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and Its Metabolite by Endometrium and Yolk Sac Placenta in Late Gestation in the Tammar Wallaby, Macropus eugenii1
a Department of Zoology, The University of Melbourne, Parkville, Victoria 3052, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and its metabolite, PGFM, by uterine tissues from tammar wallabies in late pregnancy. Endometrial explants were prepared from gravid and nongravid uteri of tammars between Day 18 of gestation (primitive streak) and Day 26.5 (term) and were incubated in Ham's F-10 medium supplemented with glutamine and antibiotics for 20 h. PGF2 and PGFM in the medium were assayed by specific, validated RIAs. Control tissues (leg muscle) did not produce detectable amounts of either PG. Both gravid and nongravid endometria secreted PGF2, and production increased significantly in both gravid and nongravid uteri towards term. PGFM was produced in small amounts by both gravid and nongravid uteri, and the rate of production did not increase. Neither oxytocin nor dexamethasone stimulated PG production in vitro in any tissue at any stage. Thus, the surge in peripheral plasma PGFM levels seen at parturition may arise from increased uterine PG production, but further study is needed to define what triggers this release.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and its synthetic analogue, cloprostenol, are potent stimulators of uterine contractions in tammars [2]. During late pregnancy, peripheral plasma concentrations of a metabolite of PGF2, PGFM, are low (< 200 pg/ml [2, 3]. However, in parturient animals there is a transient peak of PGFM in the peripheral plasma (about 1 ng/ml), with levels falling close to baseline levels within 15 min after birth [4]. This transient release of PG may be essential for birth, since treatment of late pregnant tammars with the PG synthase inhibitor indomethacin blocked parturition [5].
The PG release at birth appears to have several other functions. PGF2 injection induces a surge in plasma prolactin levels similar to that observed around birth [6]. At birth there is also a dramatic fall in plasma progesterone, either caused directly by the PG [5] or by the PG-induced prolactin peak [3, 6]. Exogenous PGF2 [7] and PGE2 [8] also induce the stereotyped parturient behavior normally seen only in the minutes around birth [7, 9].
The most likely source of the peak in plasma PGFM is from pulmonary metabolism of PGF2 released from the uterus in late gestation and labor. In tammars, injected PGF2 is rapidly metabolized to PGFM [2]. In some mammalian species, the uterus may also produce some PGFM directly [10, 11]. In a preliminary study, PGF2 content of gravid tammar uterine tissue was higher at term than 2 days earlier [12]. In many eutherian species the fetal membranes are also a major source of PG secretion. Tammars have a functional yolk sac placenta, and although it is not invasive and is relatively low in mass, it is another likely source of PG production in late gestation.
The tammar provides a valuable model system to investigate uterine hormone production. Like other marsupials, tammars have two completely separate uteri, opening by separate cervices into the vaginal system. Since this species is monotocous, only one of these uteri will contain an embryo. Comparison between the two sides within an animal thus allows investigation of effects due to a local fetal influence on endometrial function.
Oxytocic peptides and glucocorticoids are also implicated in the control of birth in tammars as in other mammals. Blocking mesotocin (the oxytocic peptide in tammars) by infusing the receptor blocker Atosiban delays birth in tammars [13]. Mesotocin may facilitate birth by directly stimulating uterine contractions, but it may also act indirectly by stimulating PG synthesis. Exogenously administered glucocorticoids can induce premature parturition [14]. The mechanism is unknown, but glucocorticoids can modulate PG production in eutherian mammals [15, 16].
This study investigates production of PGF2 in vitro by endometrial and placental explants from late pregnant tammars, and the effects of treatment with glucocorticoids and oxytocin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tammars used in this study were of Kangaroo Island stock, from a breeding colony established at Monash University. Husbandry and handling were as previously described [17, 18]. Lactating females had their pouch young removed during the breeding season to initiate active gestation, with birth expected 26.5 days later [19]. Groups of females were caught between Days 18 and 26 of gestation, and pregnancy was confirmed by laparotomy under sodium pentobarbitone anesthesia [5].
