Oxytocin Receptor mRNA Research Articles

Oxytocin receptor expression is associated with estrogen receptor status in breast tumors

The oxytocin receptor (OXTR) plays an important role in childbirth, breastfeeding, and social interactions. There is emerging evidence that OXTR is associated with the breast cancer (BC) initiation and progression. However, the mechanisms leading to a change in its expression, the diagnostic or prognostic value of the receptor in BC are currently poorly understood. Here, we have evaluated the relative level of OXTR expression in BC samples (n=107), and also investigated the effect of estradiol on its expression in MCF-7 and MDA-MB-231 cells. The level of OXTR expression was significantly lower in breast tumor tissue than in normal tissue obtained from the same patient. The expression of OXTR was dependent on the status and expression of the estrogen receptor (ER): the level of OXTR mRNA was significantly lower in ER-negative BC samples compared to ER-positive BC samples. Moreover, OXTR expression was also lower in samples from patients with luminal subtype with a low value of ER expression (0-5 score according to the IHC assay, Allred scoring) compared with samples with high ER expression (6-8 score). In luminal BC, OXTR expression was associated with the HER2 expression level: the OXTR mRNA level was higher in tumors with a HER2 IHC score of 1+ as compared to cases with the HER2 expression score of 2+, 3+. We also showed that estradiol increased the level of OXTR mRNA in MCF-7 cells, but not in ER-negative MDA-MB-231 cells. These data indicate that changes in OXTR expression in BC tissues can be caused by increased ER expression. We found no association between OXTR and T or N stages and progesterone receptor expression.

Transient-receptor-potential-vanilloid-1/Oxytocin receptor mRNA is up-regulated in Soleus muscle and brain while Oxytocin increases in bone after cold stress in mice: The Oxytonic effect of Oxytocin

Transient-receptor-potential-vanilloid-1/Oxytocin receptor mRNA is up-regulated in Soleus muscle and brain while Oxytocin increases in bone after cold stress in mice: The Oxytonic effect of Oxytocin

Exaggerated potassium current reduction by oxytocin in visceral sensory neurons following chronic intermittent hypoxia

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and systemically in response to a variety of stimuli and stressors, including hypoxia. Within the nucleus tractus solitarii (nTS), the first integration site for cardiorespiratory reflexes, OT enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. The nTS receives sensory input from baroafferent neurons that originate in the nodose ganglia. Nodose neurons express the OT receptor (OTR) whose activation elevates intracellular calcium. However, the influence of OT on other ion channels, especially potassium channels important for neuronal activity during CIH, is less known. This study sought to determine the mechanism (s) by which OT modulates sensory afferent-nTS mediated reflexes normally and after CIH. Nodose ganglia neurons from male Sprague-Dawley rats were examined after 10d CIH (6% O2 every 3min) or their normoxic (21% O2) control. OTR mRNA and protein were identified in Norm and CIH ganglia and was similar between groups. To examine OTR function, APs and potassium currents (IK) were recorded in dissociated neurons. Compared to Norm, after CIH OT depolarized neurons and reduced current-induced AP discharge. After CIH OT also produced a greater reduction in IK that where tetraethylammonium-sensitive. These data demonstrate after CIH OT alters ionic currents in nodose ganglia cells to likely influence cardiorespiratory reflexes and overall function.

