Involvement of orexin A in nocturnal melatonin secretion into the cerebrospinal fluid and the blood plasma in seasonal sheep.

In sheep, differences in orexin A (OXA) gene expression and activity are related to changes in energy demand and seasonal reproduction. However, the mechanism by which and the key place where the OXA signal is integrated with photoperiod, whose main biochemical expression is melatonin (MEL), remain unknown. We examined the effects of cisterna magna injections of OXA (0.3 µg/kg body weight) on nocturnal cerebrospinal fluid (CSF) and plasma MEL concentrations; mRNA and protein expression of two rate-limiting enzymes for MEL biosynthesis, tryptophan 5-hydroxylase-1 (TPH1) and arylalkylamine-N-acetyltransferase (AA-NAT); and OXA receptor (OX1R, OX2R) expression in the pineal gland (PG) obtained from twenty ewes during the short-day (SD) and long-day (LD) seasons. OXA increased (P < 0.001) CSF and plasma MEL concentrations regardless of the season. Plasma MEL was positively correlated (P < 0.001) with CSF MEL in the OXA-treated sheep in both seasons. OXA had no effect (P > 0.05) on TPH1 transcript or protein level but upregulated (P < 0.05) AA-NAT mRNA and protein expression in both seasons. OXA enhanced (P < 0.05) OX1R mRNA level only during the LD season. Our results show that the endocrine activity of the ovine PG is regulated by day length and non-photic signals via hypothalamic OXA. These results are important for understanding the work of the biological clock and recognizing mechanisms responsible for the adaptation of seasonal animals to the changing external environment conditions. OXA and MEL are both involved in the regulation of the sleep-wakefulness system, therefore our results can be used in the study on the circadian rhythm disorders in humans (e.g. jet lag, insomnia, seasonal depression).

Pregnancy-induced changes in the transcript levels of prolactin receptor and its suppressor in the ovine hypothalamus and adenohypophysis.

The aim of this study was to analyse changes in the abundance of prolactin (PRL) receptor (PRLR) and suppressor of cytokine signalling-3 (SOCS-3) mRNA in the ventro-/dorsomedial nucleus (VMH/DMH) and arcuate nucleus (ARC) of the hypothalamus as well as in the median eminence (ME) and adenohypophysis (AP) in sheep at 30, 60, 90 and 120 d of pregnancy compared to non-pregnant animals. In the VMH/DMH, PRLR transcripts were detected only in non-pregnant ewes. In the ARC, the abundances of PRLR mRNA were higher in pregnant sheep on days 30 (p < .01), 90 (p < .01) and 120 (p < .05) than in non-pregnant sheep. In contrast, the expression of PRLR mRNA in the ME was lower (p < .01) in pregnant ewes at days 30 and 60 than in non-pregnant ewes and was undetectable at later stages of gestation. In all studied stages of pregnancy except day 60, the abundance of PRLR mRNA was higher (p < .01) in the ARC than in the AP, while in non-pregnant sheep, there were no differences (p ≥ .05) in the transcript levels between these two tissues. In non-pregnant ewes, the abundance of SOCS-3 mRNA in the AP was lower than that in any other studied tissue (p < .05-p < .01). In conclusion, the observed changes in PRLR and SOCS-3 mRNA abundance in the hypothalamus and AP during pregnancy may be important components of the mechanisms regulating the action of PRL in energy homeostasis and neuroendocrine interactions within the hypothalamic-pituitary axis.

The Influence of Photoperiod on the Action of Exogenous Leptin on Gene Expression of Proinflammatory Cytokines and Their Receptors in the Thoracic Perivascular Adipose Tissue (PVAT) in Ewes.

