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Biomedical subjects

A P Flint

Publications and source records attributed to A P Flint.

At least 19 recordsLinked to original sources
PubMed1992

Administration of recombinant bovine interferon-alpha I at the time of maternal recognition of pregnancy inhibits prostaglandin F2 alpha secretion and causes luteal maintenance in cyclic ewes.

The antiluteolytic protein, ovine trophoblast protein-1, which is secreted by sheep embryos at about the time of the maternal recognition of pregnancy, exhibits significant structural homology with alpha interferons. Experiments were conducted to examine the effects of intra-uterine and systemic administration of a recombinant bovine interferon-alpha I (rboIFN-alpha I) upon the interoestrus interval, endometrial oxytocin receptor concentrations and secretion of prostaglandin (PG) F2 alpha in cyclic ewes. In Expt 1, each ewe had a cannula placed in the tip of a uterine horn ipsilateral to a corpus luteum, 7 days after an induced oestrus. From day 9 after oestrus until day 19, ewes received either 200 (n = 4), 667 (n = 5) or 2000 (n = 9) micrograms/24 h of rboIFN-alpha I, meclofenamic acid (n = 4) or vehicle (n = 11). Other ewes received 2000 micrograms rboIFN-alpha I/24 h (n = 5) between days 12 and 15 only. All ewes were killed on day 19. Mean luteal phase, as determined by daily plasma progesterone measurements, was significantly longer (P less than 0.01) and mean concentrations of 13,14-dihydro-15-keto PGF 2 alpha (PGFM) in plasma were lower (P less than 0.05) in ewes receiving 667 or 2000 micrograms rboIFN-alpha I between days 9 and 19, or 2000 micrograms between days 12 and 15, than in animals from other treatment or control groups. A similar protocol was used in Expt 2, in which further ewes received either 2000 micrograms rboIFN-alpha I/24 h (n = 5) or vehicle (n = 5) by bolus infusions twice a day into one uterine horn. Mean luteal phase was significantly (P less than 0.05) longer in treated than in control animals, but differences in PGFM concentrations were not significant. In Expt 3, after a synchronized oestrus, ewes received either 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 12 and 15 (n = 10), 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 9 and 15 (n = 11), i.m. injection of vehicle alone twice a day (n = 20), or continual intra-uterine infusion of 2 mg rboIFN-alpha I/day between days 12 and 15 (n = 7). The mean luteal phase of ewes receiving rboIFN-alpha I by intrauterine infusion or i.m. injection between days 9 and 15 was significantly longer than for animals from the other two groups (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Role of the oxytocin receptor in the choice between cyclicity and gestation in ruminants.

The endometrial oxytocin receptor occupies a central point in the choice between luteolysis and pregnancy in ruminants. Receptor expression determines the time at which luteolysis occurs in nonpregnant animals, and thereby determines the duration of the oestrous cycle. Inhibition of receptor expression by trophoblast interferon (IFN) blocks the process of luteal regression and leads to continued progesterone secretion and the successful growth of the conceptus. Trophoblast IFN is probably required from the time of normal luteolysis during the cycle until luteal oxytocin concentrations of pregnancy decline, thus removing the stimulatory mechanism for the release of the episodes of prostaglandin F2 alpha required for luteolysis.

Animals↗
PubMed1992

Trophoblast interferons in early pregnancy of domestic ruminants.

A type I interferon (IFN) secreted by the trophoblast of early sheep and cow embryos is thought to be responsible for the maternal recognition of pregnancy. The expression of trophoblast IFN is tissue specific and temporally controlled. However, the isolated bovine trophoblast IFN promoter did not confer tissue specificity on the expression of a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, and could not be induced by virus, unlike other type I IFNs. Trophoblast IFN acts locally within the uterus to prevent luteolysis and prolong progesterone secretion. Endometrial IFN receptors are present, and trophoblast IFN decreases expression of endometrial oxytocin receptor and increases expression of endometrial beta 2-microglobulin, MHC class I antigens and Mx (a mediator of IFN antiviral activity) only in the pregnant horn of pregnant ewes with a transected uterus. The primary effect of trophoblast IFN during early pregnancy appears to be an inhibition of oxytocin receptor expression, although studies in ovariectomized ewes suggest that luteal oxytocin may be required to facilitate the inhibition of prostaglandin F secretion by trophoblast IFN. An investigation of the isolated oxytocin receptor promoter should confirm its critical role in the maternal recognition of pregnancy.

