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Endocrinology Vol. 142, No. 6 2244-2251
Copyright © 2001 by The Endocrine Society

ARTICLES

Differential Regulation of Contractility and Nitric Oxide Sensitivity in Gravid and Nongravid Myometrium during Late Pregnancy in a Marsupial1

J. N. Ingram2, M. B. Renfree and G. Shaw

Department of Zoology, University of Melbourne, Victoria, 3010, Australia

Address all correspondence and requests for reprints to: Marilyn B. Renfree, Department of Zoology, The University of Melbourne, Victoria, 3010, Australia.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Marsupials have two anatomically separate uteri; and in macropodids (kangaroos and wallabies), there is a single ovulation from alternate ovaries in each cycle. During late pregnancy, the two uteri are differentially regulated by local hormonal influences from the corpus luteum, the fetus, and placenta on one side and by the developing Graafian follicle on the other. In this study, we report striking differences in contractile behavior of nongravid and gravid myometrium from the tammar wallaby (Macropus eugenii) in late pregnancy and immediately post partum. Nongravid myometrium, from the uterus ipsilateral to a Graafian follicle, was spontaneously active but unresponsive to the oxytocic peptide mesotocin and the smooth muscle relaxant nitric oxide. Myometrium from the contralateral, gravid uterus, which contained a conceptus and was associated with an active corpus luteum, was not spontaneously active. Gravid myometrium became increasingly sensitive to mesotocin stimulation as pregnancy progressed, and nitric oxide induced marked relaxation at all stages examined, by a guanylyl-cyclase mediated pathway. These results provide further evidence that the two uteri of marsupials are under differential control, suggesting that local endocrine and paracrine influences, derived from the ovaries, the fetus, and placenta, can regulate concurrent but distinct physiological responses in the reproductive tracts of these mammals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE tammar wallaby (Macropus eugenii), parturition is a very rapid process. Transition of the myometrium from the quiescence that is maintained during pregnancy to the highly contractile state that is required for successful parturition must occur equally rapidly (1). The tiny, altricial fetus is usually born within minutes of the female adopting the characteristic birth position (2). In the maternal peripheral circulation at this time, there is a short-lived pulse of PG F2{alpha} metabolite and the oxytocic peptide mesotocin (MT) (3, 4), and both hormones are necessary for birth (5, 6). They stimulate contractions of the gravid myometrium in vivo (7, 8, 9) and in vitro (10), and the sensitivity of the uterus to both increases in late pregnancy (8, 9, 10). The activation of stimulatory pathways is thus an important component of the onset of myometrial contractions at parturition. However, the hormonal mechanisms by which quiescence of the uterus in maintained during pregnancy in this marsupial are unknown.

In all marsupials, there are two anatomically separate uteri, each with its own cervix opening into the anterior vaginae (11). In macropodid marsupials (kangaroos and wallabies), which are monovular, ovulation occurs from alternative ovaries in successive cycles (11). The gravid uterus is associated with an active corpus luteum and developing conceptus, whereas the nongravid uterus is ipsilateral to the developing Graafian follicle, which will ovulate at the postpartum estrus. The anatomical arrangement of the utero-ovarian vasculature allows hormones released by each ovary to be preferentially delivered to the ipsilateral uterus (12). The two uteri are thus differentially controlled, depending on the proximity of the corpus luteum, the fetus, and placenta on one side or the follicle on the other (10, 11, 12, 13, 14, 15). This makes the tammar an excellent model for comparing pregnant and nonpregnant tissue, because each animal has its own internal comparison between the gravid and nongravid uterus.

Myometrial contractility in mammals is regulated by ovarian steroids. In the tammar, spontaneous contractility of both uteri is absent during seasonal and lactational quiescence and after ovariectomy, when levels of estradiol and progesterone are low (7, 8). At this time, estradiol injections increase uterine contractility, whereas progesterone treatment does not (7, 8), suggesting that estradiol controls spontaneous uterine contractility. Parturition in tammars is usually associated with a precipitous fall in progesterone from the corpus luteum and a rise in estradiol from the developing follicle in the contralateral ovary (reviewed in 1, 11). However, neither of these hormonal events is essential for parturition. The timing and success of birth is not affected either by lutectomy or by treatment with exogenous progesterone in late pregnancy, and tammars give birth normally when the rise in estradiol is inhibited by blocking follicular development (reviewed in 1).

