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Biomedical subjects

D G Porter

Publications and source records attributed to D G Porter.

At least 19 recordsLinked to original sources
PubMed1995

Partial complementary deoxyribonucleic acid cloning of equine relaxin messenger ribonucleic acid, and its localization within the equine placenta.

To determine the site of relaxin gene expression in equine placentae, a set of degenerate oligonucleotide primers was made according to the published amino acid sequence of the A- and B-chain of equine relaxin (eRXN). Total cellular RNA (tcRNA) from equine placentae at about 120 and 300 days of pregnancy was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with use of these primers. A single amplification product of approximately 430 bp was detected in each case by agarose gel electrophoresis. The PCR product was ligated into Bluescript plasmid and sequenced to confirm the identity of the clone as an eRXN cDNA fragment. Nucleic acid sequence analysis revealed a 428-bp eRXN cDNA fragment encoding for parts of the A- and B-chain and the connecting peptide (109 residues). Northern analysis of tcRNA from placentae of 120 and 300 days of pregnancy was carried out with use of antisense digoxigenin-labeled cRNA generated from the cDNA clone, and a single transcript of approximately 1 kb was detected. In situ hybridization on placental tissue at 120 and uteroplacental tissue at 300 days of pregnancy indicated that only the fetal trophoblastic cells expressed eRXN mRNA transcripts. The identity of these cells was confirmed by their positive staining with an antibody specific for equine trophoblast (cell surface) protein. Relaxin peptide was also detected immunohistochemically in samples of the same placental tissues. This is the first report of the nucleic acid sequence of eRXN. The study identified fetal trophoblast cells as the site of eRXN mRNA expression and protein secretion in the equine placenta.

Amino Acid Sequence↗
PubMed1992

Ethical scores for animal experiments.

Scientists have for too long been faced with a polarized debate concerning the ethics of animal experiments. An ethical ideal and a practical scoring system would be valuable for the moderate majority.

Animal Experimentation↗
PubMed1992

Rapid increase in relaxin gene expression in early pregnancy in the pig.

Relaxin mRNA concentrations in porcine corpora lutea were examined during the peri-implantation period and throughout pregnancy using Northern and slot blot analysis. Total RNA was extracted from corpora lutea obtained from pigs of known breeding dates and pregnancy was confirmed by embryo recovery. A 32P-labelled porcine relaxin cDNA probe identified the 1.0 kilobase relaxin transcript. Slot blots were subsequently used to quantify relaxin mRNA concentrations. Relaxin mRNA was detectable in the corpus luteum of the regular cycle and was also present at similar low levels in corpora lutea of days 10, 11 and 12 of pregnancy. In corpora lutea from day 16 of pregnancy onwards 100-fold greater quantities of relaxin mRNA were observed. The intensity remained similar in samples between days 16 and 102 of pregnancy. These studies indicate that elevated relaxin gene expression commences very early in pregnancy and is first detectable in the peri-implantation period.

Animals↗
PubMed1992

Technical note: is there a season for iodinating relaxin?

We report a loss of antibody binding in our porcine relaxin radioimmunoassay that has occurred during the coldest periods of the past three winters. We do not know the reason for this seasonal variation but we have eliminated cold deterioration of the iodide used for radioiodination and equipment failures as explanations.

Animals↗
PubMed1992

Lack of effect of relaxin on oxytocin output from the porcine neural lobe in vitro or in lactating sows in vivo.

Oxytocin was measured in incubates and perifusates of neurosecretosomes prepared from sow neural lobes (n = 50) and in incubates of isolated neural lobes (n = 5). In none of these preparations was oxytocin output affected by exposure to purified porcine relaxin (at concentrations up to 10(-7) mol l-1). Moreover, in lactating sows (n = 9), 6-10 days post partum, the administration of porcine relaxin (1.5 or 3.0 mg) intravenously, immediately before a suckling episode, did not affect the plasma oxytocin profile compared with saline treatments (within sow) nor did it alter suckling behaviour or the weight gain of the litter. In all sows, a spike (25-75 pg ml-1) of oxytocin was measured during milk ejection coincident with suckling. These results suggest that porcine relaxin does not affect oxytocin release in suckling sows in contrast to reported findings in rats. The data also support the view that porcine relaxin could be used at farrowing without adverse effects on suckling.

Animals↗
PubMed1992

Relaxin is not associated with poor milk yield in the postpartum sow.

