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.

The relationship between OXT gene polymorphisms and reproductive hormones in pregnant and lactating Awassi Ewes.

BACKGROUND: Numerous genetic loci interact intricately to control reproduction in mammals. The oxytocin gene (OXT) is a promising candidate for reproductive traits in mammals. Previously, sheep and goats have been studied for the presence of the OXT polymorphism. As of yet, no polymorphisms have been identified in the OXT gene of Awassi sheep. Thus, this study was conducted to determine the effects of OXT polymorphism and litter size on reproductive hormones in pregnant and lactating Awassi ewes. METHODS AND RESULTS: This study evaluated 232 ewes aged 3 and 4 years (123 single-progeny ewes and 109 twin-producing ewes). Serum was collected to measure reproductive hormones using ELISA kits manufactured by ELK Biotechnology. DNA was extracted from sheep blood for genotyping and sequencing to identify variations in OXT gene (exon 2, 266 bp). Genotyping analysis revealed three genotypes within 266 bp: CC, CA, and AA. Sequence analysis revealed a novel mutation in exon 2: 188 C > A. Statistical analysis showed significant associations between the 188 C > A SNP and phenotypic traits. Twin-pregnant ewes carrying CC genotypes had higher estrogen, progesterone, and follicle-stimulating hormone/luteinizing hormone levels (65.86 ± 3.87) (pg/mL), (6.51 ± 0.39) (ng/mL), and (20.22 ± 1.27) (ng/mL)/( 23.37 ± 2.14) (ng/mL) respectively, compared to CA and AA genotypes in the fourth month of twin-pregnant ewes compared to single-pregnant ewes. CONCLUSIONS: This study found that the 188 C > A SNP negatively affected reproductive hormone levels in Awassi sheep. These findings provide breeders with a new insight into the sheep OXT gene, useful for future breeding.

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.

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.

RNA-Seq Reveals Protective Mechanisms of Mongolian Medicine Molor-Dabos-4 on Acute Indomethacin-Induced Gastric Ulcers in Rats.

This study aimed to apply transcriptomics to determine how Molor-Dabos-4 (MD-4) protects healthy rats against indomethacin (IND)-induced gastric ulcers and to identify the mechanism behind this protective effect. Rats were pretreated with MD-4 (0.3, 1.5, or 3 g/kg per day) for 21 days before inducing gastric ulcers by oral administration with indomethacin (30 mg/kg). Unulcerated and untreated healthy rats were used as controls. Effects of the treatment were assessed based on the ulcer index, histological and pathological examinations, and indicators of inflammation, which were determined by enzyme-linked immunosorbent assay. Transcriptomic analysis was performed for identifying potential pharmacological mechanisms. Eventually, after identifying potential target genes, the latter were validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). After pretreatment with MD-4, gastric ulcers, along with other histopathological features, were reduced. MD-4 significantly (p < 0.05) increased the superoxide dismutase (SOD) levels in ulcers and reduced pepsin, TNF-α, and IL-6 levels. RNA-seq analysis identified a number of target genes on which MD-4 could potentially act. Many of these genes were involved in pathways that were linked to anti-inflammatory and antioxidant responses, and other protective mechanisms for the gastric mucosa. qRT-PCR showed that altered expression of the selected genes, such as Srm, Ryr-1, Eno3, Prkag3, and Eef1a2, was consistent with the transcriptome results. MD-4 exerts protective effects against IND-induced gastric ulcers by reducing inflammatory cytokines and pepsin and increasing the expression of SOD levels. Downregulation of Srm, Ryr-1, Eno3, Prkag3, and Eef1a2 genes involved in regulating arginine and proline metabolism, calcium signaling pathway, HIF-1 signaling pathway, oxytocin signaling pathway, and legionellosis are possibly involved in MD-4-mediated protection against gastric ulcers.

The Importance of Experimental Investigation of the Peripheral Oxytocin System.

Oxytocin and oxytocin receptors are synthesized in the periphery where paracrine/autocrine actions have been described alongside endocrine actions effected by central release of oxytocin from the posterior pituitary. In the female reproductive system, classical actions of uterine contraction and milk ejection from mammary glands are accompanied by actions in the ovaries where roles in steroidogenesis, follicle recruitment and ovulation have been described. Steroidogenesis, contractile activity, and gamete health are similarly affected by oxytocin in the male reproductive tract. In the cardiovascular system, a local oxytocinergic system appears to play an important cardio-protective role. This role is likely associated with emerging evidence that peripheral oxytocin is an important hormone in the endocrinology of glucose homeostasis due to its actions in adipose, the pancreas, and the largely ignored oxytocinergic systems of the adrenal glands and liver. Gene polymorphisms are shown to be associated with a number of reported traits, not least factors associated with metabolic syndrome.

Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas.

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.

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.

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior.

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8-12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.

Identification of oxytocin-related lncRNAs and assessment of their expression in breast cancer.

Oxytocin is a neuropeptide released by the central nervous system. A number of studies have demonstrated the role of this neuropeptide in the pathogenesis of breast cancer. In the present project, we have identified mRNA coding genes and long non-coding RNAs (lncRNAs) that are associated with this pathway through an in-silico strategy, and measured their expression in a cohort of Iranian females affected with this type of malignancy. Expression levels of OXTR, FOS, ITPR1, RCAN1, CAMK2D, CACNA2D and lnc_ZFP161 were significantly down-regulated in breast cancer tissues compared with nearby non-cancerous tissues. On the other hand, expression of lnc_MTX2 was higher in breast cancer tissues compared with controls. Expression of lnc_TNS1 and lnc_FOXF1 were not different between these two kinds of samples. Expression of CACNA2D was associated with mitotic rate and PR status (P values = 3.02E-02 and 2.53E-02, respectively). Expression of other oxytocin-related genes was not associated with clinicopathological parameters. FOS and ITPR1 had the highest AUC value among the oxytocin-related genes. Combination of expression profiles of all oxytocin-related genes increased the AUC value to 0.75. However, the combinatorial sensitivity and specificity values were lower than some individual genes. In the breast cancer tissues, the most robust correlations have been detected between lnc_ZFP161/ lnc_FOXF1, CAMK2D/ lnc_ZFP161 and CAMK2D / lnc_FOXF1 (r = 0.86, 0.71 and 0.64 respectively). In the non-cancerous tissues, the strongest correlation was detected between lnc_FOXF1/lnc_MTX2 and lnc_ZFP161/CAMK2D respectively (r = 0.78 and 0.65). Taken together, oxytocin-associated genes have been dysregulated in breast cancer tissues. Moreover, the correlation ratio between these genes is connected with the existence of cancer.

Oxytocin receptor antagonists as a novel pharmacological agent for reducing smooth muscle tone in the human prostate.

Pharmacotherapies for the treatment of Benign Prostatic Hyperplasia (BPH) are targeted at reducing cellular proliferation (static component) or reducing smooth muscle tone (dynamic component), but response is unpredictable and many patients fail to respond. An impediment to identifying novel pharmacotherapies is the incomplete understanding of paracrine signalling. Oxytocin has been highlighted as a potential paracrine mediator of BPH. To better understand oxytocin signalling, we investigated the effects of exogenous oxytocin on both stromal cell proliferation, and inherent spontaneous prostate contractions using primary models derived from human prostate tissue. We show that the Oxytocin Receptor (OXTR) is widely expressed in the human prostate, and co-localises to contractile cells within the prostate stroma. Exogenous oxytocin did not modulate prostatic fibroblast proliferation, but did significantly (p < 0.05) upregulate the frequency of spontaneous contractions in prostate tissue, indicating a role in generating smooth muscle tone. Application of atosiban, an OXTR antagonist, significantly (p < 0.05) reduced spontaneous contractions. Individual tissue responsiveness to both exogenous oxytocin (R2 = 0.697, p < 0.01) and atosiban (R2 = 0.472, p < 0.05) was greater in tissue collected from older men. Overall, our data suggest that oxytocin is a key regulator of inherent spontaneous prostate contractions, and targeting of the OXTR and associated downstream signalling is an attractive prospect in the development of novel BPH pharmacotherapies.

The Evolution of Oxytocin and Vasotocin Receptor Genes in Jawed Vertebrates: A Clear Case for Gene Duplications Through Ancestral Whole-Genome Duplications.

The neuronal and neuroendocrine peptides oxytocin (OT) and vasotocin (VT), including vasopressins, have six cognate receptors encoded by six receptor subtype genes in jawed vertebrates. The peptides elicit a broad range of responses that are specifically mediated by the receptor subtypes including neuronal functions regulating behavior and hormonal actions on reproduction and water/electrolyte balance. Previously, we have demonstrated that these six receptor subtype genes, which we designated VTR1A, VTR1B, OTR, VTR2A, VTR2B and VTR2C, arose from a syntenic ancestral gene pair, one VTR1/OTR ancestor and one VTR2 ancestor, through the early vertebrate whole-genome duplications (WGD) called 1R and 2R. This was supported by both phylogenetic and chromosomal conserved synteny data. More recently, other studies have focused on confounding factors, such as the OTR/VTR orthologs in cyclostomes, to question this scenario for the origin of the OTR/VTR gene family; proposing instead less parsimonious interpretations involving only one WGD followed by complex series of chromosomal or segmental duplications. Here, we have updated the phylogeny of the OTR/VTR gene family, including a larger number of vertebrate species, and revisited seven representative neighboring gene families from our previous conserved synteny analyses, adding chromosomal information from newer high-coverage genome assemblies from species that occupy key phylogenetic positions: the polypteriform fish reedfish (Erpetoichthys calabaricus), the cartilaginous fish thorny skate (Amblyraja radiata) and a more recent high-quality assembly of the Western clawed frog (Xenopus tropicalis) genome. Our analyses once again add strong support for four-fold symmetry, i.e., chromosome quadruplication in the same time window as the WGD events early in vertebrate evolution, prior to the jawed vertebrate radiation. Thus, the evolution of the OTR/VTR gene family can be most parsimoniously explained by two WGD events giving rise to the six ancestral genes, followed by differential gene losses of VTR2 genes in different lineages. We also argue for more coherence and clarity in the nomenclature of OT/VT receptors, based on the most parsimonious scenario.

