Mark Baguma-Nibasheka, Richard A. Wentworth, Lucy R. Green, Susan L. Jenkins, .... loop of cotton thread at each end, were individually sus- pended at a ...
BIOLOGY OF REPRODUCTION 58, 73-78 (1998)
Differences in the In Vitro Sensitivity of Ovine Myometrium and Mesometrium to Oxytocin and Prostaglandins E2 and F, Mark Baguma-Nibasheka, Richard A. Wentworth, Lucy R. Green, Susan L. Jenkins, and Peter W. Nathanielsz 2 Laboratory for Pregnancy and Newborn Research, Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401 tion to estrogen, whereas the mesometrial EMG response was restricted to contracture-type activity and did not exhibit tachyphylaxis [8]. These two reports [1, 8] both suggested that paracrine factors from the endometrium may be involved in the regulation of myometrial contractility. The fact that chemical sympathectomy increases contracture activity in the mesometrium but not in the myometrium [9] was taken as a further indication that the contractility of the two tissues is regulated differently. During pregnancy, both the myometrium and the mesometrium predominantly display contracture-type activity, but at labor, the myometrium switches to short, efficient contractions, whereas the mesometrium tends to remain in the contracture mode [10]. This difference in contractility pattern may be due to differences in the population of receptors for various endocrine and paracrine agents involved in the parturition process. In support of this view, recent reports show that there is an increase in the expression of mRNA for both estrogen and oxytocin receptors at labor in the ovine myometrium but not in the mesometrium [11, 12]. In the present study, a superfusion technique was used to evaluate the in vitro sensitivity of myometrium and mesometrium from nonpregnant, pregnant, and parturient sheep to oxytocin, PGE 2, and PGF 2 in order to determine whether gestational status-related changes in uterine contractile activity can be correlated with uterine responsiveness to the major uterotonic agents in this species. The use of an in vitro technique to study uterine contractility permits the evaluation of specific contractile responses in tissues obtained from animals of precisely known in vivo functional status in the absence of potentially confounding interactions that might occur in vivo.
ABSTRACT We compared the in vitro response to oxytocin, prostaglandin (PG)E 2, and PGF2,, of myometrium and mesometrium from six ovariectomized ewes and 53 ewes at 106-145 days gestational age (dGA), including 14 ewes in spontaneous or betamethasone-induced labor. Myometrial baseline activity increased from 217 + 27 mN/cm 2 of cross-sectional area (mean - SEM) in ovariectomized ewes to a plateau of 696 + 39 mN/cm 2 at 126-135 dGA. No gestation-related changes were observed in mesometrial baseline activity. Myometrial, but not mesometrial, maximum tension in response to agonists increased with gestation to a plateau at 126-135 dGA. The pD2 (negative logarithm of the ECso) values for oxytocin were similar in both tissues and did not change with gestation. During pregnancy, the myometrial pD2 of both PGs was one order of magnitude higher than the mesometrial pD2. The results indicate an increase in myometrial uterotonic receptor-mediated activity that precedes labor with no increase at labor, suggesting that in sheep, activation of the basic mechanisms responsible for strength of myometrial activity at labor occurs by 135 dGA. The greater sensitivity of the myometrium than the mesometrium to PGs supports a major role for intrauterine paracrine factors in regulating myometrial contractility. INTRODUCTION Significant differences are present in the histology, contractile activity, and receptor population of the ovine myometrium and mesometrium. These two tissues are embryologically related. The mesometrium, which is the muscular portion of the broad ligament attached to the uterus, contains smooth muscle fibers running only in the longitudinal direction and continuous with the same muscle layer in the body of the uterus [1]. The myometrium has longitudinal as well as circular muscle layers, and the two layers are known to differ in both the distribution of oxytocin receptors [2, 3] and in the response to catecholamines, oxytocin, prostaglandin (PG)F 2,, and PG synthesis inhibitors [4-7]. Most importantly, in contrast to the myometrium, the mesometrium has no endometrium juxtaposed to it. In addition, the in vivo contractile activity patterns of the mesometrium and myometrium in both ovariectomized (OVX) nonpregnant [1, 8, 9] and pregnant sheep [10] have been shown to be dissimilar. In nonpregnant sheep, contracture activity (electromyographic events lasting more than 180 sec) was greater in the mesometrium than in the myometrium [1]. With estrogen supplementation, the myometrial electromyogram (EMG) response was characterized by an increase in both noncontracture-type (less than 180 sec) and contracture-type events, followed by desensitiza-
