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Regular Article |
a Department of Large Animal Medicine & Surgery,
b Department of Anatomy & Public Health,
c College of Veterinary Medicine, and Department of Health & Kinesiology, College of Education, Texas A&M University, College Station, Texas 77843-4475
ABSTRACT
Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F2
(PGF2) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca2+ regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca2+ transients of myometrial cells loaded with a Ca2+-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca2+ transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca2+ concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF2, suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca2+ release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.
aging, calcium, oxytocin, signal transduction, uterus
INTRODUCTION
Transient endometritis is a normal consequence of breeding and results from uterine contamination with both bacteria [1, 2] and semen [3–5]. The reproductively normal mare efficiently resolves the endometritis so that the intrauterine environment is optimal for embryo survival when it enters the uterus approximately 6 days after ovulation. However, a field fertility study including more than 400 mares revealed that 15% of mares developed a persistent postmating endometritis [6] leading to prolonged inflammation of the endometrium and, ultimately, early embryonic loss. Mares that are considered to be susceptible to persistent postmating endometritis are older mares [7] that have delayed physical clearance of uterine contents [8–10], which is associated with dysfunctional uterine contractility [7]. Between 10 and 20 h following intrauterine bacterial challenge, mares that were susceptible to persistent postmating endometritis exhibited reduced frequency, intensity, and duration of uterine myoelectrical activity in vivo compared to normal mares [11]. The reasons for this decline in myometrial activity remain obscure. However, possible mechanisms include 1) changes in the release, either systemically or locally, of uterotonins such as prostaglandins or oxytocin; 2) altered production of neuromuscular or vasoactive substances that affect myometrial activity; or 3) an intrinsic change within the uterine muscle that renders it incapable of responding with normal contractile force.
The primary objective of the present study was to test the hypothesis that reduced uterine contractility of mares with delayed uterine clearance is associated with an intrinsic contractile defect within the myometrium. Secondly, we tested the hypothesis that the intrinsic myometrial contractile dysfunction is associated with reduced available intracellular Ca2+. Three groups of mares were studied: young nulliparous mares, older mares with delayed uterine clearance, and older reproductively normal mares. To remove potential confounding neural, vascular, and endometrial influences present in vivo, in vitro responsiveness of isolated longitudinal and circular uterine muscle strips were studied. Three different contractile agonists, oxytocin, prostaglandin F2 (PGF2), and KCl, with different mechanisms for activating uterine muscle contractility were utilized. In addition, oxytocin-stimulated intracellular calcium (Ca2+) transients of individual uterine muscle cells were measured using laser cytometry to examine whether reduced intracellular Ca2+ concentration may affect uterine muscle contractility in mares with delayed uterine clearance.
MATERIALS AND METHODS
Mares
A total of 27 mares were studied over 2 yr, with 15 mares in the first year and 12 mares in the second. The study was conducted from May to November 1998 and from May to November 1999. Mare care and the experimental protocol (Animal Use Protocol 8-128) were approved by the Texas A&M University Laboratory Animal Care Committee. Mares were determined to be in estrus by their behavioral response to a stallion in conjunction with evaluation of the reproductive tract using transrectal palpation and ultrasonography. Mares were categorized as young nulliparous (YN; n = 7), older and susceptible to delayed uterine clearance (OS; n = 13), or age-matched older and reproductively normal mares (ON; n = 7). Categorization was based on reproductive history, breeding soundness examination of the reproductive tract (including endometrial biopsy at the start of the study [12]), and response to each of six measures of uterine function. Uterine function was evaluated for 1) maximal height and location of free fluid within the uterus during estrus [13], 2) free fluid within the uterus during diestrus [14], 3) uterine fluid 72 h following insemination, 4) uterine clearance of a radiocolloid using nuclear scintigraphy [10], 5) uterine fluid, and 6) positive endometrial culture 96 h following bacterial challenge with Streptococcus zooepidemicus [11], which had been isolated from a mare with endometritis.