In eutherians, uterine quiescence is actively maintained during pregnancy by multiple mechanisms (16, 17, 18, 19). Recently, a role for the smooth muscle relaxant nitric oxide (NO) has been suggested by studies in a number of species (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). In these studies, NO was shown to relax myometrium from pregnant animals (rat, 23, 24 ; human, 25, 26), and isoforms of NO synthase (NOS) were found in uterine tissues (rat, 27, 28, 29, 30 ; rabbit, 31, 32). NO rapidly enters cells and stimulates the enzyme cytosolic guanylyl cyclase (cGC) leading to the production of the second messenger nucleotide cyclic GMP (cGMP). This activates a cGMP-dependent protein kinase (PKG), and muscle relaxation results from phosphorylation of various proteins involved in contraction (33). NO is also a smooth muscle relaxant in the gut of marsupials (34), and it may be similarly involved in maintaining myometrial quiescence during pregnancy.

This study investigated the role of NO in regulating myometrial contractility during late pregnancy and parturition in the tammar. The contractility of gravid and nongravid myometrium from the same pregnant female was directly compared, to determine the potential differential effects of local endocrine and paracrine factors in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female tammars of Kangaroo Island (South Australia) origin were housed in open grassed enclosures, as previously described (2, 35). Care and treatment of animals conformed to the Australian National Health and Medical Research Council guidelines, and all experiments were approved by Institutional Animal Experimentation Ethics Committees. Timed pregnancies between day 19 and term were obtained by removal of pouch young, which reactivates blastocysts in embryonic diapause (11). The day of removal of pouch young is therefore designated day zero of gestation. Birth occurs, on average, 26.4 days later, with mating about 1 h post partum (1, 2). Pregnancy was confirmed by laparotomy, and pregnant females were killed by an iv anesthetic overdose [sodium pentobarbitone, 60 mg/ml, to effect (Abbot Laboratories, Kurnell, New South Wales, Australia)]. Stages of pregnancy were confirmed with fetal growth curves using crown-rump length, head length, and weight (11). Postpartum tissue (PPd0) was collected 7.0 ± 8.1 (SD) h after birth was observed.

The reproductive tract was removed, and the gravid and nongravid uteri were separated. Uteri were opened with a longitudinal incision, and the ovaries and cervices were removed. The fetus and yolk sac membrane were carefully extracted from the gravid uterus. The opened uteri were spread out flat, and the endometrium was plucked from the underlying myometrium with forceps (36). Longitudinal strips of myometrium, approximately 2.5 mm wide and 15 mm long, were cut from the tissue and placed in ice-cold Krebs-Ringer (118 mM NaCl, 1.18 mM MgSO4, 1.18 mM KH2PO4, 5.5 mM glucose, 25 mM NaHCO3, 2.5 mM CaCl2, 4.7 mM KCl) until needed.

Myometrial contractility
Myometrial strips were tied at each end with silk suture thread. One end was attached to a hook at the bottom of a double-jacketed organ bath. The other end was attached to a force transducer (Model FT03; Grass Instruments Co., Quincy, MA). Organ baths contained 10 ml Krebs-Ringer solution that was continuously gassed with carbogen (95% O2-5% CO2) and maintained at 37 C. Four parallel organ baths allowed simultaneous recordings from two gravid and two nongravid myometrial strips from the same animal. An initial tension of 0.5 g was applied, and the myometrial strips were allowed to equilibrate for 30 min before further manipulation.

Hormones and pharmacological agents were applied to myometrial strips by the manual delivery of 20–50 µl aqueous stock solutions into the top of the organ bath, with the exception of NO gas, which was applied in 250-µl aliquots using a 1-ml gas syringe. Experiments were terminated by flushing the bath contents with 50 ml fresh Krebs/Ringer. The completeness of this flushing method was assessed using methylene blue as a visual indicator before experiments started. Concentrations of each agent are final values in the organ bath, with the exception of NO gas, which is given as moles of gas applied. Strips were stimulated with either 0.14 nM or 1.4 nM MT. In this stimulated condition, the presence of a NO-cGMP relaxation system was determined by the application of either NO gas (10 µmol) or the cGMP analog 8-bromo-cGMP (8-b-cGMP; 10 µM; ICN Pty Ltd., Aurora, OH). One strip from each gravid/nongravid pair always served as a control in these experiments and received no treatment. The dependence of NO responsiveness on cGC was determined by application of the specific cGC blocker 1H- [1,2,4]oxadiazolo-[4,3-{alpha}]quinoxalin-1-one (ODQ, 12.5 µM; ICN Pty Ltd.) (37, 38). The effect of NO was determined 10 min after this treatment.