Leptin resistance is either a condition induced by human obesity or a natural phenomenon associated with seasonality in ruminants. In the cardiovascular system, the leptin resistance state presence is a complex issue. Moreover, the perivascular adipose tissue (PVAT) appears to be crucial as a source of proinflammatory cytokines and as a site of interaction for leptin contributing to endothelium dysfunction and atherosclerosis progression. So the aim of this study was to examine the influence of the photoperiod on the action of exogenous leptin on gene expression of selected proinflammatory cytokines and their receptors in thoracic PVAT of ewe with or without prior lipopolysaccharide (LPS) stimulation. The experiment was conducted on 48 adult, female ewes divided into 4 group (n = 6 in each): control, with LPS intravenous (iv.) injection (400 ng/kg of BW), with leptin iv. injection (20 µg/kg BW), and with LPS and 30-minute-later leptin injection, during short-day (SD) and long-day (LD) seasons. Three hours after LPS/control treatment, animals were euthanized to collect the PVAT adherent to the aorta wall. The leptin injection enhanced IL1B gene expression only in the LD season; however, in both seasons leptin injection intensified LPS-induced increase in IL1B gene expression. IL1R2 gene expression was increased by leptin injection only in the SD season. Neither IL6 nor its receptor and signal transducer gene expressions were influenced by leptin administration. Leptin injection increased TNFA gene expression regardless of photoperiodic conditions. Only in the SD season did leptin treatment increase the gene expression of both TNFα receptors. To conclude, leptin may modulate the inflammatory reaction progress in PVAT. In ewe, the sensitivity of PVAT on leptin action is dependent upon the photoperiodic condition with stronger effects stated in the SD season.

Downregulation of LRb in VMH/DMH during the second half of gestation and upregulation of SOCS-3 in ARC in late-pregnant ewes - Implications for leptin resistance.

To investigate factors involved in pregnancy-induced regulation of tissue sensitivity to leptin, we determined leptin concentrations and expression levels of the long form of the leptin receptor (LRb) and suppressor of cytokine signalling (SOCS)-3 in the ventro- and dorsomedial nuclei (VMH/DMH), arcuate nucleus (ARC), median eminence (ME) and anterior pituitary (AP) in 15 Polish Longwool ewes euthanized at 30, 60, 90 and 120 days of pregnancy and before gestation (n = 3 per group). Leptin concentrations increased during the first half of pregnancy, peaked on day 60, and then declined. In the VMH/DMH, LRb mRNA levels decreased from day 60 of pregnancy; in the ARC, LRb mRNA levels remained stable before and throughout pregnancy. LRb expression in the ME was lower in the first two months of pregnancy than before pregnancy (P < 0.01) and peaked at day 90. In the AP, LRb mRNA levels were higher during mid-pregnancy (P < 0.05) than before pregnancy. SOCS-3 expression in the VMH/DMH was higher throughout gestation (P < 0.05) than before pregnancy but was undetectable at day 120. SOCS-3 transcript levels were higher in the ARC (P < 0.05) in late-pregnancy (at day 120) than in non-pregnant ewes. SOCS-3 mRNA levels in the ME were lower at days 30 and 60 (P < 0.05) than at day 120 or before pregnancy. In the AP, SOCS-3 transcription was stable throughout gestation except at day 120, when it increased (P < 0.05). The changes in plasma leptin concentrations during pregnancy, hypothalamic LRb downregulation in the VMH/DMH during the second half of gestation and SOCS-3 upregulation in the ARC in late-pregnant ewes identified here may be essential components of the mechanisms driving ovine leptin insensitivity.

Resistin regulates reproductive hormone secretion from the ovine adenohypophysis depending on season.

Work in cattle and rodents has shown that resistin, in addition to its roles in insulin resistance and inflammation, is involved in the regulation of gonadal steroidogenesis. However, the role of resistin in the regulation of reproductive processes in other species, such as seasonally breeding sheep, is completely unknown. Herein, we tested the hypothesis that resistin can influence the secretion of anterior pituitary hormones and that its effect in ewes is dependent on the day length. Thirty Polish Longwool ewes, a breed that exhibits a strong seasonal reproductive pattern, were ovariectomized with estrogen replacement using subcutaneously inserted estradiol implants. Ewes were fed ad libitum and housed under a natural photoperiod (longitude: 19°57' E, latitude: 50° 04' N). Intravenous treatments consisted of control or recombinant bovine resistin (rbresistin) in saline: (1) control (saline; n = 10), (2) low resistin dose (1.0 µg/kg BW; n = 10), and (3) high resistin dose (10.0 µg/kg BW; n = 10). Experiments were conducted during both short-day (SD) and long-day (LD) seasons using 5 sheep per group within each season. Blood samples were collected every 10 min over 4 h. Blood plasma concentrations of FSH, LH, and prolactin (PRL) were assayed using RIA. A season × dose interaction was observed for all hormonal variables measured. Greater concentrations (P < 0.001) of LH and FSH were observed during SDs than during LDs in all groups. During SDs, the high dose (10 µg/kg BW) decreased (P < 0.001) basal LH levels and amplitude (P < 0.05) of LH pulses and increased (P < 0.001) circulating concentrations of FSH. However, the low dose of resistin decreased (P < 0.001) FSH concentrations compared to those of controls. During LDs, both the low and high resistin doses increased mean concentrations of LH (P < 0.001 and P < 0.05, respectively) and FSH (P < 0.001). A high dose of rbresistin increased (P < 0.001) the mean circulating concentrations of PRL during both seasons. However, in all groups, concentrations of PRL were greater during LDs than SDs. These results demonstrate for the first time that resistin is involved in the regulation of pituitary hormone secretion and that this effect is differentially mediated during LDs and SDs.

