Insulin-like growth factors and their binding proteins define specific phases of myometrial differentiation during pregnancy in the rat

While the insulin-like growth factor (IGF) system is known to regulate uterine function during the estrous cycle, there are limited data on its role in myometrial growth and development during pregnancy. To address this issue, we defined the expression of the Igf hormones (1 and 2), their binding proteins (Igfbp 1-6), and Igf1r receptor genes in pregnant, laboring, and postpartum rat myometrium by real-time PCR. IGF family genes were differentially expressed throughout gestation. Igf1 and Igfbp1 mRNA levels were upregulated during proliferative phase (Days 6-12) of rat gestation. Igfbp3 gene expression also was elevated in proliferating smooth muscle cells (SMCs) and was highest at the time of transition between proliferative and synthetic phases (Days 12-15). Igfbp6 gene expression profile paralleled plasma progesterone (P4) concentrations, peaking during the synthetic phase (Days 17-19) and decreasing thereafter. Administration of P4 at late pregnancy (starting from Day 20) to maintain elevated plasma P4 concentrations blocked the onset of labor and prevented the fall in Igfbp6 mRNA levels. In contrast, the treatment of pregnant rats with the P4 receptor antagonist RU486 on Day 19 induced preterm labor and the premature decrease of Igfbp6 gene expression. Igfbp2 gene expression was transiently upregulated during the contractile phase of gestation (Days 21-23) solely in the gravid horn of unilaterally pregnant rats, but it was not affected in P4- or RU486-treated animals, supporting a role for mechanical stretch imposed by the growing fetuses. Igfbp5 gene was induced during postpartum involution. Our results suggest the importance of the IGF system in phenotypic and functional changes of myometrial SMCs throughout gestation in preparation for labor.     

Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: II. Effects of DES on development

The effects of diethylstilbestrol (DES) treatment on myometrial development from the prenatal to adult period were examined in rats and mice by histologic and immunocytochemical methods using anti-actin, -vimentin, and -laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells, and by morphometric procedures to assess quantitatively the effect of DES on the expression of cellular orientation in the emerging inner circular myometrial layer. Neonatal rats and mice were treated with DES from day 0 (day of birth) to day 2 with dosages known to perturb myometrial development. Neonatal treatment with DES increased the degree of circular orientation within the uterine mesenchyme, an effect detectable following as little as 24 hr of DES treatment. This effect on spatial organization of the mesenchyme was followed by an increase in the thickness of the actin-positive middle layer (prospective circular myometrium) of uterine mesenchyme during days 3-15; from day 15 onward, however, the circular myometrial layer began to fragment into irregular bundles of smooth muscle, and the longitudinal myometrial layer became thinner and more irregularly organized than controls. Vimentin localization in rats treated with DES neonatally was more intense than in controls within the circularly orientated uterine mesenchyme at 5 days. By 60 days the circular and longitudinal myometrial layers of DES-treated animals showed strands and bundles of vimentin-positive cells, which were not present in controls. Both rats and mice show comparable effects of DES treatment.     

Localization and regulation of IL-1alpha in rat myometrium during late pregnancy and the postpartum period

Interleukin-1 (IL-1) has been implicated as a participant in preterm labor that is induced by bacterial infection. Previously, we showed that serotonin-induced production of IL-1alpha by myometrial smooth muscle cells in vitro is also essential for the synthesis of interstitial collagenase. It is therefore likely that IL-1alpha production in uterine tissues has implications for both the normal physiology of involution and for the pathophysiological mechanisms of preterm labor. The objective of this study was to characterize the serotonin-induced production of IL-1alpha by myometrial cultures in vitro and to assess the production of IL-1alpha and its relationship to collagenase production in vivo during pregnancy and the postpartum period. Immunohistochemistry demonstrated IL-1alpha protein in the nuclei and cytoplasm of serotonin-treated myometrial cells. IL-1alpha levels were decreased by treatment with progesterone or IL-1-receptor antagonist but were unaffected by lipopolysaccharide. Western analysis of myometrium from pregnant rats showed low levels of IL-1alpha during midpregnancy with increased concentrations at days 21 and 22 and postpartum. IL-1alpha mRNA levels also increased from days 15 to 22. Levels of mRNA for IL-1beta also increased, although to a lesser degree than IL-1alpha. Both mRNAs decreased postpartum. Conversely, mRNA for interstitial collagenase was barely detectable at term but increased postpartum. Together, these data show that serotonin stimulates IL-1alpha production in vitro and indicate that normal myometrium from pregnant rats is an identifiable source of IL-1 during late pregnancy. The findings are consistent with the possibility that myometrial IL-1alpha participates in normal labor as well as the postpartum production of interstitial collagenase.     

