In need of a specific antibody against the oxytocin receptor for neuropsychiatric research: A KO validation study.

Antibodies are one of the most utilized tools in biomedical research. However, few of them are rigorously evaluated, as there are no accepted guidelines or standardized methods for determining their validity before commercialization. Often, an antibody is considered validated if it detects a band by Western blot of the expected molecular weight and, in some cases, if blocking peptides result in loss of staining. Neither of these approaches are unquestionable proof of target specificity. Since the oxytocin receptor has recently become a popular target in neuropsychiatric research, the need for specific antibodies to be used in brain has arisen. In this work, we have tested the specificity of six commercially available oxytocin receptor antibodies, indicated by the manufacturers to be suitable for Western blot and with an available image showing the correct size band (45-55 KDa). Antibodies were first tested by Western blot in brain lysates of wild-type and oxytocin receptor knockout mice. Uterus tissue was also tested as control for putative differential tissue specificity. In brain, the six tested antibodies lacked target specificity, as both wild-type and receptor knockout samples resulted in a similar staining pattern, including the expected 45-55 KDa band. Five of the six antibodies detected a selective band in uterus (which disappeared in knockout tissue). These five specific antibodies were also tested for immunohistochemistry in uterus, where only one was specific. However, when the uterine-specific antibody was tested in brain tissue, it lacked specificity. In conclusion, none of the six tested commercial antibodies are suitable to detect oxytocin receptor in brain by either Western blot or immunohistochemistry, although some do specifically detect it in uterus. The present work highlights the need to develop standardized antibody validation methods, including a proper negative control, in order to grant quality and reproducibility of the generated data.

The effect of macrophage polarization on the expression of the oxytocin signalling system in enteric neurons.

BACKGROUND: The aim of the current study was to investigate the effect of macrophage polarization on the expression of oxytocin (OT) and the oxytocin receptor (OTR) in enteric neurons. METHODS: In this study, we used a classic colitis model and D-mannose model to observe the correlation between macrophage polarization and OT signalling system. In order to further demonstrate the effect of macrophages, we examined the expression of OT signalling system after depletion of macrophages. RESULTS: The data showed that, in vitro, following polarization of macrophages to the M1 type by LPS, the macrophage supernatant contained proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) that inhibited the expression of OT and OTR in cultured enteric neurons; following macrophage polarization to the M2 type by IL4, the macrophage supernatant contained anti-inflammatory cytokines (TGF-ß) that promoted the expression of OT and OTR in cultured enteric neurons. Furthermore, M1 macrophages decreased the expression of the OT signalling system mainly through STAT3/NF-κB pathways in cultured enteric neurons; M2 macrophages increased the expression of the OT signalling system mainly through activation of Smad2/3 and inhibition of the expression of Peg3 in cultured enteric neurons. In a colitis model, we demonstrated that macrophages were polarized to the M1 type during the inflammatory phase, with significant decreased in the expression of OT and OTR. When macrophages were polarized to the M2 type during the recovery phase, OT and OTR expression increased significantly. In addition, we found that D-mannose increased the expression of OT and OTR through polarization of macrophages to the M2 type. CONCLUSIONS: This is the first study to demonstrate that macrophage polarization differentially regulates the expression of OT and OTR in enteric neurons.

Synthesis and in vitro characterization of oxytocin receptor targeted PEGylated immunoliposomes for drug delivery to the uterus.

Targeted delivery of therapeutics to the uterus is an important goal in the treatment of obstetric complications, such as preterm labour, postpartum hemorrhage, and dysfunctional labour. Current treatment for these obstetric complications is challenging, as there are limited effective and safe therapeutic options available. We have developed a targeted drug delivery system for the uterus by conjugating anti-oxytocin receptor (OTR) antibodies to the surface of PEGylated liposomes (OTR-PEG-ILs). The functionality of the OTR-PEG-ILs has previously been evaluated on human and murine myometrial tissues as well as in vivo in a murine model of preterm labour. The aim of this study was to report the pharmaceutical synthesis and characterization of the OTR-PEG-ILs and investigate their specific cellular interaction with OTR-expressing myometrial cells in vitro. Immunoliposomes composed of 1,2-distearoyl-sn-glycero-2-phosphocholine (DSPC) and cholesterol were prepared using an optimized method for the coupling of low concentrations of antibody to liposomes. The liposomes were characterized for particle size, antibody conjugation, drug encapsulation, liposome stability, specificity of binding, cellular internalization, mechanistic pathway of cellular uptake, and cellular toxicity. Cellular association studies demonstrated specific binding of OTR-PEG-ILs to OTRs and significant cellular uptake following binding. Evaluation of the mechanistic pathway of cellular uptake indicated that they undergo internalization through both clathrin- and caveolin-mediated mechanisms. Furthermore, cellular toxicity studies have shown no significant effect of OTR-PEG-ILs or the endocytotic inhibitors on cell viability. This study further supports oxytocin receptors as a novel pharmaceutical target for drug delivery to the uterus.