The effects of leptin on plasma concentrations of prolactin, growth hormone, and melatonin vary depending on the stage of pregnancy in sheep.

The effects of hyperleptinemia and leptin resistance during gestation are unclear. Leptin, an important neuroendocrine regulator, has anorexic effects, but its interactions with other metabolic hormones during pregnancy are unclear. We examined potential roles of leptin in regulating prolactin (PRL), GH, and melatonin plasma concentrations during pregnancy in Polish Longwool ewes. Twelve estrus-synchronized ewes carrying twins after mating were randomly assigned to receive i.v. injections of saline or recombinant ovine leptin (2.5 or 5.0 µg/kg BW). Blood samples were collected (15-min intervals over 4 h) immediately before the first injection at dusk and kept under red light. Treatments were repeated at 2-wk intervals, starting before mating and continuing from days 30 to 135 of gestation. Concentrations of plasma PRL, GH, and melatonin were determined using a validated RIA. The effects of leptin on hormone plasma concentrations varied depending on pregnancy stage and leptin dose. PRL plasma concentrations were affected at most stages of pregnancy and before gestation. In non-, very early- (day 30), and late- (day 120 and 135) pregnant ewes, exogenous leptin stimulated PRL (P < 0.001) plasma concentrations, while during the second month of gestation, it decreased PRL concentrations (P < 0.01). Leptin affected GH plasma concentrations (P < 0.05) only during the first 2 mo of pregnancy, with no effects during the second part of gestation or before pregnancy. In early-pregnant ewes (day 30 and 45), leptin decreased melatonin plasma concentrations (P < 0.05), but at day 60, leptin stimulated melatonin plasma concentrations at low (P < 0.01) and high doses (P < 0.05), with no effects in ewes after 105 d of gestation. These data indicate specific pregnancy-induced endocrine adaptations to changes in energy homeostasis, supporting the hypothesis that leptin affects PRL, GH, and melatonin release during gestation.

Comparison of the effects of pretreatment with Veramix sponge (medroxyprogesterone acetate) or CIDR (natural progesterone) in combination with an injection of estradiol-17ß on ovarian activity, endocrine profiles, and embryo yields in cyclic ewes superovulated in the multiple-dose Folltropin-V (porcine FSH) regimen.

Follicular wave status at the beginning of exogenous FSH administration is an important contributor to variability in superovulatory responses in ruminants. Studies in ewes have shown a decrease in the number of ovulations when superovulation is initiated in the presence of ostensibly ovulatory-sized ovarian follicles. Hormonal ablation of large antral follicles with the progestin-estradiol (E2-17ß) treatment significantly reduces this variability in superovulated anestrous ewes, but the effects of the treatment in cycling ewes have not yet been assessed. Sixteen Rideau Arcott × Polled Dorset ewes (November-December) received either medroxyprogesterone acetate (MAP)-releasing intravaginal sponges (60 mg) or controlled internal drug release (CIDR) devices (containing 300 mg of natural progesterone) for 14 days (Days 0-14), with a single intramuscular injection of 350 µg of E2-17ß on Day 6. The superovulatory treatment consisted of six injections of porcine FSH (Folltropin-V) given twice daily, followed by a bolus GnRH injection (50 µg intramuscular) on Day 15. There were no differences (P < 0.05) in the ovulatory responses and embryo yields between the two groups of ewes. In both subsets of animals, the next follicular wave emerged ∼2.5 days after an E2-17ß injection (P > 0.05). A decline in maximum follicle size after an E2-17ß injection was more abrupt in CIDR- compared with MAP-treated animals, and the ewes pretreated with exogenous progesterone had significantly more 3-mm follicles at the start of the superovulatory treatment. The metabolic clearance rate of exogenous E2-17ß appeared to be greater in MAP-treated ewes, but circulating concentrations of porcine FSH failed to increase significantly after each Folltropin-V injection in CIDR-treated animals. The CIDR-treated ewes exceeded (P < 0.05) their MAP-treated counterparts in serum E2-17ß concentrations during superovulation. In spite of differences in antral follicle numbers and endocrine profiles between MAP- and CIDR-treated cyclic ewes receiving E2-17ß before ovarian superstimulation, there were no differences in superovulatory responses.

