Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

In the past, most research in equine reproduction has been performed in vivo but the use of in vitro and ex vivo models has recently increased. This study aimed to evaluate the functional stability of an ex vivo hemoperfused model for equine uteri with molecular characterization of marker genes and their proteins. In addition, the study validated the respective protein expression and the aptness of the software QuPath for identifying and scoring immunohistochemically stained equine endometrium. After collection, uteri (n = 12) were flushed with preservation solution, transported to the laboratory on ice, and perfused with autologous blood for 6 h. Cycle stage was determined by examination of the ovaries for presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Samples were obtained directly after slaughter, after transportation, and during perfusion (240, 300, 360 min). mRNA expression levels of progesterone (PGR), estrogen (ESR1) and oxytocin (OXTR) receptor as well as of MKI67 (marker of cell growth) and CASP3 (marker of apoptosis) were analyzed by RT-qPCR, and correlation to protein abundance was validated by immunohistochemical staining. Endometrial samples were analyzed by visual and computer-assisted evaluation of stained antigens via QuPath. For PGR, effects of the perfusion and cycle stage on expression were found (P < 0.05), while ESR1 was affected only by cycle stage (P < 0.05) and OXTR was unaffected by perfusion and cycle stage. MKI67 was lower after 360 min of perfusion as compared to samples collected before perfusion (P < 0.05). For CASP3, differences in gene expression were found after transport and samples taken after 240 min (P < 0.05). Immunohistochemical staining revealed effects of perfusion on stromal and glandular cells for steroid hormone receptors, but not for Ki-67 and active Caspase 3. OXTR was visualized in all layers of the endometrium and was unaffected by perfusion. Comparison of QuPath and visual analysis resulted in similar results. For most cell types and stained antigens, the correlation coefficient was r > 0.5. In conclusion, the isolated hemoperfused model of the equine uterus was successfully validated at the molecular level, demonstrating stability of key marker gene expression. The utility of computer-assisted immunohistochemical analysis of equine endometrial samples was also confirmed.

Methylation of the Oxytocin, Oxytocin Receptor, and Vasopressin Gene Promoters in Tobacco Use Disorder during Cessation.

INTRODUCTION: Vasopressin (AVP) and oxytocin (OT) exert sex-specific effects on social pair bonding and stress reactions while also influencing craving in substance use disorders. In this regard, intranasal oxytocin (OT) and AVP antagonists present potential treatments for tobacco use disorder (TUD). Since transcription of both hormones is also regulated by gene methylation, we hypothesized sex-specific changes in methylation levels of the AVP, OT, and OT receptor (OXTR) gene during nicotine withdrawal. METHODS: The study population consisted of 49 smokers (29 males, 20 females) and 51 healthy non-smokers (25 males, 26 females). Blood was drawn at day 1, day 7, and day 14 of smoking cessation. Craving was assessed with the questionnaire on smoking urges (QSU). RESULTS: Throughout cessation, mean methylation of the OT promoter gene increased in males and decreased in females. OXTR receptor methylation decreased in females, while in males it was significantly lower at day 7. Regarding the AVP promoter, mean methylation increased in males while there were no changes in females. Using mixed linear modeling, CpG position, time point, sex, and the interaction of time point and sex as well as time point, sex, and QSU had a significant fixed effect on OT and AVP gene methylation. The interaction effect suggests that sex, time point, and QSU are interrelated, meaning that, depending on the sex, methylation could be different at different time points and vice versa. There was no significant effect of QSU on mean OXTR methylation. DISCUSSION: We identified differences at specific CpGs between controls and smokers in OT and AVP and in overall methylation of the AVP gene. Furthermore, we found sex-specific changes in mean methylation levels of the mentioned genes throughout smoking cessation, underlining the relevance of sex in the OT and vasopressin system. This is the first study on epigenetic regulation of the OT promoter in TUD. Our results have implications for research on the utility of the AVP and OT system for treating substance craving. Future studies on both targets need to analyze their effect in the context of sex, social factors, and gene regulation.

Non-canonical Ca2+- Akt signaling pathway mediates the antiproteolytic effects induced by oxytocin receptor stimulation in skeletal muscle.

