Oxytocin gene expression in rat uterus.

The neurohypophyseal hormone oxytocin (OT) is the most potent uterotonic agent known and is used to induce labor. Yet, endogenous circulating OT appears not to participate in the induction of labor. As shown here, the finding of OT messenger RNA and peptide in the uterus suggests a solution for this paradox. During gestation, rat uterus OT messenger RNA increased more than 150-fold and, at term, exceeded hypothalamic OT messenger RNA by 70-fold. Thus, during parturition, OT may act primarily as a local mediator and not as a circulating hormone.

Biosynthesis of immunoreactive somatostatin by hypothalamic neurons in culture.

The neuronal biosynthesis of somatostatin-like immunoreactivity (SLI) was investigated using mechanically dispersed neonatal rat hypothalamic cells kept in culture for up to 6 wk. Immunohistochemically, SLI was specifically localized to a small subpopulation of parvicellular neurons and their cell processes. By radioimmunoassay the cellular SLI content declined steadily during the first 2 wk in culture (nadir value of 60 fmol/dish at day 15) but then increased progressively to reach a maximum value of 381 fmol/dish at day 46. Gel chromatographic analysis showed this immunoreactivity to consist of forms corresponding to tetradecapeptide somatostatin (S-14), somatostatin-28 (S-28), and a 15,000-mol-wt molecule. After incubation of the cells with [3H]phenylalanine, the cellular extracts, purified by adsorption to C18 silica, contained material that bound specifically to an immobilized antisomatostatin antibody. Analysis by gel chromatography and high performance liquid chromatography of the specifically bound label provided evidence for the presence of labeled S-14, S-28, and the 15,000-mol-wt molecule. Pulse-chase experiments (20-min pulse, 20-min chase) demonstrated a transfer of radioactivity from the 15,000-mol-wt form to material corresponding to S-14 as well as to S-28. These studies demonstrate that cultured hypothalamic neurons are capable of synthesizing three somatostatin-like peptides (15,000-mol-wt SLI, S-28, S-14), one of which (15,000-mol-wt SLI) serve as a biosynthetic precursor for both S-28 and S-14. This in vitro system should provide a powerful tool for further investigation of the biosynthesis and regulation of biosynthesis of somatostatin in the hypothalamus.

Tissue-specific alterations in somatostatin mRNA accumulation in streptozocin-induced diabetes.

In streptozocin-induced diabetes in rats, there is a marked increase in the content and release of immunoreactive somatostatin (SLI) from the pancreas and upper gut. To elucidate whether these SLI changes are associated with alterations in somatostatin gene transcription, we measured somatostatin mRNA (SmRNA) accumulation in these and other SLI-producing tissues. Pancreas, stomach, jejunum, hypothalamus, and cerebral cortex were removed from control rats, 6-wk-diabetic rats, and diabetic rats treated with insulin for 6 wk. Total RNA was isolated by centrifugation through CsCl and fractionated on agarose gels. A sensitive radiodensitometric hybridization assay was used to determine SmRNA levels in absolute amounts by in vitro synthesized sense-strand RNA as a quantitative standard and antisense cRNA as a specific probe. SLI was determined by radioimmunoassay. SmRNA exhibited size heterogeneity between the different control and diabetic tissues. A 2- to 3-fold increase in total SmRNA was found in pancreas and stomach of the diabetic rats that suppressed toward normal with insulin treatment. These two tissues also exhibited significant 1.6- to 2.6-fold increases in SLI, respectively. The remaining tissues showed no diabetes-related changes in SLI or SmRNA. We conclude that in insulinopenic diabetes, tissue SLI and SmRNA accumulation undergo parallel changes; are increased in pancreas and upper gut, reflecting augmented somatostatin synthesis; are reciprocally related to insulin acting directly or indirectly on somatostatin-producing cells; and are unchanged in the lower gut and brain, suggesting tissue-specific regulation of somatostatin gene transcription in diabetes.

Novel vasopressin gene-related transcripts in rat testis.

