Advances in Experimental Medicine and Biology, 1998
2 The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neu... more 2 The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neurohypophysial system (HNS) have been the most studied of all the neuroendocrine cell-types. Despite this, our understanding of the mechanisms that underly the cell-specific expression of the peptide genes in these neurons has remained obscure. Part of the reason for this may be related to the close apposition of the OT and VP genes in the chromosomal locus, the genes being separated by as little as 3.5 kb in the mouse, and their interactions which are critical for cell-specific expression of the genes. Recent studies using intact rat OT and VP constructs in transgenic mice, and rat and mouse VP genes with CAT inserts in exon III as reporters in transgenic rats and mice, respectively, have suggested the presence of cell-specific enhancer elements in the 3' downstream (intergenic region, IGR) region of the VP gene. Evidence in favor of this view is presented from transgenic mouse studies on the expression of mouse OTand VP-CAT gene constructs. Oxytocin and vasopressin phenotypes in the magnocellular neuronal population have traditionally been assessed by either immunocytochemical or in situ hybridization histochemical methods leading to the view that these genes are never coexpressed . However, more sensitive methods show that most OT cells also express some VP mRNA, and most VP cells contain some OT mRNA. A third phenotype containing equivalent levels of both OT and VP mRNA can also be found under some conditions, thereby complicating our analysis of cell-specificity. A continuing problem hindering studies of the regulation of OT and VP gene expression in neurons, is the absence of an appropriate cell line to examine these issues. We have found that stationary slice-explant cultures allow for excellent preservation of highly differentiated magnocellular neurons in long-term culture, and that Vasopressin and Oxytocin, edited by Zingg et a/. Plenum Press, New York, 1998. 15
Proceedings of the National Academy of Sciences of the United States of America, Oct 14, 1997
Previous studies indicated that the central nervous system induces release of the cardiac hormone... more Previous studies indicated that the central nervous system induces release of the cardiac hormone atrial natriuretic peptide (ANP) by release of oxytocin from the neurohypophysis. The presence of specific transcripts for the oxytocin receptor was demonstrated in all chambers of the heart by amplification of cDNA by the PCR using specific oligonucleotide primers. Oxytocin receptor mRNA content in the heart is 10 times lower than in the uterus of female rats. Oxytocin receptor transcripts were demonstrated by in situ hybridization in atrial and ventricular sections and confirmed by competitive binding assay using frozen heart sections. Perfusion of female rat hearts for 25 min with Krebs-Henseleit buffer resulted in nearly constant release of ANP. Addition of oxytocin (10 ؊6 M) significantly stimulated ANP release, and an oxytocin receptor antagonist (10 ؊7 and 10 ؊6 M) caused dose-related inhibition of oxytocin-induced ANP release and in the last few minutes of perfusion decreased ANP release below that in control hearts, suggesting that intracardiac oxytocin stimulates ANP release. In contrast, brain natriuretic peptide release was unaltered by oxytocin. During perfusion, heart rate decreased gradually and it was further decreased significantly by oxytocin (10 ؊6 M). This decrease was totally reversed by the oxytocin antagonist (10 ؊6 M) indicating that oxytocin released ANP that directly slowed the heart, probably by release of cyclic GMP. The results indicate that oxytocin receptors mediate the action of oxytocin to release ANP, which slows the heart and reduces its force of contraction to produce a rapid reduction in circulating blood volume.
Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used t... more Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI ϭ 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form ␣-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.
We analyzed the response of uterine smooth muscle cells to interleukin-1b (IL-1b). We first showe... more We analyzed the response of uterine smooth muscle cells to interleukin-1b (IL-1b). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1b. In total, we identified 198 known genes whose mRNA levels are significantly modulated (O2.0-fold change) following IL-1b exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1b includes transcription factors and inflammatory response genes such as nuclear factor of k light polypeptide gene enhancer in B-cells (NFkB), pentraxin-related gene (PTX3), and tumor necrosis factor a-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1b elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.