Effects of space flight on GLUT-4 content in rat plantaris muscle

 The effects of 14 days of space flight on the glucose transporter protein (GLUT-4) were studied in the plantaris muscle of growing 9-week-old, male Sprague Dawley rats. The rats were randomly separated into five groups: pre-flight vivarium ground controls (PF-VC) sacrificed approximately 2 h after launch; flight groups sacrificed either approximately 5 h (F-R0) or 9 days (F-R9) after the return from space; and synchronous ground controls (SC-R0 and SC-R9) sacrificed at the same time as the respective flight groups. The flight groups F-R0 and F-R9 were exposed to micro-gravity for 14 days in the Spacelab module located in the cargo bay of the shuttle transport system – 58 of the manned Space Shuttle for the NASA mission named ”Spacelab Life Sciences 2”. Body weight and plantaris weight of SC-R0 and F-R0 were significantly higher than those of PF-VC. Neither body weight nor plantaris muscle weight in either group had changed 9 days after the return from space. As a result, body weight and plantaris muscle weight did not differ between the flight and synchronous control groups at any of the time points investigated. The GLUT-4 content (cpm/μg membrane protein) in the plantaris muscle did not show any significant change in response to 14 days of space flight or 9 days after return. Similarly, citrate synthase activity did not change during the course of the space flight or the recovery period. These results suggest that 14 days of space flight does not affect muscle mass or GLUT-4 content of the fast-twitch plantaris muscle in the rat. Received: 25 March 1997 / Accepted: 18 August 1997     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.     

Myometrial oxytocin receptors and prostaglandin in the parturition process in the rat.

Parturition in rats is associated with an abrupt and marked increase in myometrial oxytocin (OT) receptor concentrations. In this study, we investigated the role of myometrial OT receptors in the initiation and the process of parturition. We produced chronic OT receptor blockade during the last 3 days of gestation by administration of a specific OT antagonist at 100 micrograms/day and 300 micrograms/day. We also suppressed OT receptor formation by inhibiting prostaglandin synthesis with naproxen sodium at 2 mg/day and 5 mg/day. We found that chronic blockade of OT receptors inhibited the uterotonic response to OT in Day 22 and Day 23 pregnant rats in a dose-dependent manner. OT antagonist treatment did not prolong the gestation period. However, the duration of parturition, fetal mortality, and the mortality incidence were increased in rats treated with the high dose of the OT antagonist compared to controls. Naproxen sodium at both dosage levels prolonged gestation by 24 h or longer, doubled the duration of parturition, and markedly increased fetal mortality and mortality incidence. Combined OT antagonist and naproxen treatment produced adverse outcomes similar to that produced by naproxen treatment alone. Myometrial OT receptor concentrations were markedly increased in all rats immediately postpartum, ranging from 210 to 425 fmol/mg protein compared to the 50 to 100 fmol/mg found in Day 21 and Day 22 pregnant rats. Correlation analyses between OT receptor concentrations and various parameters associated with gestation and parturition showed that there was a correlation between low OT receptor concentrations and long gestation period, prolonged parturition, and high fetal mortality rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of simulated high altitude (3800 m) on reproductive function in the pregnant rat.