The analog output of the transducer was amplified (MacLab Bridge Amplifier; Analog Digital Instruments, Sydney, Australia), converted to a digital recording (MacLab Analog to Digital Converter; Analog Digital Instruments) and stored on computer (Apple MacIntosh LC, running Chart Recorder V3.2; Analog Digital Instruments). Area under the curve (A) and contraction frequency were calculated for selected regions of each recording, and pretreatment (t0) and posttreatment (t1) values were directly compared for each strip. Changes in contractility ({Delta}C) were quantified as the difference between pretreatment (At0) and posttreatment (At1) areas divided by the pretreatment area (At0) and expressed as a percentage. For percent stimulation, {Delta}C = [(At1 - At0)/At0]·100. For percent relaxation, {Delta}C = [(At0 - At1)/At0]·100). Zero indicated no change.

Data analysis
Data from nongravid and gravid myometrium are always presented together in the same figure ( Figs. 1–5GoGoGoGoGo). Nongravid data are presented in the top half of the figure, and gravid data in the bottom half. Specific examples of contractile activity (raw traces) occupy the left side of the figure, and data for all animals (histograms pooled for stage of pregnancy) occupy the right side. Comparisons within and between strips and animals were done by factorial repeated-measure ANOVA, and P values of less than 0.05 were considered significant. Quantitative data (contraction frequency or integrated area under the curves) are presented as means ± SEM.



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  		Figure 1. Spontaneous activity of myometrial strips from nongravid and gravid uteri during late pregnancy (days 19–26) and within 24 h post partum. Typical traces from an animal, on day 25 of pregnancy, show regular spontaneous contractions in the nongravid myometrium (A), whereas the gravid myometrium is quiescent (C). Nongravid tissue was spontaneously active at all stages examined, with small progressive decreases in frequency of contraction occurring from day 21 of pregnancy (B). Gravid tissue was quiescent from day 21 to day 26 of pregnancy (D). Contractile activity of nongravid tissue differed significantly from gravid tissue (*, P < 0.001). Activity of gravid tissue changed significantly over the period examined (#, P < 0.05). Error bars (B, D) are SEM, and numbers (n) indicate animals per stage (1–2 strips per uterus per animal).

 


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  		Figure 2. Response of nongravid and gravid myometrial strips to MT (0.14 nM) during late pregnancy (days 19–26) and within 24 h post partum. Typical traces from an animal on day 25 of pregnancy show no response to MT by nongravid myometrium (A), and rapid onset of strong contractions after MT treatment in gravid myometrium (C). Nongravid myometrium remained relatively unresponsive to MT in late pregnancy, with a small increase in responsiveness occurring in postpartum tissue (B). Responsiveness of gravid myometrium to MT increased progressively over the last 6 days of pregnancy and was refractory post partum (D). Responsiveness (percent stimulation above pretreatment values, histograms B and D) was expressed as the increase in area under the trace after MT treatment divided by the pretreatment area (see Materials and Methods). Response to MT changed significantly during the period examined in gravid myometrium (*, P < 0.001) and was significantly different from nongravid tissue (#, P < 0.001). Error bars (B, D) are SEM, and numbers (n) indicate animals per stage (1–2 strips per uterus per animal).

 


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  		Figure 3. Response of nongravid and gravid myometrial strips to NO during late pregnancy (days 19–26) and within 24 h post partum. Tissue was stimulated with MT (1.4 nM) before treatment with NO. Nongravid myometrium was relatively unresponsive to NO, exhibiting a small decrease in amplitude of contractions after treatment (see typical trace from a day-25 animal in A). Contractions of gravid tissue were completely abolished by NO, with activity resuming approximately 1 min after treatment (see corresponding typical trace in C). Responsiveness to NO remained approximately constant in both nongravid (B) and gravid (D) myometrium during the period examined. Responsiveness (percent relaxation below pretreatment values, histograms B and D) was expressed as the decrease in area under the trace after treatment with NO divided by the pretreatment area (see Materials and Methods). Nongravid responses to NO differed significantly from gravid responses (*, P < 0.001). Typical traces from nongravid myometrium (A), showing small relaxations in response to NO, and gravid myometrium (C), showing complete relaxation for 1 min after NO treatment, are taken from the same animal on day 25 of pregnancy. Error bars (B, D) are SEM, and numbers (n) indicate animals per stage (1–2 strips per uterus per animal).

