Biomedical subjects
Publications and source records attributed to R Ivell.
In the bovine ovary there is a delay of 4-6 days between the observed maximum of oxytocin mRNA and the peak in the luteal levels of oxytocin nonapeptide. This implies a maturation process involving components of the post-translational processing pathway. In situ hybridization shows the oxytocin gene to be transcribed exclusively in the large cells of the corpus luteum at the beginning of the estrous cycle.
cDNA clones corresponding to vasopressin gene transcripts were isolated from a lambda gt11 library made using mRNA extracted from a bovine corpus luteum of the early non-pregnant cycle. None of the characterized clones included the vasopressin-encoding exon A sequence, instead two of these clones included sequence from the first intron. Together these data and controls indicate that vasopressin gene transcription in this tissue does not yield translatable mRNA and that positive RNA-DNA hybridization signals are not necessarily evidence for local biosynthesis of the neuropeptide.
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Sequence analysis of cDNA clones derived from hypothalamic mRNA of diabetes insipidus (Brattleboro) rats shows that the vasopressin gene transcript also includes the single base deletion demonstrated in the gene. This causes a frame-shift in the C terminus of the vasopressin precursor with a reading frame open through the 3' end of the mRNA including the poly(A) sequence. Antibodies raised against a synthetic tetradecapeptide (CP-14) corresponding to the frame-shifted C terminus identified a product of mol. wt approximately 26 000 in a reticulocyte lysate system programmed with Brattleboro hypothalamic mRNA. Immunohistochemical analysis indicated that a similar precursor is also present in vivo in neurones of the Brattleboro hypothalamus. Electrophoretic analysis of vasopressin mRNA from wild-type and mutant rat tissues revealed that (i) the hypothalamic mRNA from Brattleboro rats contains a longer stretch of poly(A) sequence than the wild-type strains; (ii) vasopressin mRNA is also present in the adrenal, ovary, testis and cerebellum, at very low levels; however, (iii) the extra-hypothalamic mRNA is considerably shorter than that in the hypothalamus because of a curtailed poly(A) sequence. Thus similar vasopressin gene transcripts are subject to a tissue-specific differential polyadenylation.
The nonapeptide vasopressin is synthetized as part of a longer common precursor polypeptide, together with its carrier protein neurophysin and a glycopeptide of unknown function. The gene for this common precursor has been isolated and sequenced and shown to comprise three exons encoding, respectively, the protein domains approximately corresponding to the N-terminal leader peptide and hormone, to most of the neurophysin, and to the glycopeptide. In the Brattleboro rat, the hereditary hypothalamic diabetes insipidus, characteristic of this strain, has been shown to be caused by a single nucleotide deletion in the vasopressin gene. This leads to the much reduced synthesis of a mutant vasopressin precursor, whose C terminus is quite unlike that in wild-type rats and which does not appear to be correctly processed in vivo.
Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magnocellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on recombinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was less than 1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdissected supraoptic nucleus (SON) mean (+/- SEM) basal levels of 1.37 +/- 0.18 pg VP mRNA and 1.95 +/- 0.14 pg OT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 +/- 0.02 pg VP mRNA and 1.77 +/- 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85-fold increase in VP mRNA levels of the SON and a 1.6-fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.
In situ hybridization has been used to identify specific hypothalamic magnocellular neurons, in normal and Brattleboro rats, that contain vasopressin (VP) or oxytocin (OT) mRNA. The subnuclear distribution of identified neurons in hypothalamic magnocellular nuclei after hybridization with several probes specific for OT or VP mRNA was in direct agreement with immunocytochemical descriptions of the distribution of cells containing VP or OT hormone. The number of grains per cell suggested that Brattleboro rats contained greater levels of OT mRNA, while hybridization with VP probes produced fewer grains in tissue from Brattleboro rats compared to normal rats. This paper provides the first cell-by-cell description of VP gene expression in the Brattleboro rat and demonstrates that VP gene transcription is confined precisely to the magnocellular neurons that normally synthesize VP hormone in normal rats.
In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.
Complete cDNA sequences for the vasopressin and oxytocin precursor polyproteins have been determined for the rat, calf, human and pig (vasopressin only), indicating the essential conservation of the precursor structures throughout mammals. DNA probes specific for vasopressin or oxytocin mRNAs have been used to identify both classic (hypothalamic) and novel (thymus, corpus luteum, phaeochromocytoma) sites of hormone expression. Semiquantitative DNA/RNA hybridization suggests that in rats expression of the vasopressin and oxytocin genes is positively effected by osmotic stress, negatively by a systemically applied excess of vasopressin; in the latter experiment a reduction in the hypothalamic levels of vasopressin and oxytocin mRNAs in normal and Brattleboro rats have been observed. This suggests a feedback regulation by the hormone as a possible element in controlling the transcription of the vasopressin gene.
