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Biomedical subjects

M J Harper

Publications and source records attributed to M J Harper.

At least 73 records · Page 4Linked to original sources

Storage in vivo of [3H]prostaglandins by rabbit blastocysts.

Day 6 rabbit blastocysts that had previously been incubated with 10 nM [3H]prostaglandin E2 (PGE2), [3H] PGF2 alpha, or 0.5 microCi [3H]water were surgically transferred to the uteri of day 6 pseudopregnant recipient rabbits. At 1, 3, and 20 h after transfer, blastocysts were collected from the recipient rabbits and evaluated for retention of [3H]ligands. The transferred day 6 blastocysts were able to retain a significant proportion of the initial PGs for up to 20 h in vivo. These PGs were not metabolized while they remained in the blastocysts in vivo, as assessed by HPLC. The transferred blastocysts appeared normal and viable after 20 h in vivo, since implantation sites could be detected visually. These data support our hypothesis that blastocysts can sequester PGs from their environment in vivo and retain them unmetabolized during the critical period just before implantation.

Animals↗

[3H]Prostaglandin accumulation in vitro by baboon uterus.

Uteri from mature baboons at various stages of the menstrual cycle were collected at autopsy. Transverse slices of the uteri were incubated with [3H]prostaglandins in Tyrode's buffer. Bound and free ligands were separated by filtration. Prostaglandin (PG) accumulation by the tissue slices was evaluated as a function of incubation time, PG type and concentration, temperature, wet weight of tissue and stage of the menstrual cycle. There was no significant difference in PG accumulation in response to PG type (PGE2 or PGF2 alpha) or stage of the menstrual cycle. These results from baboon uteri were compared with those using baboon oviducts and also rabbit uteri and oviducts. Unlike the rabbit tissues, the baboon oviducts and uterine tissues did not exhibit specific net accumulation of prostaglandins.

Animals↗

Progesterone and oestradiol receptor concentration and translocation in uterine tissue of rabbits treated with indomethacin.

The purpose of this study was to determine whether endogenous prostaglandins are involved in the regulation of sexual steroid receptor concentration and cytosol to nucleus translocation in the rabbit uterus. The animals were injected s.c. with indomethacin (10 mg/kg) twice daily on days 5 and 6 and on the morning of day 7 of pregnancy or pseudopregnancy and killed 2 h after the last injection. The indomethacin treatment did not change progesterone and oestradiol serum concentrations compared with animals injected with vehicle only. No differences were observed either in progesterone receptor or oestradiol (pregnant animals only) receptor concentration and intracellular distribution in endometria of indomethacin-treated or control animals. In an extended experiment indomethacin given to oestrogen-pretreated ovariectomized rabbits did not inhibit the nuclear progesterone receptor accumulation induced by a single progesterone injection. These results suggest that there is no direct relationship between prostaglandins and sexual steroid receptors in the rabbit uterus.

Animals↗

Effect of indomethacin in vivo on prostaglandin content of several rabbit tissues.

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle in vivo. PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.

Animals↗

Prostaglandin translocation from the lumen of the rabbit uterus in vitro in relation to day of pregnancy or pseudopregnancy.