 


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  		Figure 4. Effect of the cGC blocker, ODQ (12.5 nM), on NO-induced relaxation in MT (1.4 nM) stimulated myometrial strips. Typical nongravid myometrium trace from day 23 of pregnancy (A) shows unresponsiveness to NO and ODQ. Typical gravid myometrium trace from day 25 of pregnancy (C) shows blockade of NO-induced relaxation by ODQ. Responsiveness of nongravid myometrium to NO did not differ after treatment with ODQ (B) (#, P >> 0.05). In contrast, relaxation of gravid myometrium, in response to NO, was blocked by ODQ (D). The difference between pretreatment and posttreatment values was significant for gravid tissue (*, P < 0.001). Responses of ODQ-treated gravid myometrium to NO were not statistically different from those of untreated nongravid tissue (P > 0.05). Responsiveness (percent relaxation as in Fig. 3Go) was expressed as the decrease in area under the trace after treatment with NO divided by the pretreatment area (see Materials and Methods). Error bars (B, D) are SEM, and numbers (n) indicate animals per stage (1–2 strips per uterus per animal).

 


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  		Figure 5. Effect of cGMP analog 8-bromo-cGMP (8-b-cGMP, 10 µM) on MT-stimulated (1.4 nM) contractions in nongravid and gravid myometrial strips during late pregnancy (days 19–26) and post partum. Typical traces (nongravid in A, gravid in C) are taken from the same animal on day 25 of pregnancy, showing treated strip (top) and concurrent nontreated control strip (bottom). The cGMP analog induced relaxation in both nongravid (top trace in A) and gravid tissue (top trace in C) over a 20-min period. Activity of the control trips (bottom traces in A and C) remained constant during this period. Treatment induced a similar degree of relaxation in both nongravid (histogram B) and gravid (histogram D) myometrium at all stages examined. Responsiveness (percent relaxation as in Fig. 3Go) was expressed as the decrease in area under the trace after treatment with 8-b-cGMP divided by the pretreatment area (see Materials and Methods). Error bars (B, D) are SEM, and numbers (n) indicate animals per stage (1–2 strips per uterus per animal).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Myometrial contractility was examined from days 19–26 of pregnancy and within 24 h post partum (7.0 ± 8.1 h, mean ± SD, n = 4). Nongravid tissue was spontaneously active at all stages examined (Fig. 1Go, A and B). The frequency of contractions decreased progressively over the last days of pregnancy and post partum (Fig. 1BGo). In contrast, late pregnant (days 21–26) gravid myometrium was quiescent and did not spontaneously contract. Spontaneous contractions were observed earlier in pregnancy, on days 19 and 20, and also on the day of parturition (Fig. 1DGo). Frequency of spontaneous contractions in nongravid tissue differed significantly from gravid tissue (P < 0.001).

Mesotocin (0.14 nM) initiated contractions in quiescent gravid myometrium, with significant increases in responsiveness occurring over the last 4 days of gestation (ANOVA, P < 0.001, see Fig. 2DGo). On days 25 and 26, the last days of gestation, MT sensitivity reached a maximum and induced contractions of large amplitude. A typical recording from day 25 gravid myometrium is shown in Fig. 2CGo. Sensitivity decreased dramatically in postpartum tissue (Fig. 2DGo). At higher concentrations (1.4 nM), MT induced larger responses in gravid myometrium (data not shown) and obscured stage-specific changes in sensitivity that were present at lower doses (responses did not vary significantly, P > 0.05). Nongravid myometrium was relatively insensitive to low concentrations of MT (Fig. 1BGo), with a small increase in responses occurring on the day of parturition. Higher concentrations of MT (1.4 nM) produced a more substantial increase in nongravid myometrial contractility (data not shown). At both concentrations, the response of nongravid strips differed significantly from that of gravid tissue (P < 0.001).

