The pregnant myometrium is epigenetically activated at contractility-driving gene loci prior to the onset of labor in mice.

During gestation, uterine smooth muscle cells transition from a state of quiescence to one of contractility, but the molecular mechanisms underlying this transition at a genomic level are not well-known. To better understand these events, we evaluated the epigenetic landscape of the mouse myometrium during the pregnant, laboring, and postpartum stages. We generated gestational time point-specific enrichment profiles for histone H3 acetylation on lysine residue 27 (H3K27ac), histone H3 trimethylation of lysine residue 4 (H3K4me3), and RNA polymerase II (RNAPII) occupancy by chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), as well as gene expression profiles by total RNA-sequencing (RNA-seq). Our findings reveal that 533 genes, including known contractility-driving genes (Gap junction alpha 1 [Gja1], FBJ osteosarcoma oncogene [Fos], Fos-like antigen 2 [Fosl2], Oxytocin receptor [Oxtr], and Prostaglandin G/H synthase 2 (Ptgs2), for example), are up-regulated at day 19 during active labor because of an increase in transcription at gene bodies. Labor-associated promoters and putative intergenic enhancers, however, are epigenetically activated as early as day 15, by which point the majority of genome-wide H3K27ac or H3K4me3 peaks present in term laboring tissue is already established. Despite this early exhibited histone signature, increased noncoding enhancer RNA (eRNA) production at putative intergenic enhancers and recruitment of RNAPII to the gene bodies of labor-associated loci were detected only during labor. Our findings indicate that epigenetic activation of the myometrial genome precedes active labor by at least 4 days in the mouse model, suggesting that the myometrium is poised for rapid activation of contraction-associated genes in order to exit the state of quiescence.

The selective progesterone receptor modulator-promegestone-delays term parturition and prevents systemic inflammation-mediated preterm birth in mice.

BACKGROUND: Progesterone, acting via its nuclear receptors called progesterone receptors, promotes myometrial relaxation during pregnancy, and suspension of this activity triggers labor. We previously found that 20α-hydroxysteroid dehydrogenase causes a local withdrawal of progesterone in the term and preterm myometrium by converting the progesterone into an inactive form before it accesses the progesterone receptors. OBJECTIVE: We hypothesized that a selective progesterone receptor modulator called promegestone, which is not metabolized by 20α-hydroxysteroid dehydrogenase, would sustain progesterone receptor signaling and prevent/delay term labor and preterm labor in mice. STUDY DESIGN: In the term labor mouse model, promegestone (0.2 mg/dam) or a vehicle were administered subcutaneously in timed-pregnant CD-1 mice at gestational days 15, 16, and 17 (term gestational days, 19.5). In the inflammation preterm labor model, pregnant mice received promegestone or a vehicle on gestational days 15, 16, and 17, which was 24 hours before, immediately before, and 24 hours after systemic bacterial endotoxin (50 µg intraperitoneal; lipopolysaccharide group) or vehicle (saline) administration. The maternal and fetal tissues were collected on gestational day 16 6 hours after lipopolysaccharide±promegestone injection and at term gestational day 18.75. The protein levels of 10 cytokines were measured by multiplex immunoassay in maternal plasma and amniotic fluid. Myometrial, decidual, and placental messenger RNA levels of multiple cytokines and procontractile proteins were evaluated by real-time polymerase chain reaction and confirmed by immunoblotting. RESULTS: Promegestone prevented term labor and maintained mice pregnancy postterm >24 hours. The litter size and fetal weights were not different from the controls. Promegestone prevented systemic bacterial-endotoxin-induced preterm labor in 100% of the mice, blocked uterine contractions, significantly inhibited all systemic inflammation-induced myometrial cytokines, and partially inhibited decidual and placental inflammation. Promegestone did not prevent bacterial-endotoxin-induced fetal toxicity. CONCLUSION: Promegestone a selective progesterone receptor modulator that binds progesterone receptors with high affinity and is not metabolized by 20α-hydroxysteroid dehydrogenase could completely suppress term parturition and systemic bacterial-endotoxin-induced preterm birth in mice. We suggest that such selective progesterone receptor modulators may represent a potential therapeutic approach to the prevention of preterm labor in women at high risk of preterm birth.