Transport of 3H-labeled prostaglandins (PGs) E2 and F2 alpha from the uterine lumen across the uterine wall has been studied in rabbit uteri in vitro in incubations lasting up to 180 min, in relation to sexual state of the rabbit, incubation temperature, intraluminal PG concentration, addition of metabolic inhibitors and time of incubation. PG accumulation by the tissue increased rapidly up to 30 min and then remained relatively constant. By 30 min, radioactivity was found in the external incubation medium, and this increased linearly with time. The translocation of PGF2 alpha was significantly greater in pseudopregnant than in pregnant animals on Day 6, whereas that of PGE2 was significantly higher in pregnant than in pseudopregnant animals on Day 6.8. In pregnant animals, both PGF2 alpha and PGE2 were translocated to the exterior more rapidly on Day 6.8 than on Days 5 or 6. Transport of PGs was reduced by low temperature, unaffected by metabolic inhibitors and only that of PGE2 increased with increased (5 microM) intraluminal concentrations. During incubation, the tissue remained viable as judged by T/M ratios (dpm tissue/dpm medium) for 204 thallium. Transport of [14C] sucrose was much slower than that of [14C] urea, which was greater than the fastest rates exhibited by the PGs. In general, amounts of radioactivity found in antimesometrial, mesometrial and lateral portions of the uterine wall, or in implantation and interimplantation areas did not differ, but more was found in the endometrium than the myometrium. PGF2 alpha was translocated unmetabolized to the external medium, while only two-thirds of the PGE2 was translocated unchanged, and one-third converted to PGF2 alpha. It is concluded that the rabbit uterus shows some selectivity in handling PGs in relation to stage of pregnancy.

Animals↗

Rabbit blastocysts accumulate [3H]prostaglandins in vitro.

Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with 3H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of [3H]PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of [3H]PGE2 and [3H]PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the [3H]PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. We conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

Analysis of Variance↗

Site of action of indomethacin on implantation in the rabbit.

Blastocysts recovered from oil- or indomethacin-treated donor rabbits between 5 1/2 and 6 days after insemination and hCG injection were transferred to oil- or indomethacin-treated recipients between 135 and 147 h after hCG injection. Indomethacin treatment of donor rabbits (10 mg/kg s.c.) given every 6 h during the day before transfer had no effect on subsequent implantation of the blastocysts. However, indomethacin treatment of the recipients (10 mg/kg s.c. every 6 h from 120 to 168 h after hCG) prevented implantation of all transferred blastocysts, although 6 of the 8 rabbits died between Days 9 and 16 of (pseudo)pregnancy. Restriction of the indomethacin treatment of the recipients to only 3 injections of 10 mg/kg s.c. between 128 and 140 h after hCG injection had no effect on the implantation of the transferred blastocysts. It is concluded that indomethacin exerts its inhibitory influence on implantation via an action on the endometrium rather than on the blastocyst.

Animals↗

Uterine venous, peripheral venous, and radial arterial levels of prostaglandins E and F in women with pregnancy-induced hypertension.

Patients with pregnancy-induced hypertension have higher prostaglandin (PG) F concentrations in radial arterial blood (0.39 +/- 0.03 ng/ml) than control subjects (0.24 +/- 0.03 ng/ml) and higher (PGE plus PGF) concentrations in uterine venous blood obtained at the time of cesarean section (1.62 +/- 0.18 versus 1.03 +/- 0.12 ng/ml in the control group). The present results suggest that both PGE and PGF production by the uterus in patients with pregnancy-induced hypertension is increased but that catabolism of PGF by the lung is compromised; this permits larger quantities of the vasoconstrictor PG to pass into the systemic circulation, where it may cause hypertension directly or indirectly, by association with other vasoactive substances.

Alprostadil↗

Prostaglandin production by human term placentas in vitro.

The release of PGs increases with increasing incubation time and the main PG produced is different in different tissues. This could implicate the presence of an inhibitory factor in vivo. PGE release is the same in patients in labor and not in labor, and this PG is mainly produced by the villi. Villi, membranes and decidua in tissue cultures from patients not in labor release more PGF than patients in labor, the difference for the decidual tissue being strikingly large. Conversion of labelled arachidonic acid by pieces of term placenta from women in labor during short-term incubations was mainly to PGE2 and its metabolites and PGD2.

Arachidonic Acid↗

Platelets bind to rhesus monkey corpora lutea and stimulate its prostaglandin synthesis.