Following at least 1 mo of sexual rest after classification challenges, mares that were in physiologic estrus (dominant follicle 
32 mm in diameter, serum progesterone concentration < 1 ng/ml) were inseminated with 500 million total sperm in a milk-glucose based semen extender from a single fertile stallion. Care was taken to minimize variability among mares at the time of breeding. Spermatozoa have been shown to cause an influx of polymorphonuclear cells into the uterine lumen [3, 4, 15], which may be modified by the presence of seminal plasma [3, 16]. Therefore, total sperm numbers and volume of seminal plasma in the insemination dose were the same for all mares (insemination volume of 10 ml and containing 25% [v/v] seminal plasma). Insemination was performed using aseptic technique, and broad-spectrum antibiotics were included in the semen extender to minimize bacterial contamination. Eighteen hours after insemination, mares were sedated using xylazine hydrochloride (1.1 mg/kg i.v.) and anesthetized using ketamine hydrochloride (2.2 mg/kg i.v.). General anesthesia was maintained using isoflurane while the reproductive tract was surgically removed via a ventral midline celiotomy.
Solutions and Drugs
The Krebs-Hensleit Solution (KHS) contained 131.5 mM NaCl, 5.0 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl26H2O, 2.5 mM CaCl2, 11.2 mM glucose, 13.5 mM NaHCO3, 0.003 mM propranolol, and 0.025 mM EDTA (pH 7.4). Contractile agonists were prepared as concentrated stock solutions (3 M KCl, 400 mM oxytocin, and 1.8 mM PGF2) using distilled water.
Tissue-culture flasks and dishes were obtained from Corning, Inc. (Oneonta, NY). Stock solutions of fluo-4, acetoxy-methyl ester (AM) (Molecular Probes, Inc., Eugene, OR) were prepared in dimethyl sulfoxide and diluted with medium to 3.0 µM for loading in cultured cells. All culture media, Dulbecco PBS, serum, and chemicals were reagent grade and purchased from Sigma Chemical Company (St. Louis, MO).
In Vitro Contractility of Uterine Muscle Strips
Full-thickness muscle sections were removed from the ventral uterine surface at the bifurcation of both the left and right uterine horns and maintained in chilled (4°C) KHS for transport to the laboratory. Eight uterine muscle strips, approximately 1 cm long x 0.5 cm wide, were prepared (four each from both the right and left uterine horns). Care was taken to remove all blood vessels and as much endometrial stroma as possible with the aid of a stereomicroscope (Olympus model SZX12; Olympus America, Melville, NY). The serosa was left intact on the longitudinal muscle strips to preclude loss of the thin muscle layer; therefore, comparisons between longitudinal and circular muscle function were not made in this study. Both ends of the muscle strips were secured with a loop of suture material (5-0 vicryl). Using a Filar (Olympus America) calibrated micrometer eyepiece, the length of muscle between knots was maintained at 7 mm. Wet weight of each muscle strip was also determined.
Muscle strips were mounted on two stainless-steel wires passed through the vicryl loop on each end of the muscle. One wire was attached to a force transducer (Model FT03c; Grass Instruments Co., Quincy, MA) and the other to a micrometer microdrive (Stoelting/Prior Microdrive; Stoelting Co., Wood Dale, IL) to allow the muscle strip to be stretched. The force transducer was connected to a computer equipped with a software program (MacLab Electronic Data Acquisition System; Castle Hill, Australia) for data collection. Each muscle strip was then lowered into an individual 20-ml jacketed tissue bath (Harvard Apparatus, South Natick, MA) containing KHS maintained at 37°C and bubbled with 95% O2/5% CO2. The muscle strips were stretched to create just enough tension to keep them on the wires and allowed to equilibrate (rinsed every 20 min with fresh KHS solution) for 1 h before beginning the experiment. Immediately before the experiment, the muscle strips were stretched so that baseline tension was at 2 g. In some preliminary studies, four muscle strips (two longitudinal and two circular) from eight mares (two YN, three OS, and three ON) were used to determine the maximum length-tension relationship by repeated test exposure of the muscle to 70 mM KCl at increasing muscle length. The muscle strips were stretched by 2-mm increments, resting tension recorded, and active tension generated in response to 70 mM KCl calculated (i.e., total tension - resting tension). The muscle length that resulted in the greatest active tension was considered to be Lmax. The results from these preliminary studies indicated that the length-tension relationships for longitudinal and circular muscle, respectively, were not different among the three mare groups. In addition, the setting of the muscle strips to a tension of 2 g was approximately 70–80% of Lmax for longitudinal muscle and approximately 100% of Lmax for circular muscle for all groups.