Although it has been previously demonstrated that oxytocin (OXT) receptor stimulation can control skeletal muscle mass in vivo, the intracellular mechanisms that mediate this effect are still poorly understood. Thus, rat oxidative skeletal muscles were isolated and incubated with OXT or WAY-267,464, a non-peptide selective OXT receptor (OXTR) agonist, in the presence or absence of atosiban (ATB), an OXTR antagonist, and overall proteolysis was evaluated. The results indicated that both OXT and WAY-267,464 suppressed muscle proteolysis, and this effect was blocked by the addition of ATB. Furthermore, the WAY-induced anti-catabolic action on protein metabolism did not involve the coupling between OXTR and Gαi since it was insensitive to pertussis toxin (PTX). The decrease in overall proteolysis induced by WAY was probably due to the inhibition of the autophagic/lysosomal system, as estimated by the decrease in LC3 (an autophagic/lysosomal marker), and was accompanied by an increase in the content of Ca2+-dependent protein kinase (PKC)-phosphorylated substrates, pSer473-Akt, and pSer256-FoxO1. Most of these effects were blocked by the inhibition of inositol triphosphate receptors (IP3R), which mediate Ca2+ release from the sarcoplasmic reticulum to the cytoplasm, and triciribine, an Akt inhibitor. Taken together, these findings indicate that the stimulation of OXTR directly induces skeletal muscle protein-sparing effects through a Gαq/IP3R/Ca2+-dependent pathway and crosstalk with Akt/FoxO1 signaling, which consequently decreases the expression of genes related to atrophy, such as LC3, as well as muscle proteolysis.

Differential Roles of Oxytocin Receptors in the Prefrontal Cortex and Nucleus Accumbens on Cocaine Self-Administration and Reinstatement of Cued Cocaine Seeking in Male Rats.

BACKGROUND: Little is known about the specific roles of cortical and accumbal oxytocin receptors in drug use disorders. To better understand the importance of the endogenous oxytocin system in cocaine relapse behavior, we developed an adeno-associated viral vector-expressing short hairpin (sh) RNAs to selectively degrade the rat oxytocin receptor (OxyR) mRNA in vivo. METHODS: Male (Sprague-Dawley) rats received bilateral infusions of the shRNA for the oxytocin receptor (shOxyR) or an shRNA control virus into the prefrontal cortex (PFC) or the nucleus accumbens core (NAc). Rats self-administered cocaine on an escalating FR ratio for 14 days, lever responding was extinguished, and rats were tested for cued and cocaine-primed reinstatement of drug seeking. RESULTS: OxyR knockdown in the PFC delayed the acquisition of lever pressing on an fixed ratio 1 schedule of reinforcement. All rats eventually acquired the same level of lever pressing and discrimination, and there were no differences in extinction. OxyR knockdown in the NAc had no effect during acquisition. In both the PFC and NAc, the shOxyR decreased cued reinstatement relative to shRNA control virus but was without effect during drug-primed reinstatement. OxyR knockdown in the PFC increased chamber activity during a social interaction task. CONCLUSIONS: This study provides critical new information about how endogenous OxyRs function to affect drug seeking in response to different precipitators of relapse. The tool developed to knockdown OxyRs in rat could provide important new insights that aid development of oxytocin-based therapeutics to reduce return-to-use episodes in people with substance use disorder and other neuropsychiatric disorders.

Behavioral convergence in defense behaviors in pair bonded individuals correlates with neuroendocrine receptors in the medial amygdala.

Monogamous, pair-bonded animals coordinate intra-pair behavior for spatially separated challenges including territorial defense and nest attendance. Paired California mice, a monogamous, territorial and biparental species, approach intruders together or separately, but often express behavioral convergence across intruder challenges. To gain a more systems-wide perspective of potential mechanisms contributing to behavioral convergence across two conspecific intruder challenges, we conducted an exploratory study correlating behavior and receptor mRNA (Days 10 and 17 post-pairing). We examined associations between convergence variability in pair time for intruder-oriented behaviors with a pair mRNA index for oxytocin (OXTR), androgen (AR), and estrogen alpha (ERα) receptors within the medial amygdala (MeA) and the anterior olfactory nucleus (AON), brain regions associated with social behavior. An intruder behavior index revealed a bimodal distribution of intruder-related behaviors in Challenge 1 and a unimodal distribution in Challenge 2, suggesting population behavioral convergence, but no significant correlations with neuroendocrine measures. However, OXTR, AR, and ERα mRNA in the MeA were positively associated with convergence in individual intruder-related behaviors, suggesting multiple mechanisms may influence convergence. Mice could also occupy the nest during intruder challenges and convergence in nest attendance was positively correlated with MeA OXTR. At an individual level, nest attendance was positively associated with MeA ERα. Vocalizations were positively associated with AR and ERα mRNA. No positive associations were found in the AON. Overall, neuroendocrine receptors were implicated in convergence of a monogamous pair's defense behavior, highlighting the potential importance of the MeA as part of a circuit underlying convergence.

