Biomedical subjects

R Ivell

Publications and source records attributed to R Ivell.

At least 91 records · Page 5Linked to original sources

PubMed1996

Sertoli cell lines established from H-2Kb-tsA58 transgenic mice differentially regulate the expression of cell-specific genes.

Sertoli cell lines have been established from H-2K(b)-tsA58 transgenic mice carrying an inducible temperature-sensitive SV40 T antigen in their germline. All cell lines tested for expression of Sertoli cell products by reverse transcription-polymerase chain reaction were shown to express mRNAs for alpha-inhibin, Steel factor, SGP-2, and transferrin as well as for androgen receptor and the orphan nuclear receptor SF-1. Selected cell lines were shown by immunocytochemistry to express the established Sertoli cell-specific pattern of cytoskeletal markers. The FSH receptor gene was also expressed, though downregulated by comparison with in vivo levels of expression. In some lines low expression of the luteinizing hormone receptor gene could also be detected. The gene for the transcription factor GATA-1, which is expressed specifically in Sertoli cells, was expressed only in a subset of the cell lines. Quantitative analysis of SGP-2 transcript levels by ribonuclease protection assays showed an increase at the nonpermissive temperature, whereas using a similar assay, Steel factor mRNA was shown to be expressed in amounts comparable to the in vivo situation only in two cell lines during permanent growth. In summary, cell lines that exhibit distinct Sertoli cell characteristics have been established, which may resemble different stages of phenotypic development.

Animals↗

PubMed1996

Oxytocin receptors in bovine cervix during pregnancy and parturition: gene expression and cellular localization.

OBJECTIVES: Our purpose was to determine the expression of transcripts encoding the oxytocin receptor protein in bovine cervix during pregnancy and parturition, the cellular localization of immunoreactive oxytocin receptors, and oxytocin receptors concentrations in the same tissues. STUDY DESIGN: Ribonuclease protection assay for oxytocin receptor messenger ribonucleic acid was used to determine gene expression in bovine cervical tissues obtained from 20 cows throughout pregnancy and parturition, cellular localization of oxytocin receptors was determined by immunohistochemistry, and tritiated oxytocin binding was measured in each tissue. RESULTS: Oxytocin receptor gene expression and tritiated oxytocin binding were well correlated in each instance. During pregnancy the level of oxytocin receptor messenger ribonucleic acid was very low; it was increased at term with a further, marked increase at parturition. Tritiated oxytocin binding also increased dramatically at parturition and was most abundant in the mucosal layer. Strong oxytocin receptor immunoreactivity was present in mucosal epithelial cells, and scattered muscle cells in the muscular part showed the signal. CONCLUSIONS: Our results, together with the previous finding that oxytocin stimulates prostaglandin E2 release from cervical tissue in vitro, indicate that cervical mucosal epithelial cells are targets for oxytocin at parturition and may mediate release of prostaglandin E2.

Animals↗

PubMed1996

Expression of GH, TSH beta, LH beta and FSH beta genes during fetal pituitary development in the pig.

The development of the anterior pituitary gland involves the proliferation and differentiation of ectodermal cells in Rathke's pouch to generate distinct cell types, each of which produces its corresponding trophic hormone. Studying pituitary development will therefore reveal novel aspects of organogenesis. In the present study, we examined by in situ hybridization the expression of genes for anterior pituitary hormones during development of the fetal pig pituitary. We found that the beta-subunit gene of thyroid-stimulating hormone (TSH beta) was first expressed at E40, (E = day of embryonal/fetal life), growth hormone (GH) mRNA appeared between E40 and E50, and the gonadotrophin genes (LH beta and FSH beta) were expressed at E50. The transcripts for TSH beta, LH beta and FSH beta were abundantly expressed at about E80, while GH mRNA continued to be richly expressed until after birth. The GH gene was first expressed in the mantle layer of the anterior lobe, while the TSH beta and gonadotrophin (LH beta and FSH beta) mRNAs were found in the central and the basal regions of the anterior lobe, respectively. All of these mRNAs (GH, TSH beta, LH beta, and FSH beta) remained concentrated until the end of gestation in the area where they first appeared. The distinctive pattern of developmental expression of these hormone genes in the fetal pig anterior pituitary makes this tissue an excellent system in which to study tissue-specific gene activation and regulation.