Porcine relaxin (pRXN) titers were measured in 109 plasma samples drawn between 24 and 120 h post-partum from good (n = 34), and poor (n = 25) milking sows and from sows (n = 12) exhibiting overt signs of hypogalactia located on six swine farms in Ontario. Mean plasma pRXN titers among the three groups at 24 h post-partum were not significantly different although there was considerable variation. By 48 h however, the pRXN titers had declined markedly in all groups and between 72 and 120 h, pRXN was cleared from the plasma of all sows examined except one hypogalactic animal that had a titer of 1.13 ng/mL at 96 h. These results suggest that, although pRXN may be identifiable in the corpora lutea during the puerperium, it does not contribute to either hypogalactia or poor lactational performance in sows. The data also encourage the view that pRXN could be used at farrowing without deleterious effects on suckling.

Animals↗
PubMed1991

Immunoreactive adrenocorticotrophin is present in the ovary and in particular the oocyte of several mammalian species.

Proopiomelanocorticotrophin (POMC)-derived peptides have been identified in both male and female reproductive systems. However, there have been few reports of ACTH, the major biologically active POMC product, in the mammalian ovary. We sought evidence for the presence and localization of immunoreactive (ir)-ACTH in ovaries from sheep, humans, cows, pigs, rats and cats using immunohistochemical techniques. Tissue sections were stained with diaminobenzidine following incubation with a primary antibody raised against ACTH1-24. There was positive staining for ACTH in cells scattered throughout the interstitium of ovaries from all species examined. Immunoreactive ACTH was observed in the oocytes of ovaries from humans, cows, pigs, pregnant and non-pregnant sheep, but not from cats or rats. Positive staining of oocytes was associated with all tertiary and secondary follicles, and some primary follicles. There was no apparent difference in the pattern of staining between pregnant and non-pregnant sheep. Staining for ir-ACTH was absent in ovaries from fetal sheep. We conclude that ir-ACTH is present in ovarian tissue, and in particular the oocyte, from several species of mammal. The presence of ir-ACTH within the oocyte is dependent on species and stage of follicular maturation.

Adrenocorticotropic Hormone↗
PubMed1989

Relationship between pre-partum relaxin concentrations and farrowing intervals in the pig.

Gilts were treated on Day 112 of gestation with saline or a prostaglandin (PG) F-2 alpha analogue. In control gilts there was a rise in the relaxin concentration from 48 h before the onset of delivery, peaking between 12 and 28 h pre partum followed by a steep fall. The relaxin concentrations at each 1-h time interval were analysed in relation to the farrowing interval for each gilt using correlation analysis. There was a significant (at least P less than 0.05) positive correlation between the relaxin concentration and the farrowing interval at every time period from 14 to 2 h before delivery. In contrast there was little relationship between concentrations of progesterone, oestrone and oestradiol-17 beta and farrowing intervals. The gilts treated with PGF-2 alpha analogues had steroid profiles indistinguishable from those in controls but differing relaxin secretion patterns. Relaxin concentrations peaked at 1-2 h after PGF-2 alpha injection and this was followed by a second smaller increase closer to the time of delivery in 7 out of 12 gilts. The 'two-peak' gilts had significantly higher relaxin concentrations at farrowing and took significantly longer to farrow than did the 'one-peak' gilts (P less than 0.005). These results suggest that high relaxin concentrations during the last 14 h before the onset of parturition are associated with increased farrowing times, but are not associated with any increase in neonatal mortality.

Animals↗
PubMed1988

Plasma steroid, relaxin and dihydro-keto-prostaglandin F-2 alpha changes in the minipig in relation to myometrial electrical and mechanical activity in the pre-partum period.

Recordings of electromyographic and mechanical activities of the uterus of intact and ovariectomized minipigs during the last days of pregnancy, combined with a frequent blood sampling regimen, indicated that the evolution of electrical activity was dependent upon the clearance both of progesterone and relaxin from the plasma. Increases in dihydro-keto-prostaglandin-F-2 alpha (DHKF-2 alpha) occurred only after the initial decline in progesterone, and were positively correlated with increases in electrical activity only after circulating relaxin titres had fallen. These results demonstrate that relaxin provides for the inhibition of the myometrium during the period when progesterone titres decline. The temporal relationship of the decline of progesterone and the rise in DHKF-2 alpha suggests that uterine PGF-2 alpha may not initiate luteolysis in the sow at term.

Animals↗
PubMed1988

Identification of relaxin in the placenta of the ewe.