Experimental design
In vitro contractility of uterine muscle strips was evaluated by measuring isometric tension generated in response to three different contractile agonists: KCl, oxytocin, and PGF2. Agonists were chosen because they activate contraction via different mechanisms. Potassium chloride (KCl) activates voltage-gated plasma membrane Ca2+ channels to stimulate nonreceptor-mediated contraction. In contrast, oxytocin and PGF2 activate oxytocin and prostaglandin receptors, respectively, which are located on the plasma membrane of the myometrial cell. Cumulative concentration responses for KCl (10–100 mM) and oxytocin (10-11 to 3 x 10-7 M) were evaluated sequentially using the same uterine muscle strips. Therefore, a recovery period of at least 30 min (rinsing every 5–10 min) was allowed after KCl was rinsed from the tissue baths before initiating the oxytocin response. The PGF2 (10-8 to 10-4 M) concentration response was evaluated in a similar manner using different muscle strips. The contractile responses of two longitudinal or two circular muscle strips from each mare were averaged for every agonist concentration tested and counted as one observation (n = 1).
The mean value for resting tension generated during the last 2 min before starting a concentration-response curve was calculated and considered to be the baseline tension. Data for each agonist concentration were collected as the mean tension measured during the last 4 min of a 5-min period. A 5-min period for each concentration was sufficient for contractile activity to reach a steady state. Data are reported as the percentage increase in active tension for concentration responses. Maximal active tension was determined for each agonist (calculated by subtracting resting tension from maximum tension developed), and the EC50 (i.e., the agonist concentration that produced 50% of the maximal active response) values were calculated and compared to determine if shifts in muscle sensitivity for each agonist occurred.
Intracellular Ca2+ Transients of Individual Uterine Muscle Cells
Myometrial cell culture
Full-thickness muscle sections were harvested from the ventral uterine surface at the bifurcation of either the right or left uterine horn. The serosa and endometrium were carefully dissected free of the myometrium. Myometrial tissue was placed in cold Dulbecco PBS containing penicillin and streptomycin for transport to the laboratory. Myometrial tissue was minced in Ca2+/Mg2+-free buffer with 10 mM glucose and 0.15 mg/ml of trypsin type XII-S, 0.2 mg/ml of EDTA, 0.5 ml (200 U) of collagenase type II, and 0.5 ml (200 U) of DNase I for 1.5 h at 37°C. Uterine tissues were vortexed for approximately 10 sec. The cell suspension was removed and washed in Dulbecco modified Eagle-F12 medium supplemented with 5% charcoal-stripped fetal bovine serum and 1% penicillin-streptomycin, then seeded onto two-well Coverglass slides (Nunc, Inc., Naperville, IL). Identity of pure myometrial cell preparations was verified by immunofluorescence staining with smooth muscle-specific probes, including antibodies to -smooth muscle actin and desmin. Purity of myometrial preparations was at least 95%.
Intracellular Ca2+ transients Cells were loaded with 3.0 µM fluo-4, AM for 1 h in serum- and phenol red-free medium at 37°C. Following repeated washing with serum- and phenol red-free medium, slides were placed on the stage of a Meridian Ultima laser confocal microscope (Meridian Instruments, Okemos, MI). An area of the slide containing approximately 10–15 cells was selected and scanned five times at 10-sec intervals to determine basal intracellular Ca2+ levels. Oxytocin (1, 10, and 100 nM) was then added, and scanning continued with the same sampling rate for approximately 5 min. For each concentration of oxytocin tested, the agonist-induced increase in fluo-4 fluorescence intensity was normalized to its corresponding basal level. Data reported here represent the average response for at least 20 cells per treatment group per mare (n = 1).