MATERIALS AND METHODS Animal Groups Tissues were collected from six OVX Rambouillet-Columbia sheep and from 53 ewes mated on a single occasion and of known gestational age from 106 days gestational age (dGA) to term (four sheep at 106-115 dGA, 32 at 116125 dGA, ten at 126-135 dGA, six at 136-145 dGA, seven in spontaneous term labor at 148 2 dGA, and seven in glucocorticoid-induced preterm labor at 127-129 dGA). In 40 pregnant sheep, tissue was taken either at surgery for instrumentation or at necropsy. Tissue from 13 sheep was obtained both at surgery and at necropsy. Experimental Procedures Nonpregnant ewes were synchronized in their cycles by the insertion of vaginal progesterone sponges (Carter Holt Harvey Plastic Products, Hamilton, New Zealand; containing 0.3 g progesterone) 14 days before surgery and removal of the sponges 2 days before bilateral oophorectomy was
Accepted August 20, 1997. Received May 27, 1997. 'Supported by NIH HD 21350. 2Correspondence. FAX: (607) 253-3455; e-mail: pwnl @cornell.edu
73
74
BAGUMA-NIBASHEKA ET AL.
performed. Tissues from these OVX sheep were collected at necropsy 2 wk after the operation. Tissues from pregnant ewes were obtained during surgery at 116-117 dGA or at necropsy. Surgery involved the placement of polyvinyl catheters into the fetal jugular vein and carotid arteries, and the implantation of stainless steel electrodes in the myometrium for recording electromyogram (EMG) activity, as described in detail in [13]. The induction of premature labor using glucocorticoids has been fully described in [14]. Briefly, beginning at noon on 125 dGA, betamethasone (Celestone phosphate; Schering, Berlin, Germany) was administered into the fetal jugular vein at a rate of 10 tpg/h over 48 h. Labor started within 24 h after the end of this infusion period. Labor, whether spontaneous or induced, was defined as having occurred when the EMG record showed a clear switch from myometrial contractures to labor-type contractions followed by contraction activity for at least 5 h. All surgeries and necropsies were conducted under halothane general anesthesia. Studies were approved by the Cornell University Institutional Animal Care and Use Committee. All facilities were approved by the American Association for the Accreditation of Laboratory Animal Care. Tissue Preparation The myometrial tissue was obtained from pieces of fullthickness uterine wall from the greater curvature of the uterine horn (the pregnant horn in pregnant ewes). Mesometrium was taken from the same side of the uterus at a point 2-3 cm from the junction of the broad ligament and the uterine horn. Black silk ties were used to mark the direction of the longitudinal muscle. The tissues were immediately placed in 4C modified Krebs buffer solution (composition: NaCl, 118.0 mM; NaHCO 3, 25.0 mM; glucose, 11.0 mM; KC1, 5.0 mM; CaCI 2, 1.3 mM; MgSO 4, 1.25 mM; KH 2PO 4, 1.0 mM; and containing 0.003 mM of the cyclo-oxygenase blocker indomethacin to prevent postcollection endogenous synthesis of prostaglandin that could alter the contractility of the tissue). The myometrium and mesometrium were dissected free of endometrium and blood vessels, respectively. Longitudinal strips (surface area 0.4 by 0.6 cm), sutured with a loop of cotton thread at each end, were individually suspended at a resting tension of 30 mN from force transducers (UF1; Pioden Controls, Canterbury, UK), in the superfusion system (Glaxo, Ware, Harts, UK). The procedures used to measure contractile response have been described previously in detail [15] and are reviewed here briefly. Superfusion In each study, two individual-cartridge peristaltic pumps (CR 07618-60; Ismatec, Cole-Parmer, Chicago, IL), fitted with 2.5-mm-caliber vinyl tubes, were used to perifuse eight strips of myometrium and eight of mesometrium with 37°C oxygenated (95% 02:5% C0 2) Krebs buffer of the described composition, according to the Coleman and Nials [16] technique. The flow rate of the nutrient buffer was 2.0 ml/min, and drugs were instilled into the buffer using the same pumps, which were also equipped with 0.25-mm-caliber tubing to give a flow rate one hundredth that of the buffer. The tissues were allowed an equilibration period of 60 min to develop spontaneous contractile activity before any drug was administered.