Pro-inflammatory signals induce 20α-HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal.

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α-HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection-induced) and associated activation of transcription factors: NF-кB and AP-1, common to term and preterm labour. We found that 20α-HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro-inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12-O-tetradecanoylphorbol-13-acetate (TPA, mimicking inflammation), induced 20α-HSD gene expression and increased 20α-HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up-regulation of GJA1 in the hTERT-HM cells. The NF-кB /AP-1 transcription factors mediated effects of LPS and TPA on 20α-HSD gene transcription. Both pro-inflammatory stimuli induced 20α-HSD promoter activity in LPS/TPA-treated cells which was significantly attenuated by inhibition of NF-кB (JSH: 20 µM) or AP-1 signalling (T5224: 10 µM). Deletion of NF-кB consensus sites abrogated LPS-mediated promoter induction, while removal of AP-1 sites reversed the TPA-mediated induction of 20α-HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF-кB or AP-1 induce 20α-HSD transcription and subsequent local P4 withdrawal resulting in up-regulation of GJA1 and activation of myometrium that precedes labour.

Differential expression of myometrial AP-1 proteins during gestation and labour.

Preterm labour (PTL) is a leading cause of perinatal mortality and postnatal morbidity. Contractions of the uterine muscle (myometrium) that determine the onset of labour depend on the expression of contraction-associated proteins (CAPs, i.e. connexin43) regulated by dimeric AP-1 transcription factors. Here, we examined subcellular (by immunoblotting) and tissue expression (by immunohistochemistry) of myometrial AP-1 proteins (cJUN, JUNB, JUND, cFOS, FOSB, FRA1, FRA2) throughout gestation and TL in different species (mouse, rat and human). To identify the critical AP-1 members associated with preterm birth, we studied their expression in mouse model of 'infectious' (LPS-induced) and 'sterile' (RU486-induced) PTL. We found that (1) myometrial AP-1 composition is preserved in vivo between different species (rodents and human) indicating that Fos/Jun heterodimer (i.e. FRA2/JUND) may be indispensable for labour initiation. (2) Our in vivo study using murine models of gestation shows that there is a similarity in the myometrial AP-1 protein composition during TL and pathological PTL of different aetiology suggesting the involvement of similar molecular machinery in the induction of labour. (3) This study is first comprehensive protein analysis of seven AP-1 members in human labouring versus non-labouring myometrium, showing their cellular expression and tissue distribution in relation to labour status.

Inhibition of infection-mediated preterm birth by administration of broad spectrum chemokine inhibitor in mice.

Preterm birth (PTB) is the single most important cause of perinatal and infant mortality worldwide. Maternal infection can result in PTB. We investigated the ability of a Broad Spectrum Chemokine Inhibitor (BSCI) to prevent infection-induced PTB in mice. PTB was initiated in pregnant mice by intraperitoneal injection of lipopolysaccharide (LPS; 50 µg). Half the mice received BSCI (10 mg/kg) 24 hrs prior to and immediately before LPS administration. The impact of LPS alone or LPS plus BSCI was assessed on (i) injection-to-delivery interval, foetal survival rate, placental and neonates' weight; (ii) amniotic fluid and maternal plasma cytokine levels (by Luminex assay); foetal and maternal tissue cytokine gene expression levels (by Real-Time RT-PCR); (iii) immune cells infiltration into the uterine tissue (by stereological immunohistochemistry). Pre-treatment with BSCI (i) decreased LPS-induced PTB (64% versus 100%, P < 0.05); (ii) significantly attenuated cytokine/chemokine expression in maternal tissues (plasma, liver, myometrium, decidua); (iii) significantly decreased neutrophil infiltration in the mouse myometrium. BSCI-treated mice in which PTB was delayed till term had live foetuses with normal placental and foetal weight. BSCI represents a promising new class of therapeutics for PTB. In a mouse model of preterm labour, BCSI suppresses systemic inflammation in maternal tissues which resulted in the reduced incidence of LPS-mediated PTB. These data provide support for efforts to target inflammatory responses as a means of preventing PTB.