Statistical Analysis
Concentration responses of uterine muscle were compared among the three mare groups using a mixed-regression model. The model fit mare, repetition within mare, group, and concentration of agonist tested as sources of random effects and treated the serial correlation within repetition as type 1 autocorrelation. To determine the effect of agonist concentration, we used a saturated model for the mean response by fitting a separate mean parameter for the response at each concentration of agonist tested. This is the desired method used in designed experiments during which modeling the entire dose-response curve is deemed to be unwarranted [17]. The variance components and fixed effects were calculated using the methods of restricted maximum likelihood. The data were plotted as the least-squares means ± SEM. Values for maximal active tension, EC50, and oxytocin-stimulated peak fluorescence in individual myometrial cells were compared among the three mare groups using ANOVA. The least-squares means ± SEM and P values for each comparison are presented.
RESULTS
Classification of Mares Based on Uterine Function Tests
Of the seven YN mares evaluated, one mare failed two measures of uterine function (failed to clear radiocolloid and the bacterial inoculum), and two mares failed one measure of uterine function each (failed to clear >40% of radiocolloid but passed all other uterine function tests). Furthermore, older mares were not categorized as ON if they failed more than two uterine function tests. Mean values for age and parity of the ON mares (16 ± 4 yr and 5 ± 5 foals; [mean ± SD]) and the OS mares (17 ± 3 yr and 5 ± 5 foals) were similar (P > 0.05) and greater than the respective values of the YN mares (4 ± 0.5 yr and zero foals).
General Characteristics of Longitudinal and Circular Uterine Muscle Strips and Contractility
Spontaneous contractile activity during the 1-h equilibration period was seldom displayed by the uterine muscle strips, presumably due to the propranolol in the KHS. Depolarization of longitudinal muscle with KCl or by activation with oxytocin resulted in a tonic contracture that yielded a concentration-dependent increase in tension (Fig. 1, A and B). At low concentrations of KCl and oxytocin, circular muscle exhibited phasic contractions, which converted to a tonic contracture at high concentrations of either agonist (Fig. 2, A and B). The muscle strips readily relaxed to resting tension following maximal activation by KCl. However, the return to baseline resting tension following maximal activation by oxytocin required 90–120 min and was characterized by spontaneous phasic contractions.
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Longitudinal and circular muscle responded similarly to PGF2, with an increase in phasic contractile activity at low concentrations. At high concentrations of PGF2 (usually >10 µM), an abrupt cessation of phasic contractions was observed, and no active tension was generated (Figs. 1C and 2C). Spontaneous phasic contractions began as soon as the high concentrations of PGF2 were washed from the tissue bath.
Comparison of longitudinal muscle contractility among mare groups A significant (P = 0.002) group x dose interaction was observed in the response of longitudinal muscle depolarized by KCl. The increase in active tension generated by longitudinal muscle from OS mares was less than (P < 0.05) that of longitudinal muscle from ON mares at concentrations of KCl equal to or greater than 50 mM (Fig. 3A). In addition, the increase in active tension generated by longitudinal muscle from OS mares was less than (P > 0.05) that of longitudinal muscle from YN mares at 40 mM and higher concentrations of KCl (Fig. 3A). The KCl-mediated increase in active tension generated by longitudinal muscle from ON and YN mares was similar (P > 0.05) for all concentrations evaluated (Fig. 3A). Among the three mare groups, no difference was found in the sensitivity of longitudinal muscle to KCl (P = 0.51). Maximal active tension generated by longitudinal muscle also was not different among the three mare groups (P = 0.08) (Table 1).
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When longitudinal muscle was activated using oxytocin, a significant (P = 0.02) group x dose interaction was observed. Oxytocin stimulated a greater (P < 0.05) increase in active tension of longitudinal muscle from ON mares than from longitudinal muscle of OS mares at concentrations of 10 nM or higher (Fig. 3B). At 1000 nM oxytocin, the increase in active tension of longitudinal muscle from YN mares was greater (P = 0.04) then the response in OS mares (Fig. 3B). The response of longitudinal muscle from ON and YN mares was similar (P > 0.05) for all oxytocin concentrations evaluated (Fig. 3B). Maximal active tension in response to oxytocin was not different (P = 0.15) among the three mare groups (Table 1). Likewise, the sensitivity of longitudinal muscle from the three mare groups to oxytocin was similar (P = 0.53) (Table 1).
