| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California 92350
b Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2-adrenergic receptor density and absence of neuronal nitric oxide synthase [4, 5]. Moreover, systemic changes in progesterone, the estrogen/progesterone ratio, prostaglandins, oxytocin, glucocorticoid concentrations, or uterine oxytocin receptor numbers do not necessarily precede the onset of labor across various species [6–10]. These hormones are important for increased numbers and size of gap junctions, increased numbers of oxytocin receptors, and enhanced myometrial responsiveness at term, and thus appear to play crucial roles in the progression, but not necessarily the initiation, of labor. More recently, work in a murine model has suggested that an immune mechanism, involving infection-induced local production of cytokines and prostaglandins, may contribute to the etiology of premature labor [11–14]. For this species, however, little is known about normal uterine contractile activity or the capabilities of myometrium to respond to agonists. In other species, the onset and progression of labor depend upon development of rhythmic, synchronous, and powerful contractions by the myometrium. A gradual increase in basal contraction force by the uterus occurs long before term in sheep [15–17] and the rhesus macaque [18, 19]. However, immediately preceding parturition, uterine contractile activity is augmented 2-fold or more. Moreover, in vitro myometrial responsiveness to agonists is enhanced sometime before the transition from relative quiescence to onset of labor, but neither frequency nor amplitude of contractions changes during the last 5 weeks of gestation [15]. In rats, frequent and high-amplitude contractions appear on, but not before, the day of labor—a conclusion based on in vivo electromyography [20] and intrauterine pressure recordings [21] and limited in vitro data [22–24]. Capabilities of the myometrium to respond to various agonists are also enhanced as pregnancy progresses. These in vitro studies assessed contractile activity by uterine segments in which specific layers of myometrium or endometrium were surgically removed, or they involved other methodological distinctions that preclude comprehensive examination of contractile capabilities during the peripartum period. Damage to the stromal interface could prevent interactions between relevant cell layers and interfere with either basal or agonist-induced responses. In fact, endometrium is crucial for oxytocin-stimulated uterine contractile activity in the sheep [25] and may be a more important source of prostaglandins than the rodent myometrium [26]. Thus, studies of intact uterine tissue, especially in a novel animal model, are essential for understanding both basal and agonist-mediated contractile activity during the peripartum period.
Perhaps more importantly, advances in statistical methods for rhythm analyses can now be applied to improve resolution of frequency and amplitude of contractile activity during the peripartum period. Qualitative assessment of contractility by the peripartum uterus suggested that contractile capabilities, both basal and agonist-stimulated, were enhanced at term [23]. However, changes in neither frequency nor amplitude of contractions were quantified during the period immediately before or after birth or following agonist treatment. To further understanding of uterine maturation at term, contractile capabilities by complete uterine segments were quantitatively analyzed. This novel approach was used to study murine uterine contractile capabilities because the mouse has recently been developed as a model for investigating how immune system activation may participate in infection-induced premature onset of labor [13]. Thus, the present study in the mouse tested the hypothesis that maximum in vitro contractile capabilities, as well as increased responsiveness to agonist, are achieved by uterine tissue before the onset of labor. Findings suggest that increased basal amplitude of myometrial contractions and response to agonists, rather than maturation of frequency of an endogenous oscillator, occurs during the peripartum period in the mouse.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Time-dated-pregnant mice (C3/HeN strain; Harlan Sprague Dawley, Inc., Indianapolis, IN) arrived at the vivarium on Day 12 of pregnancy (term = 19 days). Animals were housed individually in plastic cages on a 12L:12D cycle (lights-on 0600 h); food and water were provided ad libitum. Mice were killed by cervical dislocation in the morning of gestational Day 15 or 18, on the day when pups were born (Day 19 postconception, about 6 h after birth), or 1 day postpartum (Day 20 postconception); n = 11, 6, 4, and 9 per group, respectively. As a control, uterine strips from virgin nonpregnant mice were similarly harvested and contractile activity simultaneously studied (n = 13; mice were group housed). Histological analysis of hematoxylin-stained uterine tissue sections from nonpregnant mice confirmed endometrial thickening, glandular development, and columnar epithelium that was indicative of the estrous phase of the reproductive cycle. The research protocol was reviewed and approved by the Institutional Animal Care and Use Committees.