Superfusion Drugs Oxytocin as a 42.0 RpM saline solution (Butler, Columbus, OH) and PGF 2. as the 11.0 mM Lutalyse tromethamine salt (Upjohn, Kalamazoo, MI) were stored at 4C. Crystalline PGE 2, a gift from the Upjohn Company, was used to prepare a 1.0 mM stock solution of PGE 2 in absolute ethanol, which was then stored at -20°C. For each experiment, fresh serial dilution solutions of all drugs were made in PBS (NaCI, 120.0 mM; KC1, 2.7 mM; KH2 PO4 Na 2HPO 4, 10.0 mM; Sigma Chemical Company, St. Louis, MO) and were kept on ice until use on the same day. Indomethacin (Sigma) was dissolved (5 mg/ml) in absolute ethanol before addition to the Krebs buffer. Contractility Measurement The tension changes were recorded by a Data Acquisition System connected to an IBM PC computer as previously described [15]. The average tension measured during the last 10 min of the equilibration period was taken as baseline activity, following which the drugs were delivered at five cumulative molar concentrations-10--l 10-7 (oxytocin), 10-9-10 - 5 (PGE 2), and 10-8-10 - 4 (PGF2,)-covering the range from that producing no effect to that causing maximal response as determined previously [15], each dose administered for 12 min. Tension was recorded every second and integrated as mean tensile force (mN/cm 2 of crosssectional area) achieved by each strip during the last 10 min of every dosage period. (Two strips of each tissue were used as time-effect controls, perifused with only PBS, the diluent of the test drugs. Since the activity in those strips remained at baseline levels throughout the course of the experiments, those data are not included in the results.) At the end of each experiment, the lengths (at 1 g of tension) and weights of the tissue strips were measured to obtain the cross-sectional areas using the formula A = W / L x D, where A = the cross-sectional area (cm 2); W = weight (g); L = length (cm); and D = density, taken as 1.03 g/cm 3 for both tissues. Data Analysis Baseline activity, representing the contractility of the tissue before drug administration, was taken as the average tension recorded in six strips of either tissue from all the animals in each group. Maximum tension was measured as the highest contractile activity observed in response to each drug in individual animals and averaged within the groups. The characteristics (amplitude, frequency, and duration) of myometrial contractile epochs were measured during the last 10 min of the equilibration period and of the highest drug dose (10 7 M oxytocin, 10 5 M PGE 2, or 10 - 4 M PGF 2.) infusion period in two strips per drug per animal and five animals per group (four at 106-115 dGA). The increase in these characteristics from the equilibration to the high-dose infusion period was then recorded as the percentage change from baseline. (Changes in response to PGE 2 and PGF 2. were similar to those in response to oxytocin, and are therefore not shown in the results.) Individual dose-response curves (% maximum response vs. logo drug concentration) were also plotted for each animal, using a nonlinear curve-fitting program (GraphPad; ISI, Philadelphia, PA) by fitting a third-order polynomial to the data points according to the logistic equation E = Emax X [drug]s / EC5 + drugs], in which E = the effect of a given drug concentration; Emax = the maximal achieved
75
OVINE MYOMETRIAL AND MESOMETRIAL CONTRACTILITY
A
) 1000
I
MYO -
___L
__L·___
· · L····L·-
< ' 600 a
MYO MESO
A k
L
k
-10
I
X
&
a
L
4
OVX A
MESO
z
E
o
MYO,
Alfkm.I
I
X
I--
10 min -
*
a,b,c
a,b,c
a,b,c
d d
z
B
MESON
*
E 200
min_--,I
n= 6
MYO
a,b -
.