Oxytocin receptor expression in stallion testes and epididymides

Endocrine, paracrine, and autocrine factors orchestrate the development and physiology of the stallion reproductive system. Oxytocin (OXT) is one of the critical endocrine, paracrine, and autocrine factors for the male reproductive system. Previous studies have investigated OXT receptor (OXTR) expression in testes and epididymides, including humans, marmosets, macaques, swine, and sheep. This study aimed to explore (1) OXTR localization in the testes and epididymides and (2) the seasonal modification of OXTR expression in the testes. Adult stallion testis and epididymis samples were prepared using routine castration procedures. Reverse-transcription PCR was performed to detect the presence of OXTR messenger RNA (mRNA) in the testes. Western blot procedure was performed to confirm the cross-reactivity of OXTR antibody to horse OXTR. Immunohistochemistry was performed to detect OXTR protein expression in the testes and epididymides. Oxytocin receptor mRNA was detected in the stallion testes. The OXTR protein band was observed at 55 kDa. Interestingly, the relative intensity of the OXTR protein band varied between nonbreeding and breeding season. The OXTR protein level in the testes collected during the breeding season was higher than that during the nonbreeding season. Oxytocin receptor localization was observed in the cytoplasm of Type A spermatogonia and spermatid. Oxytocin receptor protein expression was also observed in the cytoplasmic area of Leydig cells and the membrane of the seminiferous tubules. The cytoplasm of principal and basal cells in the caput, corpus, and cauda was also immunolabeled with OXTR antibody. In conclusion, based on the expression of OXTR in tissues of testes and epididymides, OXT–OXTR system may be a critical factor for stallion testicular and epididymal function. In addition, according to the seasonal alteration of intensity, the OXT–OXTR system may be associated with seasonal changes in the reproductive system in stallions.

Oxytocin reduces adipose tissue inflammation in obese mice

BackgroundObesity and adipose tissue expansion is characterized by a chronic state of systemic inflammation that contributes to disease. The neuropeptide, oxytocin, working through its receptor has been shown to attenuate inflammation in sepsis, wound healing, and cardiovascular disease. The current study examined the effects of chronic oxytocin infusions on adipose tissue inflammation in a murine model of obesity, the leptin receptor-deficient (db/db) mouse.MethodsThe effect of obesity on oxytocin receptor protein and mRNA expression in adipose tissue was evaluated by Western blotting and real-time polymerase chain reaction. Mice were implanted with osmotic minipumps filled with oxytocin or vehicle for 8 weeks. At study endpoint adipose tissue inflammation was assessed by measurement of cytokine and adipokine mRNA tissue levels, adipocyte size and macrophage infiltration via histopathology, and plasma levels of adiponectin and serum amyloid A as markers of systemic inflammation.ResultsThe expression of adipose tissue oxytocin receptor was increased in obese db/db mice compared to lean controls. In adipose tissue oxytocin infusion reduced adipocyte size, macrophage infiltration, IL-6 and TNFα mRNA expression, and increased the expression of the anti-inflammatory adipokine, adiponectin. In plasma, oxytocin infusion reduced the level of serum amyloid A, a marker of systemic inflammation, and increased circulating adiponectin.ConclusionsIn an animal model of obesity and diabetes chronic oxytocin treatment led to a reduction in visceral adipose tissue inflammation and plasma markers of systemic inflammation, which may play a role in disease progression.

Social approach and social vigilance are differentially regulated by oxytocin receptors in the nucleus accumbens

Oxytocin is currently being considered as a novel therapeutic for anxiety disorders due to its ability to promote affiliative behaviors. In the nucleus accumbens (NAc) activation of oxytocin receptors (OTR) promotes social approach (time spent near an unfamiliar individual). Here, we show that stressful social experiences reduce the expression of NAc OTR mRNA, coinciding with decreases in social approach. Social stressors also increase social vigilance, characterized as orienting to an unfamiliar individual without approaching. Vigilance is a key component of behavioral inhibition, a personality trait that is a risk factor for anxiety disorders. To understand whether NAc OTR can modulate both social approach and vigilance, we use pharmacological approaches to assess the impact of activation or inhibition of NAc OTR downstream pathways on these behaviors. First, we show that in unstressed male and female California mice, inhibition of OTR by an unbiased antagonist (L-368,899) reduces social approach but does not induce social vigilance. Next, we show that infusion of Atosiban, an OTR-Gq antagonist/OTR-Gi agonist, has the same effect in unstressed females. Finally, we show that Carbetocin, a biased OTR-Gq agonist, increases social approach in stressed females while simultaneously inhibiting social vigilance. Taken together these data suggest that OTR in the NAc differentially modulate social approach and social vigilance, primarily through an OTR-Gq mechanism. Importantly, pharmacological inhibition of OTR alone is insufficient to induce vigilance in unstressed mice, suggesting that mechanisms modulating social approach may be distinct from mechanisms modulating social vigilance.

