Effect of Neurosteroids on Basal and Stress-Induced Oxytocin Secretion in Luteal-Phase and Pregnant Sheep.

Oxytocin (OT) is a neuropeptide synthesized in the hypothalamic nuclei that modulates both behavioral and reproductive functions, associated with the increased neurosteroid synthesis in the brain. Therefore, the present study tested the hypothesis that manipulation of central neurosteroid levels could affect oxytocin synthesis and release in non-pregnant and pregnant sheep under both basal and stressful conditions. In Experiment 1, luteal-phase sheep were subjected to a series of intracerebroventricular (icv.) infusions of allopregnanolone (AL, 4 × 15 µg/60 µL/30 min) for 3 days. In Experiment 2, pregnant animals (4th month) received a series of infusions of the neurosteroid synthesis blocker, finasteride (4 × 25 µg/60 µL/30 min), conducted for 3 days. In non-pregnant sheep AL alone was shown to differentially modulate OT synthesis in basal conditions, and strongly inhibit OT response to stress (p < 0.001). In contrast, in pregnant animals, basal and stress-induced OT secretion was significantly (p < 0.001) increased during finasteride infusion compared to controls. In conclusion, we showed that neurosteroids were involved in the control of OT secretion in sheep, particularly under stress and pregnancy conditions and are part of an adaptive mechanism which is responsible for protecting and maintaining pregnancy in harmful situations.

Pomegranate Seed Oil as a Source of Conjugated Linolenic Acid (CLnA) Has No Effect on Atherosclerosis Development but Improves Lipid Profile and Affects the Expression of Lipid Metabolism Genes in apoE/LDLR-/- Mice.

The aim of this study was to evaluate the anti-atherosclerotic effect of pomegranate seed oil as a source of conjugated linolenic acid (CLnA) (cis-9,trans-11,cis-13; punicic acid) compared to linolenic acid (LnA) and conjugated linoleic acid (CLA) (cis-9,trans-11) in apoE/LDLR-/- mice. In the LONG experiment, 10-week old mice were fed for the 18 weeks. In the SHORT experiment, 18-week old mice were fed for the 10 weeks. Diets were supplied with seed oils equivalent to an amount of 0.5% of studied fatty acids. In the SHORT experiment, plasma TCh and LDL+VLDL cholesterol levels were significantly decreased in animals fed CLnA and CLA compared to the Control. The expression of PPARα in liver was four-fold increased in CLnA group in the SHORT experiment, and as a consequence the expression of its target gene ACO was three-fold increased, whereas the liver's expression of SREBP-1 and FAS were decreased in CLnA mice only in the LONG experiment. Punicic acid and CLA isomers were determined in the adipose tissue and liver in animals receiving pomegranate seed oil. In both experiments, there were no effects on the area of atherosclerotic plaque in aortic roots. However, in the SHORT experiment, the area of atherosclerosis in the entire aorta in the CLA group compared to CLnA and LnA was significantly decreased. In conclusion, CLnA improved the lipid profile and affected the lipid metabolism gene expression, but did not have the impact on the development of atherosclerotic plaque in apoE/LDLR-/- mice.

Involvement of neurosteroids in the control of hypothalamic-pituitary-adrenal axis activity in pregnant sheep under basal and stressful conditions.