Poly(A)+ RNA isolated from post-mortem human hypothalami has been used to characterize the poly-protein precursors to vasopressin and oxytocin. Translation in a cell-free system and subsequent immuno-precipitation with antibodies raised against either vasopressin or neurophysin identified a product of Mr 19000 (prepro-vasopressin). A second less intense product of Mr 16500 was tentatively identified as prepro-oxytocin. A cDNA library derived from the human hypothalamic poly(A)+ RNA was screened for vasopressin and oxytocin-encoding cDNA using heterologous probes; clones encoding the two precursors were identified and found to be organized as their rat and bovine counterparts. Northern blot analysis shows that the mRNAs for the two prepro-hormones consist of approximately 840 (AVP) and approximately 700 (OT) nucleotides.
The gene for the nonapeptide neurohormone oxytocin is highly expressed in the bovine corpus luteum. Measurements of oxytocin-specific mRNA through the oestrous cycle of non-pregnant cows show that transcription is maximal accompanying ovulation and decreases rapidly thereafter. In contrast, immunohistochemistry shows neurophysin peptide levels to be greatest at mid-cycle. Low levels of oxytocin mRNA are detected in follicles and in the luteolytic half of the cycle. This mRNA is virtually absent in the corpus luteum of pregnant cattle. No cyclicity is evident in hypothalamic oxytocin mRNA levels.
The vasopressin gene from normal and diabetes insipidus (Brattleboro) rats has been isolated and sequenced. Except for a single deletion of a G residue in region coding for the neurophysin carrier protein the approximately 2300 nucleotides of both genes are identical. Blot analysis of hypothalamic RNA as well as transfection and microinjection experiments indicate that the mutant gene is correctly transcribed and spliced, however the resulting mRNA is not efficiently translated.
Messenger mRNA has been prepared from post mortem human hypothalami and translated in a cell-free system. Using specific antibodies, biosynthetic precursors have been identified to neuropeptide Y (12 kDa), somatostatin (15 kDa) and vasopressin/neurophysin (19 kDa).
Cell-free translation of rat hypothalamic mRNA and specific immunoprecipitation were used to identify a polypeptide of 16,000 apparent molecular weight as prepro-somatostatin. Quantifying these results suggested that the somatostatin-specific mRNA represented less than 0.01% of the total hypothalamic mRNA. Co-translational addition of microsomal membranes led to the in vitro synthesis of a pro-form of 14,500 molecular weight. By using antisera specifically recognizing 3 different but overlapping segments of somatostatin-28 (SRIF-28), the rat prepro-somatostatin was shown to contain antigenic determinants of this N-terminally extended somatostatin as well as of the tetradecapeptide (SRIF-14). Sequential immunoprecipitation experiments implied the existence of only a single somatostatin precursor among the rat hypothalamic translation products, which would have to be differentially processed to allow release of both SRIF-28 and SRIF-14.
Expression of the vasopressin and oxytocin genes has been described so far only in the hypothalamus. We report here that at least the oxytocin gene is highly transcribed in the bovine corpus luteum during the mid-luteal phase of the oestrous cycle. Luteal cDNA sequence analysis as well as cell-free translation studies showed that the luteal mRNA is essentially similar to that in the hypothalamus, except that in the corpus luteum the poly(A) tail of this mRNA is shorter. When calculating the relative amounts per organ, the active corpus luteum produces approximately 250 times more oxytocin mRNA than a single hypothalamus.
The gene encoding the precursor protein to the hormone oxytocin and its associated neurophysin has been isolated from a rat genomic library, and its sequence has been determined. The small gene (approximately equal to 850 base pairs) predicts a mRNA of approximately equal to 500 bases [without the poly(A) tail]. The exon-intron organization is similar to that of the vasopressin gene, with two splice sites in the protein-coding region. The first exon (A) comprises the 5' noncoding promoter region, a putative signal peptide, the nonapeptide hormone oxytocin, and the NH2-terminal, variable region of neurophysin. The second exon (B) encodes the central, conserved region of neurophysin, and the third exon (C) encodes the remaining COOH terminus of neurophysin, with an additional arginine residue at its end, presumably cleaved off during post-translational processing. A stretch of 143 nucleotides within exon B, except for a single base change, is entirely homologous to the equivalent part of the rat vasopressin gene, offering support for a gene conversion event having recently affected the two genes.
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Using a combination of in vitro methodology, including cell-free translation, two-dimensional peptide mapping and recombinant DNA techniques, the structure of the precursors of the hypothalamic nonapeptide hormones vasopressin and oxytocin has been elucidated. Both hormone precursors are model cellular polyproteins in that they comprise several different entities within the same polypeptide molecule. In each precursor, the nonapeptide hormone follows immediately the signal peptide and is, in turn, attached to its respective carrier neurophysin. The vasopressin precursor also includes a pituitary glycoprotein at its C-terminus. The posttranslational processing of the precursors to set free the nonapeptide hormones is thus a critical regulatory step, which can in part be simulated in the quasi in vivo system of the Xenopus laevis oocyte. The preprohormones to vasopressin and oxytocin illustrate well the convenience of the in vitro experimental approach in understanding the function of the peptidergic neuron.