Acciones óseas de las hormonas de la neurohipófisis / Bone actions of neuro hypophyseal hormones

La oxitocina (OXT) como la arginina-vasopresina (AVP) son dos hormonas primitivas secretadas por la hipófisis posterior. Sus receptores están mucho más ampliamente distribuidos en el organismo de lo que se pensaba originalmente, incluido el hueso. En los estudios preclínicos, la OXT ha mostrado ser anabólica para el hueso, promoviendo la osteogénesis sobre la adipogénesis y favoreciendo la actividad osteoblástica sobre la osteoclástica. Tanto los osteoblastos como los osteoclastos tienen receptores para la OXT, y los efectos de los estrógenos sobre la masa ósea en ratones está mediada por lo menos en parte por la OXT. El mecanismo preciso por el cual la activación de los receptores de oxitocina (OXTR) se traduce en un incremento de la formación ósea permanece poco claro. La AVP también podría afectar el esqueleto en forma directa. Dos de los receptores de la AVP, V1a y V2 están expresados en osteoblastos y osteoclastos. La inyección de AVP en ratones de tipo salvaje aumenta la formación osteoclastos que producen resorción y reduce los osteoblastos formadores de hueso. En forma opuesta, la exposición de precursores osteoblásticos a antagonistas de los receptores V1a o V2, incrementan la osteoblastogénesis, como también lo hace la deleción genética del receptor V1a. (AU)

Both oxytocin (OXT) and argininevasopressin (AVP) are primitive hormones secreted by the posterior pituitary gland. OXT receptors are much more widely distributed in the body than originally thought, including in bone. In preclinical studies, OXT has been shown to be anabolic for bone, promoting osteogenesis over adipogenesis and favoring osteoblastic over osteoclastic activity. Both osteoblasts and osteoclasts have receptors for OXT, and the effects of estrogen on bone mass in mice is mediated at least in part by OXT. The precise mechanism by which the activation of oxytocin receptors (OXTRs) results in an increase in bone formation remains unclear. AVP could also have direct actions on the skeleton. The two AVP receptors, V1a and V2, are expressed in osteoblasts and osteoclasts. Injection of AVP in wild-type mice increases the formation of osteoclasts increasing bone resorption, and reduces bone-forming osteoblasts. On the contrary, the exposure of osteoblastic precursors to V1a and V2 antagonists increase osteoblastogenesis, the same as the genetic deletion of the V1a receptor. (AU)

Expression of Oxytocin Receptors in Canine Mammary Tumours.

The aims of the present study were: (1) to investigate the presence of oxytocin receptors in benign and malignant canine mammary tumours (CMTs) and to evaluate the possible association between oxytocin receptor (OTR) expression and the expression of oestrogen receptor (OR) α and ORß, and (2) to examine associations between receptor expression and tumour size, clinical stage, histological subtype, tumour grading and lymph node status. Forty-three canine mammary tumour samples (19 benign, 24 malignant) were examined by immunohistochemistry to detect OTR, ORα and ORß expression. Results were expressed as total score for each receptor, calculated as the sum of the percentage of positive cells and the intensity of immunolabelling. In all of the evaluated mammary tumour samples, OTRs were identified and their expression tended to be higher in benign tumours than malignant tumours. Among the malignant tumours, the expression of OTR was significantly higher in grade I and II lesions than in grade III lesions. ORα-positive tumours had a tendency towards a higher OTR total score than ORα-negative tumours. These results report for the first time that CMTs express OTRs and their expression is associated with the presence of ORα. An interaction between oxytocin and the OTR might play a role in the development and progression of this type of neoplasia.

Perinatal bisphenol A (BPA) exposure alters brain oxytocin receptor (OTR) expression in a sex- and region- specific manner: A CLARITY-BPA consortium follow-up study.