The oxytocin-prostaglandins pathways in the horse (Equus caballus) placenta during pregnancy, physiological parturition, and parturition with fetal membrane retention

Despite their importance in mammalian reproduction, substances in the oxytocin-prostaglandins pathways have not been investigated in the horse placenta during most of pregnancy and parturition. Therefore, we quantified placental content of oxytocin (OXT), oxytocin receptor (OXTR), and prostaglandin E2 and F2 alpha during days 90–240 of pregnancy (PREG), physiological parturition (PHYS), and parturition with fetal membrane retention (FMR) in heavy draft horses (PREG = 13, PHYS = 11, FMR = 10). We also quantified OXTR and prostaglandin endoperoxide synthase-2 (PTGS2) mRNA expression and determined the immunolocalization of OXT, OXTR, and PTGS2. For relative quantification of OXT and OXTR, we used western blotting with densitometry. To quantify the prostaglandins, we used enzyme immunoassays. For relative quantification of OXTR and PTGS2, we used RT-qPCR. For immunolocalization of OXT, OXTR, and PTGS2, we used immunohistochemistry. We found that OXT was present in cells of the allantochorion and endometrium in all groups. PTGS2 expression in the allantochorion was 14.7-fold lower in FMR than in PHYS (p = 0.007). These results suggest that OXT is synthesized in the horse placenta. As PTGS2 synthesis is induced by inflammation, they also suggest that FMR in heavy draft horses may be associated with dysregulation of inflammatory processes.

Sex Differences and Estrous Influences on Oxytocin Control of Food Intake

Central oxytocin potently reduces food intake and is being pursued as a clinical treatment for obesity. While sexually dimorphic effects have been described for the effects of oxytocin on several behavioral outcomes, the role of sex in central oxytocin modulation of feeding behavior is poorly understood. Here we investigated the effects of sex, estrous cycle stage, and female sex hormones (estrogen, progesterone) on central oxytocin-mediated reduction of food intake in rats. Results show that while intracerebroventricular (ICV) oxytocin potently reduces chow intake in both male and female rats, these effects were more pronounced in males than in females. We next examined whether estrous cycle stage affects oxytocin’s food intake-reducing effects in females. Results show that ICV oxytocin administration significantly reduces food intake during all estrous cycle stages except proestrous, suggesting that female sex hormones may modulate the feeding effects of oxytocin. Indeed, additional results reveal that estrogen, but not progesterone replacement, in ovariectomized rats abolishes oxytocin-mediated reductions in chow intake. Lastly, oxytocin receptor mRNA (Oxtr) quantification (via quantitative PCR) and anatomical localization (via fluorescent in situ hybridization) in previously established sites of action for oxytocin control of food intake revealed comparable Oxtr expression between male and female rats, suggesting that observed sex and estrous differences may be based on variations in ligand availability and/or binding. Overall, these data show that estrogen reduces the effectiveness of central oxytocin to inhibit food intake, suggesting that sex hormones and estrous cycle should be considered in clinical investigations of oxytocin for obesity treatment.

Distinct changes in chronic pain sensitivity and oxytocin receptor expression in a new rat model (Wisket) of schizophrenia

Clinical studies have shown that schizophrenia is accompanied by hypoalgesia. Accordingly, we have previously reported that a chronic schizophrenia-related rat substrain (Wisket) showed decreased acute heat pain sensitivity. The aim of the present study was to determine the mechanical pain sensitivity and the effects of opioid ligands in a chronic osteoarthritic pain model generated using Wisket rats. Our previous molecular biological studies indicated that the impairment in opioid and cannabinoid receptor functions observed in these animals did not explain their altered pain sensitivity. Therefore, we aimed to investigate another endogenous antinociceptive system, i.e., the oxytocinergic system (which is also implicated in schizophrenia) via the determination the brain-region specific oxytocin receptor mRNA expression in Wisket rats. Osteoarthritis was induced in male adult control Wistar rats without any interventions and in Wisket rats after juvenile social isolation and ketamine treatment. The degree of allodynia and the effects of systemic morphine or intrathecal endomorphin-1 administration were determined. Furthermore, the expression of the oxytocin receptor mRNA was assessed in different brain structures (prefrontal cortex, striatum, diencephalon, brainstem, and olfactory bulb). A lower degree of allodynia was observed in the Wisket group compared with control animals 1 and 2 weeks after the induction of osteoarthritis, which was accompanied by a comparable degree of edema. Systemically or intrathecally applied opioids caused similar time-response curves in both groups, with apparently shorter effects in Wisket animals. The expression of the oxytocin receptor mRNA was lower in most of the brain regions (with the exception of the diencephalon) investigated in Wisket rats vs. the control animals. In summary, both acute and chronic hypoalgesia (as nonspecific symptoms in patients with schizophrenia) can be simulated in Wisket animals as endophenotypes despite the impairment of the endogenous antinociceptive systems evaluated. Thus, this model might be an appropriate tool for further investigation of the molecular basis of altered pain perception in schizophrenia.