Assessment of Uterine Contractile Activity
Methods to assess uterine contractile activity followed those described by Rhee et al. [27]. Briefly, the bicornuate uterus was extricated and incised along the antimesometrial borders, and fetuses were removed intact. Multiple 2 x 5-mm strips of complete myometrium were longitudinally cut from the mid-horn regions of each uterine horn; sample duplicates represent tissue from each of the two horns, respectively. Endometrium was left intact. Strips were mounted on calibrated isometric transducers (Radnoti Glass Corp., Monrovia, CA) in standard muscle bath preparations (10 ml total volume) that contained oxygenated Na+-Krebs buffer at 37°C and pH 7.4. After a 1-h prestretch equilibration to approximately 1 g of tension [28], tension recordings were acquired with online data acquisition software (Labview 2.2.1; National Instruments, Austin, TX). After an initial period of spontaneous contractility, oxytocin (OT; Bachem, Torrance, CA) or arginine vasopressin (AVP; Bachem) was added in bolus to the incubation bath in increasing half-log doses (from 10-10 to 10-5.5 M) at 3.33-min intervals. These two uterotonins were used for the following reasons. OT is an endogenous contractile agent in the mouse that stimulates myometrial inositol-1,4,5-trisphosphate production [29]. AVP was used as an additional novel agonist because data in the rat suggest that AVP may act independently of the OT receptor to stimulate contractions by the same signal transduction cascade [30, 31]; however, work by Chan et al. [32] suggests that a similar receptor-mediated mechanism may regulate uterine contractility. At the conclusion of each agonist dose-response curve, the Na+-Krebs solution was replaced with K+-Krebs buffer containing 120 mM KCl to verify tissue viability. KCl depolarizes the myometrium, thus stimulating Ca+ release, a process that is independent of second messenger signal transduction.
Data Analysis and Statistics
Total contractile force generated by each longitudinal uterine strip was calculated by integrating the area under the contractile response curve over time, a summative estimate of change in contraction frequency and amplitude [30]. Tension generated during each dose challenge was normalized to cross-sectional area of the uterine strip to account for differences in tissue size. Cross-sectional area was calculated as previously described [27]: wet tissue weight/length x 1.05 g/cm3 (= specific density of tissue). A dose-response curve was then constructed with tension as the ordinate and agonist concentration as the abscissa. Thus, tension generated was expressed as (grams x second)/cm2. The maximum tension generated (TMAX) and pD2, i.e., the -log[dose concentration yielding half-maximal response], were calculated using a linear curve-fitting model. Contraction frequency (a derivative of periodicity; f = 1/p) and amplitude were defined by the presence of a statistically significant rhythm (frequency and peak) as detected by fast Fourier transform-nonlinear least-squares time series analysis with shuffling analysis [33]. Fourier analysis was performed on waveforms measured during periods of spontaneous contractility and during periods of maximal response to agonist (OTMAX or AVPMAX). OTMAX and AVPMAX were defined as those periods of contractility that coincided with TMAX for OT or AVP (10-6 to 10-8 M), respectively. Statistical analysis of the primary oscillation was based on rank order; this methodology was verified by manual review of the raw data. Data for baseline activity and agonist-stimulated responses were log-transformed and then examined by one-way ANOVA (SPSS, Chicago, IL). When main effects were significant, individual comparisons were made using Bonferroni's test (P < 0.05 was considered significant). If the Levene test for homogeneity of variance was significant, then nonparametric analysis using the Kruskal-Wallis test with multiple comparisons was performed.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Regular and repetitive contractions were evident in all uterine strips (Fig. 1, top). Spontaneous oscillations differed with respect to reproductive status. Representative contractile rhythms were plotted on the same scale to emphasize differences across the peripartum period. Visually, the scale obscures small changes in amplitude and frequency; however, resolution capabilities of the Fourier analysis revealed statistically significant rhythmicity. In nonpregnant mice, contractions were infrequent and significantly lower in amplitude than those of the peripartum period. On Day 15 of pregnancy, a simple but regular waveform was established. By Day 18, secondary and harmonic rhythms occurred in more than 40% of the uterine recordings, an indication of a more complex contractile event. Postpartum, the waveform was robust but less complex.