400
) A
k
MO* a,b,c
MESO
o. 800
·L. - -1...... . . ........... bbI····
MESO
MYO _
=I
C
4 1
30 3
10 8
6 6
7 5
7 2
106-115d
116-125d
126-135d
136-145d
SPON
BETA
FIG. 2. Baseline activity (mean SEM) in myometrial (MYO) and mesometrial (MESO) strips removed from OVX ewes and ewes at different gestational ages or in spontaneous (SPON) or betamethasone-induced (BETA) labor. The values were measured from six strips of either tissue per animal. Baseline myometrial activity increased with gestational age whereas baseline mesometrial activity remained constant. Superscripts indicate baseline myometrial activity: *, significantly different from mesometrial activity of the same age group; a, different from myometrial tension in OVX group; b, different from 106-115 dGA myometrial tension; c, different from 116-125 dGA myometrial tension; and d, mesometrial tension different from OVX mesometrial tension (p < 0.05).
1
MYO ·
a
MESO
A
k Ak I
MYO -
MESO I
-
10 min -
m
-
FIG. 1. A) Representative polygraph traces from myometrial (MYO) and mesometrial (MESO) strips from one OVX sheep during the equilibration period. Point X marks 30 min after suspension in superfusion system. All tissue strips are beginning to exhibit regular discrete contractions. B)Traces from strips of tissue from one ewe at 140 dGA. Point X marks 45 min after suspension in the superfusion system. Only the myometrial strips show discrete contractions. C) Traces from the same strips in B, after the addition of 10 7 M oxytocin.
effect (top of the curve); EC50 = the drug concentration achieving 50% of the maximum effect; and exponent s = the slope factor. From these curves, drug potencies were calculated and expressed as the pD 2, which is the negative logarithm of the EC50 . Statistical Analysis A two-sample t-test on paired data was used to compare responses of myometrium and mesometrium within groups, and a one-way analysis of variance with the Student-Newman-Keuls procedure was used for comparison of the response of either tissue between groups. Differences of p < 0.05 were considered significant. Data are presented as mean + SEM. Statistical evaluation of data from the mesometrium of sheep at 106-115 dGA was not possible because mesometrium was obtained from only one animal in the group. RESULTS OVX Ewes Baseline activity The tissue strips from OVX ewes exhibited regular spontaneous contractile episodes after 26.8 + 6 min of being suspended in the superfusion chambers (Fig. 1A). The baseline activity averaged 217 ±+ 27.4
mN/cm 2 of cross-sectional area (n = 6) and was similar in both tissues (Fig. 2). Effect of agonists on contractility. Both myometrial and mesometrial strips from OVX ewes responded to oxytocin, PGE 2, and PGF 2. in a dose-dependent manner with increases in the frequency, amplitude, and duration of individual contractions. The myometrium showed a greater response than the mesometrium to PGF 2 , p < 0.05 (Fig. 3). Tissue sensitivity to agonists. The pD 2 values for each agonist were similar on myometrium and mesometrium, and were therefore pooled (oxytocin: 8.8 0.1 M; PGE 2: 7.25 ± 0.14 M; PGF2,: 6.55 + 0.15 M; Fig. 4). Pregnant and Parturient Ewes Baseline activity. During the equilibration period, myometrial strips displayed regular spontaneous discrete contractions at intervals of 137 + 9.9 sec. Each contractile episode had an average amplitude of 42 7.7 mN and lasted 44 ± 5.9 sec. This activity was established 49 + 4.2 min after suspension of the strips in the perifusion system, which was significantly later than in nonpregnant OVX ewes (p < 0.05). These characteristics of individual baseline contractile epochs, measured in two strips per animal and five animals per group, exhibited no significant correlation to the gestational status. The mesometrial strips did not manifest similar contractile activity before agonist stimulation (Fig. lB). The baseline activity in myometrial strips showed a gradual increase with gestational age, from 364 40 mN/cm 