Neurosteroids are synthesized locally in the brain, where they can modify neuronal functionality depending on the physiological state. A high correlation was demonstrated between the increasing activity of the hypothalamic-pituitary-adrenal (HPA) axis and allopregnanolone (AL) concentration in the cerebrospinal fluid in sheep during pregnancy. Therefore, the present study tested the hypothesis that blocking neurosteroid synthesis in the brain of a pregnant sheep would affect HPA axis activity under both basal and stressful conditions. Two groups of sheep in the fourth month of gestation (n = 7 each) were subjected to the following treatments: 1) intracerebroventricular (icv) infusion of vehicle for three days (C) and then icv infusion of finasteride (a total of 100 µg/240 µL/day) for three days (F), one week apart, and 2) icv infusion of vehicle for three days and application of stressful stimuli (isolation and partial movement restriction) on the third day (S), and subsequently icv infusion of finasteride for three days and application of stressful stimuli on the third day (SF), one week apart. On the third days of the experiment, a 4-h push-pull perfusion of the infundibular nucleus/median eminence and blood sampling were performed. Mean perfusate corticotropin-releasing hormone (CRH), plasma adrenocorticotropin (ACTH) and cortisol concentrations were significantly higher in sheep treated with finasteride, stress and finasteride in combination with stress compared to controls. The highest hormone concentrations in Groups F, S and SF, were recorded during the first 60 min; however, significant increases in CRH and ACTH levels were observed in Group SF towards the end of the experiment. It can be concluded that neurosteroids may be an essential component of the mechanism controlling HPA axis activity in pregnant sheep, not only under stress-free conditions, but more importantly, also by inhibiting the neuroendocrine response to stressors.

Allopregnanolone reduces neuroendocrine response to acute stressful stimuli in sheep.

The verified hypothesis assumed that centrally administered neurosteroid, allopregnanolone (AL), could affect basal and/or stress-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis in sheep. Four groups (n = 6 each) of luteal-phase sheep were intracerebroventricularly infused for 3 days with a vehicle without stress (control); a vehicle treated with stressful stimuli (isolation and partial movement restriction) on the third day; AL (4 × 15 µg/60 µL/30 min, at 30-min intervals) treated with stressful stimuli, and AL alone. Simultaneously, the push-pull perfusion of the infundibular nucleus/median eminence and plasma sample collection were performed. After the experiment, the sheep were killed to collect the hypothalamic and anterior pituitary (AP) tissues. Stressful stimuli evoked an increase in the expression of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the hypothalamic paraventricular nucleus (PVN), and AVP receptor (V1b) and proopiomelanocortin (POMC) mRNA in the AP; the concentrations of perfusate CRH, and plasma adrenocorticotropic hormone (ACTH) and cortisol compared to controls. Conversely, the expression of the CRH receptor (CRHR1) mRNA in the AP was downregulated. AL decreased the expression of CRH and AVP mRNA in the PVN, and AVPRV1b and POMC mRNA in the AP in stressed sheep, compared to only stressed ones. There was also a reduction in perfusate CRH, and plasma ACTH and cortisol concentrations. AL alone decreased the expression of CRHR1 mRNA in the AP, and plasma cortisol concentration at the beginning of the collection period compared to controls. In conclusion, AL may function centrally as a suppressor of HPA axis activity in stressed sheep.

Obestatin stimulates the somatotrophic axis activity in sheep.

The effects of obestatin (an anorexigenic peripheral peptide) on somatotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on the activity of the somatotrophic axis in peripubertal female sheep. Animals were randomly divided into two groups: control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four hourly infusions on three consecutive days were performed. The blood samples were collected on day 0 and on day 3. Immediately after the end of experiment sheep were slaughtered. Parts of the brains were fixed in situ for further immunohistochemical analysis, while the remaining brains were frozen for Real Time RT-qPCR analysis. Substantial changes in the activity of the somatotrophic axis were observed in obestatin-infused sheep. In those animals obestatin evoked an increase in growth hormone-releasing hormone (GHRH) mRNA expression and a decrease in somatostatin mRNA expression in the anterior hypothalamic area. Moreover, a decrease in somatostatin immunoreactivity in the periventricular nucleus and an increase in somatostatin immunopositive fibers in the median eminence were noted. Changes in the GHRH and somatostatin activity are associated with an increase in growth hormone (GH) gene expression and in the amount of GH immunoreactive material stored in the somatotrophic pituitary cells. Consequently, an increase in GH concentration in the peripheral blood, due to an increase in the number of pulses was observed. It was revealed that obestatin affects the somatostatin/GHRH/GH system at the level of protein synthesis, accumulation and release. It is suggested that obestatin participates in the mechanism modulating somatotrophic axis activity at the central level by stimulating GH release through suppression of somatostatin output. Thereby, it can be concluded that obestatin may be involved in the modulation of growth processes in sheep.