Animals were held under Department of Conservation and Natural Resources permits. All experiments were conducted with ethics committee approval in accordance with the National Health and Medical Research Council Guidelines for the Use of Animals in Biomedical Research and the Guidelines for the Use of Native Fauna in Biomedical Research. These guidelines are in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Tissue Incubations
Pregnant females were killed by an overdose of sodium pentobarbitone (60 mg/ml; Abbott Labs., Kurnell, NSW, Australia) and the reproductive tract was dissected out and opened. Before Day 23, the embryo is not attached and can be readily rolled from the uterus. After this stage, the yolk sac placenta becomes folded around the increasingly rugose endometrial layer; however, this attachment is superficial, allowing the endometrium and yolk sac membranes to be separated cleanly [17]. The endometria were gently plucked from the myometrium using fine forceps, and the tissue was chopped using a razor blade into pieces 0.5–1.0 mm across. These explants were incubated in 5 ml Ham's F-10 medium (Sigma Chemical Co., St. Louis, MO) with 5 IU/ml penicillin-G and 5 mg/ml streptomycin at 37°C under 5% CO2 in air in 6-well x 5-ml culture plates (Nunc, Roskilde, Denmark; cat. no. 1-52795). The explants (10–20 mg wet weight per well) were held at the medium surface on polycarbonate membranes (Costar, Cambridge, MA; cat. no. 110409) supported by stainless steel mesh grids. After a 3-h equilibration period, the medium was changed. At 24 h the medium was removed and stored frozen at -80°C until assayed for PG content. Tissue from each well was stored frozen until assayed for protein content. Six replicate incubations were prepared for each endometrium: two without added hormone, two with dexamethasone (100 ng/ml), and two with oxytocin (20 ng/ml). Incubations of leg muscle were made on each day to serve as a control. These doses were based on previous studies. Endogenous cortisol concentrations are around 50–100 ng/ml, and dexamethasone is approximately 20 times as potent as cortisol in binding to tammar glucocorticoid receptors in vitro (unpublished results). Endogenous mesotocin concentrations peak at 1–2 ng/ml at birth [20], and oxytocin concentrations of 20 ng/ml are sufficient to stimulate tetanic spasm of myometrium in an organ bath [21].
PGF2 Assay
A specific PGF2 RIA was validated for this study. The assay buffer was 0.1 M tricine (Sigma), pH 8.0, with 1 g/l NaCl, 1 g/l gelatine, and 0.1 g/l NaN3. The antiserum, kindly provided by Dr. D. Wong (CSIRO, Prospect, NSW, Australia), was used at a dilution of 1:100 in assay buffer. Tracer was [3H]PG F2 (Amersham, Arlington Heights, IL; TRK464) was made up in assay buffer. Aliquants of culture medium were assayed directly. Assay tubes (11 x 55 mm; Nunc) contained 200 µl sample or standard in the range 62.5–16 000 pg/ml, 100-µl tracer containing approximately 5000 cpm, and 100 µl antiserum (1:100 dilution). The tubes were vortexed and incubated for at least 4 h at 4°C. Preliminary tests showed that equilibrium was achieved by about 2 h. Then 100 µl human gamma globulins (10 mg/ml in buffer; CSL Ltd., Parkville, Victoria, Australia) and 1 ml 20% PEG-6000 (Ajax Chemicals, Auburn, NSW, Australia) were added at 4°C, vortexed, and incubated for a further 30 min before separation of bound and free tracer by centrifugation for 10 min at 800 x g. The supernatant was aspirated, and 1 ml of scintillation fluid was added to the pellet, which was then counted in a scintillation counter.
Assay sensitivity was less than 125 pg/ml. Interassay variation was 9.3%, and intraassay variation was 4%. Authentic PGF2 added to medium was recovered quantitatively, and serial dilutions of medium containing high levels of PGF2 produced a curve parallel to the standard curve.