Could nitric oxide be a mediator of action of oxytocin on myocardial injury in rats? (Biochemical, histological and immunohistochemical study).

Oxytocin (OT) was revisited recently as a hormone of cardiovascular system with several new functions in cardiovascular regulation. But less is known about its role in acute myocardial injury (MI). The aim of our study was to investigate the possible protective effect of OT on the biochemical, histological and immunohistochemical changes of MI induced by isoprenaline (ISO) in adult male albino rats and studying the possible role of nitric oxide (NO) in its action. Forty male albino rats were divided into 5 groups: control rats (Group I), acute MI rats (Group II), rats pretreated with OT prior to induction of MI (Group III), rats injected with a combination of OT and atosiban (ATO, OT receptor antagonist) prior to induction of MI (Group IV). In Group V, a combination of OT and nitric oxide synthase inhibitor (L-NAME) were injected to the rats prior to induction of MI. The heart wall in all groups were taken and processed for histological, immunohistochemical, morphometrical and biochemical studies. We concluded that OT has antioxidant, anti-inflammatory and anti-apoptotic effects on MI and its effects is mediated through NO.

Distribution of nonapeptide systems in the forebrain of an African cichlid fish, Astatotilapia burtoni.

Nonapeptides and their receptors have important functions in mediating social behavior across vertebrates. Where these nonapeptides are synthesized in the brain has been studied extensively in most vertebrate lineages, yet we know relatively little about the neural distribution of nonapeptide receptors outside of mammals. As nonapeptides play influential roles in behavioral regulation in all vertebrates, including teleost fish, we mapped the distributions of the receptors for arginine vasotocin (AVT; homolog of arginine vasopressin) and isotocin (IST; homolog of oxytocin/mesotocin) throughout the forebrain of Astatotilapia burtoni, an African cichlid fish with behavioral phenotypes that are plastic and reversible based on the immediate social environment. We characterized the distribution of the AVT V1a2 receptor (V1aR) and the IST receptor (ITR) using both immunohistochemistry for protein detection and in situ hybridization for mRNA detection, as well as AVT and IST using immunohistochemistry. Expression of the neuropeptide receptors was widely distributed throughout the fore- and midbrain, including the proposed teleost homologs of the mammalian amygdala complex, striatum, hypothalamus, and ventral tegmental area. We conclude that although the location of nonapeptide synthesis is restricted compared to tetrapod vertebrates, the distribution of nonapeptide receptors is highly conserved across taxa. Our results significantly extend our knowledge of where nonapeptides act in the brains of teleosts to mediate social transitions and behavior.

Immunoreactivity of oxytocin receptor and transient receptor potential vanilloid type 1 and its correlation with dysmenorrhea in adenomyosis.

OBJECTIVE: We sought to investigate the expression and localization of oxytocin receptor (OTR) and transient receptor potential vanilloid type 1 (TRPV1) in women with and without adenomyosis. STUDY DESIGN: Ectopic and homologous eutopic endometrium from 50 women with adenomyosis and endometrium from 18 women without adenomyosis were used for immunohistochemical analysis of OTR and TRPV1. Microscopic evaluation assessed the presence and localization of OTR and TRPV1 throughout the menstrual cycle in both eutopic endometrial and endometriotic tissues of women with adenomyosis and compared them with normal endometrium. RESULTS: Compared with normal endometrium, immunoreactivity of OTR and TRPV1 were significantly increased in ectopic endometrium. Both OTR and TRPV1 immunoreactivity were positively correlated with the severity of dysmenorrhea and found to be significant predicators for dysmenorrhea severity. CONCLUSION: These findings suggest that OTR and TRPV1 may be involved in dysmenorrhea and its severity in adenomyosis and may be potential therapeutic targets.

Intrauterine oxytocin system. Compartmental distribution of oxytocin and oxytocin receptors in rat endometrial epithelium.

At term, uterine epithelial cells express oxytocin (OT) as well as the OT receptor (OTR). Like other epithelial cells, uterine epithelial cells are polarized and sort secretory and membrane components to the apical or the basolateral cell surface. We have studied the subcellular localization of OT-like immunoreactivity (OT-IR) and OTR-IR in rat uterine epithelium by immuno-gold labelling of ultrathin frozen sections. Our observations indicate that OT and OTR are both distributed preferentially to the apical surface of rat uterine epithelial cells. OT-IR showed a 6-fold apical versus basolateral preference and was localized in apical secretory vesicles, suggesting that uterine OT is released by apical exocytosis. OTR-IR was localized to the apical surface with a 9-fold apical versus basolateral preference and was found specifically in association with apical microvilli. The present findings represent the first example of a G protein-coupled receptor that is preferentially localized on the microvillar compartment and support the concept of an autocrine uterine OT system at the apical side of the uterine epithelium.