SOX4 exerts contrasting regulatory effects on labor-associated gene promoters in myometrial cells.

The uterine muscular layer, or myometrium, undergoes profound changes in global gene expression during its progression from a quiescent state during pregnancy to a contractile state at the onset of labor. In this study, we investigate the role of SOX family transcription factors in myometrial cells and provide evidence for the role of SOX4 in regulating labor-associated genes. We show that Sox4 has elevated expression in the murine myometrium during a term laboring process and in two mouse models of preterm labor. Additionally, SOX4 differentially affects labor-associated gene promoter activity in cooperation with activator protein 1 (AP-1) dimers. SOX4 exerted no effect on the Gja1 promoter; a JUND-specific activation effect at the Fos promoter; a positive activation effect on the Mmp11 promoter with the AP-1 dimers; and surprisingly, we noted that the reporter expression of the Ptgs2 promoter in the presence of JUND and FOSL2 was repressed by the addition of SOX4. Our data indicate SOX4 may play a diverse role in regulating gene expression in the laboring myometrium in cooperation with AP-1 factors. This study enhances our current understanding of the regulatory network that governs the transcriptional changes associated with the onset of labor and highlights a new molecular player that may contribute to the labor transcriptional program.

Myometrial immune cells contribute to term parturition, preterm labour and post-partum involution in mice.

This study aimed to determine the mechanism of uterine activation during labour, both term (TL) and preterm (PTL). We hypothesized that the peripheral leucocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of parturition. Mouse uteri were collected (i) during gestation, TL and post-partum (PP), (ii) during PTL initiated by intrauterine infusion of LPS (125 µg) or (iii) injection of the progesterone receptor antagonist RU486 and analysed for multiple cytokine expression levels by real-time polymerase chain reaction (RT-PCR) and 23-plex Cytokine assay or enzymatically dispersed for assessment of immune cell populations. Markers of myeloid cell differentiation (Gr1, Neu7/4 and F4/80) were evaluated by FACS to define tissue macrophages (Macs), monocytes (M) and neutrophils (N) and by immunohistochemistry to detect tissue Macs and N. Our results indicate that: (1) Macs were elevated in mouse myometrium before TL (P < 0.05) followed by an increase in M and N; these changes were accompanied by an increase in multiple pro-inflammatory cytokines/chemokines genes. The expression of corresponding proteins increased PP. (2) TL and RU486-PTL models showed similar gene/protein expression profiles, (3) LPS-PTL was characterized by strong pro-inflammatory response and massive influx of N in myometrial tissues showing a pattern different from TL and RU486-PTL, (4) The PP period appears similar in all three models, with elevated myometrial cytokine levels and high infiltration of immune cells. We concluded that leucocytes infiltrate myometrium around the time of parturition implicating their potential role in labour activation (both term and preterm) and major role in PP uterine involution.

Infiltration of myeloid cells into decidua is a critical early event in the labour cascade and post-partum uterine remodelling.