Animals↗
PubMed1991

Prostaglandin-induced secretion of oxytocin and prolactin in red (Cervus elaphus) and Pere David's (Elaphurus davidianus) deer hinds: evidence for oxytocin of luteal origin.

Peripheral plasma concentrations of oxytocin in female red deer during the luteal phase of the oestrous cycle (9.3 +/- 2.1 fmol/ml) exceeded those in the follicular phase (3.1 +/- 1.4) or during seasonal anoestrus (3.2 +/- 1.3). In both red and Père David's deer hinds during the mid-luteal phase of the cycle, systemic administration of a luteolytic dose of the prostaglandin F2 alpha analogue, cloprostenol, caused the concentration of oxytocin in the peripheral circulation to rise. Mean (+/- SEM) concentrations increased from 8.1 +/- 0.7 to 97 +/- 8 fmol/ml in red and from 6.2 +/- 0.7 to 153 +/- 30 fmol/ml in Père David's hinds within 5 min of treatment. During seasonal anoestrus oxytocin secretion in response to cloprostenol was reduced to less than 10% of that during the breeding season, in both species. Cloprostenol treatment raised circulating concentrations of prolactin in both species during the breeding season, and during anoestrus in red deer only. The concentration of oxytocin in a single corpus luteum removed at laparotomy from one red deer hind at the mid-luteal phase of the cycle was 66 nmol/g wet wt; identification was authenticated by HPLC. These results suggest that the corpus luteum secretes oxytocin in the Cervidae, as established previously in the Bovidae, and that luteal oxytocin secretion is stimulated by prostaglandin.

Animals↗
PubMed1991

Evidence against a role for blastocyst-secreted oxytocin in early pregnancy maintenance in sheep.

Analysis of ovine conceptus RNA by slot blotting, Northern analysis and nested polymerase chain reaction failed to detect oxytocin-neurophysin prohormone mRNA. Probes used hybridized with both the 3' end of the prohormone mRNA and the oxytocin-coding sequence. Northern analysis of bovine and porcine conceptus RNA was also negative, and polymerase chain reaction demonstrated oxytocin-neurophysin mRNA in ovine corpus luteum, but not in human corpus luteum or decidua, or in ovine endometrium. Infusion of oxytocin into the uterine lumen in cyclic ewes between days 9 and 19 or 20 after oestrus failed to prolong the luteal phase of the cycle and had no effect on endometrial oxytocin receptor concentrations or uterine prostaglandin F secretion. Oxytocin administered systematically prevented luteolysis and reduced uterine prostaglandin F secretion. Taken together, these data suggest that blastocyst-derived oxytocin is unlikely to contribute to corpus luteum maintenance in early pregnancy. They are inconsistent with a previous report that the ovine blastocyst synthesizes and secretes oxytocin.

Animals↗
PubMed1991

Molecular biology of trophoblast interferons and studies of their effects in vivo.

Southern blotting of bovine genomic DNA indicated the presence of at least 3 bovine tIFN genes. The full DNA sequence of one of these genes, thought to be expressed in trophoblast, has been determined, including 193 bp of 5' non-coding region. The inferred amino acid sequence of bovine tIFN is more similar to ovine tIFN (80%) than to bovine IFN-alpha II (70%). The 5' flanking sequence has some similarity with bovine IFN-alpha II, and may contain a viral response element. A recombinant bovine alpha I interferon (Ciba Geigy; brIFN), resembling tIFN, extended oestrous cycle length in sheep when administered by intrauterine infusion over the period, Days 12-15 after oestrus, when maternal recognition of pregnancy occurs. Intramuscular injection was only effective at the doses used if given over a longer period (i.e. Days 9-15). Our experiments indicate that both tIFN and brIFN inhibit luteolysis by preventing a rise in endometrial oxytocin receptor concentrations, and suggest that tIFN achieves this by extending the time for which progesterone suppresses oxytocin receptor development. Further studies are required to confirm this hypothesis and to elucidate the interaction of the effects of progesterone and tIFN in endometrial cells.

Amino Acid Sequence↗
PubMed1990

Roles of prolactin and the uterus in the control of luteal regression in the Bennett's wallaby (Macropus rufogriseus rufogriseus).