We have recently demonstrated that the gene encoding the hypothalamic peptide oxytocin (OT) is hi... more 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.
It is well established that uterine oxytocin receptors (OTRs) are strongly up-regulated immediate... more 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.
As illustrated in Fig. 1 A, the novel sequence has to be inserted at the location of a dinucleoti... more As illustrated in Fig. 1 A, the novel sequence has to be inserted at the location of a dinucleotide repeat, (GT) 26 , located 89 nucleotides 5Ј to the main transcriptional initiation site. Resequencing of a newly generated phage subclone as well as Southern blot and PCR analyses (not shown) confirmed that the sequence presented here is indeed part of the genomic sequence. Since this novel sequence element is itself flanked by two dinucleotide repeats, (GT) 20 and (GT) 24 , respectively, a likely explanation is that this segment was spliced out during subcloning due to recombination between the two dinucleotide repeats. This idea is further supported by the fact that dinucleotide repeats that have the potential of forming Z-DNA structures have been shown to enhance recombination in extrachromosomal DNA up to 20-fold (1). Despite the recurrence of dinucleotide repeats around chromosomal rearrangement breakpoints, their role in mediating recombination on intact chromosomes remains, however, uncertain (2). We thank Tracy L. Bale and Daniel M. Dorsa for pointing out the error and for their help and collaboration in its correction. Pharmacology. In the article ''Tityustoxin K␣ blocks voltagegated noninactivating K ϩ channels and unblocks inactivating K ϩ channels blocked by ␣-dendrotoxin in synaptosomes'' by
The peptide hormone oxytocin is produced both in the hypothalamus and in certain peripheral organ... more The peptide hormone oxytocin is produced both in the hypothalamus and in certain peripheral organs. The extent of extra-hypothalamic hormone synthesis in the pregnant cow has not previously been examined. In this study we have analyzed different tissues from the pregnant bovine reproductive tract and corpus luteum for the presence of mRNA encoding the oxytocin peptide as well as the oxytocin receptor. In uterine tissues oxytocin mRNA could only be detected sporadically with the help of a reverse transcription-polymerase chain reaction method, implying only very low levels of expression. The caruncles showed a consistently low level of oxytocin gene expression, which appeared up-regulated at term. However, in the corpus luteum there was a significant level of oxytocin gene expression at term, particularly following the onset of labor. The transcript levels were sufficiently high to be measurable by both RNase protection assay and by Northern hybridization; these levels imply a rescue of the oxytocin gene expression seen in the corpora lutea of cyclic and early pregnant cows. At the peptide level this expression was confirmed by immunohistochemistry. A sensitive RNase protection assay was developed to detect transcripts encoding the oxytocin receptor. Transcripts were detected in most uterine tissues, including the caruncles, with highest levels in the endometrium and myometrium at term. No transcripts could be detected in the corpus luteum at any stage of pregnancy, nor in the amnion. The results suggest the possibility of local, paracrine effects of oxytocin within the uterus of the pregnant cow. The rescue of luteal oxytocin at term could act to supplement the circulating hormone of pituitary origin.
Dehydration represents a strong stimulus for the secretory activity of hypothalamic vasopressiner... more Dehydration represents a strong stimulus for the secretory activity of hypothalamic vasopressinergic neurons and induces a sustained rise in hypothalamic [Arg8]vasopressin (AVP) mRNA. We now report that, in addition to the change in mRNA accumulation, this stimulus leads to an increase in the size of the mature AVP gene transcripts. Using Northern blot analysis in conjunction with internal RNA size markers, we demonstrate here that a 6-day period of salt imbibition induced a gradual size increase of AVP mRNA from 810 to a maximum of 930 bases. This change was fully reversed by day 30 following termination of the salt treatment. Any alteration of the mRNA capping or polyadenylation sites was ruled out by S1 mapping. However, poly(A) tail removal reduced AVP mRNA of all treatment groups to exactly the same size (600 bases). It is concluded that the dehydration-induced size change is due to a greater than 50% increase of the mean steady state length of the poly(A) tail. It remains to be determined to what extent the observed increase in poly(A) tail length may have an effect on AVP mRNA stability or translational efficiency.