The intertwining roles of caveolin, oxytocin receptor, and the associated signalling pathways in prostate cancer progression.

Caveolae are invaginations in the plasma membrane of most cell types and are present in the cells of normal prostate tissue. Caveolins are a family of highly conserved integral membrane proteins that oligomerise to form caveolae and interact with signalling molecules by providing a scaffold that sequesters signal transduction receptors in close proximity to each other. Signal transduction G proteins and G-protein-coupled receptors (GPCR), including oxytocin receptor (OTR), are localised within caveolae. Only one OTR has been identified, and yet, this single receptor both inhibits and stimulates cell proliferation. As caveolae sequester lipid-modified signalling molecules, these differing effects may be due to a change in location. The cavin1 necessary for caveolae formation is lost in prostate cancer progression. With the loss of caveolae, the OTR moves out onto the cell membrane influencing the proliferation and survival of prostate cancer cells. Caveolin-1 (cav-1) is reportedly overexpressed in prostate cancer cells and is associated with disease progression. This review focuses on the position of OTRs within caveolae, and their movement out onto the cell membrane. It explores whether movement of the OTR is related to changes in the activation of the associated cell signalling pathways that may increase cell proliferation and analyse whether caveolin and particularly cavin1 might be a target for future therapeutic stratagies.

Oxytocin receptor (OXTR) is a risk gene for polycystic ovarian syndrome.

OBJECTIVE: Oxytocin (OXT) controls appetite, promotes diet-induced energy expenditure, and may protect against obesity. Furthermore, the oxytocin system controls ovarian follicle luteinization and steroidogenesis as well as adrenal steroidogenesis, which if impaired might lead to anovulation and hyperandrogenism, signs found in women with polycystic ovarian syndrome (PCOS). PCOS is a common complex endocrine disorder of reproductive-age women, and it often presents with impaired glucose metabolism, insulin resistance (IR), and type 2 diabetes (T2D). The oxytocin receptor gene (OXTR) may confer a risk for PCOS, conceivably through dysregulation of metabolism, ovarian follicle maturation, and ovarian and adrenal steroidogenesis. Therefore, we aimed to investigate whether OXTR variants confer risk for PCOS. SUBJECTS AND METHODS: In 212 Italian subjects with T2D and PCOS, we have analyzed 22 single nucleotide polymorphisms (SNPs) within the OXTR gene for linkage to and/or linkage disequilibrium (LD, i.e., association) with PCOS. We tested whether the significant risk variants were independent or part of an LD block. RESULTS: We found 5 independent variants significantly linked to/in LD with PCOS within the peninsular families. CONCLUSIONS: This is the first study to report OXTR as a novel risk gene in PCOS. Functional and replication studies are needed to confirm these results.

Oxytocin receptor single nucleotide polymorphism predicts atony-related postpartum hemorrhage.