The factors responsible for controlling luteal regression in macropodid marsupials are unclear. Experiments were designed to examine the role of the uterus and the pre-partum prolactin pulse in this process. In experiment 1, two groups of adult female Bennett's wallabies were hysterectomized (n = 3) or sham-hysterectomized (n = 3) on Day 16 of a non-pregnant oestrous cycle and daily peripheral blood samples were collected between Days 10-37. In both groups, circulating progesterone concentrations declined to below 0.96 nmol L-1 at luteolysis on Days 28-33. Circulating prolactin concentrations remained low over the period of luteal regression. In experiment 2, two groups of animals, which were maintained in the presence of a fertile male and synchronized by the removal of pouch young (RPY), were treated on Day 16 after RPY with a single injection of long-acting bromocriptine (Parlodel LA, Sandoz Ltd, Basle, Switzerland; 50 mg per animal n = 5) or vehicle (n = 4). Vehicle-treated animals gave birth on Day 28 (n = 3) or Day 29 (n = 1) with a significant elevation of prolactin on Day 28. In contrast, 4 of the 5 bromocriptine-treated animals failed to give birth and one gave birth on Day 29. No comparable prolactin pulse was detected in any of them. Circulating progesterone concentrations declined later in the bromocriptine-treated group than in the controls. These data suggest that different mechanisms lead to luteolysis in pregnant and non-pregnant animals; in pregnancy the luteolytic signal is prolactin, whereas in the non-pregnant cycle another signal of non-uterine origin is involved.

Animals↗
PubMed1990

Effect of continuous infusion of oxytocin on prostaglandin F2 alpha secretion and luteolysis in the cyclic ewe.

Circulating concentrations of 13,14-dihydro-15-keto PGF2 alpha (DHKF2 alpha), luteinizing hormone (LH) and prolactin (PRL) have been measured in cyclic ewes treated with continuous infusions of oxytocin, in order to investigate the mechanism by which the treatment delays luteal regression. Continuous infusion of oxytocin reduced prostaglandin F2 alpha (PGF2 alpha) secretion but had no detectable direct effect on LH or PRL. Oxytocin (3 nmol h-1 i.v.) given from Day 12 or 13 until Day 18 after oestrus delayed luteolysis, eight out of nine treated ewes not returning to behavioural oestrus until Day 29.1 +/- 3.2 (mean +/- s.e.m.; cycle length of control ewes 16.7 +/- 0.3 days). In the ewe in which oxytocin failed to prevent luteolysis, luteal regression had commenced before oxytocin treatment was started. In three ewes undergoing delayed luteolysis (cycle lengths, 21, 24 and 25 days) basal concentrations of PGF2 alpha (measured as DHKF2 alpha) were unchanged, but there was only one episode of PGF2 alpha secretion compared with 20 episodes in three control ewes. Prolactin secretion was pulsatile during oxytocin infusion, and levels were low following infusion in ewes with cycle length greater than 25 days while the corpora lutea were maintained. Circulating PRL concentrations were high in ewes undergoing delayed luteolysis but there was not discrete episode of PRL secretion associated with the pre-ovulatory LH surge in these animals. To investigate the possibility that the pattern of PGF2 alpha secretion was affected by depletion of oxytocin from corpora lutea, ewes previously treated with oxytocin to delay luteolysis were given a luteolytic dose of cloprostenol on Day 21 after oestrus. The amount of oxytocin secreted in response to cloprostenol was less than 10% of that seen in ewes similarly treated on Days 11-13 after oestrus. Low levels of luteal oxytocin may therefore reduce PGF2 alpha secretion in ewes undergoing delayed luteolysis.

Animals↗
PubMed1990

Structure of an interferon-alpha 2 gene expressed in the bovine conceptus early in gestation.

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-alpha 2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5' flanking region of the bovine IFN gene. Computer-aided analysis of the 5' flanking sequence demonstrated a similarity with that of bovine IFN-alpha 2 and the existence of a possible viral induction sequence.

Amino Acid Sequence↗
PubMed1990

Effects of prostaglandin F2 alpha and other potential secretagogues on oxytocin secretion and second messenger metabolism in the ovine corpus luteum in vitro.