Suitability of antral follicle counts and computer-assisted analysis of ultrasonographic and magnetic resonance images for estimating follicular reserve in porcine, ovine and bovine ovaries ex situ.

This study was conducted to determine if correlations exist between the numbers of microscopic follicles comprising ovarian follicular reserve (OFR) and antral follicle counts (AFCs), and to assess the usefulness of computerized analyses of ovarian ultrasonograms and magnetic resonance (MR) images for estimating OFR in excised porcine, ovine and bovine ovaries. As a pre-requisite to these analyses, we characterized and compared ovarian cortical histomorphology and follicle populations in the three species varying in prolificacy and overall reproductive longevity, and hence the total number of microscopic and antral follicles. Ultrasonographic and MR images were obtained at the scanner settings optimized to provide opposing contrasts between antral follicles and the ovarian stroma. Commercially available ImageProPlus® analytical software was used to calculate numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of the pixel values) along the computer-generated lines (4-6) placed in the area corresponding to the ovarian cortex. The numbers of primordial (r = 0.38, P < 0.01) and intermediate follicles (r = 0.37, P < 0.01) were correlated with the numbers of antral follicles in bovine ovarian sections. The numbers of primordial (r = 0.28, P < 0.05), intermediate (r = 0.31, P < 0.01) and primary follicles (r = 0.27, P < 0.05) correlated directly with mean NPVs of the ultrasonographic ovarian images in cattle. There was a negative correlation between primary follicle numbers and NPVs of MR images (3D FAST-SPOILED GRADIENT ECHO) of the porcine ovarian cortex (r = -0.31, P < 0.05). To summarize, the numbers of primordial and intermediate follicles could only be estimated from AFCs in cows. Using ultrasound NPVs, the numbers of primordial, intermediate and primary follicles could be directly estimated in bovine ovaries and the quantitative image attributes of MR images were useful for quantifying porcine primary follicles. The bovine ovarian model is compatible with human situation and hence future studies should be undertaken to ascertain the usefulness of AFCs and ultrasonographic image analyses for estimating OFR in women.

The utility of nanowater for ram semen cryopreservation.

Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes.

Correlations among antral follicular echotexture, apoptosis and expression of key steroidogenic enzymes in sheep.

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3ß-hydroxysteroid dehydrogenase, 3ß-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3ß-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.

A review on the effect of the photoperiod and melatonin on interactions between ghrelin and serotonin.

Ghrelin and serotonin, which exhibit rhythmic secretion profiles under feeding/fasting conditions, are sensitive to increases and decreases in the day length and form a close web of interrelationships in the regulation of energy homeostasis. Ghrelin and serotonin are biochemically and functionally linked to the suprachiasmatic nucleus, which is a circadian pacemaker, and melatonin, which is an internal transducer of photic environmental changes. Ghrelin and serotonin might be candidates for integrating photic and nonphotic signals, such as light and food availability in the central nervous system. The mechanisms that convert a light signal into a variety of physiological and behavioral rhythms remain unknown. However, we know that the conversion of light signals is necessary to maximize an animal's chances of survival and reproduction.

[The discovery of neuromedin U and its pivotal role in the central regulation of energy homeostasis]. / Odkrycie neuromedyny U i jej rola w centralnej regulacji homeostazy energetycznej.

 Neuromedin U (NMU) is a structurally highly conserved neuropeptide and has been paired with the G-protein-coupled receptors (GPCRs) NMUR1 and NMUR2, which were formerly classified in the orphan receptor family. Activation of the G protein Gq/11 subunit causes a pertussis toxin (PTX)-insensitive activation of both phospholipase C and mitogen-activated protein kinase (MAP), and activation of the Go subunit causes a PTX-sensitive inhibition of adenyl cyclase. Additionally, NMU selectively inhibits L-type high-voltage-gated Ca2+ channels in mouse hippocampus, as well as low-voltage-activated T-type Ca2+ channels in mouse dorsal root ganglia (DRG). NMU peptide and its receptors are predominantly expressed in the gastrointestinal tract and specific structures within the brain, reflecting its major role in the regulation of energy homeostasis. A novel neuropeptide, neuromedin S (NMS), is structurally related to NMU. They share a C-terminal core structure and both have been implicated in the regulation of food intake, as well as the circadian rhythms. The acute anorectic and weight-reducing effects of NMU and NMS are mediated by NMUR2. This suggests that NMUR2-selective agonists may be useful for the treatment of obesity.