Cellular mechanism underlying oxytocin-stimulated Cl- secretion in rat cauda epididymal epithelium.

The neurohypophyseal hormone oxytocin (OT) plays critical roles in lactation and parturition, while its function in male reproduction system is largely unknown. This study aims to investigate the effect of OT on regulating transepithelial ion transport in rat cauda epididymal epithelium. With the use of RT-PCR, Western blot, and immunohistochemical analysis, we found that OT receptor (OTR) was expressed and localized at the basal membrane of rat cauda epididymal epithelium. The short-circuit current (Isc) measurement showed that basolateral application of OT to the primary cultured rat cauda epididymal epithelial cells elicited an increase in Isc, which was abrogated by pretreating the epithelial cells with CFTRinh-172, a blocker of cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with the prostaglandin H synthase inhibitors indomethacin and piroxicam, or the nonselective antagonists of prostaglandin E2 (PGE2) receptor EP2 or EP4, AH-6809, and AH-23848, significantly attenuated OT-stimulated Isc response. Furthermore, the generation of PGE2 was measured using enzyme-linked immunosorbent assay, demonstrating that OT induced a substantial increase in PGE2 release from primary cultured rat cauda epididymal epithelial cells. In conclusion, activation of OTR by OT triggered PGE2 release, resulting in CFTR-dependent Cl- secretion through paracrine/autocrine pathways in rat cauda epididymal epithelium.

Colocalization of Oxtr with Prader-Willi syndrome transcripts in the trigeminal ganglion of neonatal mice.

Prader-Willi syndrome (PWS) is caused by deficient expression of the paternal copy of several contiguous genes on chromosome 15q11-q13 and affects multiple organ systems in the body, including the nervous system. Feeding and suckling deficits in infants with PWS are replaced with excessive feeding and obesity in childhood through adulthood. Clinical trials using intranasal oxytocin (OXT) show promise to improve feeding deficits in infants with PWS. The mechanism and location of action of exogenous OXT are unknown. We have recently shown in neonatal mice that OXT receptors (OXTR) are present in several regions of the face with direct roles in feeding. Here we show that the trigeminal ganglion, which provides sensory innervation to the face, is a rich source of Oxtr and a site of cellular co-expression with PWS gene transcripts. We also quantified OXTR ligand binding in mice deficient in Magel2, a PWS gene, within the trigeminal ganglion and regions that are anatomically relevant to feeding behavior and innervated by the trigeminal ganglion including the lateral periodontium, rostral periodontium, tongue, olfactory epithelium, whisker pads and brainstem. We found that peripheral OXTR ligand binding in the head is mostly intact in Magel2-deficient mice, although it is reduced in the lateral periodontium (gums) of neonatal Magel2-deficient mice compared to wild-type controls. These data suggest that OXT via orofacial OXTR may play a peripheral role to modulate sensory-motor reflexes necessary for suckling and may be part of the mechanism by which intranasal OXT shows promise for therapeutic benefit in PWS.

Role of Oxytocin/Vasopressin-Like Peptide and Its Receptor in Vitellogenesis of Mud Crab.

Oxytocin (OT)/vasopressin (VP) signaling system is important to the regulation of metabolism, osmoregulation, social behaviours, learning, and memory, while the regulatory mechanism on ovarian development is still unclear in invertebrates. In this study, Spot/vp-like and its receptor (Spot/vpr-like) were identified in the mud crab Scylla paramamosain. Spot/vp-like transcripts were mainly expressed in the nervous tissues, midgut, gill, hepatopancreas, and ovary, while Spot/vpr-like were widespread in various tissues including the hepatopancreas, ovary, and hemocytes. In situ hybridisation revealed that Spot/vp-like mRNA was mainly detected in 6-9th clusters in the cerebral ganglion, and oocytes and follicular cells in the ovary, while Spot/vpr-like was found to localise in F-cells in the hepatopancreas and oocytes in the ovary. In vitro experiment showed that the mRNA expression level of Spvg in the hepatopancreas, Spvgr in the ovary, and 17ß-estradiol (E2) content in culture medium were significantly declined with the administration of synthetic SpOT/VP-like peptide. Besides, after the injection of SpOT/VP-like peptide, it led to the significantly reduced expression of Spvg in the hepatopancreas and subduced E2 content in the haemolymph in the crabs. In brief, SpOT/VP signaling system might inhibit vitellogenesis through neuroendocrine and autocrine/paracrine modes, which may be realised by inhibiting the release of E2.