Bisphenol A (BPA) is a well-characterized endocrine disrupting chemical (EDC) used in plastics, epoxy resins and other products. Neurodevelopmental effects of BPA exposure are a major concern with multiple rodent and human studies showing that early life BPA exposure may impact the developing brain and sexually dimorphic behaviors. The CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) program was established to assess multiple endpoints, including neural, across a wide dose range. Studies from our lab as part of (and prior to) CLARITY-BPA have shown that BPA disrupts estrogen receptor expression in the developing brain, and some evidence of oxytocin (OT) and oxytocin receptor (OTR) disruption in the hypothalamus and amygdala. While BPA disruption of steroid hormone function is well documented, less is known about its capacity to alter nonapeptide signals. In this CLARITY-BPA follow up study, we used remaining juvenile rat tissues to test the hypothesis that developmental BPA exposure affects OTR expression across the brain. Perinatal BPA exposure (2.5, 25, or 2500 µg/kg body weight (bw)/day) spanned gestation and lactation with dams gavaged from gestational day 6 until birth and then the offspring gavaged directly through weaning. Ethinyl estradiol (0.5 µg/kg bw/day) was used as a reference estrogen. Animals of both sexes were sacrificed as juveniles and OTR expression assessed by receptor binding. Our results demonstrate prenatal exposure to BPA can eliminate sex differences in OTR expression in three hypothalamic regions, and that male OTR expression may be more susceptible. Our data also identify a sub-region of the BNST with sexually dimorphic OTR expression not previously reported in juvenile rats that is also susceptible to BPA.

The abnormal expression of oxytocin receptors in the uterine junctional zone in women with endometriosis.

BACKGROUND: The junctional zone (JZ), also called as the endometrial-myometrial junction, is related to peristaltic-like movements in the non-pregnant uterus. Hyperperistalsis and dysperistalsis of uterus constructions might underlie many important disorders such as dysmenorrhea, infertility, endometriosis, implantation failure. The major proteins for uterine contraction of the non-pregnant uterus may be Oxytocin (OT) and oxytocin receptor (OTR). The objective of this study was to inspect the expression of OTR in isthmic and mid-fundal parts of the uterine junctional zone at different stages of the follicular cycle in patients with and without endometriosis. METHODS: Uterine biopsies containing endometrium and junctional zone were collected from the isthmic and mid-fundal parts of the anterior wall after hysterectomy. The OTR expression was evaluated by immunohistochemistry. RESULTS: In the control uterus, OTR expression in the isthmic region was significantly higher than in the fundal region in the proliferative phase (p < 0.05) but significantly lower in the secretory phase (p < 0.05). And the expression of OTR in the proliferative phase was significantly higher than that in the secretory phase in both isthmic and fundal regions (p = 0.000 and 0.049, respectively). However, in endometriosis uteri, OTR expression in the isthmic region showed no significant difference with that in the fundal region in both proliferative and secretory phases (p = 0.597 and 0.736, respectively). In both isthmic and fundal regions, OTR expression was not significantly different between the proliferative phase and secretory phase (p = 0.084 and 0.222, respectively). OTR expression in fundal regions of revised ASRM I and II endometriosis were lower than that of revised ASRM III and IV (p = 0.049). In the fundal region of JZ, the expression of OTR in ovarian endometriosis was significantly lower than that in deep infiltrating endometriosis (p = 0.046). The expression level of OTR in the funds region is positively associated with the severity of dysmenorrhea in endometriosis group (r = 0.870, p < 0.05). Comparing to normal uteri, the expression of OTR in the secretory phase was significantly higher in the endometriosis uteri (p < 0.05). In the fundus of endometriosis uteri, OTR expression was significantly higher in both the proliferative and secretory phases (p = 0.045 and 0.028, respectively). CONCLUSION: OTR expression in the JZ of women with endometriosis changes significantly, which may result in abnormal uterine contractile activity, reducing the endometriosis-related fertility and dysmenorrhea.

Oxytocin receptors are expressed on dopamine and glutamate neurons in the mouse ventral tegmental area that project to nucleus accumbens and other mesolimbic targets.

The mesolimbic dopamine (DA) circuitry determines which behaviors are positively reinforcing and therefore should be encoded in the memory to become a part of the behavioral repertoire. Natural reinforcers, like food and sex, activate this pathway, thereby increasing the likelihood of further consummatory, social, and sexual behaviors. Oxytocin (OT) has been implicated in mediating natural reward and OT-synthesizing neurons project to the ventral tegmental area (VTA) and nucleus accumbens (NAc); however, direct neuroanatomical evidence of OT regulation of DA neurons within the VTA is sparse. To phenotype OT-receptor (OTR) expressing neurons originating within the VTA, we delivered Cre-inducible adeno-associated virus that drives the expression of fluorescent marker into the VTA of male mice that had Cre-recombinase driven by OTR gene expression. OTR-expressing VTA neurons project to NAc, prefrontal cortex, the extended amygdala, and other forebrain regions but less than 10% of these OTR-expressing neurons were identified as DA neurons (defined by tyrosine hydroxylase colocalization). Instead, almost 50% of OTR-expressing cells in the VTA were glutamate (GLU) neurons, as indicated by expression of mRNA for the vesicular GLU transporter (vGluT). About one-third of OTR-expressing VTA neurons did not colocalize with either DA or GLU phenotypic markers. Thus, OTR expression by VTA neurons implicates that OT regulation of reward circuitry is more complex than a direct action on DA neurotransmission. J. Comp. Neurol. 525:1094-1108, 2017. © 2016 Wiley Periodicals, Inc.