Animals↗

PubMed1996

Oxytocin receptors in bovine cervix: distribution and gene expression during the estrous cycle.

Oxytocin (OT) receptor (OTR) concentrations were determined in the cervix of nonpregnant cows on cycle Days 0, 3, 7-8, 17, and 19 (n = 3-4 cows each day); [3H]OT was used as the labeled ligand. Mucosal and muscle layers of the cervix were also analyzed separately for both ligand binding and expression of the OTR gene using a newly developed RNase protection assay (RAP). Cellular localization of OTR protein was determined by immunohistochemistry. All regions of cervix from cows at estrus had high concentrations of OTR; in the luteal phase, all were sharply down-regulated. At estrus the mucosal layer had about 30-fold higher concentrations than the muscle layer. OTR mRNA was readily detected by RAP in the mucosa from estrous cows, while much weaker signals were found in the muscle. On Days 7-17, the OTR mRNA signals in both mucosa and muscle were very faint or nondetectable. Thus, there was a good correlation between ligand binding and mRNA expression, which suggests that OTR concentrations are mainly regulated at the transcriptional level. The epithelial cells at the luminal surface of the mucosa were the principal site of immunoreactive OTR; muscle cells showed significantly weaker signals. Previously, OT was found to stimulate prostaglandin (PG) E2 output in vitro in bovine cervical tissues. Since PGE2 is capable of softening the cervix, our findings suggest that OT may have a novel physiological function to cause softening of the bovine cervix mediated by the release of PGE2.

Animals↗

PubMed1996

Relaxin-like factor gene is highly expressed in the bovine ovary of the cycle and pregnancy: sequence and messenger ribonucleic acid analysis.

Relaxin-like factor (RLF) is a new member of the insulin/insulin-like growth factor family of hormones and growth factors, which appears to be predominantly expressed in the Leydig cells of the testis. An analysis of male and female bovine tissues indicated that in the cow the RLF gene is also highly expressed in the female, mainly in the follicular theca interna and in the corpus luteum, with a pattern of gene expression very similar to that for the related peptide relaxin in other species. Sequence analysis of bovine testicular and luteal RLF and cDNA shows that the same gene is expressed in both male and female gonads. Because the bovine RLF sequence, like those from pigs, humans, and mice, retains, the putative receptor binding motif described for relaxin, it seems plausible that RLF might functionally substitute for relaxin in the cow, in which the latter peptide appears not to be significantly expressed.

Amino Acid Sequence↗

PubMed1996

Body temperature (37 C) specifically down-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture.

To study the role of scrotal vs. body temperature in epididymal function we established a simplified cell culture system from the dog epididymis in which the cells showed a pattern of gene expression similar to that in the human epididymis and retained many characteristics of epididymal epithelial cells. The cultured cells had an epithelial-type cytoskeleton, nuclear androgen receptor protein, and a striking temperature responsiveness. Exposure of the cells to a culture temperature of 37 C, compared to 33 C, had a fast and irreversibly suppressive effect on the levels of an abundant epididymal messenger RNA (mRNA), CE5, which represents the canine counterpart of the human CD52/HE5 mRNA, encoding a major glycosylphoshatidylinnositol (GPI)-anchored sperm membrane glycopeptide. The temperature effect on the mRNA was a direct and specific one, not mediated by temperature influences on the testis and not affecting other epididymal mRNAs. Exposure of the cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (25 micrograms/ml culture medium) and cycloheximide (2 micrograms/ml) suggested that the steady state levels of CD52/CE5 mRNA may be controlled posttranscriptionally by changing the half-life of this specific mRNA in response to an extracellular temperature stimulus.

Animals↗

PubMed1996

Molecular cloning and expression of the relaxin-like factor from the mouse testis.

A complementary DNA encoding the mouse homolog of the testicular relaxin-like factor was cloned from a subtractive complementary DNA library, comprising clones preferentially expressed in the testes of mutant w/wv mice. The predicted amino acid structure conforms with that for other members of the insulin, insulin-like growth factor, and relaxin hormone family, with A, B, and C (connecting) domains. Northern and in situ transcript hybridization showed that the gene is exclusively expressed at a high level in the Leydig cells of both mutant and wild type testes and is up-regulated at puberty. The level of the specific messenger RNA is such as to imply that this novel member of the insulin family represents a major secretory product of Leydig cells, with a potential to be a part of a novel systemic feedback loop to other organs, including the brain.

