Kiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility†.

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values (BVs) for fertility was studied in three groups (n = 8), based on their previous post-partum period: POS cows, which had spontaneous ovarian cycles (POS-CYC) and NEG cows, which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status, and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridization of hypothalamic GNRH1, Kiss1, TAC3, and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminence (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB were similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status and may lead to deficient GnRH and gonadotropin secretion.

Brown adipose tissue thermogenesis in polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with increased obesity with a greater propensity to weight gain and a lack of sustainable lifestyle interventions. Altered brown adipose tissue (BAT) thermogenesis is a potential contributor to obesity in PCOS. BAT activity and modulation have not been studied in PCOS. This observational study explored BAT thermogenesis and its associations in women with and without PCOS. PARTICIPANTS AND METHODS: Cutaneous temperature was recorded from supraclavicular (indicator of BAT activity) and upper arm regions using dataloggers (SubCue, Calgary, Canada) in a cross-sectional substudy, nested within a randomized control trial, of community-recruited premenopausal women with (n = 47, Rotterdam diagnostic criteria) and without (n = 11) PCOS. RESULTS: Complete temperature data were available in 44 PCOS (mean age: 30.0 ± 6.2, mean BMI: 29.3 ± 5.5) and 11 non-PCOS (mean age: 33.0 ± 7.0, mean BMI: 25 ± 3) women. Women with PCOS had lower supraclavicular skin temperature compared to controls overall (33.9 ± 0.7 vs 34.5 ± 1, P < 0.05) and during sleep (34.5 ± 0.6 vs 35.2 ± 0.9, P < 0.001). In the PCOS group, supraclavicular skin temperature overall and over sleep and waking hours correlated inversely with testosterone (r = -0.41 P < 0.05, r = -0.485 P < 0.01 and r = -0.450 P < 0.01 respectively). Testosterone levels explained approximately 15%, 30% and 20% of the variability in supraclavicular skin temperature overall and over sleep and waking hours in women with PCOS, respectively. CONCLUSION: Women with PCOS have lower BAT activity compared to controls. BAT thermogenesis is negatively associated with androgen levels in PCOS.

Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Caloric restriction causes a homeostatic reduction in thermogenesis. We aimed to determine whether exercise could counteract this. We studied four groups of normal-weight ewes ( n = 5), including control sedentary fed ad libitum, exercise fed ad libitum (30 min/d, 5 d/wk), diet-restricted (70% of ad libitum food intake), and combined diet and exercise. Temperature probes implanted in sternal and retroperitoneal adipose tissue and skeletal muscle measured thermogenesis. After the 4-wk intervention, hypothalami were collected for in situ hybridization, and fat and muscle biopsies were collected for real-time PCR and Western blotting. Combined diet and exercise reduced adiposity ( P < 0.05). Caloric restriction alone reduced overnight temperatures in sternal and retroperitoneal fat ( P < 0.05), which was counteracted by exercise ( P < 0.05). Exercise did not induce expression of cellular markers of browning in adipose tissue. There was no effect of diet or exercise on skeletal muscle thermogenesis. Combined diet and exercise increased the expression of neuropeptide Y and agouti-related protein in the hypothalamic arcuate nucleus ( P < 0.05), consistent with reduced adiposity. Gene expressions of key hypothalamic appetite-regulating peptides were not associated with altered thermogenesis. We demonstrate that exercise counteracts the inhibitory effect of caloric restriction to restore thermogenesis in adipose tissue of sheep.-Fuller-Jackson, J.-P., Clarke, I. J., Rao, A., Henry, B. A. Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Lipopolysaccharide reduces gonadotrophin-releasing hormone (GnRH) gene expression: role of RFamide-related peptide-3 and kisspeptin.

RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15µgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.

Triiodothyronine Administration in a Model of Septic Shock: A Randomized Blinded Placebo-Controlled Trial.