Historical Overview: Vasopressin Receptors: A Historical Survey S. Jard. Biosynthesis of Vasopres... more Historical Overview: Vasopressin Receptors: A Historical Survey S. Jard. Biosynthesis of Vasopressin and Oxytocin. Cell-Specific Gene Expression in Oxytocin and Vasopressin Magnocellular Neurons H. Gainer. Hypothalamic Transcription Factors and the Regulation of the Hypothalamo-Neurohypophysial System J.P.H. Burbach, et al. POU Domain Factors in Neural Development M.D. Schonemann, et al. Local Protein Synthesis in Magnocellular Dendrites: Basic Elements and Their Response to Hyperosmotic Stimuli Dan Ma, J.F. Morris. Vasopressin Gene Expression in Rat Choroid Plexus A. Chodobski, et al. Neuronal Activity and Release. Electrophysiological Distinctions between Oxytocin and Vasopressin Neurons in the Supraoptic Nucleus W.E. Armstrong, J.E. Stern. Mechanisms of Neuroendocrine Cell Excitability G.I. Hatton, Zhenhui Li. Properties of the Transient K+ Current in Acutely Isolated Supraoptic Neurons from Adult Rat T.E. Fisher, C.W. Bourque. Gaba b Receptors and Supraoptic Neuronal Activity Q.J. Pittman, et al. Local Glutamatergic and Gabaergic Synaptic Circuits and Metabotropic Glutamate Receptors in the Hypothalamic Paraventricular and Supraoptic Nuclei J.G. Tasker, et al. Progesterone Regulates Hypothalamic Oxytocin mRNA Levels through Gamma Aminobutyric Acid A. Thomas, J.A. Amico. Differential Effects of the Neurosteroid Pregnenolone Sulphate on Oxytocin and Vasopressin Neurones in Vitro J.B. Wakerley, C.M. Richardson. Regulation of Vasopressin Release by Ionotropic Glutamate Receptor Agonists D.J. Morsette, et al. Role of N-Methy-D-Aspartate (NMDA) Receptors in Vasopressin and Oxytocin Responses to Emotional Stimuli K. Yagi, et al. Electrophysiological Studies of Oxytocin Neurons in Organotypic Slice Cultures P. Jourdain, et al. Inhibitory Actions of Nociceptin (Orphanin FQ) on Rat Supraoptic Nucleus Oxytocin and Vasopressin Neurones in Vitro N. Doi, et al. New Aspects of Firing Pattern Autocontrol in Oxytocin and Vasopressin Neurones F. Moos, et al. Intrahypothalamic Vasopressin Release: An Inhibitor of Systemic Vasopressin Secretion? M. Ludwig, G. Leng. Differential Central and Peripheral Release of Vasopressin and Oxytocin in Response to Swim Stress in Rats M. Engelmann, et al. Pregnancy and Aging: Two Model Systems with Altered Release Patterns of Oxytocin and Vasopressin within the Hypothalamus I. Neumann, et al. Effect of Age and Testosterone on the Vasopressin Response to Dehydration in F344BNF1 Male Rats J. Catudioc-Vallero, et al. Evidence for Nitric Oxide (NO) Actions throughout the Forebrain Osmoresponsive Circuit S.M. Luckman. Gaseous Neurotransmitter Modulation of Vasopressin and Oxytocin Release M.L. Forsling, A. Grossman. Membrane Excitability in the Neurohypophysis R.A. Wilke, et al. Effects. Vasopressin's Depolarizing Action on Neonatal Rat Spinal Lateral Horn Neurons May Involve Multiple Conductances M. Kolaj, L.P. Renaud. Vasopressin Action in the Mammalian Cerebral Cortex R. Diaz Brinton, et al. Oxytocin, Vasopressin, and the Neuroendocrine Basis of Pair Bond Formation T.R. Insel, et al. Central Oxytocin Neurotransmission: Receptor Characterisation and Role in Modulating Limbic Circuits in the Peripartum Period C.D. Ingram, et al. Targeted Reduction of Oxytocin Expression Provides Insights into Its Physiological Roles W.S. Young III, et al. Phenotypic Expression of and Oxytocin Peptide Null Mutation in Mice J.T. Winslow, et al. Antiproliferative Effect of Oxytocin through Specific Oxytocin Receptors in Human