Aging↗

PubMed1996

Oxytocin: cellular and molecular approaches in medicine and research.

In May 1995 the third Hanseatic Endocrine Conference at Stade, Germany, attracted 140 scientists from all over the world to summarize the current knowledge on one hormone--oxytocin. This article presents the major findings of the meeting with the realisation that oxytocin provides major model systems with which to elaborate a whole series of novel endocrinological paradigms, as well as being the example of choice for establishing revolutionary new techniques, which will no doubt spread to studies of other hormone systems. The papers from this symposium will be published in full.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and in vivo expression of the bovine steroidogenic acute regulatory protein.

Differential screening was used to select clones from a bovine luteal cDNA library, which were specifically expressed in late luteal stages. One of these clones encoded the bovine homologue of the Steroidogenic Acute Regulatory protein (StAR). The bovine StAR gene is transcribed as 3kb and 1.8kb transcripts, which differ in their sites of 3' polyadenylation. Both transcripts are highly expressed in corpora lutea of mid to late cycle and through pregnancy, but only at low levels in the early cycle. Northern analysis showed expression only in the corpus luteum and in the adrenal gland, but not in any other tissue examined. Within the protein coding region of the bovine StAR gene, there is a marked 124 base homology to the 5' non-coding region of another luteal transcript, TIMP-1, suggesting a possible common regulatory function for this sequence.

Adrenal Glands↗

PubMed1995

Structure and expression of the bovine oxytocin receptor gene.

The gene for the bovine oxytocin receptor has been sequenced using a combination of clones derived from a bovine endometrial cDNA library from estrus and a bovine genomic DNA library, with confirmation of structure using reverse transcription PCR programmed by term myometrial RNA. The receptor belongs to the seven transmembrane domain family and predicts a protein of 391 amino acids. A comparison of the genomic sequence with the cDNA structure, as well as reverse transcription polymerase chain reaction (RT-PCR) analysis, shows there are two introns, one in the 5'noncoding region that appears to be differentially spliced in the bovine uterus and a conserved intron within the open reading frame between the regions encoding the transmembrane domains VI and VII. Northern blot analysis indicated three major transcripts in myometrium and endometrium in vivo at approximately 6.5 kb, 3.5 kb, and 2.0 kb. In situ hybridization analysis of uterine tissue at term showed highest mRNA concentrations in the endometrial epithelium, particularly in the deep glands, a pattern confirmed also at the immunohistochemical level by monoclonal antibodies raised against a human amino-terminal peptide. Further confirmation of the identity of the receptor was obtained by transient transfection of a reconstituted receptor construct into COS-7 cells. The expressed receptor was shown to have identical pharmacological properties in respect to various oxytocin analogs to the natural bovine endometrial receptor.

Amino Acid Sequence↗

PubMed1995

Oxytocin and oxytocin receptor gene expression in the reproductive tract of the pregnant cow: rescue of luteal oxytocin production at term.

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.

Animals↗

PubMed1995

Regulation of oxytocin expression in the bovine corpus luteum. Orphan receptors and the oxytocin promoter.

The bovine oxytocin gene is massively up-regulated during the early development of the corpus luteum. Oxytocin transcription is induced in a highly synchronous fashion in the granulosa cells of the dominant follicle at the time of ovulation. The possibility to isolate large numbers of differentiating granulosa-luteal cells from exactly defined stages of development allows the investigation of the factors controlling oxytocin expression in vivo by molecular and cell biology methods. Using primary cultures of bovine granulosa cells the synergistic activation of oxytocin transcription by the cAMP pathway and stimulation of IGF-I or insulin receptors could be established. Analysis of transcription factors isolated from the nuclei of bovine granulosa cells and corpus luteum led to the identification of the tissue-specific orphan receptor SF-1 binding to the promoter of the actively transcribed oxytocin gene. The luteinizing bovine granulosa cells provide the only easily accessible experimental system established so far in which the endogenous oxytocin gene is expressed. Although the link between increased cAMP level and receptor tyrosine kinase activation on the one hand and the induction of oxytocin transcription on the other has not been established yet, these experiments constitute one of the few direct approaches to investigate the complexity of events that regulate oxytocin expression in vivo.

