PubMed Health⌕ Search

Biomedical subjects

R Ivell

Publications and source records attributed to R Ivell.

At least 109 records · Page 6Linked to original sources
PubMed1994

hCG-induced changes in LH/CG receptor mRNA transcript levels in the testis of adult hypophysectomized, ethane dimethyl sulphonate-treated rats.

To elucidate the molecular mechanisms involved in the development of precursor cells into functional Leydig cells, effects of hCG on the expression of the luteinizing hormone (LH)/chorionic gonadotrophin (CG) receptor gene were investigated in the testis of adult hypophysectomized, ethane dimethyl sulphonate (EDS)-treated rats. The alkylating drug EDS specifically destroys Leydig cells in the adult rat testis. In hypophysectomized rats, in the absence of LH, this is not followed by the formation of new Leydig cells, leaving a testis in which Leydig cell precursors are presumed to be the only cell type that possess LH receptors. The absence of Leydig cells was confirmed by determining the gene expression of two key steroidogenic enzymes, 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) and P-450 17 alpha-hydroxylase/C17-20 lyase (P-45017 alpha) in these animals. Northern blot analysis revealed that hypophysectomy, followed by EDS treatment decreased the total amount of LH/CG receptor mRNA and drastically changed the pattern of LH/CG receptor mRNA transcripts. In the testis of intact adult rats, the expression of the LH/CG receptor gene results in multiple mRNA transcripts with molecular sizes of approximately 7.0, 4.2, 2.5, 1.8 and 1.2 kb. The most abundant species observed in the testis of adult hypophysectomized, EDS-treated rats was the 1.8 kb mRNA transcript, whereas the levels of the other mRNA transcripts were decreased. To stimulate the development of precursor cells into functional Leydig cells, hypophysectomized, EDS-treated rats were treated with high doses of hCG (100 IU) for different periods of time.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

The proopiomelanocortin gene is expressed as both full-length and 5'truncated transcripts in rodent Leydig cells.

A reverse transcription polymerase chain reaction (RT-PCR) assay was developed to distinguish between full-length transcripts and functionally aberrant, truncated transcripts of the proopiomelanocortin (POMC) gene. This assay was applied to RNA obtained from the testes of rats and mice, to Percoll-purified primary mouse Leydig cells, and to various Leydig-derived cell lines. The results indicate a very low level of expression for the full-length transcripts in whole rodent testis, in mouse Leydig cells and in the MA10 cell line. In Leydig cells, the major transcripts of the POMC gene were the shorter, truncated transcripts where the regions corresponding to exons 1 and 2 are missing.

Animals↗
PubMed1994

Structure of the alpha-inhibin gene and its regulation in the ruminant gonad: inverse relationship to oxytocin gene expression.

The genes for the alpha subunit of inhibin and for the nonapeptide hormone oxytocin are both expressed in the granulosa cells of the ruminant follicle as well as in the Sertoli cells of the ruminant testis. Northern hybridization of mRNA from both ovary and testis indicate that in both gonads the expression of the two genes is inversely regulated. In the luteinizing granulosa cells, in vitro as in vivo, the alpha-inhibin gene is down-regulated when the oxytocin gene is up-regulated. In the Sertoli cells of the bull and sheep testis, the situation is similar, with the alpha-inhibin gene being up-regulated in the prepubertal gonad and down-regulated concomitantly with an up-regulation of the oxytocin gene in early puberty. The gene for the bovine alpha-inhibin subunit was cloned and characterized. Assessment of transcriptional initiation by primer extension and ribonuclease protection assays showed that several different sites were used in both granulosa cells and testis. Transient transfection of primary bovine granulosa cells with alpha-inhibin/luciferase gene constructs indicated that a major promoter element resided in the region -178 to -245 respective to the methionine start codon of translation, a region that contains a cAMP response element. The ability of forskolin to up-regulate the transcription of transfected gene constructs also depended on the integrity of this region. In contrast, transfection of TM4 cells led to transcriptional initiation from an unusual site in the alpha-inhibin gene and to a lack of forskolin regulation. Comparison of the alpha-inhibin and oxytocin genes indicates that although both can be up-regulated by FSH or by forskolin within the same cells, different mechanisms of signal transduction are involved to explain the temporal differences in expression. Together the results indicate that a differentiation step occurring in Sertoli cells at early puberty and in granulosa cells at luteinization involves comparable regulation of genes through the sequential action of different cAMP-linked transcription factors.