OBJECTIVES: Triiodothyronine concentration in plasma decreases during septic shock and may contribute to multiple organ dysfunction. We sought to determine the safety and efficacy of administering triiodothyronine, with and without hydrocortisone, in a model of septic shock. DESIGN: Randomized blinded placebo-controlled trial. SETTING: Preclinical research laboratory. SUBJECTS: Thirty-two sheep rendered septic with IV Escherichia coli and receiving protocol-guided sedation, ventilation, IV fluids, and norepinephrine infusion. INTERVENTIONS: Two hours following induction of sepsis, 32 sheep received a 24-hour IV infusion of 1) placebo + placebo, 2) triiodothyronine + placebo, 3) hydrocortisone + placebo, or 4) triiodothyronine + hydrocortisone. MEASUREMENTS AND MAIN RESULTS: Primary outcome was the total amount of norepinephrine required to maintain a target mean arterial pressure; secondary outcomes included hemodynamic and metabolic indices. Plasma triiodothyronine levels increased to supraphysiological concentrations with hormonal therapy. Following 24 hours of study drug infusion, the amount of norepinephrine required was no different between the study groups (mean ± SD µg/kg; placebo + placebo group 208 ± 392; triiodothyronine + placebo group 501 ± 370; hydrocortisone + placebo group 167 ± 286; triiodothyronine + hydrocortisone group 466 ± 495; p = 0.20). There was no significant treatment effect on any hemodynamic variable, metabolic parameter, or measure of organ function. CONCLUSIONS: A 24-hour infusion of triiodothyronine, with or without hydrocortisone, in an ovine model of septic shock did not markedly alter norepinephrine requirement or any other physiological parameter.

High cortisol responses identify propensity for obesity that is linked to thermogenesis in skeletal muscle.

Subjects characterized as cortisol high responders (HRs) consume more calories after stress, but it is unknown whether cortisol responsiveness predicts a propensity for obesity. Female sheep with either high or low cortisol responses to adrenocorticotropin (ACTH) were identified. Body composition was similar in HRs and cortisol low responders (LRs), but the HRs had greater (P<0.01) adiposity than did the LRs (40.5±0.7 vs. 35.8±1.4%) after high-energy feeding, despite comparable food intake. Postprandial thermogenesis in muscle temperature was 0.8 ± 0.08°C higher in the LRs than in the HRs (P<0.01), whereas feeding-induced changes in fat temperature were similar. Leptin and insulin sensitivity were similar in the HRs and LRs. Feeding lowered (P<0.001) the respiratory control ratio in muscle (HRs 9.2±0.8-5.2±1.2; LRs 8.4±0.5-5.2±0.7), indicative of increased uncoupled respiration. Also in muscle, the feeding-induced increases in uncoupling protein (UCP)-3 (fold increase: HRs, 2.4; LRs, 2.0), ryanodine 1 receptor (RyR1; fold increase: HRs 3.1; LRs 2.1), and sarcoendoplasmic reticulum Ca(2+)-dependent ATPase (fold increase: HRs 1.5; LRs 1.6) were equivalent in the HRs and LRs. Sequencing of mitochondrial DNA revealed no haplotypic differences between the 2 groups. We conclude that predisposition to obesity can be predicted by cortisol responsiveness to an ACTH challenge and that the response is due to innate differences in muscle thermogenesis.

Evidence that Neurokinin B Controls Basal Gonadotropin-Releasing Hormone Secretion but Is Not Critical for Estrogen-Positive Feedback in Sheep.

BACKGROUND: Loss-of-function mutations in genes encoding kisspeptin or neurokinin B (NKB) or their receptors cause infertility. NKB is coproduced in kisspeptin neurons in the arcuate nucleus (ARC), and these neurons also produce the NKB receptor (NK3R), allowing autosynaptic function. We tested the hypothesis that NKB action in ARC kisspeptin neurons is aligned with increased pulsatile secretion of luteinizing hormone (LH) and/or activation of the estrogen-induced LH surge in ewes. METHODS: Using in situ hybridization and immunohistochemistry, we examined NKB expression in kisspeptin neurons during the ovine estrous cycle. We infused kisspeptin, senktide (an NK3R agonist), or dynorphin into the lateral ventricle during the luteal phase of the estrous cycle to determine effects on pulsatile LH secretion. Finally, we examined the effect of an NK3R antagonist (MRK-08) in ovariectomized ewes. RESULTS: NKB (Tac3) mRNA expression in mid-ARC kisspeptin neurons was elevated during the mid-to-late follicular phase of the estrous cycle. The number of NKB-immunoreactive cells and NKB/kisspeptin terminals in the median eminence was similar during the estrous cycle. Kisspeptin and senktide increased LH pulse frequency and mean LH levels. Central MRK-08 infusion eliminated the LH pulses but did not prevent an estrogen-positive feedback on LH secretion. CONCLUSIONS: NKB expression in ARC kisspeptin neurons is upregulated during the late follicular phase of the estrous cycle, when the pulsatile secretion of gonadotropin-releasing hormone (GnRH)/LH is maximal. When GnRH/LH secretion is minimal, central senktide infusion induces LH secretion, similar to the response to kisspeptin. Although the increase in LH in response to senktide appeared surge-like, we did not observe any change in the surge following NK3R antagonist treatment. We conclude that NKB plays a role in increasing basal GnRH/LH pulsatility in the follicular phase of the cycle but is not essential for estrogen-induced positive feedback.