Although the arginine vasopressin (AVP) gene is predominantly expressed in specific hypothalamic neurons, immunoreactive AVP (irAVP) has also been identified in peripheral tissues, including testis. To determine whether hypothalamic and testicular ir-AVPs derive from the same or different transcripts, we examined testicular AVP gene-related transcripts by Northern blot analysis, polymerase chain reaction (PCR), and RNase mapping. We show that the testis contains three distinct AVP gene-related transcripts of 0.67, 0.83, and 2.0 kilobases (kb) which differ in size from the 0.81-kb hypothalamic AVP mRNA. As demonstrated by deadenylation, this size heterogeneity is not due to differences in the length of the poly(A) tails. By the use of exon-specific probes we determined that the 0.67- and the 0.83-kb transcripts contain exons B and C, but not A. In contrast, the 2-kb transcript is the only testicular transcript that contains an exon A-related sequence. By application of the PCR technique, we confirm the existence of testicular transcripts in which exons B and C are spliced together, but exon A is excluded. Our findings indicate that the 0.67- and 0.83-kb mRNA results from a differential splicing event that excludes exon A and that the 2-kb mRNA probably originates from the expression of an AVP-related gene. Since the nonapeptide AVP is encoded by exon A, testicular irAVP cannot arise from the translation of any of the exon B- and C-containing transcripts, and any mRNA that has so far been identified by AVP exon C probes is unrelated to the biosynthesis of AVP-immunoreactive products.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and region-specific regulation of the oxytocin receptor gene in rat brain.

The neuropeptide oxytocin (OT) exerts its various neurotransmitter functions via specific OT receptors (OTRs) that have been localized to distinct brain regions, including the ventromedial hypothalamus, the bed nucleus of stria terminalis, the amygdala, the subiculum, the hippocampus, and the olfactory nuclei. In the present study, we have characterized OTR gene expression by Northern blot and by semiquantitative RT-PCR in these brain regions and studied its regulation in response to estrogen (E2), progesterone, and the antiestrogen tamoxifen. We find that all regions analyzed express two messenger RNA (mRNA) bands (6.7 and 4.8 kb) that hybridize to a rat OTR complementary DNA probe and that correspond in size to two of the three OTR mRNA bands expressed in rat uterus. Analysis by RT-PCR, with two different primer pairs, did not reveal any structural differences between the coding regions of uterine and brain OTR mRNA. E2 treatment and gestation led to an 8-fold and a 6.5-fold increase in OTR mRNA levels, respectively. Progesterone was without effect, if administered alone, and did not influence the E2-induced rise in OTR mRNA. The E2 effect was restricted to E2-sensitive regions, such as the hypothalamus, and was not observed in the subiculum or the olfactory nuclei. Tamoxifen had a dual effect: on the one hand, it acted as a partial agonist in raising OTR mRNA levels in the hypothalamus of ovariectomized animals; on the other hand, it suppressed the E2-induced OTR mRNA rise in E2-sensitive brain regions. Although the present data do not exclude the possible existence of OTR subtype(s) in brain, they show that the uterine-type OTR gene is expressed in all major OTR-containing brain regions. Moreover, they show that region-specific regulation of OTR gene expression underlies the previously observed region-specific steroid regulation of central OT binding sites.

Expression of the oxytocin gene in rat placenta.

The placenta is an endocrinologically active organ and expresses an important number of polypeptide hormone genes. Although oxytocin (OT)-like immunoreactivity has been detected in placental extracts by RIA, the precise nature and origin of placental OT has remained unclear. In the present study, we examined OT gene expression in rat placental tissue at various stages of gestation using northern blot analysis, polymerase chain reaction, in situ hybridization, HPLC, and immunocytochemistry. Northern blot analysis of RNA extracted from rat placenta revealed a single type of OT gene transcript (0.66 kilobases) which differed in size from hypothalamic OT transcripts (0.75 kilobases). Deadenylation of placental and hypothalamic messenger RNA (mRNA) showed that this size difference was due to differences in poly(A) tail lengths. Polymerase chain reaction amplification of placental and hypothalamic complementary DNAs using four different exon-specific primers provided no evidence for the existence of any additional structural differences between hypothalamic and placental OT-gene transcripts. Quantitative evaluation of northern blots showed that OT mRNA abundance per microgram of total RNA was stage specific and declined by a factor of 6 from day 14 to day 21 of gestation. In contrast to the marked variation of mRNA abundance, the OT peptide content, as measured by RIA, underwent no significant change during the time period studied and varied between 0.37-0.51 ng/g wet tissue wt. Characterization of placental OT immunoreactivity by HPLC and gel filtration identified two peaks of immunoreactivity: one peak (70% of immunoreactivity) corresponded to synthetic OT; whereas the other peak (Mr 11,000, 30% of immunoreactivity) represented a noncovalent association between OT and another molecule, consistent with the formation of a neurophysin/OT complex. By in situ hybridization and immunocytochemistry, we localized OT mRNA and OT immunoreactivity to cells of the trophoblastic epithelium covering the septa of the labyrinth as well as to cytotrophoblastic elements and giant cells of the maternally derived basal zone of the placenta. Placental OT may act locally, may interact with uterine OT receptors, or may play a role in fetal development.