Neuroblastoma and Astrocytoma Cell Lines P. Cassoni, et al. Phosphorylation of Proteins Induced in a Murine Pre-T Cell Line by Neurohypophysial Peptides H. Martens, et al. Signalling. Molecular Pharmacology of Human Vasopressin Receptors M. Thibonnier, et al. Molecular Mechanisms Regulating the Effects of Oxytocin on Myometrial Intracellular Calcium B.M. Sanborn, et al. Receptors: Expression and Regulation. Genomic and Non-Genomic Mechanisms of Oxytocin Receptor Regulation H.H. Zingg, et al. The Molecular Basis of Oxytocin and Oxytocin Receptor Gene Expression in Reproductive Tissues R. Ivell, et al. Transcriptional Regulation of the Oxytocin Receptor Gene T.L. Bale, D.M. Dorsa. Female Reproduction in Mice Lacking the Prostaglandin F Receptor: Roles of Prostaglandin and Oxytocin Receptors in Parturition Y. Sugimoto, et al. Expression of the Oxytocin Receptor and Oxytocin Gene in Human Oocytes and Preimplantation Embryos S. Stock, C. Osterlund. Vasopressin Regulates Adrenal Functions by Acting through Different Vasopressin Receptor Subtypes E. Grazzini, et al. All Three Vasopressin Receptor Sub-Types Are Expressed by Small-Cell Carcinoma W.G. North, et al. Receptors: Structure/Function. Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor H.M. Sadeghi, et al. Molecular Aspects of Vasopressin Receptor Function T. Schoneberg, et al. Mapping Peptide Antagonist Binding Sites of the Human V1a and V2 Vasopressin Receptors B. Mouillac, et al. Structure/Function Studies on…
where he created the Inserm Unit 344 that he directed for 15 years as a Senior Director of Resear... more where he created the Inserm Unit 344 that he directed for 15 years as a Senior Director of Research at Inserm followed by an appointment as a University Hospital Professor (1993). During this time, Paul played a key role in a project to bring together several laboratories on the Necker campus, and, in 2007, succeeded in creating the Research Center 'Growth and Signaling' (Inserm Unit 845) that he directed until 2010. This structure has become a department of the present Institute Necker Enfants Malades (INEM, Inserm U1151) of which Paul was a member until 2014 as a Professor Emeritus. Besides science and research,
Oxytocin is a neuropeptide that regulates physiology and behaviors associated with reproduction a... more Oxytocin is a neuropeptide that regulates physiology and behaviors associated with reproduction as well as other social and nonsocial behaviors. The actions of oxytocin are mediated by a single G protein-coupled receptor distributed in many peripheral tissues and specific brain areas. Oxytocin secreted into the circulation stimulates uterine contractions during labor and stimulates milk ejection during lactation. Oxytocin released within the brain modulates social recognition, sexual behavior, maternal care, and maternal bonding as well as pair bonding in monogamous species. Appetite, mood, and anxiety are also affected by oxytocin. Paralleling its effects on social behavior in animals, oxytocin has been implicated in facilitating trust, in the processing of social information, and in intuiting emotional states of others. It has also been suggested that it plays a role in etiology, and may represent a potential treatment, of social deficits in psychiatric disorders such as autism spectrum disorder. Most recently, specific genetic and epigenetic changes in the oxytocin receptor gene have been correlated with specific behavioral traits, including autism. In this chapter we review the oxytocin system and its role in regulating physiology and behavior.