Ovarian appearance, uterine contents (including numbers of fetuses, size and weight), and levels of gonadotropic hormones were studied in pregnant rats exposed to simulated altitude (3800 m). Comparisons were made at 11, 20 and 21 days of gestation between sea level controls rats bred at sea level and placed at reduced barometric pressure on confirmation of pregnancy (SHA). Although fewer fetuses were present in SHA rats on day 11 of gestation, litter size at days 20 and 21 was similar to controls. Body weight and length of SHA fetuses were significantly reduced by day 21 of gestation while placental weights remained similar to controls. The follicle-stimulating hormone (FSH) content in the pituitaries of SHA rats was significantly reduced at days 11 and 21 of pregnancy as compared with controls; luteinizing hormone (LH) content was lower at day 20 than at day 11 among SHA rats. Plasma gonadotropin levels were similar between SHA and control rats all all three stages of gestation. It is suggested that, despite lack of placental adaptation and altered endocrine functions, exposure of rats to hypoxic conditions only during gestation permits better maintenance of pregnancy than reported in rats with longer exposure to high altitude.     

Myometrial apoptosis: activation of the caspase cascade in the pregnant rat myometrium at midgestation

In the present study, we determined the contribution of myometrial hyperplasia, hypertrophy, and apoptosis to uterine growth during pregnancy. The changes in two endogenous markers of cell replication, proliferating cell nuclear antigen (PCNA) protein expression and bromodeoxyuridine (BrdU) incorporation, were studied. Myocyte hypertrophy was assessed by measuring the protein:DNA ratio. The expression levels of antiapoptotic regulatory proteins (BCL2 and BCL2L1) and enzymes involved in apoptosis (caspases 3, 6, 7, 9, and 10) were assessed by immunoblotting throughout gestation and postpartum. Myometrial cell apoptosis was determined by TUNEL staining and DNA fragmentation assays. Both BrdU incorporation and PCNA labeling were elevated in early pregnant myometrium and decreased dramatically after midgestation, with a simultaneous increase in cellular hypertrophy. Levels of BCL2 were high during early gestation, followed by significantly elevated levels of BCL2L1 at midgestation. The expression of caspase 10 in myometrial samples declined from a high nonpregnant level to a complete loss at early gestation. The cleaved forms of caspases (CC) 3, 6, 7, and 9, as well as poly(ADP-ribose)polymerase-1, were undetectable in the myometrial samples at early or late gestation but were transiently elevated at midgestation. Immunohistochemical staining of CC3 confirmed the activation of the caspase cascade, but TUNEL-positive staining or the increase in DNA fragmentation was not detected. Collectively, two distinct phases of myometrial growth were observed: myocyte hyperplasia associated with an increase in antiapoptotic proteins during the first half of gestation, and cellular hypertrophy during the second part of gestation. The transition between these phases was associated with transient activation of the caspase cascade that triggered the differentiation of uterine smooth muscle.     

Dense-cored membrane structures in rat myometrial smooth muscle cells: increase in number during parturition

Membrane-bound structures containing electron-dense material were found in the cytoplasm of the smooth muscle cells of rat myometrium. Most of these structures were observed as membrane-bound vesicles with a variable diameter in the range of 50-90 nm. Some of these were present underneath the cell membranes among the clusters of caveolae and some were found deep in the interior of the smooth muscle cell. The number of such structures progressively increased with gestation reaching the maximum during parturition and decreasing to a minimal level 2 days postpartum, suggesting that the occurrence of these structures is of physiological relevance in parturition. Ovariectomy of the pregnant rats had no influence upon the temporal distribution of these structures within the gestation period, indicating that hormones of ovarian origin are not responsible for the maintenance of these structures during their disappearance after parturition.     

Regulation of calcitonin gene-related peptide receptors in the rat uterus during pregnancy and labor and by progesterone.

Calcitonin gene-related peptide (CGRP) is a potent smooth muscle relaxant in a variety of tissues. We recently demonstrated that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine (125)I-CGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We found that (125)I-CGRP binding to membrane preparations from uteri was elevated during pregnancy and decreased during labor and postpartum. Changes in immunoreactive CGRP receptors were similar to the changes in (125)I-CGRP binding in these tissues, suggesting pregnancy-dependent regulation of CGRP receptor protein. CGRP receptors were elevated by Day 7 of gestation, and a precipitous decrease in these receptors occurred on Day 22 of gestation prior to the onset of labor. Both (125)I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells. Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17beta. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day 22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by (125)I-CGRP-binding, Western immunoblotting, and immunolocalization methods. These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased at term. These receptors are localized to the myometrial cells, and progesterone is required for maintaining CGRP receptors in the rat uterus. Thus, the inhibitory effects of CGRP on uterine contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy.     

