala (Phascolarctos cinereus) anterior pituitary-gonadal axis with analogues of GnRH

The aim of the present study was to determine whether analogues of gonadotrophin-releasing hormone (GnRH) could be used to both induce an acute testosterone response and suppress anterior pituitary function in male koalas, and induce a luteal phase in female koalas. Experiment 1 characterised the steroidogenic response of male koalas to administration of 30 microg (4.3 microg kg(-1)) natural-sequence GnRH. Intra-muscular injection of natural-sequence GnRH induced the release of LH and testosterone with peak concentrations at 30 min (3.7 +/- 1.9 ng mL(-1)) and 2 h (5.4 +/- 0.5 ng mL(-1)), respectively. In Experiment 2, a single injection of the GnRH antagonist acyline (100 microg (14.3 microg kg(-1)) or 500 microg (71.4 microg kg(-1))) did not influence the testosterone response to subsequent injections of natural-sequence GnRH. In Experiment 3, 4 microg (~0.67 microg kg(-1)) of the GnRH agonist buserelin induced a luteal phase in five female koalas based on a LH surge, secretion of progestogen, and a normal-length oestrous cycle. The findings have shown that (1) natural-sequence GnRH can be used to test gonadotroph cell function and determine the testosterone-secreting capacity of male koalas, (2) the GnRH antagonist, acyline, at the dose rates used, does not suppress the pituitary-testis axis in male koalas, and (3) the GnRH agonist, buserelin, induces a normal luteal phase in female koalas.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P<0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P<0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the codon reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin secretion.