Advances in Experimental Medicine and Biology, 1998
2 The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neu... more 2 The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neurohypophysial system (HNS) have been the most studied of all the neuroendocrine cell-types. Despite this, our understanding of the mechanisms that underly the cell-specific expression of the peptide genes in these neurons has remained obscure. Part of the reason for this may be related to the close apposition of the OT and VP genes in the chromosomal locus, the genes being separated by as little as 3.5 kb in the mouse, and their interactions which are critical for cell-specific expression of the genes. Recent studies using intact rat OT and VP constructs in transgenic mice, and rat and mouse VP genes with CAT inserts in exon III as reporters in transgenic rats and mice, respectively, have suggested the presence of cell-specific enhancer elements in the 3' downstream (intergenic region, IGR) region of the VP gene. Evidence in favor of this view is presented from transgenic mouse studies on the expression of mouse OTand VP-CAT gene constructs. Oxytocin and vasopressin phenotypes in the magnocellular neuronal population have traditionally been assessed by either immunocytochemical or in situ hybridization histochemical methods leading to the view that these genes are never coexpressed . However, more sensitive methods show that most OT cells also express some VP mRNA, and most VP cells contain some OT mRNA. A third phenotype containing equivalent levels of both OT and VP mRNA can also be found under some conditions, thereby complicating our analysis of cell-specificity. A continuing problem hindering studies of the regulation of OT and VP gene expression in neurons, is the absence of an appropriate cell line to examine these issues. We have found that stationary slice-explant cultures allow for excellent preservation of highly differentiated magnocellular neurons in long-term culture, and that Vasopressin and Oxytocin, edited by Zingg et a/. Plenum Press, New York, 1998. 15
Proceedings of the National Academy of Sciences of the United States of America, Oct 14, 1997
Previous studies indicated that the central nervous system induces release of the cardiac hormone... more Previous studies indicated that the central nervous system induces release of the cardiac hormone atrial natriuretic peptide (ANP) by release of oxytocin from the neurohypophysis. The presence of specific transcripts for the oxytocin receptor was demonstrated in all chambers of the heart by amplification of cDNA by the PCR using specific oligonucleotide primers. Oxytocin receptor mRNA content in the heart is 10 times lower than in the uterus of female rats. Oxytocin receptor transcripts were demonstrated by in situ hybridization in atrial and ventricular sections and confirmed by competitive binding assay using frozen heart sections. Perfusion of female rat hearts for 25 min with Krebs-Henseleit buffer resulted in nearly constant release of ANP. Addition of oxytocin (10 ؊6 M) significantly stimulated ANP release, and an oxytocin receptor antagonist (10 ؊7 and 10 ؊6 M) caused dose-related inhibition of oxytocin-induced ANP release and in the last few minutes of perfusion decreased ANP release below that in control hearts, suggesting that intracardiac oxytocin stimulates ANP release. In contrast, brain natriuretic peptide release was unaltered by oxytocin. During perfusion, heart rate decreased gradually and it was further decreased significantly by oxytocin (10 ؊6 M). This decrease was totally reversed by the oxytocin antagonist (10 ؊6 M) indicating that oxytocin released ANP that directly slowed the heart, probably by release of cyclic GMP. The results indicate that oxytocin receptors mediate the action of oxytocin to release ANP, which slows the heart and reduces its force of contraction to produce a rapid reduction in circulating blood volume.
Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used t... more Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI ϭ 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form ␣-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.
We analyzed the response of uterine smooth muscle cells to interleukin-1b (IL-1b). We first showe... more We analyzed the response of uterine smooth muscle cells to interleukin-1b (IL-1b). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1b. In total, we identified 198 known genes whose mRNA levels are significantly modulated (O2.0-fold change) following IL-1b exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1b includes transcription factors and inflammatory response genes such as nuclear factor of k light polypeptide gene enhancer in B-cells (NFkB), pentraxin-related gene (PTX3), and tumor necrosis factor a-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1b elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.