|
Frequency Contraction rates in uterine strips from pregnant mice were significantly greater than in uteri from nonpregnant controls (Fig. 1, bottom left; P < 0.05). Contraction frequency was enhanced more than 50% in uterine strips from postpartum versus nonpregnant mice. Peripartum, contraction frequency was not different among groups between 1 day before and 1 day after delivery (Day 18 versus the day of birth or 1 day postpartum).
Amplitude Contraction amplitude was significantly enhanced in uterine strips from pregnant and postpartum mice compared to nonpregnant controls (Fig. 1, bottom right). Uteri from Days 15 and 18 of pregnancy produced more than 9 times as much force as tissue from nonpregnant mice. Contraction amplitude in postpartum uteri demonstrated a further 4-fold increase in contraction amplitude above that in pregnant uteri and nearly a 35-fold increase above that in nonpregnant uteri.
Contractile Response to Agonist
The contractile response to OT, in terms of integrated area under the curve, was dose dependent (Fig. 2, left). This response was biphasic in all groups (obscured in the graph for the nonpregnant group due to scale) with peak contractile activity, i.e., TMAX, induced by doses of OT that ranged from 10-6 to 10-8 (OTMAX). In uterine strips from pregnant and peripartum mice, TMAX was significantly elevated at all time points compared to that for nonpregnant mice (Fig. 2, top right). The pD2, an indication of tissue sensitivity to OT, was not different in uterine strips from peripartum and nonpregnant mice but was enhanced on Day 15 of pregnancy as compared to the values in all other groups (Fig. 2, bottom right). Because pD2 is not altered during the peripartum period, a shift in OT sensitivity most likely does not account for agonist-mediated changes in frequency and amplitude.
|
Waveform complexity, including frequency and amplitude of contractions, varied with respect to agonist treatment across the peripartum period. In response to OTMAX, secondary waveforms and harmonics were common in Day 15 and postpartum groups (Fig. 3, top). With OTMAX, waveforms were more regular and less complex at 18 days of pregnancy than at 15 days and in nonpregnant controls. AVPMAX treatment produced the same effect as OT on waveform character and harmonics (data not shown).
|
Frequency of contractions after agonist treatment In response to OTMAX, frequency of contractions increased, on average, by 60% from basal levels; the most pronounced increase occurred on Day 18 (106%). Contraction frequency was significantly elevated in pregnant and postpartum uterine strips as compared to nonpregnant controls (Fig. 3, middle left). Peak frequency of contractions occurred on Day 18. The same pattern of contraction frequency responses was found with AVPMAX treatment (Fig. 3, bottom left).
