Margaret Blackberry

Expression of orexin receptors in the brain and peripheral tissues of the male sheep

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.

Use of a new drug delivery formulation of the gonadotrophin-releasing hormone analogue Deslorelin for reversible long-term contraception in male dogs

In the present study, we tested the effect of treatment with a slow-release implant containing the gonadotrophin-releasing hormone agonist Deslorelin(TM) (Peptech Animal Health Australia, North Ryde, NSW, Australia) on pituitary and testicular function in mature male dogs. Four dogs were treated with Deslorelin (6-mg implant) and four were used as controls (blank implant). In control dogs, there were no significant changes over the 12 months of the study in plasma concentrations of luteinising hormone (LH) or testosterone, or in testicular volume, semen output or semen quality. In Deslorelin-treated dogs, plasma concentrations of LH and testosterone were undetectable after 21 and 27 days, testicular volume fell to 35% of pretreatment values after 14 weeks and no ejaculates could be obtained after 6 weeks. Concentrations returned to the detectable range for testosterone after 44 weeks and for LH after 51 weeks and both were within the normal range after 52 weeks. Semen characteristics had recovered completely by 60 weeks after implantation. At this time, the testes and prostate glands were similar histologically to those of control dogs. We conclude that a single slow-release implant containing 6 mg Deslorelin has potential as a long-term, reversible antifertility agent for male dogs.

Seasonality in male Australian cashmere goats: Long term effects of castration and testosterone or oestradiol treatment on changes in LH, FSH and prolactin concentrations, and body growth

Abstract Australian cashmere goat bucks exhibit marked seasonal changes in gonadotrophin secretion and growth. We investigated the mechanisms underlying these changes by testing the effects of castration and gonadal steroid replacement in grazing bucks at 29 °S, 153 °E over a period of 2 years. Three year old bucks were either left intact ( n = 6), castrated ( n = 6), or castrated and treated with testosterone propionate ( n = 5) or oestradiol-17β ( n = 5). Oestradiol- 17β (E 2 ) was provided by 2 subcutaneous Silastic ® implants while testosterone propionate (TP) was provided by implanting four 23.5 mg pellets subcutaneously every 12 weeks. Intact bucks exhibited marked circannual cycles of plasma LH, FSH, prolactin and testosterone concentration, body mass, paired testicular mass and male odour. Castration resulted in long term increases in LH and FSH concentration and abolition of the circannual pattern of change in these hormones without affecting prolactin concentrations. It also reduced the magnitude of seasonal changes in body mass. Treatment of castrates with TP, which produced peripheral concentrations of testosterone in the lower physiological range (mean 1.2 ± 0.08 μg L −1 ), had no effect on plasma concentrations of LH, FSH or prolactin. On the other hand treatment with E 2 , which produced E 2 concentrations in the mid physiological range (mean 12.3 ± 1.08 ng L −1 ), re-imposed a circannual pattern in plasma LH and FSH, albeit significantly different from that of intact bucks. It had no effect on prolactin concentrations. Treatment with both steroids resulted in growth cycles of intermediate magnitude between those of intact bucks and castrates. We conclude that the circannual cycle of gonadotrophin concentrations in goat bucks is dependent upon the presence of the testis, and suggest that circulating metabolites of testosterone, including oestradiol, are more important than testosterone itself in the maintenance of this cycle. We also suggest that the seasonal growth cycle observed in these bucks is driven by factors other than prevailing nutrition, probably photoperiod, and is mediated by both steroid-dependent and independent mechanisms.

Decrease in voluntary feed intake and pulsatile luteinizing hormone secretion after intracerebroventricular infusion of recombinant bovine leptin in mature male sheep.

The aim of the present study was to determine whether leptin might play a role in the gonadotrophic response of mature merino rams to changes in the level of nutrition in rams fed ad libitum. Recombinant bovine leptin was infused intracerebroventricularly and voluntary food intake (VFI) and luteinizing hormone (LH) pulse frequency were measured. In Experiment 1, rams (n = 5) were infused for 24 h per day for 5 days with vehicle or with leptin (0.04, 0.4 and 4.0 microg h(-1)). All doses decreased both VFI and LH pulse frequency. In Experiment 2, rams were infused for 24 h per day for 5 days with vehicle (n = 10) or leptin (4 pg h(-1); n = 5); a sub-group of 5 controls was pair-fed to the leptin-infused group to control for effects of changes in feed intake. LH pulse frequency was reduced equally in both the leptin-infused and pair-fed groups. Leptin did not affect other systems controlled by the hypothalamic-pituitary axis. Thus, rather than stimulate LH secretion, intracerebral leptin specifically inhibits it by reducing food intake, so it is unlikely that effects of nutrition on the reproductive axis in mature rams involves leptin as a single blood-borne signal. A range of nutritional or metabolic inputs may be needed, and perhaps interconnections between neural centres that control appetite and reproduction.