Effect of cigarette smoking on mRNA and protein levels of oxytocin receptor and on contractile sensitivity of uterine myometrium to oxytocin in pregnant women.

OBJECTIVES: Although smoking is the most important modifiable risk factor associated with preterm delivery, the underlying mechanism by which smoking stimulates premature uterine contractions is still poorly understood. In the present study, we investigated whether cigarette smoking affects the contractile sensitivity of uterine myometrium to oxytocin in pregnant women. STUDY DESIGN: Cigarette smoking habits of pregnant women were evaluated by direct interviews and by measuring exhaled carbon monoxide (CO). We isolated myometrial strips from pregnant smokers and non-smokers and evaluated uterine contractile sensitivity to oxytocin. Gene expression levels of oxytocin receptors (OTR) were compared between myometrial strips obtained from smokers and non-smokers by real-time PCR. OTR protein levels in the myometrium were evaluated by Western blotting. RESULTS: The reported number of cigarettes smoked per day by the interviewee significantly correlated with the concentration of exhaled CO. Oxytocin sensitivity increased significantly in smokers (n=6) compared with non-smokers (n=11). Real-time PCR analysis did not reveal any significant difference in OTR mRNA expression between smokers and non-smokers. Western blotting revealed that OTR level was significantly increased in smokers compared with non-smokers. Both number of cigarettes smoked per day and the concentration of exhaled CO correlated with oxytocin sensitivity. CONCLUSION: Our findings suggest that smoking increases oxytocin sensitivity of pregnant myometrium by increasing OTR levels even though OTR mRNA expression remains unaltered, thereby increasing the risk of preterm delivery in women who smoke during pregnancy. The sensitivity is dependent on number of cigarettes smoked per day.

The effects of exogenous melatonin and melatonin receptor blockade on aggression and estrogen-dependent gene expression in male California mice (Peromyscus californicus).

Photoperiodic regulation of aggression has been well established in several vertebrate species, with rodents demonstrating increased aggression in short day photoperiods as compared to long day photoperiods. Previous work suggests that estrogens regulate aggression via rapid nongenomic pathways in short days and act more slowly in long days, most likely via genomic pathways. The current study therefore examines the role of melatonin in mediating aggression and estrogen-dependent gene transcription. In Experiment 1, male California mice were housed under long day photoperiods and were treated with either 0.3 µg/g of melatonin, 40 mg/kg of the melatonin receptor antagonist luzindole, or vehicle for 10 days. We found that melatonin administration significantly increased aggression as compared to mice receiving vehicle, but this phenotype was not completely ameliorated by luzindole. In Experiment 2, male California mice were injected with either 1mg/kg of the aromatase inhibitor letrozole or vehicle, and oxytocin receptor (OTR), estrogen receptor alpha (ERα), and c-fos gene expression was examined in the bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). In the BNST, but not MPOA, OTR mRNA was significantly downregulated following letrozole administration, indicating that OTR is an estrogen-dependent gene in the BNST. In contrast, ERα was not estrogen dependent in either brain region. In the MPOA, OTR mRNA was inhibited by melatonin, and luzindole suppressed this effect. C-fos and ERα did not differ between treatments in any brain region examined. These results suggest that it is unlikely that melatonin facilitates aggression via broad spectrum regulation of estrogen-dependent gene expression. Instead, melatonin may act via regulation of other transcription factors such as extracellular signal regulated kinase.

Analysis of transcriptional levels of the oxytocin receptor in different areas of the central nervous system and behaviors in high and low licking rats.