NO completely inhibited MT-stimulated (1.4 nM MT) contractions in gravid myometrium, with activity resuming approximately 60 sec after treatment (Fig. 3CGo). In contrast, nongravid tissue was relatively insensitive to NO (Fig. 3AGo), responding with a small decrease in the frequency and amplitude of contractions. The sensitivity of gravid myometrium to NO remained approximately constant throughout late pregnancy and on the day of parturition (Fig. 3DGo) and did not vary significantly at any stage of pregnancy (P >> 0.05). Similarly, nongravid responsiveness (Fig. 3BGo) did not change significantly during the period examined (P >> 0.05). The marked difference in NO-induced relaxation between gravid and nongravid myometrium was significant (P < 0.001).

The inhibitor of cyclic guanylyl cyclase (ODQ) blocked the effect of NO on gravid tissue (Fig. 4Go), as evidenced by a significant reduction in the relaxation induced by NO after treatment (P < 0.001). In contrast, the minimal NO responsiveness of nongravid tissue was not altered by ODQ (P >> 0.05). Furthermore, the NO responsiveness of gravid myometrium after ODQ treatment was reduced to levels that were statistically indistinguishable from those of either treated or untreated nongravid tissue (P > 0.05).

The cGMP analog, 8-b-cGMP, reduced MT-stimulated contractility in both gravid and nongravid myometrium (Fig. 5Go, A and C, top recordings). The effect had a long latency, producing marked relaxation over a 20-min period. Activity in parallel gravid and nongravid control strips remained constant over the same time period (Fig. 5Go, A and C, bottom recordings). Responsiveness to 8-b-cGMP varied significantly with stage of pregnancy in both gravid (ANOVA, P < 0.05) and nongravid (ANOVA, P < 0.05) myometrium. Gravid tissue was most sensitive on days 23 and 24 pregnancy, with reduced responses on days 25 and 26 and post partum (Fig. 5DGo). Nongravid myometrial sensitivity was lowest on days 25 ands 26 pregnancy but rose to a maximum post partum (Fig. 5BGo). The relaxation seen overall in gravid uteri was slightly, but significantly, higher than in nongravid uteri (P < 0.05) and probably reflects the greater MT-induced activation of the gravid myometrium.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The contractility of the myometrium from the gravid uterus during late pregnancy in the tammar is markedly different from that of the contralateral, nongravid uterus. Spontaneous activity, which is present at all stages examined in nongravid tissue, is absent in the gravid myometrium from day 21 of pregnancy until parturition on day 26.5. In contrast, the sensitivity to MT, which remains low in nongravid tissue throughout late pregnancy, increases in the gravid myometrium on days 23 and 24, to reach a maximum on the last days of pregnancy (days 25 and 26). Within 24 h of parturition, gravid tissue is refractory to MT stimulation, whereas a small increase in sensitivity is observed in nongravid tissue. Similarly, NO has very little effect on nongravid myometrium, but it completely inhibits MT-stimulated contractions of gravid tissue. These results support previous studies, which show that local influences regulate differential effects between the gravid and nongravid uterus of the pregnant tammar (reviewed in 11, 15).

In eutherian mammals, changes in myometrial contractility during pregnancy and at the time of parturition are effected by the regulation of multiple pathways in uterine smooth muscle. The electrophysiology of muscle cells is dramatically altered during pregnancy, to limit excitability (16, 18); and elements of the signal transduction pathways for stimulatory factors are down-regulated, whereas those for inhibitory pathways are emphasized (19). These changes are reversed at parturition, so that stimulatory influences dominate, myometrial contractions ensue, and fetal expulsion occurs (18). Oxytocin receptors increase dramatically at the end of pregnancy, preparing this important stimulatory pathway for the initiation of parturition (human, 39 ; sheep, 40 ; rat, 41). In the tammar, MT receptors are also up-regulated in gravid myometrium at the end of pregnancy (10, 14), suggesting that similar mechanisms may be operating in this species.