Renal oxytocin receptor messenger ribonucleic acid: characterization and regulation during pregnancy and in response to ovarian steroid treatment.

Although the neurohypophyseal hormone oxytocin (OT) is best know for its role in reproduction, OT also stimulates natriuresis at physiological plasma levels. This effect is mediated via specific renal OT receptors (OTRs). In the present study, we have characterized rat renal OTR gene transcripts and assessed their regulation during gestation and in response to gonadal steroid treatment. Using a specific rat OTR probe, two major OTR messenger RNA (mRNA) bands [6.7 and 4.8 kilobases (kb)] were detected in renal extracts, corresponding to two of the three bands present in rat uterus. In contrast to the dramatic rise of OTR mRNA levels at term in the uterus and pituitary, renal OTR mRNA levels underwent a strong more than 3-fold decrease at term. Binding studies using a iodinated specific OT antagonist revealed a concomitant decrease in renal OT-binding sites. On the other hand, estrogen (E2) treatment led to an increase in renal OTR mRNA levels, as is also the case in the uterus and pituitary. However, the predominant E2-induced mRNA species were shorter (3.6 and 3.2 kb) than those present in control rat kidneys (6.7 and 4.8 kb). Analysis by reverse transcriptase-PCR and 5'- and 3'-directed complementary DNA probes indicated that the E2-induced OTR mRNA transcripts possessed the same coding region, but contained a shortened 3'-untranslated region. Binding studies showed that E2 treatment also led to an increase in renal OT-binding sites, suggesting that the shortened OTR transcripts encoded a functional receptor. The present study indicates that the uterine-type OTR gene is expressed in rat kidneys, but that the mechanisms controlling the expression of this gene in the two tissues are markedly different. The differential tissue-specific regulation of OTR gene expression may represent a mechanism by which circulating OT can assume a multifunctional role in both reproduction and sodium homeostasis.

Developmental expression of the rat somatostatin gene.

The developmental expression of the somatostatin (SRIF) gene was investigated in rat brain and stomach, two SRIF-rich tissues. The accumulation of mRNA encoding SRIF was determined in these organs during fetal and early (1-4 weeks) postnatal development using a sensitive radiodensitometric cDNA hybridization assay and a cloned preprosomatostatin cDNA. A single band of mRNA which hybridized specifically to the rat SRIF cDNA was detected in both tissues examined throughout ontogenesis, suggesting that the same SRIF gene is expressed in these tissues in the developing as well as in the adult rat. Whereas SRIF mRNA was undetectable in fetal stomach and rose gradually only after birth, brain SRIF mRNA was already detectable by day 7 of embryonic life and reached concentrations corresponding to those in the adult brain by embryonic day 20. These marked differences may reflect basic differences in the developmental regulation of SRIF gene expression in neural vs. nonneural tissues or may be related to the onset of functional activity in the organs studied.

Oxytocin receptor messenger ribonucleic acid: characterization, regulation, and cellular localization in the rat pituitary gland.