We have recently demonstrated that the gene encoding the hypothalamic peptide oxytocin (OT) is hi... more 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.
It is well established that uterine oxytocin receptors (OTRs) are strongly up-regulated immediate... more 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.
As illustrated in Fig. 1 A, the novel sequence has to be inserted at the location of a dinucleoti... more As illustrated in Fig. 1 A, the novel sequence has to be inserted at the location of a dinucleotide repeat, (GT) 26 , located 89 nucleotides 5Ј to the main transcriptional initiation site. Resequencing of a newly generated phage subclone as well as Southern blot and PCR analyses (not shown) confirmed that the sequence presented here is indeed part of the genomic sequence. Since this novel sequence element is itself flanked by two dinucleotide repeats, (GT) 20 and (GT) 24 , respectively, a likely explanation is that this segment was spliced out during subcloning due to recombination between the two dinucleotide repeats. This idea is further supported by the fact that dinucleotide repeats that have the potential of forming Z-DNA structures have been shown to enhance recombination in extrachromosomal DNA up to 20-fold (1). Despite the recurrence of dinucleotide repeats around chromosomal rearrangement breakpoints, their role in mediating recombination on intact chromosomes remains, however, uncertain (2). We thank Tracy L. Bale and Daniel M. Dorsa for pointing out the error and for their help and collaboration in its correction. Pharmacology. In the article ''Tityustoxin K␣ blocks voltagegated noninactivating K ϩ channels and unblocks inactivating K ϩ channels blocked by ␣-dendrotoxin in synaptosomes'' by
The peptide hormone oxytocin is produced both in the hypothalamus and in certain peripheral organ... more The peptide hormone oxytocin is produced both in the hypothalamus and in certain peripheral organs. The extent of extra-hypothalamic hormone synthesis in the pregnant cow has not previously been examined. In this study we have analyzed different tissues from the pregnant bovine reproductive tract and corpus luteum for the presence of mRNA encoding the oxytocin peptide as well as the oxytocin receptor. In uterine tissues oxytocin mRNA could only be detected sporadically with the help of a reverse transcription-polymerase chain reaction method, implying only very low levels of expression. The caruncles showed a consistently low level of oxytocin gene expression, which appeared up-regulated at term. However, in the corpus luteum there was a significant level of oxytocin gene expression at term, particularly following the onset of labor. The transcript levels were sufficiently high to be measurable by both RNase protection assay and by Northern hybridization; these levels imply a rescue of the oxytocin gene expression seen in the corpora lutea of cyclic and early pregnant cows. At the peptide level this expression was confirmed by immunohistochemistry. A sensitive RNase protection assay was developed to detect transcripts encoding the oxytocin receptor. Transcripts were detected in most uterine tissues, including the caruncles, with highest levels in the endometrium and myometrium at term. No transcripts could be detected in the corpus luteum at any stage of pregnancy, nor in the amnion. The results suggest the possibility of local, paracrine effects of oxytocin within the uterus of the pregnant cow. The rescue of luteal oxytocin at term could act to supplement the circulating hormone of pituitary origin.
Dehydration represents a strong stimulus for the secretory activity of hypothalamic vasopressiner... more Dehydration represents a strong stimulus for the secretory activity of hypothalamic vasopressinergic neurons and induces a sustained rise in hypothalamic [Arg8]vasopressin (AVP) mRNA. We now report that, in addition to the change in mRNA accumulation, this stimulus leads to an increase in the size of the mature AVP gene transcripts. Using Northern blot analysis in conjunction with internal RNA size markers, we demonstrate here that a 6-day period of salt imbibition induced a gradual size increase of AVP mRNA from 810 to a maximum of 930 bases. This change was fully reversed by day 30 following termination of the salt treatment. Any alteration of the mRNA capping or polyadenylation sites was ruled out by S1 mapping. However, poly(A) tail removal reduced AVP mRNA of all treatment groups to exactly the same size (600 bases). It is concluded that the dehydration-induced size change is due to a greater than 50% increase of the mean steady state length of the poly(A) tail. It remains to be determined to what extent the observed increase in poly(A) tail length may have an effect on AVP mRNA stability or translational efficiency.