Animals↗
PubMed1994

Molecular cloning and characterization of a novel human sperm antigen (HE2) specifically expressed in the proximal epididymis.

A novel human cDNA encoding a small secretory glycopeptide specifically expressed within the caput and proximal corpus regions of the human epididymis, HE2, has been cloned and sequenced. Southern hybridization analysis showed it to be derived from a single-copy gene. A subclone containing the coding region of the mature peptide was used to generate the putatively encoded peptide as a 10,000 M(r) recombinant fusion protein in a bacterial expression system. This recombinant HE2 protein, as well as an oligopeptide deduced from the open-reading frame, was used to raise polyclonal antisera. Immunohistochemistry and immunofluorescence showed that the predicted HE2 peptide is present in the epididymal epithelium and on the sperm surface, where it adopts a typical antigenic pattern with a subacrosomal equatorial distribution on the sperm head.

Amino Acid Sequence↗
PubMed1994

Oxytocin gene expression and oxytocin immunoactivity in the ovary of the common marmoset monkey (Callithrix jacchus).

Oxytocin was identified in ovaries recovered on Day 5 (+/- 1) of the luteal phase from three female marmoset monkeys (Callithrix jacchus). With use of a reverse transcription-polymerase chain reaction assay, expression of mRNA for oxytocin and oxytocin receptor was detected in both luteal tissue and in the ovarian remnant. Evidence for ovarian synthesis of oxytocin was provided by immunohistochemistry, which showed positive staining for oxytocin and neurophysin in the cytoplasm of the luteal cells. Some luteal cells had a more intensely stained perinuclear region than others for oxytocin immunoreactivity, whereas the staining for neurophysin was evenly distributed. Granulosa and theca cells of antral follicles also showed positive staining for oxytocin immunoreactivity; no reactivity was found in fibroblast or endothelial cells. Oxytocin immunoreactivity was also detected in the luteal tissue of all animals by immunoassay, with values ranging from 2.8 to 12.1 ng/g wet weight. The oxytocin concentration for the ovarian remnant was either very low (0.55-0.75 ng/g wet weight) or nondetectable (< 0.5 ng/g wet weight). Local production of oxytocin within the ovary was suggested by the measurement of higher oxytocin concentrations in the blood from ovaries containing corpora lutea compared with peripheral blood. Collectively, these results provide evidence for ovarian biosynthesis of oxytocin and suggest the possibility of a paracrine role in the regulation of primate ovarian function.

Animals↗
PubMed1994

Gene expression in the dog epididymis: a model for human epididymal function.

The physiology of the epididymis is an integral part of the maturation process by which human spermatozoa acquire the ability to reach and fertilize an oocyte. Because of the high degree of species specificity exhibited by the epididymal proteins involved in sperm maturation, we have assessed tissue from several alternative species for their suitability as a model for human epididymal physiology. Of these, the dog appears to offer an appropriate system. Northern hybridization using cDNA probes specific for human epididymal genes established that, irrespective of dog breed, the canine equivalents of the epididymis-specific HE1, HE4 and HE5 mRNAs were expressed highly in the canine epididymis. cDNA cloning and sequencing confirmed that the canine gene products, CE1, CE4 and CE5 were indeed true structural homologues of their human counterparts. Finally, tissue culture conditions were established wherein all three specific canine genes remained up-regulated after 5 days of culture. Thus, the prerequisite criteria for the development of a system which models human epididymal physiology are to a large degree fulfilled by this canine culture system.

Amino Acid Sequence↗
PubMed1994

Regional variation of specific gene expression in the dog epididymis as revealed by in-situ transcript hybridization.