2 of cross-sectional area at 106-115 dGA to a plateau of 696 + 39 mN/cm2 at 126-135 dGA, whereas the mesometrial baseline activity averaged 430 + 34 mN/cm 2 and was not correlated with gestational status (Fig. 2). In this respect, the myometrial baseline activity was significantly higher than the mesometrial basal activity after 125 dGA and during labor. Effect of agonists on contractility. Both myometrial and mesometrial strips responded to oxytocin, PGE2 , and PGF2 . in a dose-dependent manner, but whereas the mesometrial strips showed a progressive increase in tension with occasional contractions only at the highest drug concentrations
76
BAGUMA-NIBASHEKA ET AL. 2500-
Oxytocina,
a,c
a,c
2000 1500
8
1000
7
500
6
M 2500-
0
E
5
5
o W
10-
PGE 2
2000
0D
Oxytocin
9
a,c
0
C t
10
+
ac
ac
ac
ac
1500-
o 1000 ,
PGF,
500 0
Co
Z E
2500
200
PGF2 a,b,c
2000 -
a,b,c
1500
abc
*
9
ac
1000 67
500
5
0 n= 5 4
4 1
29 3
10 8
5 5
7 5
72
OVX
106-115d
116-125d
126-135d
136-145d
SPON
BETA
FIG. 3. Maximum tension (mean ± SEM) in myometrial (solid bars) and mesometrial (open bars) strips from OVX ewes and ewes at different ages of gestation or in spontaneous (SPON) or betamethasone-induced (BETA) labor, in response to oxytocin, PGE 2, or PGF2,,. The values were measured from two strips of either tissue per drug per animal. In all cases, myometrial maximum tension increased at 126-135 dGA and subsequently remained constant. Superscripts indicate myometrial maximum tension: *, significantly different from mesometrial tension of the same age group; #, different from same group myometrial tension in response to PGE2 or + PGF 2 ,; a, different from OVX myometrial tension; b, different from 106115 dGA myometrial tension; c, different from 116-125 dGA myometrial tension (p < 0.05).
(Fig. 1C), the myometrial strips from pregnant ewes exhibited increases in the frequency, amplitude, and duration of individual contractions (Figs. 1C and 5). Myometrial strips from pregnant ewes not in labor responded to all agonists with greater increases in the duration than in the frequency of contractions, and those from ewes in labor responded with greater increases in frequency than in duration (Figs. 5 and 6). In general, the highest contractility in both tissues and at all stages of gestation was obtained in response to oxytocin, and the lowest to PGF2 , although this was statistically significant only at 116-125 dGA and during induced labor (Fig. 3). After 125 dGA, the maximum response of the myometrium to the agonists was at least twice as great as that of the mesometrium. Whereas the maximum response of the mesometrium to all agonists did not show any gestation- or labor-related changes, that of the myometrium showed a significant increase at 126 dGA in all cases (from 966 + 72 mN/cm 2 of cross-sectional area at 116125 dGA to 2004 157 mN/cm 2 at 126-135 dGA for oxytocin) (Fig. 3). Tissue sensitivity to agonists. The pD 2 values of both tissues did not change with gestation and were similar for oxytocin: 8.6 0.1 M in myometrium and mesometrium
n=5 4 OVX
4 1
27 3
10 8
5 5
7 5
7 2
106-115d
116-125d
126-135d
136-145d
SPON
BETA
FIG. 4. pD 2 values (negative logarithm of concentrations giving 50% of maximal stimulation, mean + SEM) for oxytocin, PGE 2, or PGF2,, on myometrial (solid bars) and mesometrial (open bars) strips from OVX ewes and ewes at different ages of gestation or in spontaneous (SPON) or betamethasone-induced (BETA) labor. The values were obtained as described in Figure 4. The pD 2 s of both tissues did not change with gestation and were similar for oxytocin in myometrium and mesometrium. The myometrial pD2 of both PGs was higher than the mesometrial at all stages for which statistical comparison was possible. *, Myometrial pD2 significantly different from mesometrial pD 2 of the same age group; a, mesometrial pD2 significantly different from OVX mesometrial pD 2 ; b, mesometrial pD 2 significantly different from 116-125 dGA mesometrial pD 2 (p < 0.05).