The hypothalamic neuropeptide oxytocin (OT) stimulates the release of several pituitary hormones, including ACTH, LH, and PRL. Although specific OT receptors have been identified in anterior pituitary membranes, the structure and cellular localization of these binding sites have not been elucidated. We previously cloned a rat OT receptor (OTR) gene and showed that its expression in rat uterus results in several transcripts ranging in size from 2.9-6.7 kilobases. In this study we show, by using Northern blot analysis, reverse transcriptase-polymerase chain reaction, and ultrastructural in situ hybridization that the same OTR gene is also expressed in the pituitary, where it gives rise to a 6.7- and a 4.8-kilobase messenger RNA. Ultrastructural in situ hybridization combined with immunogold labeling indicated that pituitary OTR gene expression is highly cell-specific and restricted to lactotrophs. In accordance with this finding, only the lactotroph-derived cell line MMQ expressed the OTR gene among several pituitary cell lines tested. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis indicated a dramatic increase in pituitary OTR gene expression at the end of gestation and after estrogen treatment. Our results suggest that the OT effect on lactotrophs is direct, whereas OT actions on gonadotrophs and corticotrophs are either indirect or mediated via different receptors. Moreover, our findings imply that OT exerts its full potential as a physiological PRL-releasing factor only towards the end of gestation, and that therefore the role of OT as a hypothalamic PRL-releasing factor may so far have been underestimated.

Uterine oxytocin gene expression. I. Induction during pseudopregnancy and the estrous cycle.

We have recently demonstrated that the gene encoding the hypothalamic peptide oxytocin (OT) is highly expressed in the rat endometrial epithelium during the last 4 days of pregnancy. Here, we show that uterine OT gene expression is also induced during the proestrous phase of the estrous cycle and after induction of pseudopregnancy. In mature female rats, OT mRNA levels increased more than 10-fold between diestrus and proestrus and remained elevated at estrus. The levels attained at estrus corresponded to about 1/20th of the levels present at term. In immature rats rendered pseudopregnant by treatment with pregnant mare serum and hCG, uterine OT mRNA levels rose steadily and reached a maximum on day 14 of pseudopregnancy, corresponding to about 1/8th of the levels observed on day 21 of normal pregnancy. Oil-induced decidualization of the left uterine horn prolonged pseudopregnancy and maintained OT mRNA levels in both uterine horns until day 19 of pseudopregnancy. These changes were tissue specific, as hypothalamic OT mRNA levels remained essentially unaffected. The present findings demonstrate that either spontaneous or induced changes in endogenous steroid levels are capable of eliciting important changes in uterine, but not hypothalamic, OT gene expression.

Uterine oxytocin gene expression. II. Induction by exogenous steroid administration.

As we have recently shown, the gene encoding the hypothalamic nonapeptide oxytocin (OT) is expressed in the rat endometrial epithelium during late pregnancy and the estrous phase of the estrous cycle. To investigate the role of ovarian steroids in the regulation of uterine OT gene expression, Silastic capsules containing estradiol or progesterone were implanted into immature ovariectomized rats. Exposure to estradiol alone for 2 days caused a significant rise in OT mRNA. Administration of progesterone alone was without effect. However, a strong synergism was observed when the two hormones were applied together; progesterone potentiated the effect of estradiol by a factor of 7. In animals treated with steroids for 4 days, the removal of either the estradiol or progesterone capsule after day 2 led to a decrease in the total amount of OT mRNA accumulation, implying that the continued action of both steroids was required to achieve maximal OT mRNA levels. Immunocytochemical analysis demonstrated that the main site of steroid-induced uterine OT gene expression is the endometrial epithelium, the same site where endogenously induced OT gene expression occurs at the end of pregnancy. The OT mRNA levels achieved after 4 days of treatment with both steroids were comparable to those achieved at estrus or during pseudopregnancy, but corresponded to less than 20% of the levels present in the uterus on day 21 of pregnancy. These data suggest that in the uterus, the synergistic action of ovarian steroids represents an important, but probably not exclusive, regulator of OT gene expression.

Regulation of islet somatostatin secretion and gene expression: selective effects of adenosine 3',5'-monophosphate and phorbol esters in normal islets of Langerhans and in a somatostatin-producing rat islet clonal cell line 1027 B2.