Effects of 17 beta-estradiol on myometrial gap junctions and pregnancy in the rat

The effects of estradiol treatment on the development of myometrial gap junctions and premature labour were investigated using timed pregnant rats. In control animals myometrial gap junctions were infrequent between days 17 and 20 of pregnancy, but began to develop on day 21 and were at maximum frequency, size, and membrane area on day 22 during delivery. Gap junctions were completely absent from the myometrium 48 h after delivery. Animals treated with 500 micrograms 17 beta-estradiol/day starting on day 16 of pregnancy developed numerous myometrial gap junctions and delivered their pups prematurely on day 19. Similarly, treatment with 50 micrograms estradiol/day resulted in the development of myometrial gap junctions on day 20 of pregnancy and premature labour. However, treatment with various doses of estradiol up to and including 500 micrograms/day for 3 days beginning 1 day before delivery was not able to maintain the presence of myometrial gap junctions during the postpartum period. These results support the hypothesis that estradiol stimulates the development of myometrial gap junctions and that the presence of gap junctions in the myometrium is a requirement for the occurrence of term, as well as preterm labour. Furthermore, it is evident from this study that the postpartum regression of myometrial gap junctions is not dependent on the decrease in estradiol.     

Role of myometrial constriction in the induction of wavy ribs in rat fetuses

Furosemide, a loop diuretic, was orally administered to pregnant Crj: CD (SD) rats at a dose of 300 mg/kg once on day 16 of gestation. Cartilage bone double staining of day 17 fetuses revealed a delay in ossification. Characteristic rib deformities were not seen until day 18 of gestation. Concomitant with this observation were reductions in the amount of amniotic fluid, compared to controls, on day 18 of gestation and thereafter. When isoxsuprine HCl (2 mg/kg, b.i.d.), a uterine muscle relaxant, was additionally injected ip to pregnant rats on days 17-19 of gestation, the incidence of wavy ribs was significantly (P less than or equal to .05) less than that seen after treatment with furosemide alone. These observations indicate that the myometrial constriction plays a significant role in the induction of furosemide-induced wavy ribs.     

Effects of antiprogesterones on myometrial cell-to-cell coupling in pregnant guinea pigs.

We used intracellular microelectrodes to investigate the effects of the antiprogesterone (AP) compounds RU 486 and ZK 299 on cell-to-cell coupling in the guinea pig myometrium during pregnancy. The input resistance (Ro) of myometrial cells was high in nonpregnant tissues (44.6 +/- 6.39 M omega), but decreased by midgestation (Day 44 or 45 of gestation; 22.9 +/- 3.17 M omega), and was lowest at term (between 17.7 +/- 2.90 m omega and 13.1 +/- 4.34 M omega on Days 59-69). Treatment with the AP RU 486 or ZK 299 in three groups of midgestational animals reduced Ro to a similar level within 24 h. Lucifer Yellow (LY) was injected into smooth muscle cells as a direct but qualitative measure of metabolic coupling. In term and AP-treated animals, LY spread rapidly to neighboring cells within 60 sec, but little spread occurred in midgestational control tissues and no spread was seen even after 10 min in nonpregnant tissues. This correlation of decreased Ro (implying increased electrical coupling) with the development of extensive spread of LY indicates increased electrical and metabolical coupling between myometrial cells during labor. These data show that myometrial smooth muscle cells of guinea pigs are moderately well coupled before the onset of labor, and the coupling increases further, just prior to spontaneous delivery or due to treatment with APs. These events may be required for synchronizing and coordinating the electrical, metabolic, and contractile activity of labor.     

Effects of space flight on the immunohistochemical demonstration of connexin 26 and connexin 43 in the postpartum uterus of rats

The effect of space flight in a National Aeronautics and Space Administration shuttle was studied in pregnant rats. Rats were launched on day 11 of gestation and recovered on day 20 of gestation. Pregnancy was allowed to proceed to term and rats delivered vaginally on days 22-23, although flight animals required more labour contractions to complete the delivery process. Pups were placed with foster dams and connexin 26 and 43 were examined in the uterus of flight animals approximately 3 h after delivery. Space flight did not affect uterine connexin 26, localized primarily in epithelial cells of the endometrium, but decreased connexin 43, the major gap junction protein in the myometrium. It is suggested that decreased connexin 43 alters synchronization and coordination of labour contractions, resulting in a requirement for more contractions to complete the delivery process.     

Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: I. Normal development

Myometrial development from the prenatal to adult period was examined in rats and mice 1) by histologic and immunocytochemical methods with anti-actin, -vimentin, and -laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells; and 2) by morphometric procedures to assess quantitatively the expression of cellular orientation in the emerging inner circular myometrial layer. Uterine mesenchymal cells initially were uniformly vimentin-positive, undifferentiated, and randomly oriented during the late fetal period. By the early neonatal period, three mesenchymal layers became recognizable histologically, the middle one of which (prospective circular myometrium) developed distinct circular orientation and differentiated into a layer composed of actin-positive smooth muscle cells. The cells of the inner mesenchymal layer initially exhibited radial orientation. By 10 days postpartum, the outer longitudinal mesenchymal layer differentiated into bundles of smooth muscle cells representing the longitudinal myometrium. The inner mesenchymal layer remained vimentin-positive and differentiated into the randomly ordered endometrial stroma. The cells of the middle and outer mesenchymal layers that were destined to form myometrium initially expressed vimentin throughout and then coexpressed vimentin and actin, but with time vimentin staining disappeared in the maturing smooth muscle cells as they expressed actin.     

Muscle volume, MRI relaxation times (T2), and body composition after spaceflight.

Postflight changes in muscle volume, calf muscle transverse relaxation time, and total body composition were measured in 4 crewmembers after a 17-day mission and in 14-16 crewmembers in multiple shuttle/Mir missions of 16- to 28-wk duration. During the 17-day mission, all muscle regions except the hamstrings significantly decreased 3-10% compared with baseline. During the shuttle/Mir missions, there were significant decreases in muscle volume (5-17%) in all muscle groups except the neck. These changes, which reached a new steady state by 4 mo of flight or less, were reversed within 30-60 days after landing. Postflight swelling and elevation of calf muscle transverse relaxation time persisted for several weeks after flight, which suggests possible muscle damage. In contrast to the 17-day flight, in which loss in fat, but not lean body mass, was found (25), losses in bone mineral content and lean body mass, but not fat, were seen after the longer shuttle/Mir missions. The percent losses in total body lean body mass and bone mineral content were similar at approximately 3.4-3.5%, whereas the pelvis demonstrated the largest regional bone loss at 13%.     

Expression and localization of the contractile prostaglandin F receptor in pregnant rat myometrium in late gestation, labor, and postpartum

A polyclonal antibody was raised against amino acids 7-18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to alpha-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.     

Differences in the gestational pattern of mRNA expression of the Rnd family in rat and human myometria

Uterine myometrial contractility remains a poorly characterized area of research in reproductive physiology. Rnd1, a novel member of the GTP-binding Rho protein family, inhibits Ca(2+)-sensitization by specifically interfering with a RhoA/Rho-activated kinases-dependent mechanism in smooth muscle. In addition to Rnd1, there are two other members, Rnd2 and Rnd3, in the Rnd family of Rho proteins. In the present comparative study of myometrial contractility in rats and humans, we found that all three Rnd mRNAs were expressed in nonpregnant rat myometrium and in nonpregnant human myometrial tissues. Although all three mRNA levels increased significantly after gestation in rat myometria, only Rnd1 expression was significantly greater after gestation in human samples. In the ovariectomized rat, administration of estrogen and/or progesterone increased the expression of all Rnd mRNAs. These results suggest that universal Rnd family up-regulation during pregnancy in rats may have an important role for negative-feedback control of uterine contraction during gestation by inhibiting RhoA-mediated increase in Ca(2+) sensitivity of contractile elements. Such increases in Rnd levels may be due to augmented levels of reproductive steroids in rats. Our data also point to gestational differences between rats and humans in Rnd isoform patterns.     

Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy

Regulation of voltage-gated K(+) (K(v)) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating K(v) current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitation followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.