PGFM Assay
PGFM was assayed by an RIA already established in our laboratory for plasma [2, 4], and validated for culture media used in these assays. Assay sensitivity was < 16 pg/ml, interassay variance was 10.3%, and intraassay variation was 3.3%.
Protein Assay
Protein in endometrial tissue was assayed using a modified version of the Bio-Rad protein assay (Bio-Rad Labs., Richmond, CA). Samples of tissue were digested in 1 M sodium hydroxide overnight at 37°C. A standard protein solution was prepared by dissolving BSA in 1 M sodium hydroxide on the day of each assay. Aliquots (100 µl) from each standard and unknown solution were pipetted into a 96-well dish in quadruplicate or triplicate, respectively, and the pH of each aliquot was lowered by the addition of 10 µl of hydrochloric acid. Aliquots (200 µl) of 1:3 diluted Bio-Rad Dye Reagent Concentrate (Bio-Rad; cat. #500-0006) were added to each well, and then the absorbance at 605 nm was measured on a Bio-Rad 650 plate reader. Assay sensitivity was below 10 µg/ml, and within- and between-assay variation was less than 2%.
Statistics
Data are presented as means ± SEM (n). Data were grouped by stage: Days 18–22 (early organogenesis); Days 23–24; and Days 25–26 (term—viable neonates can be obtained at Day 25, although they are usually born on Day 26), and production rates were log-transformed before conducting repeated-measures ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production
Production of PGF2 and PGFM by control tissue (leg muscle) was below assay sensitivity. Endometrial explants from all stages produced substantial amounts of PGF2 (Fig. 1). The production rate of PGF2 by gravid endometrium increased almost 3-fold between Days 18–22 and Days 25–26 (p < 0.003). Production rates of PGF2 by nongravid endometria were more variable than those of gravid endometria (Fig. 1). The average for Days 23–34 was elevated by very high values in 2 of the 5 animals in this group. However, nongravid endometria produced significantly less PGF2 than did gravid endometria within animals overall (p < 0.05), and particularly at Days 25–26 (p < 0.01).
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PGF2 production by the yolk sac membranes increased even more dramatically than production by gravid endometrium (Fig. 2), with a significant rise between Days 18–22 and Days 23–24, and again between Days 23–24 and Days 25–26.
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Treatment with oxytocin or dexamethasone had no effect on PGF2 production in any of the tissues studied (p > 0.1; Fig. 1).
PGFM Production
Both gravid and nongravid endometria produced PGFM over the incubation period (Fig. 2), but the amount produced was less than 30% of the PGF2 production. Gravid endometrium produced significantly more PGFM than nongravid endometrium (p < 0.05), but there was no significant variation with stage of pregnancy for either side (p > 0.1; Fig. 2).
Unlike endometrium, yolk sac membrane did not produce measurable amounts of PGFM (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by gravid endometrium and yolk sac placenta at term is consistent with their being the source of PGs needed for parturition. This increase in the gravid endometrium appears to be due to a local feto-placental influence since there is no comparable increase in the contralateral nongravid uterus, which is exposed to the same systemic influences. In sheep, increased PG synthesis results from decreased progesterone and increased estrogen concentrations. In tammars, however, plasma progesterone levels usually fall immediately after birth, rather than before [5, 14]. Premature decreases in progesterone due to surgical removal of the corpus luteum, or maintenance of high plasma progesterone levels by exogenous administration does not affect the timing of birth [24–26]. The timing of the increase in PG synthesis parallels the rise in plasma estradiol concentrations as a follicle matures for the postpartum estrus [27, 28], but this estradiol rise is not needed for birth [27, 29].