BACKGROUND: Postpartum hemorrhage remains a key contributor to overall maternal morbidity in the United States. Current clinical assessment methods used to predict postpartum hemorrhage are unable to prospectively identify about 40% of hemorrhage cases. Oxytocin is a first-line pharmaceutical for preventing and treating postpartum hemorrhage, which acts through oxytocin receptors on uterine myocytes. Existing research indicates that oxytocin function is subject to variation, influenced in part by differences in the DNA sequence within the oxytocin receptor gene. One variant, rs53576, has been shown to be associated with variable responses to exogenous oxytocin when administered during psychological research studies. How this variant may influence myometrial oxytocin response in the setting of third stage labor has not been studied. We tested for differences in the frequency of the oxytocin receptor genotype at rs53576 in relationship to the severity of blood loss among a sample of individuals who experienced vaginal birth. METHODS: A case-control prospective design was used to enroll 119 postpartum participants who underwent vaginal birth who were at least 37 weeks of gestation. Cases were defined by either a 1000 mL or greater blood loss or instances of heavier bleeding where parturients were given additional uterotonic treatment due to uterine atony. Controls were matched to cases on primiparity and labor induction status. Genotype was measured from a maternal blood sample obtained during the 2nd postpartum month from 95 participants. Statistical analysis included bivariate tests and generalized linear and Poisson regression modeling. RESULTS: The distribution of the genotype across the sample of 95 participants was 40% GG (n = 38), 50.5% AG (n = 48) and 9.5% AA (n = 9). Blood loss of 1000 mL or greater occurred at a rate of 7.9% for GG, 12.5% for AG and 55.6% for AA participants (p = 0.005). Multivariable models demonstrated A-carriers (versus GG) had 275.2 mL higher blood loss (95% CI 96.9-453.4, p < 0.01) controlling for parity, intrapartum oxytocin, self-reported ancestry, active management of third stage or genital tract lacerations. Furthermore, A-carrier individuals had a 79% higher risk for needing at least one second-line treatment (RR = 1.79, 95% CI = 1.08-2.95) controlling for covariates. Interaction models revealed that A-carriers who required no oxytocin for labor stimulation experienced 371.4 mL greater blood loss (95% CI 196.6-546.2 mL). CONCLUSIONS: We provide evidence of a risk allele in the oxytocin receptor gene that may be involved in the development of postpartum hemorrhage among participants undergoing vaginal birth, particularly among those with fewer risk factors. The findings, if reproducible, could be useful in studying pharmacogenomic strategies for predicting, preventing or treating postpartum hemorrhage.

Involvement of Oxytocin and Progesterone Receptor Expression in the Etiology of Canine Uterine Inertia.

An altered oxytocin and progesterone receptor (OXTR and PGR, respectively) expression was postulated in canine uterine inertia (UI), which is the lack of functional myometrial contractions. OXTR and PGR expressions were compared in uterine tissue obtained during C-section due to primary UI (PUI; n = 12) and obstructive dystocia (OD, n = 8). In PUI, the influence of litter size was studied (small/normal/large litter: PUI-S/N/L: n = 5/4/3). Staining intensity in immunohistochemistry was scored for the longitudinal and circular myometrial layer and summarized per dog (IP-Myoscore). Mean P4 did not differ significantly between PUI (n = 9) and OD (n = 7). OXTR and PGR expressions (ratios) were significantly higher in PUI (OXTR: p = 0.0019; PGR: p = 0.0339), also for OXTR in PUI-N versus OD (p = 0.0034). A trend for a higher PGR IP-Myoscore was identified (PUI-N vs. OD, p = 0.0626) as well as an influence of litter size (lowest PGR-Myoscore in PUI-L, p = 0.0391). In conclusion, PUI was not related to higher P4, but potentially increased PGR availability compared to OD. It remains to be clarified whether OXTR is upregulated in PUI due to a counterregulatory mechanism to overcome myometrial quiescence or downregulated in OD due to physiological slow OXTR desensitization associated with an advanced duration of labor. Identified OXTR differences between myometrial layers indicate the need for further research.

Effects of maternal deprivation and environmental enrichment on anxiety-like and depression-like behaviors correlate with oxytocin system and CRH level in the medial-lateral habenula.