Ghrelin-mediated appetite regulation in the central nervous system.

The gut hormone and neuropeptide ghrelin was initially identified in the periphery as a compound released in the bloodstream in response to a negative energetic status. In the central nervous system (CNS), ghrelin mainly acts on the hypothalamus and the limbic system, with its best-known biological role being the regulation of appetitive functions. Recent research has shown that ghrelin is not an indispensable factor in the regulation of food intake. However, it plays a key role in the metabolic changes of lipids, mainly those involving hypothalamic NOS, AMPK, CaMKK2, CPT1 and UCP2 proteins. Ghrelin participates in the regulation of memory processes and the feeling of pleasure resulting from eating, both of which are metabolism-dependent and may be essential for the successful achievement of adaptive appetitive behavior. Ghrelin exerts its biological effect through a complicated network of neuroendocrine links, including the melanocortin and endocannabinoid systems. The activity of ghrelin is connected with circadian and annual fluctuations, which depend on seasons and food availability.

In vitro evidence that leptin suppresses melatonin secretion during long days and stimulates its secretion during short days in seasonal breeding ewes.

Recent observations in seasonal-breeding mammals indicate that the hypothalamus is programmed to become leptin resistant during long days (LD) and leptin sensitive during short days (SD). These observations support the possibility that photoperiod mediates at least part of its effects on melatonin secretion through changes in leptin sensitivity. Herein we examined the interaction of season and recombinant ovine leptin (oleptin) on melatonin secretion by pineal explants in short-term culture. Glands were collected after sunset from eight ewes during LD (March, April, May, June) and from an additional eight ewes during SD (September, October, November, December). Glands were transected saggitally and coronally into quarters, with each equilibrated in 2.5ml of DMEM for 120min, followed by a 3h incubation in medium containing either 0 or 50ng/ml of oleptin. Treatment with oleptin reduced (P<0.001) melatonin secretion compared to controls during LD by approximately 22% at 2, 2.5 and 3h of culture. However, in cultures from glands collected during SD, oleptin stimulated (P<0.078) melatonin secretion approximately 50% compared to control. These effects were consistent throughout each respective season. We conclude that the secretion of melatonin from the ovine pineal gland is negatively responsive to leptin during LD, whereas leptin may stimulate melatonin secretion during SD.

Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent.

We have shown recently that fasting permits leptin to modulate both luteinizing hormone (LH) and insulin secretion in cows. In rodents, leptin causes divergent effects on LH and insulin release that are dose dependent. To test the hypothesis that leptin effects on LH and insulin secretion in fasted cows are dose related, we examined the effects of various doses of recombinant ovine leptin (oleptin) in mature cows. Twenty ovariectomized beef cows, each bearing an estradiol implant to maintain basal estradiol concentrations, were used. All cows were fasted for 60 hr with free access to water and were assigned randomly to one of four groups (n = 5/group): 1) saline control; 2) leptin, 0.2 microg/kg; 3) leptin, 2.0 microg/kg; and 4) leptin, 20 microg/kg body wt. Blood samples were collected at 10-min intervals for 6 hr on Days 0 and 2, with saline or oleptin injected intravenously immediately after the first intensive sample on Day 2 (54 hr). Leptin caused a dose-related increase (P < 0.001) in mean concentrations of circulating LH. Stimulation of LH release by leptin was significant at the lowest (141% of control) and middle (122% of control) doses used, but no increase was observed for the highest dose. Increased mean concentrations of LH appeared to result from an augmentation of basal secretion, as pulse characteristics were not affected. After 54 hr of fasting, plasma insulin concentrations were lowered (P < 0.01) in all treatment groups compared to Day 0. After leptin injections, plasma insulin concentrations increased (P < 0.01) and reached highest concentrations during the first hour of sampling. However, this increase was sustained for several hours only in the intermediate (2.0 microg/kg) dose group. Collectively, our results show that leptin has potent positive effects on both LH and insulin secretion in fasted cows, but the anterior pituitary and endocrine pancreas appear to become downregulated in the presence of excess ligand.