Up-regulation of endometrial oxytocin receptor is associated with the timing of luteolysis in heifers with two and three follicular waves†.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation=day 0), Holstein heifers were randomized into two groups: cytobrush group (n=9) had an endometrial sample collected every 48h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n=4) or at the end of luteolysis (n=5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.

Coping Style of Pigs Is Associated With Different Behavioral, Neurobiological and Immune Responses to Stressful Challenges.

Based on the animal’s reaction to environmental challenges, consistent but different coping styles can be identified, which in turn may have consequences for health and welfare. Therefore, profound knowledge of the complex interrelationships between individual behavioral response patterns, underlying neurobiological mechanisms and immunological effects is required. The aim of this study was to examine whether pigs with different coping styles exhibit distinct behavioral, neurobiological and immune responses to stressful situations. Therefore, pigs (n = 40) were classified as proactive, reactive or intermediate animals according to a repeatedly-performed backtest, and behavioral, neuroendocrine and immune alterations were analyzed without any stress before weaning on day 28 and after a stress treatment on day 32. Our results show that the behavioral responses in an open-field/novel-object test characterized proactive pigs as more active. There were no significant differences in adrenocorticotropic hormone and cortisol concentrations between pigs with different coping characteristics. However, we found that proactive pigs displayed significantly increased plasma noradrenaline levels in response to stress, which may reflect a higher sympathetic reactivity of these animals. Furthermore, the present study revealed coping style differences in mRNA expression of mineralocorticoid, glucocorticoid, oxytocin and arginine vasopressin receptors and the immediate early gene c-fos in stress-related brain regions. While proactive pigs responded to stress with higher mRNA expression of arginine vasopressin, mineralocorticoid and glucocorticoid receptors, reactive pigs displayed higher oxytocin receptor and c-fos mRNA expression, indicating different neurobiological mechanisms of distinct coping styles in response to stressful challenges. Moreover, we also found humoral immune differences between proactive, intermediate and reactive animals. Proactive pigs had a higher total serum IgA concentration before and after stress treatment, with a significant increase in response to stress compared to reactive and intermediate pigs. In contrast, stress-induced IgM concentrations only increased in reactive and intermediate animals, suggesting that the effects of coping style on humoral immunity may differ depending on the specific function of the immunoglobulin classes. In conclusion, this multidisciplinary study expands the concept of coping style in farm animals, particularly in terms of individual stress reactivity and disease susceptibility, and thus contributes to the understanding of the biology of animal welfare.

Oestrogen and androgen receptor activation contribute to the masculinisation of oxytocin receptors in the bed nucleus of the stria terminalis of rats.

Oxytocin (OT) often regulates social behaviours in sex-specific ways, and this may be a result of sex differences in the brain OT system. Adult male rats show higher OT receptor (OTR) binding in the posterior bed nucleus of the stria terminalis (pBNST) than adult female rats. In the present study, we investigated the mechanisms that lead to this sex difference. First, we found that male rats have higher OTR mRNA expression in the pBNST than females at postnatal day (P)35 and P60, which demonstrates the presence of the sex difference in OTR binding density at message level. Second, the sex difference in OTR binding density in the pBNST was absent at P0 and P3, but was present by P5. Third, systemic administration of the oestrogen receptor (ER) antagonist fulvestrant at P0 and P1 dose-dependently reduced OTR binding density in the pBNST of 5-week-old male rats, but did not eliminate the sex difference in OTR binding density. Fourth, pBNST-OTR binding density was lower in androgen receptor (AR) deficient genetic male rats compared to wild-type males, but higher compared to wild-type females. Finally, systemic administration of the histone deacetylase inhibitor valproic acid at P0 and P1 did not alter pBNST-OTR binding density in 5-week-old male and female rats. Interestingly, neonatal ER antagonism, AR deficiency, and neonatal valproic acid treatment each eliminated the sex difference in pBNST size. Overall, we demonstrate a role for neonatal ER and AR activation in setting up the sex difference in OTR binding density in the pBNST, which may underlie sexual differentiation of the pBNST and social behaviour.