To investigate cAMP-dependent regulation of somatostatin secretion and gene expression in the islets of Langerhans, we have correlated the effects of forskolin, theophylline, and (Bu)2cAMP (dbcAMP) on the secretion of somatostatin-like immunoreactivity (SLI), cAMP generation, and somatosatin mRNA (S-mRNA) accumulation by cultured rat islet cells and a rat somatostatin-producing islet tumor cell line (1027 B2). Additionally, we have compared these effects with those of phorbol esters. Forskolin induced large acute increases in cAMP levels in islet cells, whereas theophylline produced modest sustained elevations in cAMP. During 4-h exposure to islets cells, forskolin, theophylline, and dbcAMP produced time- and dose-related increases of up to 14-fold in SLI release and up to 5-fold in S-mRNA levels. The rate of increase in S-mRNA paralleled secretion and occurred with the following order of potency: forskolin greater than dbcAMP greater than theophylline. The analog 1,9-dideoxyforskolin, which is unable to activate adenylyl cyclase, produced a small increase in SLI release without affecting S-mRNA. The effects of short term increases in islet cAMP levels and SLI release on long term changes in S-mRNA accumulation were investigated in a 48-h study with forskolin. Pretreatment of islet cells for 30 min with forskolin evoked large acute increases in cAMP levels and SLI release. S-mRNA rose in a biphasic pattern, with an acute increase at 30 min followed by a secondary increase at 12-48 h. In 1027B2 cells, forskolin and theophylline generated large increases in cAMP levels. Despite this, the two agents as well as dbcAMP produced only slight (20-35%) stimulation of SLI release and S-mRNA accumulation. Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate evoked dose-dependent stimulation of SLI secretion of up to 4-fold from islet cells without altering S-mRNA. Both secretion and S-mRNA were unresponsive to phorbol esters in 1027 B2 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxytocin receptor gene expression in the rat uterus during pregnancy and the estrous cycle and in response to gonadal steroid treatment.

It is well established that uterine oxytocin receptors (OTRs) are strongly up-regulated immediately before parturition as well as in response to estrogen (E2) administration. Progesterone (P4), on the other hand, induces a rapid down-regulation. We recently cloned the rat OTR gene and characterized its expression in the rat uterus. In this study, we examined the regulation of OTR messenger RNA (mRNA) levels in rat uterus during pregnancy, the estrous cycle, and in response to gonadal steroid treatment. OTR mRNA levels increased more than 25-fold during gestation: 4.5-fold during the first 21 days and 6-fold within 24 h between day 21 and the onset of parturition. Uterine OTR mRNA levels fell rapidly by 85% within 24 h following parturition. By in situ hybridization, OTR mRNA was localized specifically to the longitudinal and circular layers of the myometrium but was not detected in the endometrium. During the estrous cycle, OTR mRNA levels increased 2-fold between metestrus and proestrus, whereas oxytocin (OT) binding rose more than 10-fold within this same interval. Treatment of ovariectomized rats with E2 lead to a significant increase in both OTR mRNA levels (4.4-fold) and OT binding (< 6-fold). Cotreatment with P4 strongly reduced OT binding by 75% (P < 0.01) but did not significantly affect the E2-induced rise in OTR mRNA (11% decrease, P > 0.1). Our data suggest that the increased expression of OT binding sites observed at the onset of labor and at proestrus is mediated, at least in part, by an E2-induced up-regulation of OTR gene expression. However, it also appears that OTR mRNA levels are not the sole determinants of uterine OT binding. Specifically, P4-mediated OTR down-regulation cannot be explained by an effect on OTR mRNA accumulation and may involve novel mechanisms acting at translational or posttranslational levels.

Activation of the mouse oxytocin promoter by the orphan receptor RORalpha.

Although an increasing number of nuclear orphan receptors have recently been identified, the number of known naturally occurring genes that are directly regulated by orphan receptors is still small. We have shown previously that the gene encoding the neuropeptide oxytocin (OT) is negatively regulated by the orphan receptors chicken ovalbumin upstream transcription factor I (COUP-TFI) and II. Here we show that the mouse OT gene promoter is activated by RORalpha, a representative of the ROR/RZR orphan receptor subfamily. Using promoter/chloramphenicol acetyltransferase reporter constructs in heterologous transfection assays, we determined that RORalpha action induces a <6-fold increase in promoter activity. By 5' and 3' deletion analysis, DNase footprint analysis and electrophoretic mobility shift assays, we found that RORalpha action is mediated by two 14 bp regions centered at 160 and 180 nucleotides upstream of the transcriptional initiation site. Both sites contain significant sequence identities with an established ROR recognition sequence. Mutations in either or both of these sites reduce significantly RORalpha-induced activation of the OT promoter. In view of the strong transcriptional activation exerted by RORalpha on the OT gene promoter and the widespread distribution of different members of the ROR/RZR family, interactions between ROR/RZR isoforms and the OT gene may form part of the multifactorial regulatory mechanisms that control OT gene expression in different tissues.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter.