Animals↗

PubMed1995

Structure and organization of the bovine oxytocin receptor gene.

A DNA probe specific for the V and VI transmembrane domains of the bovine oxytocin receptor was initially prepared by reverse transcription PCR, and its structure and specificity confirmed by DNA sequencing. This probe was then used to screen a bovine genomic DNA library in bacteriophage lambda, and three positive clones were purified, subjected to restriction analysis and relevant fragments sequenced. Parallel to this, a cDNA library prepared using bovine endometrial RNA at the time of ovulation was screened by PCR employing the same primers as above. The longest cDNA clone was also fully sequenced. This clone still lacked, however, a substantial stretch of 5'sequence. The full transcript structure, and hence also the exon-intron organization, was then obtained by RT-PCR using primer oligonucleotides deduced from the cloned genomic sequence. All nucleotide sequence information was derived from a combination of two independent genomic clones, a cDNA clone and several independent RT-PCR reactions programmed by myometrial RNA, all in both strand orientations. The structural organization of the bovine oxytocin gene essentially conforms to that described for the human gene. Unlike the human gene, however, the 5'non-coding region of the primary transcript is interrupted by only a single intron, with a further intron in the coding region separating the sequences encoding the transmembrane domains VI and VII. The difference between this structure and that for the human gene suggests the existence of a differential splicing of 5' non-coding sequences.

Amino Acid Sequence↗

PubMed1995

Oxytocin and the timing of parturition. Influence of oxytocin receptor gene expression, oxytocin secretion, and oxytocin-induced prostaglandin F2 alpha and E2 release.

Oxytocin receptor (OTR) gene expression was studied in various tissues of the reproductive tract of pregnant cows and compared with ligand binding activity. Myometrium, intercaruncular endometrium, caruncular endometrium, cotyledons and fetal membranes, as well as the uterine cervix of pregnant cows expressed the bovine OTR gene. Receptor concentrations, measured by ligand binding to crude microsomal pellets, were comparable to OTR mRNA signal strength in all instances indicating that the receptor protein formation is probably regulated at the transcriptional level. During bovine pregnancy OTR gene expression was initiated at different times depending on the tissue. The expression of the gene for OT peptide was not found in any of the bovine uterine tissues but was found in the corpora lutea at term and during parturition and then at relatively low levels. Therefore endogenous OT is derived almost exclusively from the pituitary during bovine pregnancy. OT secretion occurred in a pulsatile manner during pregnancy; a significant increase in pulse amplitude was observed during the last days before delivery and a large surge was associated with active labor and delivery. We postulate that the temporal order of OTR gene expression in the uterine and intrauterine tissues is a factor in the synchronization of the events that eventually lead to the onset of parturition. Because OT receptor mediates different actions in different tissues OT has multiple functions in the mechanism of parturition. The peptide initiates and maintains myometrial contractions, it stimulates release of PGF2 alpha from the endometrium and fetal membranes and, as demonstrated in this study, OT induces PGE2 release from cervical tissues in an OTR dependent manner. We conclude that in pregnant cows, OT participates both in the events that prepare the reproductive tract for birth and initiate the birth process.

Animals↗

PubMed1995

Oxytocin: a follicular luteinisation factor in the marmoset monkey.