Interrelationships between metabolic hormones, leptin and ghrelin, and oil-related contaminants in control of oxytocin and prostaglandin F release by feline ovaries.

We examined the effects of metabolic hormones leptin and ghrelin, and the oil-related environmental contaminants toluene and xylene on the release of ovarian hormones by gravid and non-gravid cats, as well as the functional interrelationships between metabolic hormones and contaminants. Ovarian fragments of non-gravid cats were cultured with and without leptin and toluene. Next, ovarian fragments of either non-gravid or gravid animals were cultured with and without ghrelin and xylene. Oxytocin (OT) and prostaglandin F (PGF) release was measured using ELISA. We confirm ovarian OT and PGF production by feline ovary, demonstrate the involvement of leptin and ghrelin in controlling OT and PGF release, show the direct influence of toluene and xylene on feline ovarian secretory activity, indicate the ability of leptin and ghrelin to mimic and promote the main contaminant effects, demonstrate that oil-related contaminants can prevent and even invert the effects of leptin and ghrelin on the ovary, and suggest the gravidity-associated changes in ability of ghrelin to promote xylene action on PGF (but not to OT), but not in basic ovarian OT and PGF release and their response to ghrelin or xylene.

Comparative Transcriptome Analysis Identifying the Different Molecular Genetic Markers Related to Production Performance and Meat Quality in Longissimus Dorsi Tissues of MG × STH and STH Sheep.

Crossbred sheep have many prominent traits, such as excellent production performance and high-quality meat, when compared to local sheep breeds. However, the genetic molecular markers related to these characteristics remain unclear. The crossbred MG × STH (small-tailed Han sheep (STH) × Mongolian sheep (MG)) breed and the STH breed were selected to measure production performance and meat quality. We used 14 indexes of production performance and meat quality, which in the MG × STH population showed significant differences compared to the STH breed. Subsequently, the longissimusdorsi from the two sheep were subjected to comparative transcriptomic analyses to identify differentially expressed genes (DEGs) related to production performance and meat quality. A total of 874 DEGs were identified between the two sheep groups. A total of 110 unique DEGs related to sheep production performance and meat quality were selected as the candidate DEGs. We found 6 production-performance-related and 30 meat-quality-related DEGs through a correlation analysis, including SPARC, ACVRL1, FNDC5 and FREM1. The expression levels of 11 DEGs were validated by real-time PCR, and the results were in accordance with the results of the comparative transcriptomic and correlation analyses. These results will assist in understanding sheep heterosis and molecular marker-assisted selection.

Oxytocin and oxytocin receptor alterations, decreased survival, and increased chemoresistance in patients with pancreatic cancer.

BACKGROUND: Oxytocin (OXT) and its receptor (OXTR) is associated with cancer. The present study was to investigate the correlation between the genetic expression alterations of OXT and OXTR and the outcomes in patients with pancreatic cancer (PC). METHODS: Information regarding OXT and OXTR genetic alterations and changes in gene expression were retrieved from the Cancer Genome Atlas (TCGA) databases and analyzed using the cBioPortal online tool. We assessed the correlation of overall survival and disease/progression-free months to either OXT or OXTR genetic alterations and changes in gene expression using Kaplan-Meier and Cox regression analyses. Quantitative PCR (qPCR) was conducted to assess the mRNA expression levels of OXT and OXTR in human PC cell lines. RESULTS: Five percent of PC cases showed mRNA upregulation in the OXT gene. These PC cases also showed genetic alterations and changes in gene expression of OXTR. The median months of survival and disease-free survival were lower for PC cases with genetic alterations and changes in gene expression in the OXT and OXTR genes as compared to those without such alterations. qPCR data showed that OXT and OXTR mRNA expression were 1-fold and 10-fold higher, respectively in PANC-1 cell lines as compared to L3.6pl cell lines in direct negative correlation with responsiveness to gemcitabine. CONCLUSIONS: These data suggest that OXT and OXTR may potentially be important in PC progression, chemoresistance, and patient survival, and potentially could have prognostic and therapeutic implications in a subset of PC patients.