Function and localization of oxytocin receptors in the reproductive tissue of rams.

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.

Previous maternal experience potentiates the effect of parturition on oxytocin receptor mRNA expression in the paraventricular nucleus.

In sheep, central oxytocin release at parturition induces maternal behaviour which is thought to be mediated by changes in the expression of central oxytocin receptors. The distribution, effects of parturition, previous maternal experience and hormonal status on the distribution of an oxytocin receptor was investigated using immunocytochemistry and in situ hybridization. In ewes with no previous maternal experience, parturition induced significant increases in oxytocin receptor mRNA expression in the anterior olfactory nucleus, medial preoptic area, ventromedial hypothalamus, lateral septum, medial amygdala, bed nucleus of the stria terminalis and diagonal band of Broca. In maternally experienced ewes, parturition induced additional increases in two areas, the paraventricular nucleus and the Islands of Calleja. The changes in progesterone and oestrogen that occur during late pregnancy and parturition appear to contribute to increases in expression in the anterior olfactory nucleus, Islands of Calleja, medial preoptic area, ventromedial hypothalamus, bed nucleus of the stria terminalis and diagonal band of Broca, but not in the paraventricular nucleus, lateral septum and medial amygdala. These results demonstrate that progesterone and oestrogen priming enhance oxytocin receptor mRNA expression in a number of regions in the olfactory system, hypothalamus and limbic brain. These effects appear to be independent of maternal experience. Parturition increases oxytocin receptor mRNA expression in all the areas influenced by hormonal priming and the lateral septum, medial amygdala and paraventricular nucleus. Maternal experience also enhances expression of oxytocin receptor mRNA in the paraventricular nucleus and the Islands of Calleja. Because the paraventricular nucleus is the main source of oxytocin release in the brain, this upgrading of autoreceptors as a result of maternal experience may serve to enhance release of this peptide in projection sites regulating maternal behaviour.

Oxytocin receptor couples to the 80 kDa Gh alpha family protein in human myometrium.

One of the primary functions of the oxytocin receptor is to modulate intracellular calcium levels in myometrium. The oxytocin receptor has been purified and cloned. Although it has been suggested that oxytocin receptor couples with a GTP-binding regulatory protein (G-protein), the identity of this G-protein remains unclear. To elucidate the mechanism of oxytocin receptor signalling, we used the oxytocin-receptor-G-protein ternary complex preparation from human myometrium, and evaluated oxytocin-mediated activation of [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) binding and [alpha-32P]GTP photoaffinity labelling to a G-protein. Binding of [35S]GTP[S] and the intensity of the [alpha-32P]GTP photoaffinity labelled protein resulting from activation of the oxytocin receptor were significantly attenuated by the selective oxytocin antagonist, desGlyNH2d(CH2)5[Tyr(Me)2,Thr4]OVT. Furthermore, the molecular mass of the specific GTP-binding protein was approximately 80 kDa; homologous with the Gh alpha family, the new class of GTP-binding proteins first identified in rat liver that couples to the alpha 1B-adrenoceptor. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the oxytocin receptor in the ternary complex preparation by anti-Gh7 alpha antibody, the Gh alpha family protein tightly coupled to the oxytocin receptor. These findings demonstrate that oxytocin receptor couples with approximately 80 kDa Gh alpha in signal mediation.

Rat oxytocin receptor in brain, pituitary, mammary gland, and uterus: partial sequence and immunocytochemical localization.

Partial complementary DNAs of an oxytocin (OT) receptor were cloned from rat brain and uterus. The complementary DNAs encoded for the same amino acid sequence, which showed a high degree of homology with the human and porcine uterine OT receptors, except for a region in the third intracellular loop. Antibodies were raised against nonoverlapping sequences of the third intracellular loop of this rat OT receptor. Using these antisera, OT receptor expression was demonstrated in the brain, pituitary, mammary gland, and uterus by immunocytochemistry. In the brain, several areas including the ventromedial hypothalamus, the bed nucleus of the stria terminalis, the ventral pallidum, the paraventricular nucleus, and the dorsal part of the supraoptic nucleus, demonstrated OT-receptor immunoreactivity. However, no immunoreactivity was detected in two areas of the brain known to contain dense OT-binding sites by receptor autoradiography studies: the ventral hippocampus and the central nucleus of the amygdala. In the pituitary, both the anterior and posterior lobes were positive for OT receptor immunoreactivity, whereas the intermediate lobe was negative. These results demonstrate that the same receptor type is expressed in both peripheral OT target tissues and the brain, and also suggest the possibility that a different OT receptor subtype may be present in some areas of the brain.