Release of oxytocin by sliced or minced sheep luteal tissue in vitro was stimulated up to 1.6- and 2.3-fold by arachidonic acid and the calcium ionophore A23187 respectively. Prostaglandin (PG) F2 alpha and the PGF2 alpha analogue cloprostenol, and other potential agonists known to be active in vivo, including noradrenaline and acetylcholine, were ineffective, as was the phorbol ester tetradecanoylphorbol acetate (TPA). The ineffectiveness of PGF2 alpha was not due to a general unresponsiveness of the tissue in vitro, as PGF2 alpha reduced LH stimulation of tissue concentrations of cyclic AMP and activated inositol lipid hydrolysis. The effect of arachidonic acid was accompanied by release from the tissue of the cytosolic enzyme lactate dehydrogenase (at arachidonic acid concentrations below those required to release oxytocin) and its effect on oxytocin and lactate dehydrogenase release was mimicked by oleic and linolenic acids; arachidonic acid was concluded to act by a non-physiological physicochemical effect without conversion to an eicosanoid. As PGF2 alpha in vitro is known to raise intracellular Ca2+ concentrations in the large luteal cells that secrete oxytocin, and as A23187 stimulates oxytocin release in vitro in the presence and absence of TPA, it is concluded that in-vitro incubation results in an artifactual blockade of the oxytocin-releasing action of PGF2 alpha at an unidentified point distal to the effect on intracellular Ca2+.

Animals↗
PubMed1989

Radioimmunoassay investigations of 20 beta-hydroxylated steroids in maturing/ovulating female rainbow trout (Salmo gairdneri).

Radioimmunoassays, combined with thin-layer chromatography, have been used to test for the presence of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta,21-trihy-droxy-4-pregnen-3-one, and 20 beta-hydroxy-4-pregnen-3-one, and for the presence of their 5-pregnene and 5 alpha-pregnane analogs, in the blood and ovarian incubation media of maturing/ovulating female rainbow trout (Salmo gairdneri). All of these steroids have previously been shown to be more or less equipotent in in vitro oocyte final maturation assays. Only two steroids were found: 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the presence of which has already been firmly established, and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one, which has not been previously identified in rainbow trout. The amounts of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one found in vivo and in vitro were, however, only 1-1.5% of those of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and therefore seem unlikely to play a significant role in the induction of oocyte final maturation.

17-Hydroxycorticosteroids↗
PubMed1989

Oxytocin infusion from day 10 after oestrus extends the luteal phase in non-pregnant cattle.

Oestrus was synchronized in 8 cyclic heifers by progesterone treatment (PRID), after which the animals were monitored for one control cycle to measure the inter-oestrous interval. Osmotic minipumps containing saline (controls, N = 3) or oxytocin (N = 5) were implanted subcutaneously on Day 10 of the second cycle, and removed 12 days later. Jugular venous blood samples were collected daily for measurement of progesterone, and every 2 days for oxytocin. In addition, blood samples were taken every 10 min from 1 h before to 3 h after minipump insertion for measurement of plasma 15-keto-13,14-dihydroprostaglandin-F-2 alpha (PGFM) and every 30 min over the same period for measurement of progesterone and oxytocin. The lengths of the first untreated cycle in both groups of heifers were 20.2 +/- 0.56 (mean +/- s.e.m.) days compared with 25.4 +/- 0.81 days after oxytocin treatment (P less than 0.001). Oxytocin plasma concentrations in treated animals rose from less than 10 pg/ml to 70-500 pg/ml by 2 h after the start of oxytocin infusion and remained elevated until treatment was withdrawn. There was no increase in PGFM concentrations immediately after minipump insertion. Plasma progesterone concentrations were similar in treated and control animals but remained at mid-luteal levels for an average of 5 days longer in treated heifers. It is concluded that continuous administration of oxytocin can extend the luteal life-span in cattle.

Animals↗
PubMed1989

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-alpha family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

Amino Acid Sequence↗
PubMed1989

Neurotransmitters and lymphatic-vascular transfer of prostaglandin F2 alpha stimulate ovarian oxytocin output in sheep.