Interface between metabolic balance and reproduction in ruminants: focus on the hypothalamus and pituitary.

This article is part of a Special Issue "Energy Balance". The interface between metabolic regulators and the reproductive system is reviewed with special reference to the sheep. Even though sheep are ruminants with particular metabolic characteristics, there is a broad consensus across species in the way that the reproductive system is influenced by metabolic state. An update on the neuroendocrinology of reproduction indicates the need to account for the way that kisspeptin provides major drive to gonadotropin releasing hormone (GnRH) neurons and also mediates the feedback effects of gonadal steroids. The way that kisspeptin function is influenced by appetite regulating peptides (ARP) is considered. Another newly recognised factor is gonadotropin inhibitory hormone (GnIH), which has a dual function in that it suppresses reproductive function whilst also acting as an orexigen. Our understanding of the regulation of food intake and energy expenditure has expanded exponentially in the last 3 decades and historical perspective is provided. The function of the regulatory factors and the hypothalamic cellular systems involved is reviewed with special reference to the sheep. Less is known of these systems in the cow, especially the dairy cow, in which a major fertility issue has emerged in parallel with selection for increased milk production. Other endocrine systems--the hypothalamo-pituitary-adrenal axis, the growth hormone (GH) axis and the thyroid hormones--are influenced by metabolic state and are relevant to the interface between metabolic function and reproduction. Special consideration is given to issues such as season and lactation, where the relationship between metabolic hormones and reproductive function is altered.

Triiodothyronine supplementation in a sheep model of intensive care.

Triiodothyronine (T3) concentrations in plasma decrease during acute illness and it is unclear if this contributes to disease. Clinical and laboratory studies of T3 supplementation in disease have revealed little or no effect. It is uncertain if short term supplementation of T3 has any discernible effect in a healthy animals. Observational study of intravenous T3 (1 µg/kg/h) for 24 h in a healthy sheep model receiving protocol-guided intensive care supports (T3 group, n=5). A total of 45 endpoints were measured including hemodynamic, respiratory, renal, hematological, metabolic and endocrine parameters. Data were compared with previously published studies of sheep subject to the same support protocol without administered T3 (No T3 group, n=5). Plasma free T3 concentrations were elevated 8-fold by the infusion (pmol/l at 24 h; T3 group 34.9±9.9 vs. No T3 group 4.4±0.3, P<0.01, reference range 1.6 to 6.8). There was no significant physiological response to administration of T3 over the study duration. Supplementation of intravenous T3 for 24 h has no physiological effect on relevant physiological endpoints in healthy sheep. Further research is required to understand if the lack of effect of short-term T3 may be related to kinetics of T3 cellular uptake, metabolism and action, or acute counterbalancing hormone resistance. This information may be helpful in design of clinical T3 supplementation trials.

Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat.

Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterized. Our aim was to use two isogenic strains of a neurotropic virus (pseudorabies, Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague Dawley rats. Retrogradely labeled neurons in the CNS were characterized by immunohistochemistry and PCR. For the latter, laser capture of individual virally labeled neurons was used. All virally labeled brain regions contained neurons projecting to either and both WAT depots. Neurons to abdominal fat were the most abundant in males, whereas females contained a greater proportion of neurons to subcutaneous via private lines and collateral branches. Retrogradely labeled neurons directed to WAT expressed estrogen receptor-α (ERα), and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats, suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic.