The natural variation in maternal care is an interesting model to analyze the physiological mechanisms that lead to differences in the mother-infant interaction. Several studies have shown differences in the expression of brain receptors such as the dopamine, estrogen and oxytocin receptors in areas classically involved in the onset and/or maintenance of maternal behavior: the medial preoptic area, the nucleus accumbens, the amygdala, the lateral septum, and the bed nucleus of the stria terminalis. The present study examined the responses of HL and LL rats in several behavioral tests and analyzes the transcription of the oxytocin receptor (OXTR) in the olfactory bulb (OB), the prefrontal cortex (FPC), the hippocampus (HP) and the striatum (ST) in different patterns of licking behavior. Our results showed that, in the second week postpartum, HL and LL mothers did not show behavioral differences in the elevated plus maze (EPM), the forced swimming test (FST) or the open field test. In the maternal aggressive behavior test, HL females showed a higher frequency of biting compared to LL females, but no significant differences in other aggressive behaviors were detected. LL mothers had higher levels of transcriptional OXTR in the OB and in the HP when compared to HL mothers. No differences in other areas were detected when compared LL and HL. These findings suggest that variations in maternal behavior may be associated with biting behavior of mothers and that OXTR participates in modulation of maternal behavior in rats, while other emotional behaviors are less related to such behavior.

Oxytocin, vasopressin and estrogen receptor gene expression in relation to social recognition in female mice.

Inter- and intra-species differences in social behavior and recognition-related hormones and receptors suggest that different distribution and/or expression patterns may relate to social recognition. We used qRT-PCR to investigate naturally occurring differences in expression of estrogen receptor-alpha (ERα), ER-beta (ERß), progesterone receptor (PR), oxytocin (OT) and receptor, and vasopressin (AVP) and receptors in proestrous female mice. Following four 5 min exposures to the same two conspecifics, one was replaced with a novel mouse in the final trial (T5). Gene expression was examined in mice showing high (85-100%) and low (40-60%) social recognition scores (i.e., preferential novel mouse investigation in T5) in eight socially-relevant brain regions. Results supported OT and AVP involvement in social recognition, and suggest that in the medial preoptic area, increased OT and AVP mRNA, together with ERα and ERß gene activation, relate to improved social recognition. Initial social investigation correlated with ERs, PR and OTR in the dorsolateral septum, suggesting that these receptors may modulate social interest without affecting social recognition. Finally, increased lateral amygdala gene activation in the LR mice may be associated with general learning impairments, while decreased lateral amygdala activity may indicate more efficient cognitive mechanisms in the HR mice.

Oxytocin and social motivation.

Humans are fundamentally social creatures who are 'motivated' to be with others. In this review we examine the role of oxytocin (OT) as it relates to social motivation. OT is synthesized in the brain and throughout the body, including in the heart, thymus, gastrointestinal tract, as well as reproductive organs. The distribution of the OT receptor (OTR) system in both the brain and periphery is even more far-reaching and its expression is subject to changes over the course of development. OTR expression is also sensitive to changes in the external environment and the internal somatic world. The OT system functions as an important element within a complex, developmentally sensitive biobehavioral system. Other elements include sensory inputs, the salience, reward, and threat detection pathways, the hypothalamic-pituitary-gonadal axis, and the hypothalamic-pituitary-adrenal stress response axis. Despite an ever expanding scientific literature, key unresolved questions remain concerning the interplay of the central and peripheral components of this complex biobehavioral system that dynamically engages the brain and the body as humans interact with social partners over the course of development.

Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cows.

The objective of the present study was to investigate if plasma progesterone (pP(4)) concentrations are dependent on luteal size, blood flow, or gene expression in luteal tissue. To induce cycles with high and low pP(4) concentrations, respectively, 20 lactating dairy cows received either a single treatment with 25 mg prostaglandin F(2α) (PGF(2α)) on Day 4 Hour 12 (PG1; n=8), or two treatments (25 mg PGF(2α) each) on Day 4 Hours 0 and 12 (PG2; n=12) of the estrous cycle (Day 1, Hour 0=ovulation). In four cows, ovulation occurred between 4 and 6d after the second PGF(2α) treatment; these cows and one lame cow were excluded from the study. In the 15 remaining cows with physiological interovulatory intervals, pP(4), area (LTA) and volume (LTV) of luteal tissue, as well as absolute (LBF) and relative (rLBF) luteal blood flow were determined on Day 9, and relative luteal P(4) (rLP(4)) as well as luteal mRNA expression of important receptors, angiogenic, vasoactive, and steroidogenic factors were quantified on Day 11 (±1) during two successive estrous cycles. Furthermore, rLP(4) was multiplied by LTV to produce a semiquantitative assessment of absolute luteal P(4) (LP(4)). There was no effect (P>0.05) of treatment (one or two PGF(2α) treatments), neither on pP(4) concentrations nor on any other parameter in the present study. Nevertheless, there was a lower LP(4) (P=0.01), LTA (P=0.03), and LTV (P=0.02), as well as tendencies of lower pP(4) (P=0.06) and LBF (P=0.09) at first compared with second diestrus. Plasma P(4) was related with LP(4) (r=0.43, P=0.04), LTA (r=0.65, P=0.0001), and LTV (r=0.43, P=0.02), but not with rLBF (r=-0.18, P=0.34). Furthermore, there was no significant correlation between gene expression of important steroidogenic factors and P(4) concentrations in luteal tissue. Results indicate that plasma P(4) concentrations in the mid-luteal phase were dependent on luteal size, but independent of blood flow and gene expression per luteal tissue unit.