Sheep placentomes were collected at the abattoir and the stage of gestation was estimated from the crown-rump length and appearance of the fetus. Samples were extracted and either freeze dried (crude extracts) or fractionated on Sephadex G-50 and CM-cellulose. Relaxin immunoreactivity (RXN-IR) was detected in all samples by a pig relaxin RIA and diluted in parallel with the standard curve. Two patterns of RXN-IR were seen after Sephadex G50 purification: (a) a single main peak of RXN-IR eluting at a position similar to pig relaxin; or (b) a 3-peak pattern with additional higher (void volume) and lower (approximately 1000) molecular weight peaks. These peaks were all found with 4 different and specific antisera. The 6000 molecular weight peak eluted at a similar position to pig relaxin on CM cellulose and inhibited electrically stimulated rat uterine contractions in vitro. The amount of relaxin measured in crude extracts of placentomes from different ewes was very variable. Most samples were within the range 0.05-11.2 ng/g wet weight of tissue (3.0 +/- 0.45 (s.e.m.), n = 44) but a few contained much higher concentrations (25.5-61.4 ng/g, n = 3). There was no obvious variation in concentration with stage of pregnancy (20 days to term). Samples of intercotyledonary endometrium, allantochorion and whole ovaries from pregnant ewes were also extracted. All contained low concentrations of RXN-IR (0.6 +/- 0.13 ng/g, n = 4; 0.6 +/- 0.29 ng/g, n = 3; 1.0 +/- 0.66 ng/g, n = 7, respectively). We conclude that relaxin-like peptides are present in the pregnant ewe and that, as the placentomes are the largest component by weight, they represent the major source.

Animals↗
PubMed1986

Identification of relaxin immunoreactivity in human follicular fluid.

Relaxin immunoreactivity has been found in samples of human follicular fluid collected from artificially stimulated pre-ovulatory follicles. The crude extract caused a reduction in the height of the contractions in a rat uterine strip bioassay. The reactive material eluted from Sephadex G50 in two major peaks. The first contained approximately 60% of the immunoreactivity and had an elution position corresponding to that of porcine relaxin, indicating a mol. wt of approximately 6000 daltons. The second peak was of a lower mol. wt, but its exact size and significance are unknown. A possible role for relaxin in the process of follicular rupture is suggested.

Biological Assay↗
PubMed1986

Relaxin and progesterone are myometrial inhibitors in the ovariectomized non-pregnant mini-pig.

Intravenous bolus injections of pig relaxin which produced short-lived peaks of the hormone equivalent in concentration to those observed at term promptly rendered the uterus almost totally quiescent and the inhibition persisted for about 2.5 h. During this time the uterus remained responsive to oxytocin. The main effect of relaxin was to reduce the frequency of intrauterine pressure cycles rather than the amplitude. In contrast progesterone, which also inhibited myometrial activity, took between 6 and 24 h to exert its maximum effect by reducing both amplitude and frequency of IUP cycles and it also abolished the responsiveness of the myometrium to oxytocin. Its actions were reversible but recovery took between 54 and 140 h. Oestradiol benzoate had no significant effect on myometrial activity in 21 out of 26 treatments. At 24 h after the 5 remaining treatments, however, myometrial activity was virtually zero. No evidence was obtained of a biphasic effect of oestradiol on myometrial activity as reported for the rat and ewe. This work demonstrates that purified pig relaxin is an active myometrial inhibitor in the oestrogen-treated ovariectomized non-pregnant pig in vivo.

Animals↗
PubMed1986

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe.

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.

Animals↗
PubMed1984

Biosynthesis of oxytocin in the corpus luteum.

In this report we demonstrate that ovine and bovine luteal cells synthesise oxytocin by way of a precursor protein similar to that found in the hypothalamus. Isolated ovine or bovine luteal cells were incubated for up to 12 h with [35S]cysteine. Neurophysin-Sepharose column separation and HPLC of cell extracts demonstrated the presence of [35S]oxytocin. Incorporation of [35S]cysteine was confirmed by performic acid oxidation. Immunoprecipitation of cell extract with anti-rat oxytocin-neurophysin followed by SDS-PAGE yielded 2 radioactive bands of 14 kDa and 11-12 kDa. Immunoprecipitation with anti-oxytocin yielded 1 band at 14 kDa. On SDS-PAGE the 14 kDa band had a similar mobility to rat-hypothalamic oxytocin precursor.

Animals↗
PubMed1984

Identification of oxytocin and vasopressin in the testis and in adrenal tissue.

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.

Adrenal Glands↗