Preweaning Paternal Deprivation Impacts Parental Responses to Pups and Alters the Serum Oxytocin and Corticosterone Levels and Oxytocin Receptor, Vasopressin 1A Receptor, Oestrogen Receptor, Dopamine Type I Receptor, Dopamine Type II Receptor Levels in Relevant Brain Regions in Adult Mandarin Voles

Although maternal separation and neonatal paternal deprivation (PD) have been found to exert a profound and persistent effects on the physiological and behavioural development of offspring, whether preweaning PD (PPD; from PND 10 to 21) affects maternal and parental responses to pups and the underlying neuroendocrine mechanism are under-investigated. Using monogamous mandarin voles (Microtus mandarinus), the present study found that PPD increased the latency to approach a pup-containing ball, decreased the total durations of sniffing and contacting a pup-containing ball and walking and increased the total duration of inactivity in both sexes. Moreover, PPD decreased serum oxytocin levels and increased corticosterone levels, but only in females. Furthermore, in both males and females, PPD decreased the expression of oxytocin receptor mRNA and protein in the medial preoptic area (MPOA), nucleus accumbens (NAcc) and medial prefrontal cortex (mPFC), but increased it in the medial amygdala (MeA) and decreased the expression of oestrogen receptor mRNA and protein in the MPOA. PPD increased the expression of dopamine type I receptor in the NAcc, but decreased it in the mPFC. PPD decreased dopamine type II receptor (D2R) in the NAcc both in males and females, but increased D2R in the mPFC in females and decreased D2R protein expression in males. Moreover, PPD decreased vasopressin 1A receptor (V1AR) in the MPOA, MeA and mPFC, but only in males. Our results suggest that the reduction of parental responses to pups induced by PPD may be associated with the sex-specific alteration of several neuroendocrine parameters in relevant brain regions.

Expression of uterine oxytocin receptors and blood progesterone, 13,14-dihydro-15-Keto-Prostaglandin F2α, and ionized calcium levels in dystocic bitches

This study aimed to examine the etiology of canine dystocia by measuring the relative expression of oxytocin receptor (OXTR) mRNA and the concentration of serum progesterone, plasma PGF2α metabolite (PGFM), and blood ionized calcium (iCa) near term and in dystocia. Altogether 58 bitches were included in this study, 41 of which underwent cesarean section (CS). The four CS groups were based on history: complete uterine inertia (CUI; n = 7), partial uterine inertia (PUI; n = 13), obstructive dystocia (OD; n = 10), and elective cesarean section (ECS; n = 11). An additional group of medically treated dystocia without CS (MD; n = 8) and a control group (C; n = 9) with normal parturition (without CS and medical treatment) were also formed. Blood samples were taken prior to CS or medical treatment. Progesterone concentrations were highest in the ECS and a significant difference (p < 0.05) was observed between the ECS and the OD and between the ECS and the combined dystocia (CUI, PUI, OD, MD) groups (COMB). Highest concentrations of PGFM was observed in the C, the difference being significant (p < 0.05) between the C and the ECS and between the C and the COMB group. The progesterone:PGFM ratio was significantly (p < 0.05) higher in the ECS than in the C and the COMB group. No significant difference (p > 0.05) was observed in iCa concentrations between the groups. Relative OXTR mRNA expression was evaluated with real-time PCR from full-thickness uterine samples taken from the incision site during CS. The expression was highest in the ECS and the difference in expression was significant (p < 0.05) between the ECS and the OD and between ECS and the combined dystocia (CUI, PUI, OD) groups (COMB2). The study supports previous reports of decreasing progesterone and increasing PGFM during prepartum luteolysis. Upregulation of OXTR occurs near term. In obstructive dystocia, a prolonged influence of oxytocin and uterine exhaustion may lead to downregulation of OXTR. Complete primary uterine inertia may have a different etiology as no clear decrease in OXTR was observed in CUI as in OD. It remains unclear if parturition ceases because of uterine inertia or if uterine inertia occurs because of ceased parturition and desensitization of receptors.