Birth is delayed in female tammars treated with an oxytocic receptor antagonist, Atosiban, in late gestation [13], suggesting that mesotocin (the endogenous oxytocic peptide in tammars) is crucial for birth. In sheep, rats, and humans, oxytocin can induce PG release from the endometrium, which may play a role in maintaining uterine contractions in birth (reviewed in [23]). However, concentrations of oxytocin 10–20 times greater than peak plasma concentrations [20] did not stimulate increased endometrial PG synthesis in vitro in this study. Although there is a substantial increase in the concentrations of mesotocin receptors in the myometrium over the last 4 days of gestation, mesotocin receptors were not detectable in the endometrium [21, 30]. In a preliminary study, we have also shown that injection of doses of oxytocin that cause strong uterine contractions do not induce a rise in peripheral plasma PGFM. This suggests that in tammars, oxytocin may induce uterine contractions at birth by a direct effect on the myometrium rather than indirectly by stimulating PG synthesis.
Glucocorticoids may also be important in regulating birth. The effects of glucocorticoids on PG synthesis are variable, and there are conflicting reports. For example, Pasmanik et al. [16] reported that dexamethasone inhibited PG synthase expression and PGF2 output in human term placental cells, but not in first-trimester cells, and Riley et al. [15] found that dexamethasone treatment suppressed output of another PG, PGE2. However, Zakar et al. [31] found that glucocorticoids specifically stimulated expression of the PG H synthase-2 (PGHS2), a key enzyme in synthesis of PGF2 and PGE2 in human term placenta. McLaren et al. [32], and Zhang et al. [33] found increased PGHS2 levels in ovine cotyledons in glucocorticoid-treated ewes. In the present study, dexamethasone had no significant effect on endometrial or placental PG production in vitro. Treatment of tammars with the glucocorticoids on Day 24 resulted in premature parturition with a normal parturient profile of progesterone fall and PGFM rise. How glucocorticoids precipitate parturition in tammars is not known, but it is unlikely to be due to a direct stimulation of endometrial or placental PG synthesis, since dexamethasone had no such effect in vitro.
PGs are required for birth in tammars, and inhibition of PG production with indomethacin in late gestation prevents birth [5]. The acute rise in plasma PGFM at birth suggests that there must be a stimulation of PGF2 release at parturition. The nature of this stimulus is unknown, but there is substantial evidence that the fetus has a major effect on the timing of birth in the macropodids [34, 35]. Thus, it seems likely that in late gestation the uterus maintains a high capacity for PG synthesis, and that at parturition a specific signal from the fetus activates a dramatic release of PGs.
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ACKNOWLEDGMENTS |
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assay. Dr. David Small kindly allowed us to use his plate reader for the protein assays. |
FOOTNOTES |
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2 Correspondence. FAX: 61 3 9344 7909; g.shaw{at}zoology.unimelb.edu.au
3 Current address: Department of Anatomy and Cell Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.
4 Current address: McFarlane Burnet Centre for Medical Research, The University of Melbourne, Parkville, Victoria 3052, Australia.
Accepted: October 7, 1998.
Received: June 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on uterine activity, and concentrations of 13,14-dihydro-15-keto-PGF2 in peripheral plasma during parturition in the tammar wallaby (Macropus eugenii). J Reprod Fertil 1983; 69:429–436. in the tammar wallaby (Macropus eugenii). J Reprod Fertil 1990; 88:335–342.This article has been cited by other articles:
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  J. N. Ingram, M. B. Renfree, and G. Shaw Differential Regulation of Contractility and Nitric Oxide Sensitivity in Gravid and Nongravid Myometrium during Late Pregnancy in a Marsupial Endocrinology, June 1, 2001; 142(6): 2244 - 2251. [Abstract] [Full Text] [PDF]
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 J.N. Ingram, G. Shaw, and M.B. Renfree Cortisol in Fetal Fluids and the Fetal Adrenal at Parturition in the Tammar Wallaby (Macropus eugenii) Biol Reprod, March 1, 1999; 60(3): 651 - 655. [Abstract] [Full Text]
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