Historical Overview: Vasopressin Receptors: A Historical Survey S. Jard. Biosynthesis of Vasopres... more Historical Overview: Vasopressin Receptors: A Historical Survey S. Jard. Biosynthesis of Vasopressin and Oxytocin. Cell-Specific Gene Expression in Oxytocin and Vasopressin Magnocellular Neurons H. Gainer. Hypothalamic Transcription Factors and the Regulation of the Hypothalamo-Neurohypophysial System J.P.H. Burbach, et al. POU Domain Factors in Neural Development M.D. Schonemann, et al. Local Protein Synthesis in Magnocellular Dendrites: Basic Elements and Their Response to Hyperosmotic Stimuli Dan Ma, J.F. Morris. Vasopressin Gene Expression in Rat Choroid Plexus A. Chodobski, et al. Neuronal Activity and Release. Electrophysiological Distinctions between Oxytocin and Vasopressin Neurons in the Supraoptic Nucleus W.E. Armstrong, J.E. Stern. Mechanisms of Neuroendocrine Cell Excitability G.I. Hatton, Zhenhui Li. Properties of the Transient K+ Current in Acutely Isolated Supraoptic Neurons from Adult Rat T.E. Fisher, C.W. Bourque. Gaba b Receptors and Supraoptic Neuronal Activity Q.J. Pittman, et al. Local Glutamatergic and Gabaergic Synaptic Circuits and Metabotropic Glutamate Receptors in the Hypothalamic Paraventricular and Supraoptic Nuclei J.G. Tasker, et al. Progesterone Regulates Hypothalamic Oxytocin mRNA Levels through Gamma Aminobutyric Acid A. Thomas, J.A. Amico. Differential Effects of the Neurosteroid Pregnenolone Sulphate on Oxytocin and Vasopressin Neurones in Vitro J.B. Wakerley, C.M. Richardson. Regulation of Vasopressin Release by Ionotropic Glutamate Receptor Agonists D.J. Morsette, et al. Role of N-Methy-D-Aspartate (NMDA) Receptors in Vasopressin and Oxytocin Responses to Emotional Stimuli K. Yagi, et al. Electrophysiological Studies of Oxytocin Neurons in Organotypic Slice Cultures P. Jourdain, et al. Inhibitory Actions of Nociceptin (Orphanin FQ) on Rat Supraoptic Nucleus Oxytocin and Vasopressin Neurones in Vitro N. Doi, et al. New Aspects of Firing Pattern Autocontrol in Oxytocin and Vasopressin Neurones F. Moos, et al. Intrahypothalamic Vasopressin Release: An Inhibitor of Systemic Vasopressin Secretion? M. Ludwig, G. Leng. Differential Central and Peripheral Release of Vasopressin and Oxytocin in Response to Swim Stress in Rats M. Engelmann, et al. Pregnancy and Aging: Two Model Systems with Altered Release Patterns of Oxytocin and Vasopressin within the Hypothalamus I. Neumann, et al. Effect of Age and Testosterone on the Vasopressin Response to Dehydration in F344BNF1 Male Rats J. Catudioc-Vallero, et al. Evidence for Nitric Oxide (NO) Actions throughout the Forebrain Osmoresponsive Circuit S.M. Luckman. Gaseous Neurotransmitter Modulation of Vasopressin and Oxytocin Release M.L. Forsling, A. Grossman. Membrane Excitability in the Neurohypophysis R.A. Wilke, et al. Effects. Vasopressin's Depolarizing Action on Neonatal Rat Spinal Lateral Horn Neurons May Involve Multiple Conductances M. Kolaj, L.P. Renaud. Vasopressin Action in the Mammalian Cerebral Cortex R. Diaz Brinton, et al. Oxytocin, Vasopressin, and the Neuroendocrine Basis of Pair Bond Formation T.R. Insel, et al. Central Oxytocin Neurotransmission: Receptor Characterisation and Role in Modulating Limbic Circuits in the Peripartum Period C.D. Ingram, et al. Targeted Reduction of Oxytocin Expression Provides Insights into Its Physiological Roles W.S. Young III, et al. Phenotypic Expression of and Oxytocin Peptide Null Mutation in Mice J.T. Winslow, et al. Antiproliferative Effect of Oxytocin through Specific Oxytocin Receptors in Human Neuroblastoma and Astrocytoma