Kisspeptin and seasonality of reproduction.

Wild and domesticated species display seasonality in reproductive function, controlled predominantly by photoperiod. Seasonal alterations in breeding status are caused by changes in the secretion of gonadotropin-releasing hormone (GnRH) that are mediated by upstream neuronal afferents that regulate the GnRH cells. In particular, kisspeptin appears to play a major role in seasonality of reproduction, transducing the feedback effect of gonadal steroids as well as having an independent (nonsteroid dependent) circannual rhythm. A substantial body of data on this issue has been obtained from studies in sheep and hamsters and this is reviewed here in detail. Kisspeptin function is upregulated during the breeding season in sheep, stimulating reproductive function, but contradictory data are found in Siberian and Syrian hamsters. The relative quiescence of kisspeptin cells in the nonbreeding season can be counteracted by administration of the peptide, leading to activation of reproductive function. Although there is a major role for melatonin in the transduction of photoperiod to the reproductive system, kisspeptin cells do not appear to express the melatonin receptor, so the means by which seasonality changes the level of kisspeptin activity remains unknown.

The GLP-1 agonist, exendin-4, stimulates LH secretion in female sheep.

Our previous studies showed that microinjection into the median eminence of the sheep of glucagon-like peptide- 1 (GLP-1) or its receptor agonist exendin-4 stimulates luteinising hormone (LH) secretion, but it is unknown whether the same effect may be obtained by systemic administration of the same. The present study measured the response in terms of plasma LH concentrations to intravenous (iv) infusion of exendin-4. A preliminary study showed that infusion of 2 mg exendin-4 into ewes produced a greater LH response in the follicular phase of the oestrous cycle than the luteal phase. Accordingly, the main study monitored plasma LH levels in response to either 0.5 mg or 2 mg exendin-4 or vehicle (normal saline) delivered by jugular infusion for 1 h in the follicular phase of the oestrous cycle. Blood samples were collected at 10 min intervals before, during and after infusion. Both doses of exendin-4 increased mean plasma LH concentrations and increased LH peripheral pulse amplitude. There was no effect on inter-pulse interval or timing of the preovulatory LH surge. These doses of exendin-4 did not alter plasma insulin or glucose concentrations. Quantitative PCR of the gastrointestinal tract samples from a population of ewes confirmed the expression of the preproglucagon gene (GCG). Expression increased aborally and was greatest in the rectum. It is concluded that endogenous GLP-1, most likely derived from the hindgut, may act systemically to stimulate LH secretion. The present data suggest that this effect may be obtained with levels of agonist that are lower than those functioning as an incretin.

Control of GnRH secretion: one step back.

The reproductive system is controlled by gonadotropin releasing hormone (GnRH) secretion from the brain, which is finely modulated by a number of factors including gonadal sex steroids. GnRH cells do not express estrogen receptor α, but feedback is transmitted by neurons that are at least 'one step back' from the GnRH cells. Modulation by season, stress and nutrition are effected by neuronal pathways that converge on the GnRH cells. Kisspeptin and gonadotropin inhibitory hormone (GnIH) neurons are regulators of GnRH secretion, the former being a major conduit for transmission of sex steroid feedback. GnIH cells project to GnRH cells and may play a role in the seasonal changes in reproductive activity in sheep. GnIH also modulates the action of GnRH at the level of the pituitary gonadotrope. This review focuses on the role that kisspeptin and GnIH neurons play, as modulators that are 'one step back' from GnRH neurons.

Postprandial heat production in skeletal muscle is associated with altered mitochondrial function and altered futile calcium cycling.