A significant difference among mare groups (P = 0.01) and a significant group x dose interaction (P = 0.0002) were also observed in the response of longitudinal muscle to PGF2. The increase in active tension generated in response to PGF2 by longitudinal muscle from ON mares was greater (P < 0.05) than that of longitudinal muscle from OS mares for 0.1–10 µM PGF2 (Fig. 3C). In addition, the PGF2-stimulated increase in active tension of longitudinal muscle from ON mares was greater (P < 0.05) than that of longitudinal muscle from YN mares at 0.3–10 µM PGF2 (Fig. 3C). The response of longitudinal muscle from OS and YN mares was similar (P > 0.05) for all concentrations of PGF2 evaluated (Fig. 3C). Maximal active tension generated in response to PGF2 by longitudinal muscle from ON mares was greater than that of OS mares (P = 0.009), but it was not greater than that of YN mares (P = 0.15) (Table 1). No difference was found in the sensitivity of longitudinal muscle to PGF2 among the three mare groups (Table 1).
Comparison of circular muscle contractility among mare groups A significant group x dose interaction (P = 0.002) was observed when circular muscle was depolarized using KCl. At high KCl concentrations, the increase in active tension generated by circular muscle from OS mares was less (P < 0.05) then the increase in active tension generated by circular muscle from either ON or YN mares (Fig. 4A). The KCl-mediated increase in active tension by circular muscle from ON and YN mares was similar (P > 0.05) for all concentrations of KCl (Fig. 4A). Maximal active tension generated by circular muscle in response to KCl did not reach statistical difference for the three mare groups (P = 0.12) (Table 2). However, circular muscle from ON mares was more sensitive to KCl than circular muscle from YN mares (P = 0.02). No shift was observed in the sensitivity to KCl between circular muscle from OS and YN mares (P = 0.08) or between that of OS and ON mares (P = 0.56) (Table 2).
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Circular muscle from ON mares responded to oxytocin with a greater (P < 0.05) increase in active tension than circular muscle from OS mares at concentrations of 3 nM and above (Fig. 4B). The oxytocin-stimulated response of circular muscle from ON mares was greater (P < 0.05) than that of YN mares only at 100 nM oxytocin (Fig. 4B). Circular muscle from OS and YN mares responded with a similar (P > 0.05) increase in active tension for all oxytocin concentrations tested (Fig. 4B). Maximal active tension to oxytocin was similar for circular muscle from YN, ON, and OS mares (P = 0.21). Likewise, no shift was observed in the sensitivity of circular muscle to oxytocin among the three mare groups (P = 0.47) (Table 2).
A significant difference among mare groups (P = 0.05) and a significant group x dose interaction (P < 0.0001) were found for PGF2-stimulated circular muscle. At PGF2 concentrations of 0.1–10 µM, circular muscle from ON mares generated a greater (P < 0.05) increase in active tension than circular muscle from OS mares (Fig. 4C). Similarly, 0.3–10 µM PGF2 stimulated a greater (P < 0.05) increase in active tension by circular muscle from ON mares than that generated by circular muscle from YN mares (Fig. 4C). At all concentrations of PGF2, circular muscle from YN and OS mares responded similarly (P > 0.05) (Fig. 4C). Maximal active tension in response to PGF2 was similar (P = 0.07) among the three mare groups (Table 2). Because active tension fell with increasing concentrations of PGF2 for circular muscle from YN and OS mares (Fig. 4C), EC50 values were not calculated and compared among the three mare groups.