Increased oxytocinergic system activity in the cardiac muscle in spontaneously hypertensive SHR rats.

IntroductionThe present study aimed to determine whether the presence of cardiac hypertrophy due to arterial hypertension is associated with a change in the activity of the oxytocinergic system in cardiomyocytes.Material and methodsThe experiments were performed on male, spontaneously hypertensive rats (SHR, n = 10) and normotensive Wistar-Kyoto rats (WKY, n = 12). Blood samples were collected from both SHR and WKY animals to asses plasma oxytocin (OT) concentration; the rats were sacrificed by decapitation. Samples of the left and right ventricles were harvested for the analysis of the OT and oxytocin receptor (OTR) protein by ELISA, and OT and OTR mRNA expression by RT-PCR. Immunohistopathological studies were performed to confirm the presence of OTR receptors in the cardiac muscle of the ventricles.ResultsPlasma OT concentration did not differ between SHR and WKY rats. In the SHR rats, the expression of OT mRNA and the OT protein level was higher in the left and the right ventricle, while OTR mRNA expression was significantly lower in both the left and the right ventricle. However, the level of OTR protein was higher only in the left ventricle of the SHR rats. The presence of OTR receptors was confirmed by immunohistochemical analysis in the muscle of the right and left ventricle.ConclusionsThe presence of arterial hypertension is associated with increased activity of the oxytocinergic system in the heart, especially in the area of the left ventricle. These findings support the important role of this system in the maintenance of cardiovascular homeostasis.

Effect of Oxytocin on Hunger Discrimination.

Centrally and peripherally administered oxytocin (OT) decreases food intake and activation of the endogenous OT systems, which is associated with termination of feeding. Evidence gathered thus far points to OT as a facilitator of early satiation, a peptide that reduces the need for a meal that has already begun. It is not known, however, whether OT can diminish a feeling of hunger, thereby decreasing a perceived need to seek calories. Therefore, in the current project, we first confirmed that intraperitoneal (i.p.) OT at 0.3–1 mg/kg reduces food intake in deprived and non-deprived rats. We then used those OT doses in a unique hunger discrimination protocol. First, rats were trained to discriminate between 22- and 2-h food deprivation (hungry vs. sated state) in a two-lever operant procedure. After rats acquired the discrimination, they were food-restricted for 22 h and given i.p. OT before a generalization test session. OT did not decrease 22-h deprivation-appropriate responding to match that following 2-h food deprivation, thus, it did not reduce the perceived level of hunger. In order to better understand the mechanisms behind this ineffectiveness of OT, we used c-Fos immunohistochemistry to determine whether i.p. OT activates a different subset of feeding-related brain sites under 22- vs. 2-h deprivation. We found that in sated animals, OT induces c-Fos changes in a broader network of hypothalamic and brain stem sites compared to those affected in the hungry state. Finally, by employing qPCR analysis, we asked whether food deprivation vs. sated state have an impact on OT receptor expression in the brain stem, a CNS “entry” region for peripheral OT. Fasted animals had significantly lower OT receptor mRNA levels than their ad libitum-fed counterparts. We conclude that OT does not diminish a feeling of hunger before a start of a meal. Instead OT's anorexigenic properties are manifested once consumption has already begun which is—at least to some extent—driven by changes in brain responsiveness to OT treatment in the hungry vs. fed state. OT should be viewed as a mediator of early satiation rather than as a molecule that diminishes perceived hunger.