This study aimed to determine whether postprandial temperature excursions in skeletal muscle are consistent with thermogenesis or altered blood flow. Temperature probes were implanted into the vastus lateralis muscle of ovariectomized ewes, and blood flow was assessed using laser-Doppler flowmetry (tissue flow) and transit-time ultrasound flowmetry (femoral artery flow). The animals were program-fed between 1100 and 1600, and temperature and blood flow were measured during intravenous administration of either isoprenaline or phenylephrine and during feeding and meal anticipation. In addition, muscle biopsies were collected prefeeding and postfeeding to measure uncoupling protein (UCP) expression and mitochondrial function, as well as indices of calcium cycling (ryanodine 1 receptor: RyR1 and sarcoendoplasmic calcium-dependent ATPases SERCA1/ SERCA2a). Isoprenaline increased femoral artery blood flow, whereas phenylephrine reduced blood flow. At high doses only, isoprenaline treatment increased heat production in muscle. Phenylephrine treatment did not alter muscle temperature. Meal anticipation was evoked in fasted animals (previously program-fed) that were housed beside animals that were fed. Increases in muscle temperature were elicited by feeding and meal anticipation, without changes in blood flow during either paradigm. Analyses of respiration in isolated mitochondria indicated that the postprandial increase in heat production was associated with an increase in state 4 respiration, without increased UCP1, UCP2, or UCP3 expression. Feeding increased the expression of RyR1 and SERCA2a. We conclude that excursions in muscle temperature may occur independent of blood flow, suggesting that postprandial heat production is driven by altered mitochondrial function and changes in calcium cycling.

Seasonal variation in the gonadotropin-releasing hormone response to kisspeptin in sheep: possible kisspeptin regulation of the kisspeptin receptor.

Kisspeptin signaling in the hypothalamus appears critical for the onset of puberty and driving the reproductive axis. In sheep, reproduction is seasonal, being activated by short days and inhibited by long days. During the non-breeding (anestrous) season, gonadotropin-releasing hormone (GnRH) and gonadotropin secretion is reduced, as is the expression of Kiss1 mRNA in the brain. Conversely, the luteinizing hormone response to kisspeptin during this time is greater. To determine whether the GnRH response to kisspeptin is increased during anestrus, we utilized hypophysial portal blood sampling. In anestrus ewes, the GnRH and LH responses to kisspeptin were greater compared to the breeding season (luteal phase). To ascertain whether this difference reflects a change in Kiss1r, we measured its expression on GnRH neurons using in situ hybridization. The level of Kiss1r was greater during the non-breeding season compared to the breeding season. To further examine the mechanism underlying this change in Kiss1r, we examined Kiss1r/GnRH expression in ovariectomized ewes (controlling for sex steroids) during the breeding and non-breeding seasons, and also ovariectomized non-breeding season ewes with or without estradiol replacement. In both experiments, Kiss1r expression on GnRH neurons was unchanged. Finally, we examined the effect of kisspeptin treatment on Kiss1r. Kiss1r expression on GnRH neurons was reduced by kisspeptin infusion. These studies indicate the kisspeptin response is indeed greater during the non-breeding season and this may be due in part to increased Kiss1r expression on GnRH neurons. We also show that kisspeptin may regulate the expression of its own receptor.

Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding.

OBJECTIVE: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals. MATERIALS AND METHODS: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion. RESULTS: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep). CONCLUSIONS: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals.

Onset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin.

The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal oestrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18-25 days postpartum on the day of treatment (D0). Groups of eight cows received saline (Sal) vehicle by intramuscular injection at 8:00 and 16:00 h (Sal-Sal), Kp at 8:00 h and vehicle at 16:00 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The ovaries of the cows were examined daily by ultrasonography between D4 and D14. Blood samples were collected from a tail vessel at 0, 2, 4, 8, 10 and 12 h relative to the time of the first injection for luteinizing hormone (LH) and follicle-stimulating hormone assay. Additional samples were collected daily from D4 until D14 and D19, 22, 26 and 29 for progesterone assay. LH surge-like responses were observed in cows treated with Kp at 8:00 h. Ovulation was consistently induced by Kp within 48 h when a dominant ovarian follicle of at least 10 mm in diameter was observed (8/14) but in no cases (6/14) during a new wave of ovarian follicular development comprising follicles <10 mm in diameter. The subsequent ovulatory cycle was of normal length in most cases as compared with short 8- to 12-day cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing oestrous cycles of normal length, if administered when a mature dominant follicle is present in the ovaries. LAY SUMMARY: Cow fertility is important for efficient, profitable dairy farming. Cows that take too long after calving to become fertile are problematic. We tested a synthetically made, long-acting hormone called kisspeptin (Kp) to advance the time that cows become fertile after calving. Twenty-four dairy cows that had been calved for 3-4 weeks were used. One group of eight cows received an injection of Kp at the morning milking, another eight cows received Kp at both the morning and afternoon milking, while the last group of eight cows served as untreated controls. Kp treatment caused a desirable hormone response from the cows' brain. Normal oestrous cycles resulted, but only when a mature follicle was present in the ovary. Further study is required to analyse whether the use of a long-acting Kp drug could be used as an effective treatment for stimulating dairy cows to become more fertile after calving.