Amplitude of contractions after agonist treatment Amplitude responses to agonist progressively decreased as term approached; under agonist stimulation, amplitude increased 5-fold from basal levels in the nonpregnant group but decreased by almost 75% in the postpartum group (data in Fig. 3 compared to those in Fig. 1). In response to either OTMAX or AVPMAX, mean contraction amplitudes were significantly greater in pregnant and postpartum uterine strips than in nonpregnant controls (Fig. 3, middle and bottom left). At Day 15 of gestation, contraction amplitude was enhanced more than 500% by OTMAX or AVPMAX treatment in comparison to the uterine response in nonpregnant mice. This response to OTMAX was reduced by Day 18 compared to that in the Day 15 group, and continued to decline on the day when pups were born to levels equivalent to the level in uterine strips from nonpregnant mice. By 1 day postpartum, peak contraction amplitude was achieved. In addition, responses to AVPMAX were similar to that induced by OTMAX with one exception: mean contraction amplitude in response to AVPMAX was augmented in uterine strips from all pregnant and postpartum groups relative to nonpregnant controls. Manual analysis in which baseline was defined as tension prior to treatment showed a diminished peak height response to OT agonist (data not shown; peak height is analogous to amplitude in the Fourier analysis).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Findings in other animal models support the concept that maturation of an endogenous pacemaker within the uterus may precede the onset of parturition. In rats [22, 23], sheep [15], and rhesus monkeys [34, 35], spontaneous contractile activity is evident in the pregnant uterus in vivo. Further indication that this uterine activity is independent of neural or endocrine drive is suggested by synchrony in electrical and, presumably, contractile activity in myometrial tissue grafts from pregnant and nonpregnant ewes [16]. Similarly, in the present study, the presence of rhythmic contractions in all uterine strips (a strength of the Fourier analysis to statistically resolve a low-amplitude periodicity) suggests that each myometrial cell may be a component of an endogenous pacemaker. Development of high-frequency and high-amplitude contractions is the predominant sign that labor has been initiated in these species. Since agonist-induced uterine contractile activity is also augmented prior to the onset of labor [15, 23], the evidence suggests that mechanical capabilities for synchronous and powerful myometrial contractions are established before the onset of labor in the murine model.
In addition to maturation of an endogenous drive of uterine contractions, mechanisms regulating contractile capabilities in the myometrium are in transition as pregnancy nears term. Synthesis and trafficking of connexin 43 to the plasmalemma, and incorporation into gap junction plaques in uterine smooth muscle, parallel development of coordinated contractions [36, 37]. The enhanced cellular communication at term resulting from gap junction assembly may contribute to the rise in amplitude of contractions in peripartum uteri on the day of birth seen in the present study.
With regard to agonist treatments, enhanced frequency and amplitude of contractions in response to OTMAX or AVPMAX preceded the onset of labor. Peak contraction frequency response to OTMAX or AVPMAX was induced on Day 18 of pregnancy. An augmented frequency response to both agonists suggests that oscillatory capabilities by the endogenous uterine pacemaker had matured by Day 18, 1 day before the onset of labor. No further changes in this aspect of pacemaker function were evident because the enhanced frequency response to agonist relative to that in the nonpregnant uterus was sustained on the day after parturition.
By contrast, the contraction amplitude response varied with respect to agonist and reproductive status. Both OT and AVP enhanced uterine contraction amplitude on Days 15 and 18 of pregnancy compared to that in uterine strips from nonpregnant mice. Sensitivity to OT, i.e., pD2, also peaked on Day 15. With respect to contractile capabilities on Day 15, however, the response to OTMAX was reduced on Day 18 and further declined on the day of delivery to the level in the nonpregnant uterus. The peripartum decline in response to agonist treatment in vitro may reflect the loss of phasic contractions resulting from maximizing integrated contractile tension, OT receptor occupancy, or depletion of receptor numbers due to endogenous OT secretion at term. These data are consistent with the role that OT plays in the progression, but not onset, of labor [38, 39]. The suggestion has also been made that an inhibitory process, such as macrophage production of inducible nitric oxide synthase, may restrain the capability of uterine tissue to produce high-amplitude contractions [40]. Peak numbers of resident uterine macrophages are achieved on Day 15 of gestation in this species and decline in the peripartum uterus [41]. This putative restraining mechanism is likely to involve blunting of the response to OT on days preceding parturition but not the response to in vitro AVP treatment, since enhanced contraction amplitude responses to AVPMAX were sustained after Day 15 of pregnancy relative to those in nonpregnant tissue. Actions of AVP may possibly be independent of an OT receptor-mediated mechanism as suggested in the rat [30, 31]. Therefore, the uterus appears fully capable of synchronous, high-amplitude contractions in response to agonist at least 1 day before parturition.