Leucocyte infiltration in the decidua (maternal-foetal interface) before, during and after term (TL) and preterm labour (PTL) was studied in mouse. We also investigated the mechanism of peripheral leucocyte recruitment into decidua by analysing the tissue cytokine profiles. Decidual tissues were collected during late gestation, TL and post-partum (PP). PTL was initiated on gestational day 15 by intrauterine injection of Lipopolysaccharide (LPS, 125 µg) or progesterone signalling antagonism by RU486. Animals were killed during PTL or PP. Decidua basalis was analysed using FACS and immunohistochemistry. Markers of myeloid cell differentiation (Gr1, Ly6G, Neu7/4, F4/80) were assessed to define tissue monocytes (M), neutrophils (N) and macrophages (Macs). Flow cytometry revealed a significant (P < 0.05) increase in decidual Macs prior to TL; M and N numbers increased during TL and further increased during PP, which correlated with immunohistochemistry data. Massive influx of N, but not Macs and M, was detected by FACS during LPS-PTL (P < 0.05) but not RU486-PTL. Highest levels of N infiltration into the decidua occurred PP in both LPS and RU486 groups. Decidual infiltration during TL and RU486-PTL was accompanied by an increase in pro-inflammatory cytokines (IL1b and IL6) and CCL2 chemokine; LPS-PTL showed increases in multiple cytokines. PP period following TL and PTL was associated with further up-regulation of multiple cytokines/chemokines (P < 0.05). Our data suggest a programme of myeloid cells involvement in parturition with the pre-partum influx of Macs into the decidua contributing to the progression of labour, whereas the later influx of M and N contribute to PP decidual involution.

MYB and ELF3 differentially modulate labor-inducing gene expression in myometrial cells.

Spontaneous uterine contractions are initiated when smooth muscle cells (SMCs) within the uterine muscle, or myometrium, transition from a functionally dormant to an actively contractile phenotype at the end of the pregnancy period. We know that this process is accompanied by gestational time point-specific differences in the SMC transcriptome, which can be modulated by the activator protein 1 (AP-1), nuclear factor kappa beta (NF-κß), estrogen receptor (ER), and progesterone receptor (PR) transcription factors. Less is known, however, about the additional proteins that might assist these factors in conferring the transcriptional changes observed at labor onset. Here, we present functional evidence for the roles of two proteins previously understudied in the SMC context-MYB and ELF3-which can contribute to the regulation of labor-driving gene transcription. We show that the MYB and ELF3 genes exhibit elevated transcript expression levels in mouse and human myometrial tissues during spontaneous term labor. The expression of both genes was also significantly increased in mouse myometrium during preterm labor induced by the progesterone antagonist mifepristone (RU486), but not during infection-simulating preterm labor induced by intrauterine infusion of lipopolysaccharide (LPS). Furthermore, both MYB and ELF3 proteins affect labor-driving gene promoter activity, although in surprisingly opposing ways: Gja1 and Fos promoter activation increases in the presence of MYB and decreases in the presence of ELF3. Collectively, our study adds to the current understanding of the transcription factor network that defines the transcriptomes of SMCs during late gestation and implicates two new players in the control of labor timing.

Combination of histochemical analyses and micro-MRI reveals regional changes of the murine cervix in preparation for labor.

The cervix is responsible for maintaining pregnancy, and its timely remodeling is essential for the proper delivery of a baby. Cervical insufficiency, or "weakness", may lead to preterm birth, which causes infant morbidities and mortalities worldwide. We used a mouse model of pregnancy and term labor, to examine the cervical structure by histology (Masson Trichome and Picrosirius Red staining), immunohistochemistry (Hyaluronic Acid Binding Protein/HABP), and ex-vivo MRI (T2-weighted and diffusion tensor imaging), focusing on two regions of the cervix (i.e., endocervix and ectocervix). Our results show that mouse endocervix has a higher proportion of smooth muscle cells and collagen fibers per area, with more compact tissue structure, than the ectocervix. With advanced gestation, endocervical changes, indicative of impending delivery, are manifested in fewer smooth muscle cells, expansion of the extracellular space, and lower presence of collagen fibers. MRI detected three distinctive zones in pregnant mouse endocervix: (1) inner collagenous layer, (2) middle circular muscular layer, and (3) outer longitudinal muscular layer. Diffusion MRI images detected changes in tissue organization as gestation progressed suggesting the potential application of this technique to non-invasively monitor cervical changes that precede the onset of labor in women at risk for preterm delivery.