Mesotocin receptors in gravid myometrium increase progressively from day 23 of pregnancy to term, whereas they remain low in nongravid tissue (10, 14). These receptor profiles are reflected in the pattern of MT sensitivity reported in the current study. During this period of increasing responsiveness of gravid myometrium, however, spontaneous activity remains low (7, 8), suggesting that inhibitory influences must be operating in vivo. In eutherian species, the smooth muscle relaxant NO provides a paracrine inhibition of uterine contractions during pregnancy (21). The complete inhibition of contractions of the gravid myometrium during late pregnancy, reported in this study, suggests a similar role in the tammar.

In eutherians, NO induces smooth muscle relaxation by stimulating the activity of cGC. This increases intracellular levels of cGMP and leads to subsequent relaxation of the muscle (33). In tammars, relaxation of gravid myometrium was induced by a cGMP analog, and blocking cGC with ODQ blocked NO-induced relaxation. These manipulations clearly indicate the presence of a classic NO/cGMP relaxation system in the tammar gravid myometrium. In contrast, although the nongravid myometrium relaxed in response to cGMP, it was relatively insensitive to NO during the period examined. Thus, NO sensitivity specific to gravid tissue seems to be regulated at the level of cGC, which is active in gravid myometrium and inactive or absent in nongravid tissue. Similarly, in eutherian mammals, pregnancy is associated with the induction of NO sensitivity in gravid myometrium (human, 25 ; rat, 24), resulting in increased levels of cGMP in the tissue (25).

Just before parturition in humans (25) and rats (24), sensitivity of the myometrium to NO and synthesis of NO by uterine tissues decreases (28, 32). Decreases in both NO sensitivity and NO production contribute to the increased myometrial contractility at parturition. In the tammar, sensitivity to relaxation induced by cGMP was highest in gravid myometrium on days 23 and 24, declining on the last days of pregnancy (days 25 and 26) and post partum. This pattern was reflected less markedly by gravid myometrial sensitivity to NO, suggesting down-regulation of the relaxation response system at parturition. However, the observed changes were small, with decreases of 20% and 10% for cGMP and NO, respectively. In contrast, human myometrial sensitivity to NO decreases by 85% at term (25). Withdrawal of myometrial quiescence at parturition in the tammar may thus depend on reductions in NO production. The construction of NO and cGMP dose-response curves for gravid myometrium (see 24) and an analysis of NO production by the uterus during pregnancy would be required to clarify this issue.

In physiological systems, NO rapidly reacts with water to form nitrites and nitrates and so, must be produced within 100–150 µm of target cells to be effective (42). The inducible, CA2+-insensitive form of NOS (iNOS) has been implicated in the regulation of myometrium contractility in eutherian mammals. In humans (32) and rats (28), iNOS levels are high in myometrial smooth muscle cells during pregnancy, and the levels decline at parturition. In the rabbit, iNOS in the decidua of the endometrium may represent the source of NO for myometrial relaxation (30). In this study, we showed that NO is a potent inhibitor of contractility in the gravid uterus, but the source of NO was not identified. However, the specific induction of NO sensitivity in gravid tissue only, potentially via up-regulation of cGC, suggests a physiological role during pregnancy. The production of NO within the uterine environment of the tammar is thus an area for further study. In addition, the NO sensitivity of myometrium from both uteri during the nonpregnant cycle should be examined to determine the specific role of the fetus and its placenta vs. systemic and/or pregnancy-specific ovarian factors (9, 14, 43).

In late pregnancy in the tammar, the nongravid uterus is ipsilateral to a maturing Graafian follicle and is exposed via the unilateral uteroovarian vasculature to locally elevated levels of estradiol (12, 44) and other factors involved in ovulation and the postpartum estrus (discussed in 42). The gravid uterus, in contrast, is ipsilateral to an active CL and is exposed to locally elevated levels of progesterone and relaxin. It is also exposed to hormones and other factors derived from the fetus and placenta, such as cortisol (45) and PGs (46, 47). These unilateral endocrine and paracrine influences must be responsible for the differences between nongravid and gravid tissue reported here. Estradiol, which regulates myometrial contractility in the tammar (7, 8), is most likely responsible for spontaneous activity in the nongravid uterus during late pregnancy. However, it does not regulate contractility of the gravid uterus at the time of parturition (48). Relaxin is present in high levels in the tammar CL in late pregnancy (49), and porcine relaxin suppresses uterine contractions in vitro (8). Relaxin may be involved in maintaining quiescence of the gravid myometrium in vivo. Sensitivity to MT and NO in gravid tissue, and its regulation during late pregnancy and at the time of parturition, may rely on fetal and placental factors, rather than ovarian steroids, because progesterone withdrawal is not necessary for successful birth (50).