The medial-lateral habenula (LHbM)'s role in anxiety and depression behaviors in female mice remains unclear. Here, we used neonatal maternal deprivation (MD) and post-weaning environmental enrichment (EE) to treat female BALB/c offspring and checked anxiety-like and depression-like behaviors as well as the corticotropin-releasing hormone (CRH), oxytocin receptor (OTR), estrogen receptor-beta (ERß) levels in their LHbM at adulthood. We found that MD enhanced state anxiety-like behaviors in the elevated plus-maze test, and EE caused trait anxiety-like behaviors in the open field test and depression-like behaviors in the tail suspension test. The immunochemistry showed that MD reduced OT immunoreactive neuron numbers in the hypothalamic paraventricular nucleus but increased OTR levels in the LHbM; EE increased CRH levels in the LHbM but decreased OTR levels in the LHbM. The additive effects of EE and MD maintained the behavioral parameters, OT-ir neuronal numbers, CRH levels, and OTR levels similar to the additive of non-MD and non-EE. The correlation analysis showed that CRH levels correlated with synaptic connection levels, OTR levels correlated with nucleus densities, and ERß levels correlated with Nissl body levels and body weights in female mice. Neither MD nor EE affected ERß levels in the LHbM. Together, the study revealed the relationships between behaviors and neuroendocrine and neuronal alterations in female LHbM and the effects of experiences including MD and EE on them.

Preserved oxytocin-induced myometrium contraction and sensitivity to progesterone inhibition following rat uterus thermal insult. Impact on fertility.

Women seeking improved fertility often undergo diagnostic hysteroscopy that could cause uterine thermal injury with unclear impact on uterine contraction, embryo implantation and fertility. We tested whether uterine thermal insult adversely affects myometrium function and contraction related receptors, channels, junctional proteins and remodeling enzymes. Female Sprague-Dawley rats were anesthetized, the left uterine horn was infused with 85 ℃ hot saline (thermal Insult) and the right horn was infused with 25℃ warm saline (control) for 3 min. After 7-days recovery, uterine strips were prepared for tissue histology and measurement of contraction, and mRNA and protein levels of oxytocin receptor, progesterone (P4) receptor A (PR-A), membrane K+ channel TREK-1, junctional protein connexin-43 (CX-43) and matrix metalloproteinases MMP-2 and MMP-9. Uterine tissue histology showed cellular swelling and inflammatory cell infiltration immediately following thermal insult, and recovery with no difference from control 7-days later. KCl (96 mM) and oxytocin (10-13-10-7 M) caused significant contraction that was not different in thermal insult vs control uterine strips. Pretreatment with P4 (10-5 M) for 1 h caused marked inhibition of KCl and oxytocin contraction that was insignificantly greater in thermal vs control uterus. RT-PCR showed decreases in oxytocin receptor, PR-A, TREK-1, CX-43, MMP-2 and MMP-9 mRNA in thermal vs control uterus. Western blots showed decreases in oxytocin receptor, no change in TREK-1 and increased PRA, CX-43, MMP-2, and MMP-9 protein levels in thermal vs control uterus. To assess the impact on fertility, female rats were housed with male rats, and on gestational day 19, the litter size, pup weight and crown-rump length, and placenta weight were not different in thermal vs control uterus. Thus, after thermal insult-induced immediate inflammation and reduced heat-sensitive mRNA expression, the uterus undergoes a recovery and adaptation process involving preserved oxytocin-induced contraction, P4 inhibition and TREK-1 channels. The uterus self-healing process appears to require improved PR-A signaling, intercellular communication via CX-43 and tissue remodeling by MMP-2 and MMP-9. The uterine thermal recovery processes could be essential for maintaining fertility and future pregnancy outcome.

Identification of genes associated with human-canine communication in canine evolution.

The dog (Canis familiaris) was the first domesticated animal and hundreds of breeds exist today. During domestication, dogs experienced strong selection for temperament, behaviour, and cognitive ability. However, the genetic basis of these abilities is not well-understood. We focused on ancient dog breeds to investigate breed-related differences in social cognitive abilities. In a problem-solving task, ancient breeds showed a lower tendency to look back at humans than other European breeds. In a two-way object choice task, they showed no differences in correct response rate or ability to read human communicative gestures. We examined gene polymorphisms in oxytocin, oxytocin receptor, melanocortin 2 receptor, and a Williams-Beuren syndrome-related gene (WBSCR17), as candidate genes of dog domestication. The single-nucleotide polymorphisms on melanocortin 2 receptor were related to both tasks, while other polymorphisms were associated with the unsolvable task. This indicates that glucocorticoid functions are involved in the cognitive skills acquired during dog domestication.