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction.

BACKGROUND: Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. METHODS: Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. RESULTS: Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001's bioactivity: MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.

Central infusion of leptin does not increase AMPK signaling in skeletal muscle of sheep.

In sheep, central leptin infusion reduces food intake and increases energy expenditure in adipose tissue and skeletal muscle. The mechanisms for these peripheral effects of central leptin in sheep are not known but, on the basis of rodent studies, may involve AMPK. In mice, central leptin acutely increases both skeletal muscle AMPK activation and glucose uptake. Thus, to investigate whether these effects exist in higher-order mammals, ovariectomized Corriedale ewes (n = 4 per group) received a continuous lateral ventricular infusion (60 µl/h) of either leptin (50 µg/h) or artificial cerebrospinal fluid (aCSF; CON) for 8 days. Tritiated glucose (3-(3)H-glucose) was infused intravenously for calculation of whole body glucose turnover during both acute (6 h) and chronic (7-8 days) leptin/aCSF infusion. Muscle biopsies were also obtained. Leptin infusion reduced (P < 0.05) food intake and body weight, and it also increased plasma epinephrine concentration at 6 h and 7 days, suggesting increased sympathetic nerve activity. Despite this, and in contrast to rodent studies, central leptin infusion did not increase skeletal muscle AMPKα Thr(172) phosphorylation or ACCß Ser(221) phosphorylation. Surprisingly, the glucose rate of appearance (glucose Ra) and rate of disappearance (glucose Rd) were reduced by both acute and chronic leptin infusion. Direct infusion of the AMPK activator 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) into the femoral artery increased skeletal muscle AMPK phosphorylation. In conclusion, although central leptin infusion in sheep caused the predicted reduction in food intake and increases plasma epinephrine concentration, it had no effect on AMPK activation in skeletal muscle and actually reduced glucose disposal. This suggests that there are species differences in the peripheral responses to central leptin infusion.

Glucagon-like peptide-1 control of GnRH secretion in female sheep.

The role of glucagon-like peptide-1 (GLP-1) on gonadotropin-releasing hormone (GnRH) secretion was investigated in ovariectomised (OVX) ewes, in which GnRH and luteinising hormone (LH) secretion had been restrained by treatment with oestrogen and progesterone. Guide tubes for microinjection were placed above the median eminence (ME) and the animals were allowed to recover for 1 month. Jugular venous blood samples were taken via cannulae at 10 min intervals. Vehicle (50 nL) was injected into the ME at 2 h, followed by injection of GLP-1 ((7-36)-amide - 0.5 or 1 nmol) or its receptor agonist, exendin-4 (0.5 nmol) at 4 h (n = 5). Plasma LH levels were quantified as a surrogate measure of GnRH secretion. GLP-1 microinjection into the ME elicited a large amplitude LH pulse in jugular plasma, the effect was greater at the higher dose. Exendin-4 microinjection caused a large, sustained increase in plasma LH levels. To determine how GLP-1 might exert an effect on GnRH secretion, we employed double labelled in situ hybridisation, with RNAScope, for co-localisation of the GLP-1 receptor (GLP-1R) in GnRH, Kisspeptin and NPY cells in the hypothalami of three ewes in the luteal phase of the estrous cycle. GLP1R expression was clearly visible but the receptor was not expressed in GNRH1 or NPY expressing neurons and was visualised in <5% of KISS1 expressing neurons. We conclude that GLP-1 may act at the level of the secretory terminals of GnRH neurons in the ME to stimulate GnRH secretion, the pathway through which such effect is manifested remains unknown.