Intracellular Ca2+ Transients of Individual Myometrial Cells
The amplitude of the initial intracellular Ca2+ transients monitored by fluo-4 fluorescence intensity measurement in myometrial cells isolated from the three mare groups exhibited a similar response (P = 0.83) when stimulated with 10 nM oxytocin (relative increase in fluorescence: YN mares, 1.7 ± 0.1; ON mares, 1.6 ± 0.3; and OS mares, 1.6 ± 0.2). Likewise, no difference (P = 0.78) was observed in the amplitude of the initial fluo-4 fluorescence intensity of myometrial cells among the three groups when myometrial cells were activated by 100 nM oxytocin (relative increase in fluorescence: YN mares, 1.8 ± 0.2; ON mares, 1.8 ± 0.3; and OS mares, 2.0 ± 0.2). These results suggest that the initial oxytocin-activated intracellular Ca2+ transient was similar among mare groups.
DISCUSSION
The major finding in the present study is the detection of an intrinsic defect in myometrial function of mares with delayed uterine clearance. For all agonists tested, the increase in active tension was lower for myometrium from OS mares than for myometrium from ON mares. Mechanistically, the reduced ability to generate tension did not appear to be related to a change in a specific receptor-mediated response, because the increases in tension generated in response to high concentrations of both oxytocin and prostaglandin were lower than the respective responses by myometrium from ON mares (Figs. 3, B and C, and 4, B and C). Furthermore, the reduced increase in active tension was also evident in nonreceptor-mediated contraction utilizing KCl (Figs. 3A and 4A). To further dissect the mechanism for the contractile dysfunction of myometrium from OS mares, we tested whether the contractile defect was associated with reduced intracellular Ca2+ concentration. We report that oxytocin-stimulated intracellular Ca2+ transients were similar for myometrial cells from OS and ON mares. Therefore, the cellular defect for the contractile dysfunction of myometrium from OS mares may lie beyond the calcium regulatory system. Possible alterations include reductions in calmodulin protein expression, myosin light-chain kinase activity, or myofibrillar protein content [18].
In other species [19, 20], oxytocin stimulates myometrial prostaglandin production that may play an autocrine/paracrine role to modulate myometrial contractility. In cyclic, reproductively normal mares, prostaglandin F metabolites are elevated following in vivo oxytocin release [21], and an in vitro study revealed that oxytocin-stimulated endometrium released PGF2 [22]. However, oxytocin-stimulated myometrial cell prostaglandin production has not been reported for the mare. In mares susceptible to postmating endometritis, lower levels of prostaglandin metabolites were measured systemically following in vivo oxytocin release, as well as after exogenous oxytocin treatment, compared to levels in reproductively normal mares [23]. Therefore, in the present study, it is possible that reduced oxytocin-stimulated myometrial PGF2 production contributed to the reduced contractile response of oxytocin-stimulated myometrium from OS mares.
It has been suggested that structural changes within the uterus of mares with delayed uterine clearance may result from repeated, prolonged stretching of the myometrium during pregnancy. De Lille et al. [24] reported that compared to normal mares, mares with delayed uterine clearance responded to oxytocin treatment with enhanced intrauterine pressure, measured in vivo, if they had been pretreated with sedatives that activate -adrenergic receptors on the myometrium. They suggested that denervation supersensitivity of the myometrium may result from damage to uterine nerve fibers caused by repeated, prolonged stretching of the uterus during pregnancy. Other structural changes that have been reported to occur within the uterus with successive pregnancies include increased myometrial collagen and elastin content in the nongravid human uterus [25]. In guinea pigs, an increase in elastic tissue of the blood vessels within the intermuscular layers of the myometrium [26], which may alter uterine blood flow, has been reported. Changes in uterine blood flow and structure of the uterine vasculature or nerve supply were not addressed in the present study, but parity did not appear to have a role in the defective myometrial contractility of OS mares, because the number of previous pregnancies were similar for OS and ON mares.
In other species, an increase in collagen deposition within the endometrium and myometrium is associated with advancing age and may interfere with uterine function [27, 28]. Delayed uterine clearance is associated with older broodmares [7], and increased collagen deposition within the endometrium of older mares also is well documented [12]. The most abundant collagen isoforms found in uterine muscle include type I collagen, which is thought to provide structural stability, and type III collagen, the relative proportion of which is thought to correlate with tissue pliability [29, 30]. Theoretically, increased myometrial collagen or altered collagen isoform ratios may stiffen the extracellular matrix of the myometrium and reduce the effectiveness of myometrial cells to tension generation. We examined whether increased age caused a decrease in myometrial function, but the similar KCl response of uterine muscle isolated from YN and ON mares indicates that the intrinsic capability of the uterine muscle to generate tension was equivalent (Figs. 3A and 4A). Therefore, increased age is not necessarily associated with the decreased intrinsic contractility seen in myometrium from OS mares.