Regulation of embryo survival in cattle

Evidence is presented that bovine somatotrophin (bST) treatment of lactating dairy cows enhances both expression of oviductal insulin-like growth factor II (IGF-II) mRNA and endometrial insulin-like growth factor binding protein 3 (IGFBP-3) mRNA between day 3 and day 7 of the oestrous cycle. mRNA encoding growth hormone (GH) receptor in endometrial tissues increased between day 3 and day 7 of the oestrous cycle. The changes induced by bST treatment may contribute to stimulation of embryo development and increase pregnancy rates in lactating dairy cows. Additive effects of bST and rb interferon tau (rbIFN-tau) to inhibit phorbol ester induction of prostaglandin F2alpha secretion in immortalized bovine endometrial cells indicates that there is interplay between their signal transduction pathways. Non-lactating dairy cows were killed at day 17 after oestrus to evaluate the effects of pregnancy status (cyclic versus pregnant) and bST (bST versus control) treatment on endometrial gene expression. Distinctly different mRNA and protein responses were detected between cyclic and pregnant cows that were related to luteolytic-antiluteolytic drive (that is expression of progesterone receptor, oxytocin receptor, oestradiol receptor alpha and prostaglandin GH synthase 2 (PGHS-2)). The bST-induced changes in PGHS-2 protein (+), oxytocin receptor mRNA (+) and oestrogen receptor alpha protein (+) may potentially affect the mechanisms associated with maintenance of pregnancy. Two experiments were conducted to evaluate whether ovarian follicular suppression induced by biodegradable deslorelin implants would reduce either early or late embryo losses. A 450 microg deslorelin implant used to induce ovulation in a timed insemination programme decreased subsequent follicular development and tended to reduce early embryo losses, whereas a 2.1 mg deslorelin implant failed to reduce late embryonic losses when inserted on day 27 of pregnancy.

Enhanced Response to Oxytocin in Myometrium of Proven Breeder Compared to Virgin Rats Does Not Depend on Augmented Expression of Oxytocin Receptors

ObjectiveOur overall hypothesis is that pregnancy's mechanical and hormonal stimuli induce permanent changes in the properties of uterine tissue. We have shown previously that the response to oxytocin of the non‐pregnant (NP) myometrium is more pronounced in proven breeder than in virgin animals. Our objective here was to investigate some of the possible mechanisms that could produce such difference: 1) Altered number of oxytocin receptors (OXTR); 2) Altered production of intracellular prostaglandins; 3) Altered availability of voltage gated calcium channels (VGCC).MethodVirgin (V) and proven breeder (PB) NP female 18 week old rats were staged and euthanized at proestrus. One uterine horn was dissected in strips suspended in a tissue bath containing Krebs solution (in mM, NaCl 117, KCl 4.7, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, CaCl2 2.5, glucose 11, pH= 7.4) at 37°C and bubbled with 95% O2 and 5% CO2. Dose response curves to oxytocin (10E‐10 to 10E‐5 M) were recorded in the presence of indomethacin or after exposure to a 0 CaCl2 modified Krebs. The other uterine horn was flash frozen in liquid NO2 and later used for ELISA (kit from Wuhan Fine Biotech, Wuhan China) and qRT‐PCR experiments (Taqman® gene expression assays (Life Technologies) and QuantStudio™ 5 real‐time PCR system (Applied Biosystems, Grand Island, NY) were used to examine the gene of interest mRNA expression using the comparative CT method (Schmittgen and Livak, 2008).ResultsIn the ELISA experiments, NP samples showed a barely significant (32±14%, P=0.040) elevation of OXTR in V (n = 8) vs PB myometrium. This is in open contradiction to our previous motility data. To investigate whether this inversion trend in protein level is matched at the mRNA level, we evaluated OXTR gene expression in NP strips (n=5, in quadruplicates). No significant difference was detected between V and PB expression of β‐actin (P= 0.099). Instead, a significant difference was found in the OXTR gene expression (P&lt;0.01) and the 2−ΔΔCt (RQ) was 0.18 (Min RQ=0.12, Max RQ=0.27, indicative of an 8.3 to 3.7 folds decrease in the OXTR mRNA level in PB vs V samples. On the other hand, in the presence of 10 μM indomethacin (COX inhibitor) or in the absence of extracellular calcium the differences in the responses to oxytocin in V and PB myometrial strips were attenuated (−98% and −96% respectively). This suggest important roles of second messengers activated pathways in the more robust effects of oxytocin in NP PB strips.ConclusionsWhile our previous functional experiments suggested that PB myometrium might contain a higher number of OXTR, ELISA and PCR experiments imply that the opposite might be true. The gene expression of the OXTR is frankly stronger in V, while the actual protein expression is not significantly different between V and PB. A better explanation for our pharmacological observations involves differences in the activity of channels and intracellular mechanisms activated by oxytocin, some of which we presented here and are currently under investigation.Support or Funding InformationThis study was funded by an MWU intramural research grant to MP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3127 The effect of common genetic variants in the oxytocin receptor gene on oxytocin response.