Questions about pacemaker maturation and enhanced intercellular communication during the peripartum period focus attention on the endogenous oscillator within the uterus that regulates contractions. This uterine oscillator differs from the neuroplexus pacemaker in the gastrointestinal tract that drives smooth muscle contractile activity [5, 40, 42]. Common to both systems, however, are resident immune cells. In particular, the macrophage is located in the stromal layer of cells that serves as an interface between endometrium and myometrium. It has been suggested that production of nitric oxide, cytokines, and prostaglandins by immune cells modulates myometrial contractile activity or responses to agonist during gestation and parturition [8]. In the context of present findings, subsequent investigations need to focus on the last days of parturition in the murine model to determine whether the immune system may contribute to the regulation of amplitude of contractile activity [41].
In summary, an endogenous oscillator is present in virgin and gravid murine uteri. Peak response to agonists before birth for contraction frequency, and postpartum for contraction amplitude, fails to suggest a role for changes in the capability of the uterus to respond to agonists in the process of parturition. Although increased contraction frequency may be crucial for the progression of labor, a predominant increase in basal contraction amplitude on the day of birth raises the possibility that amplitude, not frequency, of contractile activity reflects the transition from quiescence to powerful synchronous contractions during the initiation of parturition.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence. FAX: 909 824 4029; syellon{at}som.llu.edu
Accepted: May 27, 1999.
Received: November 4, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2-Adrenergic receptors in guinea pig myometrium in late pregnancy: evidence for a predominantly postjunctional location. Am J Obstet Gynecol 1990; 162:831–836.[Medline]. Biol Reprod 1998; 58:73–78. do not acutely stimulate isolated murine myometrial contractile activity. Am J Obstet Gynecol 1994; 169:1424–1427.This article has been cited by other articles:
|
|
 |
|
  M. Kawamata, Y. Tonomura, T. Kimura, Y. Sugimoto, T. Yanagisawa, and K. Nishimori Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E992 - E999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A L Griffiths, K M Marshall, J Senior, C Fleming, and D F Woodward Effect of the oestrous cycle, pregnancy and uterine region on the responsiveness of the isolated mouse uterus to prostaglandin F2{alpha} and the thromboxane mimetic U46619. J. Endocrinol., March 1, 2006; 188(3): 569 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Takayanagi, M. Yoshida, I. F. Bielsky, H. E. Ross, M. Kawamata, T. Onaka, T. Yanagisawa, T. Kimura, M. M. Matzuk, L. J. Young, et al. Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice PNAS, November 1, 2005; 102(44): 16096 - 16101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B G White, S J Williams, K Highmore, and D J MacPhee Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour Reproduction, January 1, 2005; 129(1): 115 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Matthew, S. Kupittayanant, T. Burdyga, and S. Wray Characterization of Contractile Activity and Intracellular Ca2+ Signalling in Mouse Myometrium Reproductive Sciences, May 1, 2004; 11(4): 207 - 212. [Abstract] [PDF]
|
|
|
|
 |
|
 A. M. Mackler, T. C. Ducsay, C. A. Ducsay, and S. M. Yellon Effects of Endotoxin and Macrophage-Related Cytokines on the Contractile Activity of the Gravid Murine Uterus Biol Reprod, October 1, 2003; 69(4): 1165 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Yellon, A. M. Mackler, and M. A. Kirby The Role of Leukocyte Traffic and Activation in Parturition Reproductive Sciences, September 1, 2003; 10(6): 323 - 338. [Abstract] [PDF]
|
|
|
|
|
|
A.M. Mackler, L.M. Green, P.J. McMillan, and S.M. Yellon Distribution and Activation of Uterine Mononuclear Phagocytes in Peripartum Endometrium and Myometrium of the Mouse Biol Reprod, May 1, 2000; 62(5): 1193 - 1200. [Abstract] [Full Text]
|
|
|
|
|
|
A.M. Mackler, G. Iezza, M.R. Akin, P. McMillan, and S.M. Yellon Macrophage Trafficking in the Uterus and Cervix Precedes Parturition in the Mouse Biol Reprod, October 1, 1999; 61(4): 879 - 883. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