The in vitro production of different prostaglandins (PGs) was studied in rhesus monkey corpora lutea incubated with labeled arachidonic acid. The percentage of conversion of the arachidonic acid to all the PGs identified was similar, but the addition of platelet lysates caused at least a sevenfold stimulation of all PGs by the corpora lutea, the greatest stimulation in production occurring in the area corresponding to PGE2 and 15-keto-PGF2 alpha. Adipose tissue by itself produced more PGs than corpus luteum (CL); but, in contrast, such production was inhibited by addition of platelet lysates to the levels of conversion of the unstimulated CL. 125I-labeled platelets bound specifically to monkey CL, but not to adipose tissue. The interaction of the platelets with the CL in the formation of PGs may be of significance in the regulation of the luteal life span.

Adipose Tissue↗

Prostaglandin release by zygotes and endometria of pregnant rabbits.

When 4-day rabbit zygotes were incubated for 1 h at 37 degrees C in vitro, very little prostaglandin (PG) was released into the medium, and the concentration of PGs in the zygotes after incubation was also low. The release of prostaglandin E (PGE) and prostaglandin F (PGF) into the medium, and their concentration in the zygotes after incubation, increased sharply on Days 6 and 7 of pregnancy, reaching, by Day 7, values close to 200 ng of each PG released in 1 h per mg of protein. By contrast, endometrial samples on Days 4 and 5 of pregnancy released more PGF and less PGE than the zygotes of the same ages on a per mg of protein basis, and on Days 6 and 7, less of both PGs. Furthermore, endometrial concentrations of PGs after incubation, except for PGF on Day 4, were always lower than values for zygotes. Endometrial concentrations of PGs on Day 6 were lower before than after incubation. Although there was a slight upward trend in PG release by endometrial samples with increasing length of pregnancy, the changes were minimal and, in the case of PGE, none of the mean values exceeded 1 ng per mg of protein. In 7-day blastocysts, high levels of both PGF and PGE were found in the blastocoelic fluid, and these did not change during the 1-h incubation. The release of PGF and PGE during in vitro incubation of ruptured and washed Day 6 blastocysts was stimulated by arachidonic acid, and that of PGF, but not PGE, inhibited by indomethacin. The release of PGE, but not of PGF, from Day 6 blastocysts was inhibited by low temperature, and the same conditions inhibited release of both PGF and PGE from endometrial cell suspensions. It seems that both blastocysts and endometria have capability to synthesize PGs, the blastocysts being particularly active in this regard on Days 6 and 7 of pregnancy. It is hypothesized that, in vivo, Day 6 and 7 blastocysts release large quantities of PGs which trigger some of the local endometrial changes associated with pregnancy.

Animals↗

A reliable method for isolating endometrial epithelial cells from rabbits, and preliminary studies of prostaglandin uptake.

Endometrial epithelial cells were isolated enzymatically from intact, everted uteri of mature rabbits and then by sedimentation at unit gravity. Electron-microscopic examination of these cells taken from estrous and pseudopregnant animals showed that the preparation contained almost pure endometrial cells in the form of "plaques" with little contamination with other cell types. The cells were judged viable on the basis of the trypan blue exclusion test and the presence of motile cilia. It was observed that pinocytotic activity was greatest at estrus, and declined thereafter, while release of secretory granules increased during pseudopregnancy. There was also a marked increase in the number of free ribosomes and mitochondria as pseudopregnancy progressed (estrus less than 4 day less than 6 day less than 7 day). The cisternae of the rough endoplasmic reticulum were least at 6 days and the Golgi stacks greatest in size and number at 7 days. The uptake of prostaglandin (PG) E2 and F2 alpha by epithelial cells isolated from rabbits at estrus and at 4, 6, and 7 days of pseudopregnancy was then determined. The magnitude of PG accumulation was found to vary among the different groups. At low extracellular concentrations, accumulation of both PGE2 and PGF2 alpha was greatest in the estrous state, much less at 4 days, and least at 6 days of pseudopregnancy. At 7 days of pseudopregnancy, the uptake of PGs was similar to those values obtained at 4 days. These studies indicate that both PGE2 and PGF2 alpha are accumulated by epithelial endometrial cells as a result of a mechanism(s) that is more complex than a saturable, specific receptor-dependent binding system.