OBJECTIVES/SPECIFIC AIMS: Previous studies suggest that genetic variants in the oxytocin receptor (OXTR) may alter oxytocin dose requirement for labor induction and may increase risk for preterm labor and neurodevelopmental disorders. However, the mechanisms of actions of these variants remain unknown. The goal of this study was to functionally characterize common missense and noncoding variants in OXTR. First, we aimed to determine the effects of missense variants on two major aspects of receptor function: calcium signaling and β-arrestin recruitment. Second, we used allelic expression imbalance assays in an effort to identify regulatory single nucleotide polymorphisms (SNPs) in noncoding regions of OXTR that alter OXTR mRNA expression. METHODS/STUDY POPULATION: We used the Exome Aggregation Consortium database to identify the 12 most prevalent missense single nucleotide variants in OXTR. To determine the functional effects of these variants, we transfected human embryonic kidney cells (a common model system used to study receptor function) with wild type OXTR, variant OXTR, or empty vector control. We used the calcium-sensitive dye Fluo4 to quantify intracellular calcium flux in response to oxytocin treatment, and used bioluminescence resonance energy transfer assays to measure recruitment of the signaling partner β-arrestin to the receptor. To investigate potential effects of noncoding SNPs on OXTR mRNA expression, we quantified allele-specific expression of OXTR in human uterine tissue obtained from participants at the time of Cesarean section. We used next-generation sequencing (Illumina MiSeq) to count alleles of a reporter SNP in OXTR exon 3. RESULTS/ANTICIPATED RESULTS: Of the 12 most prevalent missense single nucleotide variants, four were predicted to be deleterious by PolyPhen variant annotation software. We anticipate that these variants will alter receptor signaling through calcium or β-arrestin pathways. We further observed that a reporter SNP in OXTR exon 3 exhibits significant allelic expression imbalance in a subset of our myometrial tissue samples, indicating that OXTR expression may be regulated by a functional SNP. Our current work focuses on discovering the functional SNPs in OXTR responsible for the pattern of allelic expression imbalance seen in mRNA. In the future, we will seek to explore the effects of these variants on uterine function by using genome editing of uterine smooth muscle cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that both missense and noncoding variants may affect OXTR expression and function. Future studies may suggest that OXTR sequencing, genotyping, or expression analysis would be useful to identify individuals likely to respond or fail to respond to safe doses of oxytocin for labor induction. Personalizing approaches for labor induction in this way would increase the safety of oxytocin and potentially reduce maternal morbidity and mortality.

Oxytocin pathway gene networks in the human brain

Oxytocin is a neuropeptide involved in animal and human reproductive and social behavior. Three oxytocin signaling genes have been frequently implicated in human social behavior: OXT (structural gene for oxytocin), OXTR (oxytocin receptor), and CD38 (oxytocin secretion). Here, we characterized the distribution of OXT, OXTR, and CD38 mRNA across the human brain by creating voxel-by-voxel volumetric expression maps, and identified putative gene pathway interactions by comparing gene expression patterns across 20,737 genes. Expression of the three selected oxytocin pathway genes was enriched in subcortical and olfactory regions and there was high co-expression with several dopaminergic and muscarinic acetylcholine genes, reflecting an anatomical basis for critical gene pathway interactions. fMRI meta-analysis revealed that the oxytocin pathway gene maps correspond with the processing of anticipatory, appetitive, and aversive cognitive states. The oxytocin signaling system may interact with dopaminergic and muscarinic acetylcholine signaling to modulate cognitive state processes involved in complex human behaviors.