Pituitary and testicular endocrine responses to exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone in male dogs treated with GnRH agonist implants

The present study tested whether exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) can stimulate LH and testosterone secretion in dogs chronically treated with a GnRH superagonist. Twenty male adult dogs were assigned to a completely randomised design comprising five groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other four groups received a 6-mg implant containing a slow-release formulation of deslorelin (d-Trp6-Pro9-des-Gly10-LH-releasing hormone ethylamide). The same four control dogs were used for all hormonal challenges, whereas a different deslorelin-implanted group was used for each challenge. Native GnRH (5 microg kg(-1) bodyweight, i.v.) was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 microg kg(-1) bodyweight, i.v.) was injected on Days 16, 26, 41 and 101. On all occasions after Day 25-26 postimplantation, exogenous GnRH and LH elicited higher plasma concentrations of LH and testosterone in control than deslorelin-treated animals (P < 0.05). It was concluded that, in male dogs, implantation of a GnRH superagonist desensitised the pituitary gonadotrophs to GnRH and also led to a desensitisation of the Leydig cells to LH. This explains, at least in part, the profound reduction in the production of androgen and spermatozoa in deslorelin-treated male dogs.

Dose-Response Studies for Pituitary and Testicular Function in Male Dogs Treated with the GnRH Superagonist, Deslorelin

We tested the effect of dose of GnRH superagonist on pituitary and testicular function in a study with four groups of four male dogs. The Controls received blank implants and the other three groups received implants containing 3, 6 or 12 mg deslorelin (D-Trp6-Pro9-des-Gly10-GnRH ethylamide). In all deslorelin-treated groups, there was initially an acute increase in plasma concentrations of LH and testosterone, followed by declines such that both hormones became undetectable after approximately 12 days. There was a dose-response in some of these early aspects of the hormone profiles. With respect to long-term effects of treatment, the 12-mg dose had significantly greater effects than the smaller doses for the duration of minimum testicular volume [366 +/- 77, mean +/- SEM (3 mg), 472 +/- 74 (6 mg), and 634 +/- 59 (12 mg) days], absence of ejaculate [416 +/- 88 (3 mg), 476 +/- 83 (6 mg), and 644 +/- 67 (12 mg) days], undetectable plasma concentrations of LH and testosterone [367 +/- 64 (3 mg), 419 +/- 72 (6 mg), and 607 +/- 69 (12 mg) days], the delay until complete recovery of LH and testosterone secretion [394 +/- 65 (3 mg), 484 +/- 72 (6 mg) and 668 +/- 47 (12 mg) days], and the delay until testes had regrown to normal volume [408 +/- 77 (3 mg), 514 +/- 74 (6 mg), 676 +/- 59 (12 mg) days]. The time taken to restore full ejaculates was also longest for the 12-mg dose: 716 +/- 67 (12 mg) days vs 440 +/- 66 (3 mg) and 538 +/- 83 (6 mg) days after implantation. There was no correlation between delay to recovery of normal ejaculate quality and body mass. We conclude that the dose-response relationship with deslorelin implants is not expressed with respect to the degree of suppression of reproduction, but on the maximum duration of suppression and thus to delay until recovery.

Photoperiodic control of the concentration of luteinizing hormone, prolactin and testosterone in the male emu (Dromaius novaehollandiae), a bird that breeds on short days.

The objective of this study was to establish, for a short‐day breeding bird, the male emu, whether the breeding season is principally controlled by changes in photoperiod, and to investigate the endocrine mechanisms involved. Two groups of adult males were subjected to three alternating periods of 150–185 days of 14 h light/day (LD) and 10 h light/day (SD) terminating in a 360‐day period of LD or SD. Transfer from LD to SD led to increases in plasma concentrations of luteinizing hormone (LH) and testosterone, after 82 ± 8 and 73 ± 3 (SEM) days, and an increase in prolactin concentrations after 115 ± 12 days. Concentrations of LH and testosterone began to decrease before transfer back to LD, at a time when prolactin concentrations were approaching peak values. Transfer from LD to 360 days of SD resulted in increases in LH and testosterone concentrations, and these terminated after an increase in prolactin concentrations. After transfer from SD to 360 days of LD, plasma concentrations of LH and testosterone began to increase, after delays of 222 ± 24 and 225 ± 13 days, and were high at the end of the study, while prolactin values remained depressed throughout. These observations clearly show that seasonal breeding in the emu is directly controlled by changes in photoperiod. The dynamics of the hormonal responses to change of photoperiod suggest that, despite being short‐day breeders, the photoregulation of breeding in emus involves mechanisms that are currently accepted for birds, rather than mechanisms that have been proposed for short‐day breeding mammals. The initiation of breeding in emus is due to dissipation of photorefractoriness by short days which leads to an increase in the secretion of gonadotrophins to levels that are sufficient to support full reproductive condition. The termination of breeding, while days are still short, is due to the antigonadotrophic action of prolactin which, unusually for birds, increases while the days are still short. In conclusion, breeding activity in male emus is strongly controlled by photoperiod. Emus are short‐day breeders, but the central mechanisms that regulate the secretion of reproductive hormones seem to be similar to those previously proposed for long‐day breeding birds. The pattern of prolactin secretion in emus suggests an important role for this hormone in the termination of the breeding cycle.