The two separate uteri of the tammar are clearly under differential control during late pregnancy. Results of this study, reporting differences in spontaneous contractility and sensitivity to MT and NO between gravid and nongravid myometrium, provide new evidence for this phenomenon. Such differential regulation must rely on local influences rather than systemic ones (which would affect both uteri) and allows the responses of the two separate uteri of these unique mammals to be matched to the unilateral requirements of their concurrent reproductive programs: pregnancy and parturition in the gravid uterus and ovulation and fertilization in the contralateral, nongravid uterus.


   Acknowledgments
 
We thank Dr. Bryan Dumsday for helpful discussions regarding NO pharmacology, loans of equipment, and gifts of reagents; and also Dr. Laura Parry and Anne Duns for help with animals. Permit number RP95–088 to obtain and hold tammars was provided by the Victorian Department of Natural Resources and Environment and the South Australian Department for Environment, Heritage and Aboriginal Affairs.


   Footnotes
 
1 This work was supported by Grant A096502716 (to G.S. and M.B.R.) from the Australian Research Council. Back

2 Present address: Sobell Department of Neurophysiology, Institute of Neurology, Queen Square, London, WC1N 3BG, United Kingdom. Back

Received July 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Renfree MB, Shaw G 1996 Reproduction of a marsupial: from uterus to pouch. Anim Reprod Sci 42:393–403[CrossRef]
  2. Renfree MB, Fletcher TP, Blanden DR, Lewis PR, Shaw G, Gordon K, Short RV, Parer-Cook E, Parer D 1989 Physiological and behavioural events around the time of birth in macropodid marsupials. In: Jarman P, Hume ID, Grigg G (eds) Kangaroos, Wallabies and Rat Kangaroos. Surrey Beatty & Sons Pty Ltd, Sydney, pp 323–337
  3. Lewis PR, Fletcher TP, Renfree MB 1986 Prostaglandin in the peripheral plasma of tammar wallabies during parturition. J Endocrinol 111:103–109[Abstract/Free Full Text]
  4. Parry LJ, Guymer FJ, Fletcher TP, Renfree MB 1997 Release of an oxytocic peptide in parturition in the marsupial Macropus eugenii. J Reprod Fertil 107:191–198
  5. Renfree MB, Shaw G, Fletcher TP 1994 Evidence for the essential role of prostaglandins for parturition in a marsupial Macropus eugenii. J Reprod Fertil 102:433–446[Abstract/Free Full Text]
  6. Renfree MB, Parry LJ, Shaw G 1996 Infusion with an oxytocin receptor antagonist delays parturition in a marsupial. J Reprod Fertil 108:131–137[Abstract/Free Full Text]
  7. Young IR 1977 Relationship of hormonal and reproductive status to myometrial activity in the tammar wallaby. Theriogenology 8:209[CrossRef]
  8. Young IR 1978 The physiology of parturition in the tammar wallaby, Macropus eugenii. Ph.D. thesis, Murdoch University, Perth, Western Australia
  9. Shaw G 1983 Effect of PGF2{alpha} on uterine activity, and concentrations of 13,14-dihydro-15-keto-PGF2{alpha} in peripheral plasma during parturition in the tammar wallaby. J Reprod Fertil 69:429–436[Abstract/Free Full Text]
  10. Parry LJ, Bathgate RAD, Shaw G, Renfree MB, Ivell R 1997 Evidence for a local fetal influence on myometrial oxytocin receptors during pregnancy in the tammar wallaby Macropus eugenii. Biol Reprod 56:200–207[Abstract]
  11. Tyndale-Biscoe CH, Renfree MB 1987 Reproductive Physiology of Marsupials. Cambridge University Press, Cambridge
  12. Towers PA, Shaw G, Renfree MB 1986 Urogenital vasculature and local steroid concentrations in the uterine branch of the ovarian vein of the female tammar wallaby Macropus eugenii. J Reprod Fertil 78:37–47[Abstract/Free Full Text]
  13. Renfree MB, Tyndale-Biscoe CH 1973 Intrauterine development after diapause in the marsupial. Dev Biol 32:28–40[CrossRef][Medline]
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