(Fig. 4). The pD 2 of both PGs was approximately one order of magnitude higher on the myometrium than on the mesometrium at all stages for which statistical comparison was 0.1 M possible (PGE 2: 7.6 + 0.1 M myometrium, 6.9 mesometrium; PGF 2,: 6.9 0.05 M myometrium, 5.9 + 0.04 M mesometrium). Ewes in Betamethasone-lnduced Labor All the measured parameters in both myometrium and mesometrium from ewes in betamethasone-induced premature labor, which was at 127-129 dGA, were similar to those of the corresponding tissues from ewes in spontaneous labor (Figs. 2-5). DISCUSSION Our results reveal several differences in the contractility of uterine tissues obtained from pregnant and nonpregnant ewes. Whereas both the myometrium and mesometrium from OVX sheep exhibited regular spontaneous contractile episodes within 26.8 6 min of suspension in the muscle bath, the myometrium of pregnant ewes took significantly (at least 10 min) longer to show similar activity, and the
77
OVINE MYOMETRIAL AND MESOMETRIAL CONTRACTILITY
A 156 L iA.L Al
179
__
_
A
_
A
L A
_
·
k
B E
FIG. 5. Percentage increases from baseline activity (mean +_SEM) in the amplitude, frequency, and duration of individual contractions in myometrial strips from OVX ewes and ewes at different ages of gestation or in spontaneous (SPON) or betamethasone-induced (BETA) labor, in response to oxytocin. The values were measured during the last 10 min of the equilibration period and of the 10 -7 M oxytocin infusion period in two strips per animal and five animals per group (four at 106-115 dGA). Strips from ewes in labor showed a greater increase in the frequency and a smaller increase in the duration of contractions. *, Changes in frequency and duration significantly different from changes in other groups (p < 0.05).
mesometrium only demonstrated discrete contractions after drug stimulation. Baseline activity and maximum tension in response to oxytocin and PGE 2 were seen to be similar in myometrium and mesometrium from OVX sheep but were higher in myometrium than in mesometrium from pregnant ewes and increased with gestational age in the myometrium but not the mesometrium. This finding, when coupled with the observation that the myometrial pD 2 values of both PGs were higher than the mesometrial only during pregnancy, indicates that both inherent contractility and sensitivity to agonists increase in the myometrium but not in the mesometrium during pregnancy. Significantly, in vivo studies reveal that the mesometrium, unlike the myometrium, shows little tendency to switch from contractures to contractions at labor [10]. Recently, Zhang and colleagues [17] conducted a study of gene expression for cytosolic phospholipase A2 and prostaglandin endoperoxide synthase, two enzymes essential for PG biosynthesis, in ovine uterine tissues, in which they showed differences in mRNA and protein levels, indicating that during late pregnancy, the endometrium may be a more important source of PGs than the myometrium. Because of the very short half-life of PGs, their ability to act on distant sites is quite limited. It is therefore possible that such paracrine factors produced by the intrauterine tissues during pregnancy and acting directly on the myometrium may fail to reach the mesometrium in sufficient bioactive quantities to effect similar modulation. Another difference noted between pregnant and nonpregnant uteri was that whereas during pregnancy the myometrial response to oxytocin and both PGs was always greater than the mesometrial response, the contractility of the myometrium from OVX sheep was greater than that of the mesometrium only in response to PGF 2,. The implication here is that during pregnancy, the myometrium possesses more receptors than the mesometrium for all three agonists tested, but the major receptor is PGF 2, during the nonpregnant state. This might indicate a greater role for PGF 2, in regulating the contractility of the nonpregnant myometrium than for the other agonists, in contrast tthe situation during pregnancy, in which oxytocin and PGE2
156
L
179
1
L
C 156
l
179
I -
10 min --
I
F!G. 6. Polygraph traces showing A) spontaneous contractile epochs and contractions in response to B) 10- M oxytocin and C) 10-7 M oxytocin in two myometrial strips from pregnant sheep (ewe #156 at 11 7 dGA and not in labor; ewe #179 in spontaneous labor at 147 dGA). The strip from the ewe not in labor responded to oxytocin with increased amplitude and duration of contractions whereas that from the ewe in labor shows an increase in amplitude and frequency.