Downregulation of oxytocin receptors in right ventricle of rats with monocrotaline-induced pulmonary hypertension.

AIM: pulmonary hypertension (PH) in the rat leads to right ventricular (RV) hypertrophy, inflammation and increased natriuretic peptide (NP) levels in plasma and RV. Because the release of nitric oxide (NO) and atrial natriuretic peptide (ANP) is a function of the oxytocin receptor (OTR), we examined the effect of PH on gene and protein expression of OTR, NP (A, atrial; B, brain) and receptors (NPRs), nitric oxide synthases (NOS), interleukin (IL)-1ß, IL-6 and tumour necrosis factor-α in the hypertrophied RV in a model of PH. METHODS: RV hypertrophy was induced in male Sprague-Dawley rats with monocrotaline (MCT; 60 mg kg(-1) ) and was confirmed by the presence of an increased RV weight and RV-to-[left ventricle (LV) and septum] ratio. RESULTS: in the RV of MCT-treated rats, a approximately 40% reduction in OTR mRNA and protein was observed compared with the RV of control rats. This reduction was associated with increased transcripts of ANP and BNP in both ventricles and a corresponding increase in NP receptor mRNA expression for receptors A, B and C. Protein expression of inducible NOS was increased in the RV, whereas endothelial NOS transcripts were increased only in the LV of MCT-treated rats. In the RV of MCT-treated rats, downregulation of OTR was also associated with increased mRNA expression of IL-1ß and IL-6. CONCLUSION: our results show that downregulation of the OTR in the RV of MCT-treated rats is associated with increased expression of NP and their receptors as well as IL-1ß and IL-6. This reduction in OTR in RV myocardium may have an impact on cardiac function in the MCT-induced model of PH.

Estradiol upregulates the expression of oxytocin receptor in colon in rats.

The study was designed to investigate the effect of estradiol on the excitatory effect of oxytocin (OT) on colon motility. Female Wistar rats were used, and some of them were ovariectomized (OVX) and treated with vehicle or estradiol (E(2)). A plastic balloon made of condom was inserted into colon to monitor the change of colonic pressure in vivo. Longitudinal muscle strips of distal colon were prepared to monitor the spontaneous contraction of colon in vitro. Expression of OT receptor (OTR) was investigated by Western blot analysis. Expression of OTR mRNA was detected by RT-PCR. Immunohistochemistry was used to locate OTR. In OVX rats, pretreatment of E(2) (4-100 microg/kg sc) dose-dependently increased the excitatory effect of OT on colon motility both in vivo and in vitro and increased the expression of OTR and OTR mRNA in colon. Systemic administration of OT excited the colon motility in vivo in rats at perioda of proestrus and estrus but did not influence it at diestrus period, when the concentration of plasma E(2) was lowest in the estrous cycle. Pretreatment of atosiban, the specific OTR antagonist, and TTX, the blocker of voltage-dependent sodium channel on nerve fiber, attenuated the excitatory effect of OT on colon motility. OTR was located in myenteric plexus of colon. These results suggested that E(2) increased the excitatory effect of OT on colon motility by upregulating the expression of OTR in myenteric plexus.

Oxytocin receptor pattern of expression in primary lung cancer and in normal human lung.