In-situ transcript hybridization was used to characterize the regional distribution of three marker gene transcripts which are expressed abundantly in the canine epididymis. The gene products CE1, CE4 and CE5, which are the canine equivalents of the human homologues HE1, HE4 and HE5, are shown to be expressed in a tissue-specific and regionally characteristic pattern in the epididymal epithelium. CE1 mRNA was expressed very weakly in the efferent ducts but was expressed at a high level throughout the caput and corpus regions of the epididymis, decreasing somewhat in the distal cauda region. CE4 mRNA was not detectable in the efferent ducts and was only expressed moderately in the remainder of the epididymis, with greatest levels in the caput and proximal cauda regions, and decreasing in the distal cauda. CE5 mRNA showed the most marked regional variation in levels with little or no mRNA detectable in the caput and proximal corpus regions, but increasing dramatically in the distal corpus and cauda. In the transition region of the central corpus, the CE5 mRNA appeared to be expressed intermittently, giving a mottled signal appearance over the epididymal epithelium. The patterns of mRNA distribution for the three marker genes in the dog epididymis were, therefore, essentially similar to those for the equivalent human homologues, providing further support for the suitability of the dog epididymis as a model for the human.

Animals↗
PubMed1994

The orphan receptor SF-1 binds to the COUP-like element in the promoter of the actively transcribed oxytocin gene.

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand-dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissue-specific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF-1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF-1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.

Animals↗
PubMed1994

Proopiomelanocortin gene expression during pig pituitary and brain development.

The expression of proopiomelanocortin (POMC) mRNA, which plays an important role in neural, endocrine, neuroendocrine and immune systems, was studied by in situ hybridization during the development of pituitary in the domestic pig. The POMC gene was activated as early as fetal day 30 (E30). The signal for POMC mRNA in the anterior lobe progressively increased from E30 to E80 and then remained at relatively constant level. In contrast, POMC transcripts in the intermediate lobe first appeared at E40 and steadily increased during development. POMC transcripts in the brain were first found at E40 and were scattered in the arcuate nucleus (AN) and nucleus medialis thalami (NMT). At E50 the extra-pituitary POMC mRNA was located not only in the AN and NMT but also in the fasciculus tegmenti and entorhinal cortex. In the posterior lobe, no signal was detected. The specific pattern of expression of the pig POMC gene in the pituitary and in specific regions of the central nervous system suggests important roles for POMC in fetal development. These results also suggest that POMC is excellent for studying the expression and regulation of pituitary hormone genes because of its tissue-specific regulation and developmental pattern.

Animals↗
PubMed1994

A novel biological aspect of ovarian oxytocin: oxytocin gene expression in cumulus/luteal cells and the effect of oxytocin on embryogenesis in fertilized oocytes.

Recently, several reports have demonstrated the presence of oxytocin (OT) in the corpus luteum of mammalian species. However, the biological role of ovarian OT remains obscure. This study was performed to examine OT gene expression in cumulus cells of mice and humans, and in human corpus luteum, and the role of OT in early embryogenesis. OT gene and OT mRNA were analyzed by reverse transcription-polymerase chain reaction, with single-strand-conformation polymorphism and heteroduplex procedures. OT-treated in-vitro-fertilized mouse oocytes were cultured and the rate of blastocyst development estimated. An immunohistochemical study was also carried out to detect OT on the surface of the mouse oocytes.

Animals↗
PubMed1994

Over-expression of oxytocin in the testes of a transgenic mouse model.

The bovine oxytocin gene has been expressed in the testes of two independent transgenic mouse lines. Hybridization and RNase protection analysis showed that the oxytocin transgene was transcribed from the normal functional promoter in the Sertoli cells of the seminiferous tubules in a developmentally regulated manner. Immunohistochemistry indicated that both oxytocin and neurophysin epitopes were expressed together in the Sertoli cells at stages I-V and X-XII of the cycle of the seminiferous epithelium. Furthermore, analysis with high-performance liquid chromatography showed that there was a tenfold increase in the amount of amidated oxytocin present in testicular extracts from the transgenic mice. However, there appeared to be no detectable effect of this overproduction of hormone on testicular morphology or fertility parameters. A significant decrease by 50% was detected only in the levels of intratesticular testosterone and dihydrotestosterone. The results point to a local paracrine role for oxytocin in the modulation of Leydig cell function.

Animals↗
PubMed1994

Post-transcriptional block in oxytocin gene expression within the seminiferous tubules of the bovine testis.