appear to be relatively stronger agonists. Further studies are required to evaluate the profile of PG receptors during the period of gestation and labor in both the myometrium and mesometrium. The myometrial response to agonists during pregnancy displayed some distinctive characteristics. Although all myometrial strips responded with increases in the amplitude, frequency, and duration of individual contractions, strips from ewes not in labor showed a greater increase in the duration than in the frequency of contractions, while those obtained during labor showed a greater increase in frequency than in duration. This phenomenon appears to agree with in vivo electromyographic recordings, which show that during most of pregnancy, myometrial activity is predominantly of the contracture (long duration) type but changes to the contraction (high frequency) mode at labor [10]. It is interesting, however, that the inherent behavior of this tissue is retained even in vitro. One striking observation was that the maximum response of the myometrium to oxytocin and both PGs tested showed an increase at 126 dGA and subsequently remained constant, with no further increase even at labor. Spontaneous (baseline) myometrial contractility also plateaued at this gestational age. This implies that in sheep, myometrial activation (the increase in the expression of genes encoding for agonist receptors, ion channels, and gap junctions, as defined by Challis and Lye [18]), is not abrupt but occurs well before labor, and therefore that any changes at labor must be more related to the down-regulation of inhibitory factors and/or to increased stimulation by uterotonic agents than to increased uterine responsiveness to those uteroton-
78
BAGUMA-NIBASHEKA ET AL.
ics. One previous indication that myometrial provision for the labor process begins so long before labor was the finding of Garfield and coworkers [19] that gap junctions begin to appear in the myometrium at this time. Further increases in gap junctions at the end of pregnancy may serve to produce better in vivo coordination without any increase in force. It is notable that this stage of pregnancy (126-135 dGA) is the time when fetal plasma cortisol levels begin to rise [20], indicating that the fetal hypothalamo-pituitary-adrenal axis is attaining functional maturity (presumably in response to the concurrent increase in maternal and fetal plasma PGE 2 concentration [21]), in preparation for the initiation of parturition. It is therefore possible that myometrial activation begins at this time in response to the rising hormone levels. In summary, the results indicate an increase in myometrial uterotonic receptor-mediated activity that precedes labor with no increase at labor, suggesting that in sheep, activation of the basic mechanisms responsible for strength of myometrial activity at labor occurs by 135 dGA. The greater sensitivity of the myometrium than the mesometrium to prostaglandins supports the view that intrauterine paracrine factors may play a major role in regulating myometrial contractility.
6. 7. 8.
9.
10.
11. 12.
13.
ACKNOWLEDGMENTS
14.
We gratefully acknowledge the excellent technical assistance of Lynn Buchwalder, Patricia de Vera, Jan Derks, and James Owiny and the helpful comments of Gordon Smith.
15.
REFERENCES 1. Yarrington G, Figueroa JP, Massmann A, Kassis I, Nathanielsz PW. An analysis of the characteristics of the electromyogram recorded from the mesometrium in the ovariectomized nonpregnant ewe and its similarities with and differences from the electromyogram obtained from the myometrium: the effect of estrogens. Am J Obstet Gynecol 1986; 155:1160-1164. 2. El Alj A, Bonoris E, Cynober E, Germain G. Heterogeneity of oxytocin receptors in the pregnant rat myometrium near parturition. Eur J Pharmacol 1990; 186:231-238. 3. El Alj A, Winer N, Lallaoui H, Delansorne R, Ferre F, Germain G. Progesterone and mifepristone modify principally the responses of circular myometrium to oxytocin in preparturient rats: comparison with responses to acetylcholine and to calcium. J Pharmacol Exp Ther 1993; 265:1205-1212. 4. Chow EHM, Marshall JM. Effects of catecholamines on circular and longitudinal uterine muscle of the rat. Eur J Pharmacol 1981; 76:157165. 5. Crankshaw DJ. The sensitivity of the longitudinal and circular muscle
16.