In order to assess if oxytocin- and vasopressin-induced mitogenic effects detected on small-cell lung carcinoma (SCLC) cell lines could be transposed on primary SCLC, the aim of the present work was to identify mediators of these mitogenic actions on primary tumours samples. This was addressed on normal human lung tissue, on SCLC and on non-SCLC (NSCLC). Herein, we observe, in normal human lung, that OTR is colocalized with vascular endothelial cells of the lung and is not expressed by lung cells of epithelial nature. We detected mRNA amplification of V1aR, V2R and of a V2R variant. We observed that 86% of SCLC biopsies analyzed expressed at least the OTR and that 71% expressed the OTR, the V1aR and the V2R altogether. Comparatively, 50% of NSCLC biopsies tested expressed at least the OTR and 32% expressed the OTR, the V1aR and the V2R altogether. The occurrence of the V1bR/V3R is of 28 and 18% for SCLC and NSCLC, respectively. Nevertheless, for the SCLC biopsies analyzed in this study, V1bR/V3R expression correlates, in all cases, with the expression of all the other neurohypophysial peptide receptors. Our results suggest that neurohypophysial peptide antagonists may offer promise as a potential new therapeutic modality for the treatment of lung cancer expressing at least one of the neurhypophysial peptide receptor subtypes.

Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: a polymerase chain reaction study.

BACKGROUND/AIM: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. MATERIAL AND METHODS: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malmö University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70 degrees C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A(+) mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. RESULTS: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. CONCLUSION: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown.

Effects of the estrous cycle, pregnancy and interferon tau on expression of cyclooxygenase two (COX-2) in ovine endometrium.

In sheep, the uterus produces luteolytic pulses of prostaglandin F2alpha (PGF) on Days 15 to 16 of estrous cycle to regress the corpus luteum (CL). These PGF pulses are produced by the endometrial lumenal epithelium (LE) and superficial ductal glandular epithelium (sGE) in response to binding of pituitary and/or luteal oxytocin to oxytocin receptors (OTR) and liberation of arachidonic acid, the precursor of PGF. Cyclooxygenase-one (COX-1) and COX-2 are rate-limiting enzymes in PGF synthesis, and COX-2 is the major form expressed in ovine endometrium. During pregnancy recognition, interferon tau (IFNtau), produced by the conceptus trophectoderm, acts in a paracrine manner to suppress development of the endometrial epithelial luteolytic mechanism by inhibiting transcription of estrogen receptor alpha (ERalpha) (directly) and OTR (indirectly) genes. Conflicting studies indicate that IFNtau increases, decreases or has no effect on COX-2 expression in bovine and ovine endometrial cells. In Study One, COX-2 mRNA and protein were detected solely in endometrial LE and sGE of both cyclic and pregnant ewes. During the estrous cycle, COX-2 expression increased from Days 10 to 12 and then decreased to Day 16. During early pregnancy, COX-2 expression increased from Days 10 to 12 and remained higher than in cyclic ewes. In Study Two, intrauterine infusion of recombinant ovine IFNtau in cyclic ewes from Days 11 to 16 post-estrus did not affect COX-2 expression in the endometrial epithelium. These results clearly indicate that IFNtau has no effect on expression of the COX-2 gene in the ovine endometrium. Therefore, antiluteolytic effects of IFNtau are to inhibit ERalpha and OTR gene transcription, thereby preventing endometrial production of luteolytic pulses of PGF. Indeed, expression of COX-2 in the endometrial epithelia as well as conceptus is likely to have a beneficial regulatory role in implantation and development of the conceptus.

Prolonged progesterone treatment of endometrial epithelial cells modifies the effect of estradiol on their sensitivity to oxytocin.

Estradiol (E2), progesterone (P4), and oxytocin (OT) are important for the initiation of luteolysis in ruminants but the mechanisms involved are still poorly understood. The objective of this study was to determine if duration of exposure of bovine endometrial epithelial cells to P4 affected the response of the cells to E2. Endometrial epithelial cells, from cows at Days 1-3 of the estrous cycle, were cultured for 10, 17, and 21 days in the presence or absence of P4 (100 ng ml(-1)). After culture, each group of cells was incubated for a further 6, 12, 24 or 48 h with or without E2 (100 pg ml(-1)) and then incubated for 6 h with different doses of OT (2, 20, and 200 ng ml(-1)). E2 enhanced OT-stimulated PGF2 alpha secretion in cells cultured with P4 for 17 or 21 days, with a maximum effect after 24-h exposure, but not in cells cultured with P4 for 10 days. To determine the mechanism of action of E2, COX-1 and COX-2 were measured by Western blotting and OTR number was measured by saturation analysis. OT increased COX-2 (P<0.05), but there was no significant effect of E2 on the expression of either COX-1 or COX-2. E2 did, however, increase (P<0.001) the OTR number in cells cultured with P4 for 21 days, whereas it inhibited OTR in cells cultured for 10 days. These data show that E2 can stimulate PGF2 alpha secretion by increasing OTR expression in bovine endometrial cells in vitro, but only after exposure to P4.