Stretch-induced uterine myocyte differentiation during rat pregnancy: involvement of caspase activation.

Proliferation, differentiation, and apoptosis are three major processes by which the pregnant uterus maintains homeostasis to accommodate the growing fetus. We demonstrated previously that caspase activation in the pregnant rat myometrium at midgestation coincides with the transition from uterine hyperplasia to hypertrophy. We hypothesized that this transition was induced by stasis of myometrial blood flow (and subsequent hypoxia/ischaemia insult) resulting from acute myometrial stretch induced by a growing embryo. Therefore, we measured the expression of active caspase 3 and two hypoxia markers (transcription factor HIF1A and pimonidazole hydrochloride) in pregnant rat myometrium. To investigate the effect of gravidity we used unilaterally pregnant rats. Caspase 3 was activated only in the gravid horn of the unilaterally pregnant animals on Gestational Days 12-15. This activation was associated with high levels of HIF1A and pimonidazole immunostaining, which were limited to the circular myometrial layer of the gravid horn, indicative of hypoxia within this tissue. To isolate the effect of myometrial stretch applied by the growing fetus, we inserted an expandable polymer tube (intra-uterine expandable tube [IUET]) into the empty horn of Day 13 and Day 20 unilaterally pregnant rats. Tissue was collected 2, 14, and 24 h later. In the IUET-stretched empty horn, cleaved caspase 3 was activated at midgestation (Day 14), but not at late gestation (Day 21). We speculate that hypoxia resulting from mechanical stretch may activate caspase 3 within the pregnant myometrium only in the context of a specific endocrine environment.

Monocyte chemoattractant protein-1 (CCL-2) integrates mechanical and endocrine signals that mediate term and preterm labor.

Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated positively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone (P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by pretreatment with P4 (1 microM); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2 protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and thus may represent a novel target for therapeutic prevention of preterm labor in humans.

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.

The expression of transforming growth factor beta in pregnant rat myometrium is hormone and stretch dependent.

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.

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.

Expression and localization of alpha-smooth muscle and gamma-actins in the pregnant rat myometrium.

The myometrium undergoes dramatic changes as pregnancy progresses through phases of proliferation, hypertrophy, contractile state and labor. In this study, we showed that the composition of the muscle actin isoforms, a major component of the myometrial contractile apparatus and cytoskeleton, was modified during pregnancy. The expression of smooth muscle alpha-actin (Acta2, which we abbreviate as alpha-SM-actin) and gamma-actin mRNAs and proteins was examined by real-time polymerase chain reaction and Western immunoblot, and was localized with immunohistochemistry, in the nonpregnant, pregnant, and postpartum rat uterus. Both alpha-SM-actin (vascular specific actin isoform) and gamma-actin (predominant in visceral smooth muscle) were detected in the rat myometrium. Myometrial expression of alpha-SM-actin mRNA and protein was high throughout pregnancy. The transcript and protein levels of gamma-actin were increased significantly in the second part of gestation (31.8-fold increase for mRNA and 16.7-fold increase for protein relative to nonpregnant). The localization of gamma-actin was markedly altered during pregnancy. In early gestation, myometria from empty and gravid uterine horns of the unilaterally pregnant rats showed abundant gamma-actin immunostaining in the longitudinal layer but weak staining in the circular layer. Gamma-actin immunostaining increased in only the circular layer of the gravid horn after midgestation and remained low in the empty one. Gamma-actin protein translocated to the membranous region of uterine myocytes at late gestation. The temporal alteration in gamma-actin expression and localization at late gestation suggested that this change in myometrial composition of contractile proteins is important to adequately prepare the myometrium for the development of optimal contractions during labor.