Changes in expression of signal transduction-related genes, and formation of aggregates of GPER1 and OXTR receptors in mucopolysaccharidosis cells.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by storage of glycosaminoglycans (GAGs), however, various modulations of the course of these diseases were identified recently due to impairment of different cellular processes. Here, using transcriptomic analyses in cells derived from patients suffering from eleven types of MPS, we demonstrated that expression of dozens to hundreds of genes coding for proteins involved in signal transduction processes is significantly changed in MPS cell relative to controls. When studying membrane estrogen receptor 1 (GPER1) and oxytocin receptor (OXTR) in more detail, we unexpectedly found formation of aggregates of GPER1 in MPS I, and those of OXTR in both MPS I and MPS II cells. The presence of these aggregates did not correlate with levels of expression of GPER1 and OXTR genes and levels of corresponding gene products. On the other hand, the aggregates disappeared in cells treated with enzymes which are otherwise deficient in MPS I and MPS II, causing efficient degradation of GAGs. We demonstrated that GPER1 and OXTR aggregates might be formed due to interactions with GAGs rather than arising from changes of levels of these proteins in cells.

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5 mg, respectively) during mid-luteal phase (12 and 10 days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4 h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Unravelling the role of 17ß-estradiol on advancing uterine luteolytic cascade in cattle.

In cattle, 17ß-estradiol (E2) stimulates prostaglandin F2α (PGF2α) synthesis, which causes luteolysis. Except for the well-established upregulation of oxytocin receptor gene (OXTR), molecular mechanisms of E2-induced PGF2α release in vivo remain unknown. We hypothesized that E2-induced PGF2α release requires de novo transcription of components of the PGF2α synthesis machinery. Beef cows (n = 52) were assigned to remain untreated (Control; n = 10), to receive 50% ethanol infusion intravenously (Placebo; n = 21), or 3 mg E2 in 50% ethanol infusion intravenously (Estradiol; n = 21) on day 15 (D15) after estrus. We collected a single endometrial biopsy per animal at the time of the treatment (0h; Control B0h group), 4 hours (4h; Placebo B4h group and Estradiol B4h group), or 7 hours (7h; Placebo B7h group and Estradiol B7h group) post-treatment. Compared to the Placebo group, the Estradiol group presented significantly greater 13,14-dihydro-15-keto-PGF2α concentrations between 4h and 7h and underwent earlier luteolysis. At 4h, the qPCR analysis showed a lower abundance of ESR1, ESR2 and aldo-keto reductase family 1 member B1 (AKR1B1) genes in the Estradiol B4h group, and a greater abundance of OXTR compared to the Placebo B4h group. Similarly, the E2 treatment significantly reduced the abundance of AKR1B1, and AKR1C4 in the Estradiol B7h group, compared to the placebo group. Overall, E2-induced PGF2α release and luteolysis involved an unexpected and transient downregulation of components of the PGF2α-synthesis cascade, except for OXTR, which was upregulated. Collectively, our data suggest that E2 connects newly-synthesized OXTR to pre-existing cellular machinery to synthesize PGF2α and cause luteal regression.

Oxytocin suppresses epithelial cell-derived cytokines production and alleviates intestinal inflammation in food allergy.

Food allergy is a growing healthcare problem worldwide, but prophylactic options and regulatory therapies are limited. Oxytocin (OXT), conventionally acknowledged as a hormone, was recently proven to have potent anti-inflammatory and immunomodulatory activities in certain diseases. Here, we reported the novel function and its underlying mechanisms of OXT on food allergy in vivo and in vitro. We showed that the levels of OXT were elevated in ovalbumin (OVA)-allergic mice and patients with food allergy. In HT-29 cells, OXT inhibited the production of the epithelial cell-derived cytokines thymic stromal lymphopoietin (TSLP), interleukin (IL)-25 and IL-33 by suppressing NF-κB signaling, in which ß-arrestin2 participated. These functions of OXT were abolished by oxytocin receptor (OXTR) depletion. Treating OVA-induced BALB/c mice with OXT suppressed TSLP, IL-25 and IL-33 production and attenuated systemic anaphylaxis and intestinal inflammation. OXTR-/- mice showed extreme increases in TSLP, IL-25 and IL-33 levels as well as severe systemic anaphylaxis and intestinal inflammation. In conclusion, through OXTRs, OXT has a promising antiallergic effect on experimental food allergy by suppressing epithelial TSLP, IL-25 and IL-33 production via inhibiting NF-κB signaling and upregulating ß-arrestin2 expression. Our study provides a new therapeutic perspective for food allergy in humans.