Although nonreceptor-mediated contractility of circular muscle was similar for YN and ON mares, circular muscle from YN mares failed to generate as much tension to high concentrations of the receptor-mediated agonists oxytocin and PGF2 (Fig. 4, B and C) as myometrium from ON mares. Because both oxytocin and PGF2 activate plasma membrane receptors, it is reasonable to speculate that uterine muscle from reproductively naive, young mares contains fewer plasma membrane receptors than uterine muscle from older, multiparous mares. That hypothesis, however, is inconsistent with similar oxytocin-stimulated intracellular Ca2+ transients in uterine muscle cells from both YN and ON mares. The cell-signaling mechanisms for oxytocin and PGF2-mediated contraction are similar, as shown by simultaneous measurement of tension development and intracellular Ca2+ levels in human myometrial strips [31]. Yet, in primary myometrial cell cultures, subtle differences in postreceptor-second messenger signaling events between these two uterotonins have been demonstrated [32]. The reduced contractile response of myometrium from YN mares, as compared to that of ON mares, would appear to result from a divergent receptor-second messenger signaling event downstream from Ca2+ release. However, whether the same mechanism explains the reduced response of myometrium from YN mares to oxytocin and PGF2 should be investigated in future work.
Myometrial function was evaluated in this study during a time when inflammatory stimuli should have been maximal and a contractile defect was most likely to be evident. A strong inflammatory response peaks from 4 to 24 h after breeding [4]. Furthermore, defective in vivo electrical activity of the myometrium following intrauterine bacterial challenge was greatest between reproductively normal mares and mares susceptible to postbreeding endometritis at 10 and 20 h after uterine inoculation [11]. Our goal was to evaluate uterine muscle function without the compounding effects of uterotonic substances released from the endometrium. In these studies, we cannot rule out the possibility that during the 18 h following breeding and before muscle was harvested, release of endometrial substances, such as nitric oxide [33, 34], prostaglandins [35], and other inflammatory cytokines [36], caused changes within the muscle cell that persisted in the absence of the endometrium. Changes such as suppression of myometrial gap junction numbers or function [37], altered phosphorylation state of contractile proteins, or expression of muscle cell enzymes integral to contractile function [35] may have contributed to the reduced contractile response of the myometrium isolated from OS mares. Because myometrial function was not evaluated before insemination, we cannot evaluate the role of endometrial uterotonins in the contractile defect of myometrium from OS mares in the present study.
In summary, this study demonstrated an intrinsic contractile dysfunction in the myometrium from mares with delayed uterine clearance that was not dependent on age or parity. Furthermore, the contractile defect was not receptor dependent and did not result from altered regulation of intracellular Ca2+ concentration.
ACKNOWLEDGMENTS
The authors wish to thank Ms. Emoke Racz for assisting with the isolation and care of the myometrial cell cultures.
FOOTNOTES
First decision: 20 December 2000.
1 Supported by the Grayson-Jockey Club Research Foundation, the Texas Equine Research Fund, and a USDA-Animal Health Formula grant. Preliminary results from this study were presented at the Annual Conference of the Society for Theriogenology, September 1999, Nashville, TN. 
2 Correspondence. FAX: 979 847 8863; srigby{at}cvm.tamu.edu
Accepted: April 17, 2001.
Received: November 28, 2000.
REFERENCES
release during the estrous cycle and early pregnancy in pony mares. J Reprod Fertil 1997; 109:137-144 release in mares resistant and susceptible to persistent mating-induced endometritis. In: Proceedings of the 7th International Symposium on Equine Reproduction; 1998; Pretoria, South Africa. Abstract 95 . Eur J Endocrinol 1995; 133:467-474
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