17.
18. 19. 20. 21.
layers of the rat's myometrium to oxytocin in vitro during pregnancy. Can J Physiol Pharmacol 1987; 65:773-777. Tuross N, Mahtani M, Marshall JM. Comparison of effects of oxytocin and prostaglandin F2. on circular and longitudinal myometrium from the pregnant rat. Biol Reprod 1987; 37:348-355. Anderson GF, Kawarabayashi T, Marshall JM. Effect of indomethacin and aspirin on uterine activity in pregnant rats: comparison of circular and longitudinal muscle. Biol Reprod 1981; 24:359-372. Massmann GA, Figueroa JP, Nathanielsz PW. Further characterization of the electromyographic activity of the myometrium and mesometrium in nonpregnant sheep under estrogen supplementation. Biol Reprod 1991; 45:605-610. Massmann GA, Figueroa JP, Hoffman G, Nathanielsz PW Role of the sympathetic innervation on uterine electromyographic activity in nonpregnant ovariectomized sheep under estrogen supplementation. Biol Reprod 1992; 46:483-491. Figueroa JP, Massmann A, Pimentel G, Nathanielsz PW. Characteristics of the electromyogram recorded from the mesometrium of the pregnant ewe from 106 days' gestation to delivery: similarities with and differences from the electromyogram obtained from the myometrium. Am J Obstet Gynecol 1987; 157:991-998. Wu WX, Myers DA, Nathanielsz PW. Changes in estrogen receptor messenger ribonucleic acid in sheep fetal and maternal tissues during late gestation and labor. Am J Obstet Gynecol 1995; 172:844-850. Wu WX, Nathanielsz PW Changes in oxytocin receptor messenger RNA in the endometrium, myometrium, mesometrium, and cervix of sheep in late gestation and during spontaneous and cortisol-induced labor. J Soc Gynecol Invest 1994; 1:191-196. Nathanielsz PW, Bailey A, Poore ER, Thorburn GD, Harding R. The relationship between myometrial activity and sleep state and breathing in fetal sheep throughout the last third of gestation. Am J Obstet Gynecol 1980; 138:653-659. Derks JB, Giussani DA, Van Dam LM, Jenkins SL, Winter JA, Zhao XF, Hammond GL, Nathanielsz PW. Differential effects of betamethasone and dexamethasone fetal administration on parturition in sheep. J Soc Gynecol Invest 1996; 3:336-341. Garcia-Villar R, Green LR, Jenkins SL, Wentworth RA, Coleman RA, Nathanielsz PW. Evidence for the presence of AH 13205-sensitive EP 2-prostanoid receptors in the pregnant baboon but not in the pregnant sheep myometrium near term. J Soc Gynecol Invest 1995; 2:612. Coleman RA, Nials AT. Novel and versatile superfusion system. Its use in the evaluation of some spasmogenic and spasmolytic agents using guinea-pig isolated tracheal smooth muscle. J Pharmacol Methods 1989; 21:71-86. Zhang Q, Wu WX, Brenna JT, Nathanielsz PW. The expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase in ovine maternal uterine and fetal tissues in late gestation and labor. Endocrinology 1996; 4010-4017. Challis JRG, Lye SJ. Parturition. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 985-1031. Garfield RE, Rabideau S, Challis JRG, Daniel EE. Hormonal control of gap junction formation in sheep myometrium during parturition. Biol Reprod 1979; 21:999-1007. Bassett JM, Thorburn GD. Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 1969; 44:285-286. Thorburn GD. The placenta, prostaglandins and parturition: a review. Reprod Fertil Dev 1991; 3:277-294.