Cell Lines P. Cassoni, et al. Phosphorylation of Proteins Induced in a Murine Pre-T Cell Line by Neurohypophysial Peptides H. Martens, et al. Signalling. Molecular Pharmacology of Human Vasopressin Receptors M. Thibonnier, et al. Molecular Mechanisms Regulating the Effects of Oxytocin on Myometrial Intracellular Calcium B.M. Sanborn, et al. Receptors: Expression and Regulation. Genomic and Non-Genomic Mechanisms of Oxytocin Receptor Regulation H.H. Zingg, et al. The Molecular Basis of Oxytocin and Oxytocin Receptor Gene Expression in Reproductive Tissues R. Ivell, et al. Transcriptional Regulation of the Oxytocin Receptor Gene T.L. Bale, D.M. Dorsa. Female Reproduction in Mice Lacking the Prostaglandin F Receptor: Roles of Prostaglandin and Oxytocin Receptors in Parturition Y. Sugimoto, et al. Expression of the Oxytocin Receptor and Oxytocin Gene in Human Oocytes and Preimplantation Embryos S. Stock, C. Osterlund. Vasopressin Regulates Adrenal Functions by Acting through Different Vasopressin Receptor Subtypes E. Grazzini, et al. All Three Vasopressin Receptor Sub-Types Are Expressed by Small-Cell Carcinoma W.G. North, et al. Receptors: Structure/Function. Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor H.M. Sadeghi, et al. Molecular Aspects of Vasopressin Receptor Function T. Schoneberg, et al. Mapping Peptide Antagonist Binding Sites of the Human V1a and V2 Vasopressin Receptors B. Mouillac, et al. Structure/Function Studies on…
where he created the Inserm Unit 344 that he directed for 15 years as a Senior Director of Resear... more where he created the Inserm Unit 344 that he directed for 15 years as a Senior Director of Research at Inserm followed by an appointment as a University Hospital Professor (1993). During this time, Paul played a key role in a project to bring together several laboratories on the Necker campus, and, in 2007, succeeded in creating the Research Center 'Growth and Signaling' (Inserm Unit 845) that he directed until 2010. This structure has become a department of the present Institute Necker Enfants Malades (INEM, Inserm U1151) of which Paul was a member until 2014 as a Professor Emeritus. Besides science and research,
Oxytocin is a neuropeptide that regulates physiology and behaviors associated with reproduction a... more Oxytocin is a neuropeptide that regulates physiology and behaviors associated with reproduction as well as other social and nonsocial behaviors. The actions of oxytocin are mediated by a single G protein-coupled receptor distributed in many peripheral tissues and specific brain areas. Oxytocin secreted into the circulation stimulates uterine contractions during labor and stimulates milk ejection during lactation. Oxytocin released within the brain modulates social recognition, sexual behavior, maternal care, and maternal bonding as well as pair bonding in monogamous species. Appetite, mood, and anxiety are also affected by oxytocin. Paralleling its effects on social behavior in animals, oxytocin has been implicated in facilitating trust, in the processing of social information, and in intuiting emotional states of others. It has also been suggested that it plays a role in etiology, and may represent a potential treatment, of social deficits in psychiatric disorders such as autism spectrum disorder. Most recently, specific genetic and epigenetic changes in the oxytocin receptor gene have been correlated with specific behavioral traits, including autism. In this chapter we review the oxytocin system and its role in regulating physiology and behavior.
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