[Oxytocin receptor expression is associated with estrogen receptor status in breast tumors]. / Ékspressiia oksitotsinovogo retseptora assotsiirovana so statusom éstrogenovogo retseptora v opukholiakh molochnoi zhelezy.

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.

Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway.

Oxytocin receptor (OXTR) is involved in social behaviors, thermoregulation, and milk ejection, yet little is known about its role in breast cancer. To investigate the role of OXTR in mammary gland development and tumorigenesis, a transgenic mouse model of OXTR overexpression (++Oxtr) was used. Overexpression of OXTR-induced progressive mammary hyperplasia, unexpected milk production, and tumorigenesis in females. OXTR-induced mammary tumors showed ERBB2 upregulation and mixed histological subtypes with predomination of papillary and medullary carcinomas. OXTR overexpression led to an activation of prolactin (PRL)/p-STAT5 pathway and created a microenvironment that promotes mammary-specific tumorigenesis. PRL inhibitor bromocriptine (Br) could mitigate OXTR-driven mammary tumor growth. The study demonstrates Oxtr is an oncogene and a potential drug target for HER2-type breast cancer.

Oxytocin receptor is a promising therapeutic target of malignant mesothelioma.

Malignant mesothelioma (MM) is one of the most aggressive tumors. We conducted bioinformatics analysis using Cancer Cell Line Encyclopedia (CCLE) datasets to identify new molecular markers in MM. Overexpression of oxytocin receptor (OXTR), which is a G-protein-coupled receptor for the hormone and neurotransmitter oxytocin, mRNA was distinctively identified in MM cell lines. Therefore, we assessed the role of OXTR and its clinical relevance in MM. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and OXTR mRNA expression using The Cancer Genome Atlas (TCGA) datasets. The function of OXTR and the efficacy of its antagonists were investigated in vitro and in vivo using MM cell lines. Consistent with the findings from CCLE datasets analysis, OXTR mRNA expression was highly increased in MM tissues compared with other cancer types in the TCGA datasets, and MM cases with high OXTR expression showed poor overall survival. Moreover, OXTR knockdown dramatically decreased MM cell proliferation in cells with high OXTR expression via tumor cell cycle disturbance, whereas oxytocin treatment significantly increased MM cell growth. OXTR antagonists, which have high selectivity for OXTR, inhibited the growth of MM cell lines with high OXTR expression, and oral administration of the OXTR antagonist, cligosiban, significantly suppressed MM tumor progression in a xenograft model. Our findings suggest that OXTR plays a crucial role in MM cell proliferation and is a promising therapeutic target that may broaden potential therapeutic options and could be a prognostic biomarker of MM.

Effects of gonadal status and the estrogen milieu on hypothalamic oxytocin gene expression and serum oxytocin levels in female rats.

Oxytocin (OT) and its receptor (OTR) play various roles in the central and peripheral regulation of appetite and body weight. Previously, we have shown that the administration of OT markedly decreased appetite and body weight gain in ovariectomized (OVX) obese rats. In addition, recent studies have shown that the endogenous OT system is also affected by endogenous or exogenous estrogen. In this study, we showed that ovariectomy decreased rats' hypothalamic OT/OTR mRNA and serum OT levels, but did not affect their visceral fat OTR mRNA levels. The chronic administration of estradiol (E2) abrogated these ovariectomy-induced changes; i.e., it increased the rats' hypothalamic OT/OTR mRNA and serum OT levels, and may be associated with reductions in food intake and body weight gain. In addition, acute E2 administration increased the rats' hypothalamic OTR mRNA and serum OT levels, but did not affect their hypothalamic OT mRNA levels. Taken together, these results suggest that endogenous OT and/or OTR expression might be positively regulated by E2 and that the suppressive effects of E2 on appetite and body weight gain might be mediated, at least in part, by the OT system. Thus, we consider that OT might be a target hormone to pursue subsequent interventions of menopause for menopause-induced metabolic disorders.