Northern blot hybridization showed that bovine and sheep testis, unlike testes from other mammals, contain moderate levels of an apparently normal oxytocin gene transcript. In situ hybridization localized this mRNA to within the seminiferous tubules, possibly in the Sertoli cells. Conflicting with this result, immunohistochemistry showed that both oxytocin and the syngeneic neurophysin I epitopes are both clearly restricted to the Leydig cells, being expressed here at a low level. Since illegitimate transcription from spurious start sites can lead to a lack of translation product, the integrity of the major ruminant testicular transcripts of the oxytocin gene was checked using differential hybridization. RNase protection and multiple polymerase chain reaction assays. All tests showed the transcripts to have a normal, translatable composition and to be transcribed from the conventional 5' initiation site. Therefore, the block in oxytocin gene expression within the tubules is probably due to a lesion at the post-transcriptional level. The low level peptide expression in the Leydig cells can probably be attributed to the presence of functional transcripts in these cells, which are below the level of significant detection for the in situ hybridization assay.

Animals↗
PubMed1993

A major mRNA of the human epididymal principal cells, HE5, encodes the leucocyte differentiation CDw52 antigen peptide backbone.

HE5, a very abundant human epididymal gene product, was cloned by a differential screening procedure which employed testis as the primary negative control tissue. Sequencing of the HE5 cDNA showed it to be identical to that encoding the peptide backbone of the human leucocyte differentiation antigen CDw52. Since human genomic DNA contained only one cross-hybridizing fragment, this implies that both products were transcribed from the same gene. Northern blot analysis and in situ transcript hybridization revealed a highly tissue- and cell-type-specific expression pattern for HE5, showing the gene product to originate from epithelial cells of the epididymal and deferent duct. The possible identity of the HE5 product with the CDw52 antigen suggests a link between the immune and reproductive systems.

Amino Acid Sequence↗
PubMed1993

Region-specific variation of gene expression in the human epididymis as revealed by in situ hybridization with tissue-specific cDNAs.

Three tissue-specific gene probes that had been isolated by differential screening from a human epididymal cDNA library--HE1, HE2, and HE5--were employed to investigate regional specializations in the human epididymis. All 3 cDNAs were derived from major transcripts of the epithelial cells lining the epididymal duct. Each mRNA species, however, exhibited a discrete longitudinal pattern of hybridization with maxima in different regions of this organ, suggesting regional specializations of gene expression. The HE5 mRNA, which was recently shown to encode the peptide backbone of the human leukocyte differentiation antigen CDw52, showed maximum levels in the distal corpus epididymidis and in the vas deferens, whereas the HE2 mRNA was found predominantly in the caput and proximal corpus sections of the epididymis. HE1 mRNA was found in high amounts in all parts of the epididymis, displaying a 2-peak expression pattern with maxima in the distal caput and distal corpus of the epididymal duct.

Aged↗
PubMed1993

Tissue-specific gene expression as an indicator of epididymis-specific functional status in the boar, bull and stallion.

cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue-specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HE1-homologue was up-regulated markedly only at puberty, the HE4-homologue was already present at moderate levels prepubertally. The distribution of the HE1-homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3-4-week-old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4-homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.

Animals↗
PubMed1993

Arginine vasotocin gene expression and hormone synthesis during ontogeny of the chicken embryo and the newborn chick.

Chicken embryos at different developmental stages (embryonal day (E) 6 to 21) and chicks at posthatch day 1 (D1) were monitored for the development of their hypothalamo-neurohypophysial system as indicated by the kinetics of arginine vasotocin (AVT) gene expression via mRNA concentration and brain AVT content. Our data concerning the onset of gene expression support previous results from our laboratory and others about an early activation of the AVT gene transcriptional and translational activity around E6. We could detect measurable amounts of AVT in chicken embryo brains at E6 and an exponential increase during further development until D1. Dot blots of hypothalamic RNA extracts indicated that AVT gene transcript concentrations rose between E12 and E17 and slightly dropped thereafter. Northern hybridization showed that this drop was caused by a decrease of full length message and an increase of smaller transcripts during late embryonal and D1 stages, probably an AVT mRNA specific degradation phenomenon. The dissociation between the increase of AVT concentration and AVT mRNA concentration visible at the D1 stage might be due to accumulation and storage of AVT in the magnocellular neurons, preferentially in their axon terminals in the neurohypophysis. Blood samples taken from E14 onwards revealed a constant increase in plasma osmolality and plasma AVT concentration. Our data suggest that, in the chicken, AVT seems to be required early during embryonal development, either for osmoregulatory or further unknown functions.

Animals↗
PubMed1993

Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: evidence for a soluble form of the receptor in human thyroid.

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

Animals↗