Dynamics of the responses in secretion of luteinising hormone, leptin and insulin following an acute increase in nutrition in mature male sheep

In Merino rams, an increase in the plane of nutrition increases the frequency of luteinising hormone (LH) pulses within a few days and this response is correlated with changes in the circulating concentrations of metabolic hormones. To analyse early dynamic aspects of these responses, we studied mature rams fed with diets that contained either low or high amounts of energy and protein. Jugular blood was sampled every 20 min for 96 h, including a control period of 24 h before the change of diet in the high-diet group. In the high-diet group, a significant increase in LH pulse frequency was first detected 6 h after the increase in nutrition on Day 1 and the frequency remained significantly elevated throughout the 72-h treatment period, except for a 12-h period on Day 2. Following the change of diet, insulin concentrations increased within 3 h and leptin concentrations increased within 7 h, after which time the concentrations of both hormones remained high. Dietary treatment did not affect the concentrations of thyroxine or insulin-like growth factor-I, but the high diet increased the concentrations of tri-iodothyronine. These observations are consistent with insulin and leptin playing a role in the early activation of the gonadotrophin-releasing hormone-LH axis by nutritional inputs.

Relationships between plasma concentrations of leptin and other metabolic hormones in GH-transgenic sheep infused with glucose.

To study the regulation of leptin secretion in sheep, we infused glucose (0.32 g/h/kg for 12 h) into GH-transgenic animals (n = 8) that have chronically high plasma concentrations of ovine GH and insulin, but low body condition and low plasma leptin concentrations, and compared the responses with those in controls (n = 8). In both groups, the infusion increased plasma concentrations of glucose and insulin within 1 h and maintained high levels throughout the infusion period (P < 0.0001). Compared with controls, GH-transgenics had higher concentrations of insulin, IGF-1, GH (all P < 0.0001) and cortisol (P < 0.05), but lower GH pulse frequency (P < 0.0001). Overall, leptin concentrations were lower in GH-transgenics than in controls (P < 0.01). A postprandial increase in leptin concentrations was observed in both groups, independently of glucose treatment, after which the values remained elevated in animals infused with glucose, but returned to basal levels in those infused with saline, independently of transgene status. In both GH-transgenics and controls, glucose infusion did not affect the concentrations of GH, IGF-1, or cortisol. In conclusion, GH-transgenic and control sheep show similar responses to glucose infusion for leptin and other metabolic hormones, despite differences between them in body condition and basal levels of these hormones. Glucose, insulin, GH, IGF-1 and cortisol are probably not major factors in the acute control of leptin secretion in sheep, although sustained high concentrations of GH and IGF-1 might reduce adipose tissue mass or inhibit leptin gene expression.

Role of glucose, fatty acids and protein in regulation of testicular growth and secretion of gonadotrophin, prolactin, somatotrophin and insulin in the mature ram

This study tested whether the effects of nutrition on gonadotrophin secretion and testicular growth in mature rams are due to increases in the supply of glucose, fatty acids (FA) or amino acids. Responses to protein (casein) and glucose, alone or in combination, were compared with responses to lupin grain and responses to a combination of protein, glucose and FA (acetate, propionate and vegetable oil). Glucose and casein were infused intra-abomasally whereas lupins and FA were added to the diet. Lupin feeding decreased blood growth hormone (GH) concentrations, but increased pulsatile luteinizing hormone (LH) secretion and increased the concentrations of follicle-stimulating hormone (FSH), prolactin, glucose and insulin. These effects were associated with testicular growth. Glucose or casein increased insulin concentrations and decreased GH concentrations, but did not affect gonadotrophins or testicular growth. There was no synergism between casein and glucose. Responses elicited by adding FA to the glucose+casein treatment were similar to those observed with lupins. In conclusion, the reproductive axis does not seem to be closely linked with dietary intakes of amino acids or with circulating concentrations of glucose, insulin or GH. However, the energetic components of the diet, particularly the fatty acids, appear to play a key role in the reproductive responses to changes in nutrition.