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer.

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively. Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.

Oxytocin receptor gene expression in rat mammary gland: structural characterization and regulation.

The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.

Mapping oxytocin receptor gene expression in the mouse brain and mammary gland using an oxytocin receptor-LacZ reporter mouse.

The hypothalamic nonapeptide oxytocin (OT) has an established role as a circulating hormone but can also act as a neurotransmitter and as a neuromodulator by interacting with its central OT receptor (OTR). To understand the role of the OTR in the mouse brain we investigated the expression of the OTR gene at the cellular level. We targeted the lacZ reporter gene to the OTR gene locus downstream of the endogenous OTR regulatory elements. Using lactating mouse mammary gland as a control for OTR promoter directed specificity of lacZ gene expression, X-gal histochemistry on tissue sections confirmed that gene expression was restricted to the myoepithelial cells. We also identified for the first time in mice the expression of the OTR gene in neighbouring adipocytes. Further, investigation in the mouse brain identified numerous nuclei containing neurons expressing the OTR gene. Whilst some of these regions had been described for rat or sheep, the OTR-LacZ reporter mouse enabled the identification of novel sites of central OTR gene expression. These regions include the accessory olfactory bulb, the medial septal nucleus, the posterolateral cortical amygdala nucleus, the posterior aspect of the basomedial amygdala nucleus, the medial part of the supramammillary nucleus, the dorsotuberomammillary nucleus, the medial and lateral entorhinal cortices, as well as specific dorsal tegmental, vestibular, spinal trigeminal, and solitary tract subnuclei. By mapping the distribution of OTR gene expression, depicted through histochemical detection of beta-galactosidase, we were able to identify single OTR gene expressing neurons and small neuron clusters that would have remained undetected by conventional approaches. These novel sites of OTR gene expression suggest additional functions of the oxytocinergic system in the mouse. These results lay the foundation for future investigation into the neural role of the OTR and provide a useful model for further study of oxytocin functions in the mouse.

Oxytocin mRNA: increase of polyadenylate tail size during pregnancy and lactation.

Earlier studies indicate that rat hypothalamic oxytocin (OT) mRNA accumulation rises gradually during pregnancy and remains elevated throughout the lactation period. Here we show that, in addition, hypothalamic OT mRNA undergoes a structural change during this period. On gel electrophoresis, the size of rat OT mRNA increased from 750 bases (control) to a maximum of 820 bases (7th day of lactation). Removal of the poly(A) tail by the RNase H methods revealed that this size increase can be fully accounted for by a prolongation of the polyadenylate tail. There is considerable evidence for a role of the poly(A) tail segment in augmenting mRNA stability and, possibly, translational efficiency. It is thus conceivable that the demonstrated regulation of OT mRNA poly(A) tail length represents an additional level of OT gene control during pregnancy and lactation.

Nuclear orphan receptors COUP-TFII and Ear-2: presence in oxytocin-producing uterine cells and functional interaction with the oxytocin gene promoter.

We have previously demonstrated that the oxytocin (OT) gene is expressed in the rat uterine epithelium and that its expression is upregulated in vivo and in vitro by estrogen. This hormonal regulation is mediated by a hormone response element (HRE) located in the OT gene promoter. Here we show that the same OT-HRE is also capable of interacting with two novel members of the orphan nuclear receptor family, rat COUP-TFII and Ear-2, and that this interaction antagonizes the estrogenic induction of the OT promoter. By Northern blot analysis and immunocytochemistry, using specific cDNA probes and antibodies, respectively, we demonstrate furthermore that both orphan receptors are expressed in uterine epithelial cells. Therefore, the present findings indicate that uterine OT gene expression is under stimulatory as well as inhibitory influences which are both mediated by the same HRE. More detailed analysis of the sequences necessary for estrogen receptor action and for orphan receptor action, using site-directed mutagenesis, revealed that the specific recognition sequences are overlapping but distinct: whereas the (imperfect) palindromic structure of the HRE constitutes the estrogen response element (ERE), orphan receptor action relies on an underlying direct TGACC repeat which forms part of the OT-HRE structure and overlaps with the estrogen response element.