Animals↗

Prostaglandin accumulation by isolated uterine endometrical epithelial cells from six-day pregnant rabbits.

Endometrial epithelial cell plaques were enzymatically isolated from rabbit uteri 6 days after artificial insemination. These cells were judged viable by exclusion of [3H]mannitol, trypan blue and [57Co]EDTA, and contained receptors for the ligand, [3H]quinuclidinyl benzilate (QNB). The cell plaques were evaluated for their ability to accumulate prostaglandins (PGs) as a function of PG concentration and type (E2, F2 alpha), temperature, pH, incubation time and cell protein. The PG accumulation was unsaturable, thereby suggesting that the cells, as isolated, and under the conditions of these experiments, do not have identifiable PG receptors or detectable facilitated transport mechanisms. Cells preloaded with PG rapidly released PG after dilution into buffer containing either no or 50 microM unlabeled PG. We suggest that PGs can be accumulated, by endometrial epithelial cells, in the nonionized (nonelectrolyte) form which dissolves (partitions) into the lipid bilayer; the amount of PG accumulated could reflect environmental concentration (levels in the uterine secretions).

Animals↗

Activity of peroxidase and delta 5-3 beta-hydroxysteroid dehydrogenase in the ovary of LH-treated cyclic female rats.

Rats were treated with LH at 08:00 h on the first day of dioestrus or on Days 1 and 2 of dioestrus. Peroxidase activity increased within 3 h in females injected with LH on Day 1 and was associated with a depletion of ascorbate that lasted until the afternoon of Day 1. Values of both substances then returned to basal values by the morning of Day 2. A second LH injection on Day 2 produced effects similar to those seen after LH on Day 1. Females treated with LH did not display greater delta 5-3 beta-HSD activity than did controls on Day 1 after one LH injection, but did on Day 2 after two LH injections. Nonetheless, the changes were only modest by comparison with those of peroxidase. The peroxidase-ascorbate system appears to be involved in the mechanism responsible for the increased secretion of ovarian progesterone resulting from LH stimulation.

3-Hydroxysteroid Dehydrogenases↗

Studies on peroxidase-catalyzed formation of progesterone.

Subcellular fractions of pregnant rabbit ovary at day six of gestation catalysed the formation of progesterone from pregnenolone (3 beta-hydroxy-5-pregnen-20-one). The microsomal fraction was found to have maximum activity. The enzyme needed hydrogen peroxide for its activity. In vitro studies using horseradish peroxidase also showed similar conversion and this was stimulated 200% in the presence of ascorbic acid. Ascorbate thus appears to perform a catalytic role in this conversion. The importance of peroxidase in luteal steroidogenesis is indicated.

Animals↗

Effects of tension and transmural stimulation on prostaglandin production by estrous rabbit oviductal isthmus.

The effect of longitudinal and circular stretch on the amounts of Prostaglandin F (PGF) and Prostaglandin E (PGE) found in the fluid bathing rabbit oviductal isthmus has been investigated. It was found that the amounts of PGE and PGF measured in the bathing fluid of longitudinally or circularly stretched tissues were negatively correlated to the maturity of the animal. Prostaglandin E increased with time in the tissues under longitudinal and circular tension. Prostaglandin F also increased with time under longitudinal tension but remained fairly constant under circular tension. Increasing the load from 0.5 to 2.0 g had no significant effect on PGE found under longitudinal or circular tension or on PGF found under longitudinal tension. Under circular tension, PGF found increased. Transmural stimulation at 20 Hz increased PGE 8-fold over control values while PGF increased only 1 to 3-fold. It is suggested that distension of the rabbit oviductal isthmus results in increased PGF production, which could be important in ovum transport.

Animals↗