The mechanisms of lymphatic-vascular transfer across the ovarian vascular pedicle were studied in anaesthetized sheep 8-15 days after ovulation. [3H]Prostaglandin F2 alpha (PGF2 alpha), [14C]mannitol and [36Cl]Na were infused continuously into either a uterine lymphatic or a uterine vein and the kinetics of transfer into the adjacent utero-ovarian vein or ovarian plasma were studied. Transfer occurred according to the sequence [36Cl] greater than [14C] greater than [3H] indicating that PGF2 alpha is not transferred by rapid diffusion, as with [36Cl]Na, nor by a paracellular route, as with [14C]mannitol, but by a slower process probably involving facilitated diffusion. Transfer into the adjacent utero-ovarian vein or ovarian blood was greater when compounds were infused into a uterine lymphatic than into a uterine vein. Substantially more [3H]PGF2 alpha occurred in the adjacent corpus luteum than either of the other compounds after a lymphatic infusion. Intra-lymphatic infusion of PGF2 alpha stimulated the release of ovarian oxytocin but the effect was not confined to the adjacent ovary. Intravenous (jugular) infusion of PGF2 alpha failed to stimulate ovarian oxytocin secretion whereas close-arterial infusion into the ovaries was effective, and the possibility was investigated that any systemic effect of PGF2 alpha was mediated through neural mechanisms. Noradrenaline and acetylcholine were both effective in causing the release of ovarian oxytocin when infused close-arterially into the ovary. With infusions of acetylcholine, ovarian oxytocin secretion rate was increased over fivefold without any change in posterior pituitary release. Noradrenaline and acetylcholine produced a concomitant fall in ovarian blood flow, and neurotransmitter-induced ischaemia may have played a role in ovarian oxytocin release. The finding that PGF2 alpha infused into a uterine lymphatic stimulates ovarian secretion of oxytocin, and that the effect is bilateral whereas PGF2 alpha accumulation in ovarian tissue is unilateral, implies that its mechanism of action may not be solely directed at the luteal cell.

Acetylcholine↗
PubMed1989

Post-translational processing of oxytocin-neurophysin prohormone in the ovine corpus luteum: activity of peptidyl glycine alpha-amidating mono-oxygenase and concentrations of its cofactor, ascorbic acid.

Peptidyl glycine alpha-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of oxytocin synthesis, was measured in extracts of ovine corpora lutea throughout the oestrous cycle. Activity of PGA was low early in the cycle but increased between days 2 and 10 (from 2.3 to 9.0 pmol/mg protein per h) and remained high until day 15. Thereafter, activity declined rapidly at structural luteolysis and was low in corpora albicantia collected 18 and 20 days after ovulation (1.28 and 1.07 pmol/mg protein per h respectively). Luteal concentrations of ascorbic acid, a cofactor for PGA, were high (4.7 mumol/g wet wt tissue) by day 4 after oestrus; concentrations fell rapidly after day 15 (to 2.1 mumol/g on day 16). Concentrations of ascorbic acid were also high in the pituitary gland and in the adrenal medulla and cortex. Concentrations of oxytocin in luteal tissue, which were low (0.3 nmol/g wet wt) on day 2 after oestrus, were highest (2.73 nmol/g) on day 6 and declined thereafter (0.56 nmol/g on day 10, 0.08 nmol/g on day 15 and not detectable on days 18 and 20). Concentrations of oxytocin, progesterone, PGA and protein were measured in subcellular fractions obtained after density gradient centrifugation of extracts of corpora lutea collected on days 6, 7 and 12 of the oestrous cycle, and on day 7 from an anaesthetized ewe before and after treatment with the prostaglandin F2 alpha analogue, cloprostenol. PGA colocalized with particle-associated oxytocin in fractions of density 1.049-1.054 g/ml. Exogenous [3H]oxytocin and [3H]progesterone and endogenous progesterone localized in fractions of density 1.035 g/ml. Oxytocin and PGA were depleted from fractions of density 1.049-1.054 g/ml following cloprostenol treatment in vivo. Fractionation of extracts of ovine corpora lutea by high-performance liquid chromatography (HPLC) followed by radioimmunoassay and radioreceptor assay for oxytocin demonstrated the presence of at least two cross-reacting substances with elution characteristics distinct from oxytocin. Concentrations of these peptides increased as the cycle progressed. These compounds differed from the putative C-terminally extended post-translational processing intermediates, oxytocinyl-glycine, oxytocinyl-glycine-lysine and oxytocinyl-glycine-lysine-arginine, as indicated by their elution positions on HPLC and the specificities of the assays used to detect them, and no conclusions could be drawn on which post-translational processing step was rate-limiting in oxytocin synthesis. These data are consistent with the suggestion that post-translational processing of oxytocin-neurophysin prohormone takes place in secretory granules in luteal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