The oxytocin receptor.

Novel sites of oxytocin receptor expression have recently been detected, including breast cancer cells, bone cells, myoblasts, cardiomyocytes and endothelial cells. These discoveries have greatly expanded the possible spectrum of oxytocin action beyond its classic role as an inducer of uterine contractions and milk ejection. Additional advances in the understanding of oxytocin receptor structure-function relationships, receptor trafficking and novel receptor-linked signaling cascades have made this receptor an attractive model for the study of G-protein-linked receptor function. Finally, the tocolytic efficiency of the oxytocin receptor antagonist atosiban, recently approved for clinical use in Europe, has opened new avenues for the prevention and treatment of preterm labor.

Parathyroid hormone-related protein treatment of pregnant rats delays the increase in connexin 43 and oxytocin receptor expression in the myometrium.

Myometrial quiescence during pregnancy is maintained by progesterone, which suppresses the expression of labor-associated genes such as connexin 43 (Cx43) and the oxytocin receptor (OTR). Parathyroid hormone-related protein (PTHrP) is a smooth muscle relaxant that inhibits myometrial contractions and therefore may act in synergy with progesterone to maintain myometrial quiescence during late pregnancy. We investigated the possibility that PTHrP, like progesterone, could act to suppress the expression of labor-associated genes. Pregnant rats were treated starting on Day 19 with daily i.p. injections of 100 microg/kg PTHrP (human synthetic fragment 1-34). On Day 22 of gestation, there was a significant reduction in the expression of Cx43 (mRNA and protein) and OTR (mRNA) in the myometrium of PTHrP-treated animals, whereas on Day 23 (labor) the expression of both Cx43 and OTR was unchanged by PTHrP treatment. Treatment of pregnant rats with PTHrP did not affect the time of delivery, concentrations of progesterone in maternal plasma, or levels of c-fos, fra-2, or parathyroid hormone/PTHrP receptor mRNA on any gestational day. Because PTHrP treatment delayed the dramatic increase in the expression of Cx43 and OTR, it may be an important factor in the maintenance of the quiescent state of the myometrium at a time when the concentrations of progesterone in maternal circulation decrease. PTHrP treatment did not prevent the increase in Cx43 and OTR gene expression on Day 23 or the timing of labor, suggesting that the effects of PTHrP signaling are overridden with the onset of labor.

Transcriptional regulation of the bovine oxytocin receptor gene.

The oxytocin receptor (OTR) is expressed in the cow uterus at high levels at estrus and at term of pregnancy. This expression appears to be controlled mostly at the transcriptional level and correlates with increasing estrogen concentration and progesterone withdrawal. Approximately 3200 base pairs of the upstream region of the bovine OTR gene were cloned and analyzed using a combination of bioinformatic, electrophoretic mobility shift (EMSA), and transfection analyses. Using nuclear proteins from high- and low-expressing tissues, EMSA indicated no significant quantitative or qualitative changes in specific DNA-protein binding, suggesting that transcription is probably controlled by signalling systems targeting constitutive factors. Using various cell types, including primary and immortalized ruminant endometrial epithelial cells, as hosts for transfection of promoter-reporter constructs showed that endogenous activity resided only in the longest, i.e., 3.2-kb, construct but not in those shorter than 1.0 kb. While estrogen appears to be important in vivo, no effect of estradiol was found on any construct directly; only when the longest 3.2-kb construct was used in combination with some cotransfected steroid receptor cofactors, e.g., SRC1e, was an estradiol-dependent effect observed. A putative interferon-responsive element (IRE) was found at approximately -2,400 from the transcription start site. This element was shown to bind mouse IRF1 and IRF2 as well as similar proteins from bovine endometrial and myometrial nuclear extracts. This element also responded to these factors when cotransfected into various cell types. The bovine equivalents to IRF1 and IRF2 were molecularly cloned from endometrial tissue and shown to be expressed in a temporal fashion, supporting the role of interferon-tau in maternal recognition of pregnancy. Of many factors tested or analyzed, these components of the IFN system are the only ones found to significantly influence the transcription of the bovine OTR gene.