The distributions of oxytocin (OT) and the oxytocin receptor (OTR) have been characterised in preovulatory follicles of the marmoset monkey and are described in relation to the process of luteinisation using a combination of in vitro and in vivo techniques, including immunohistochemistry and granulosa cell culture. Ovaries were collected in the periovulatory phase before and 22h after exogenous gonadotropin (hCG) treatment, but prior to ovulation. Before hCG treatment, OT immunoreactivity (OT-ir) was found mostly in granulosa cells of antral follicles, especially in the layers nearest the antrum. In contrast, oxytocin receptor immunoreactivity (OTR-ir) was observed principally in basal granulosa cells of antral follicles. Progesterone receptor (PR) and 3 beta hydroxysteroid dehydrogenase (3 beta HSD), markers for luteinisation, were negative for all ovarian tissues collected before hCG treatment. After hCG treatment, almost all granulosa cell layers in antral follicles stained positively for both OT-ir and OTR-ir, most prominently in preovulatory follicles and especially in the cumulus oophorus. This increase in staining was associated with an induction of PR and 3 beta HSD activity in the granulosa cells of preovulatory follicles. OT production by granulosa cells in culture was stimulated by the addition of hCG, but only in cultures derived from preovulatory follicles; whereas FSH was without any effect. Addition of extrinsic OT to the cultures elicited an increase in progesterone production only for the granulosa cells derived from preovulatory follicles. Together, the results of the in vivo and in vitro studies point to an involvement of OT in the process of leuteinisation in the marmoset monkey.

Animals↗

PubMed1995

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

Recently, several authors have reported the existence of oxytocin (OT) in mammalian granulosa-luteal cells after ovulation. The purpose of this study was to examine the evidence for gene expression and localization of OT and OT receptor (OTR) in the cumulus cells surrounding the oocytes. Cumulus cells with mature oocytes were obtained from experimental and clinical in vitro fertilization-embryo transfer (IVF-ET) programs. OT and OTR gene expression was analyzed with reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR/single strand conformation polymorphism (SSCP). OT gene expression was detected in mouse and human cumulus cells. The results of RT-PCR/SSCP showed that the structure of OT mRNA in cumulus cells was similar to that in the hypothalamus. Furthermore, OTR gene expression was clearly demonstrated in human cumulus cells, and a weak positive signal was observed in human oocytes. Immunocytochemical staining of OTR was clearly detected in human cumulus cells. The rate of mouse blastocyst development was significantly higher in the group cultured with OT than that without OT. These results are the first observations of simultaneous OT and OTR gene expression in cumulus cells, suggesting that ovarian OT might have some physiological role in the early stage of embryo development.

Animals↗

PubMed1994

Two orphan receptors binding to a common site are involved in the regulation of the oxytocin gene in the bovine ovary.

The peptide hormone oxytocin is highly expressed in the hypothalamus within only a small number of magnocellular neurons. However, it is also expressed in a much larger number of cells in the bovine corpus luteum at high levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studies, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been identified as the bovine homologue of the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The second protein is here characterized as the bovine homologue of a tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The relative expression of these two factors during luteal development correlates with the level of luteal oxytocin gene expression, with SF-1 being the factor binding to the promoter of the oxytocin gene when this promoter is activated. Cotransfection experiments using the murine testicular cell line TM4 show that SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly by transducing a stimulatory signal to the RNA polymerase.

Animals↗

PubMed1994

Major human epididymis-specific gene product, HE3, is the first representative of a novel gene family.

Differential screening of a human epididymal cDNA library led to the isolation and characterization of a major epididymis-specific cDNA clone family, referred to as HE3. More detailed sequence and PCR analysis identified two different but homologous gene transcripts, HE3 alpha and HE3 beta. The former represents an mRNA of ca. 1 kb, encoding a putative small secretory polypeptide of 14903 MW. The HE3 beta transcript was only found as incomplete 3' fragments. Analysis of human genomic DNA by Southern blotting suggested the presence in the human genome of at least three independent HE3-related genes. Isolation of genomic clones for the HE3 alpha gene showed this to contain a single intron of 1.4 kb in the 5' noncoding region. Although genomic clones corresponding to HE3 beta could not be found, a third highly homologous gene, HE3 gamma, was identified as a potential pseudogene. Neither nucleotide nor encoded amino acid sequences of the HE3 gene family are related to any other known sequence in the central databases, and thus represents a novel human gene family, with at least three nonallelic members. Northern hybridization analysis showed that HE3 gene products are specifically expressed in the human epididymis, and not in any other tissue examined. Furthermore, except for the pig, no other nonprimate species has been identified to express homologous sequences in the epididymis. RNase protection assays showed that both the HE3 alpha and HE3 beta, but not the HE3 gamma genes, are expressed in the human epididymis.

Amino Acid Sequence↗