Mouse Myometrium Research Articles

Ovarian Stimulation Affects the Population of Mouse Uterine NK Cells at Early Pregnancy

The aim of this study was to determine the influence of ovarian stimulation on endometrial mouse NK cell population. For superovulation, the female adult NMRI mice were injected i.p. with 10 IU of the pregnant mare serum gonadotropin followed 48 h later by an i.p. injection of 10 IU human chorionic gonadotropin hormone. Ovarian stimulated and nonstimulated mice were mated with fertile male. The presence of vaginal plug proved natural pregnancy, and this day was considered as day one of pregnancy. Tissue samples were prepared from the uterine horn and spleen of both groups of study on 7th day of pregnancy. Serum estradiol-17β and progesterone were measured at the same time. The tissue cryosections were prepared and double stained for CD 161 and CD3 markers, and NK cells population was analyzed. Relative frequency of NK cells was significantly lower in stroma and myometrium in hyperstimulated mice compared with the control group. However, no difference was seen in percentage of NK cells in spleen. The ovarian stimulation influences the proportion of uterine NK cells and may affect the embryo implantation.

A novel mouse model that closely mimics human uterine leiomyomas

To develop a predictive mouse model for uterine fibroids. Human fibroid cells xenografted to immunodeficient mice. University and industrial research center. Immunodeficient scid/beige mice. Subcutaneous and intrauterine injection of fibroid-derived cells, SV40 transformation of primary cells by lentiviral transduction, proliferation determined by immunohistochemistry, FISH. Characterization of primary and immortalized cells by Western blot and soft agar assay, determination of invivo tumorigenicity, comparative histology and immunohistochemistry, fluorescence in situ hybridization. Tumorigenicity of primary myoma cells disappears upon invitro culture. Transformation and immortalization does not restore or conserve the invivo growth potential of cultured cells. Injection of primary cells into myometrium of mice leads to xenografts with a leiomyoma-like histology. Primary myoma cells are suited to generate fibroid-like xenografts for studying pathogenesis without genetic modifications. In contrast, invitro culture abolishes transplantability, and neither transformation nor immortalization is sufficient to restore tumorigenic capacity.

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.

The microRNA (miR)-199a/214 Cluster Mediates Opposing Effects of Progesterone and Estrogen on Uterine Contractility during Pregnancy and Labor

Progesterone (P(4)) and estradiol-17β (E(2)) play critical and opposing roles in regulating myometrial quiescence and contractility during pregnancy and labor. Although these contrasting hormonal effects are likely mediated via differential regulation of inflammatory and contractile genes, the underlying mechanisms remain incompletely understood. Recently we discovered that targets of the microRNA (miR)-200 family, transcription factors zinc finger E-box binding homeobox (ZEB)-1 and ZEB2, serve as P(4)/progesterone receptor-mediated regulators of uterine quiescence during pregnancy. In the present study, we found that levels of the clustered miRNAs, miR-199a-3p and miR-214, were significantly decreased in laboring myometrium of pregnant mice and humans and in an inflammatory mouse model of preterm labor, whereas the miR-199a-3p/miR-214 target, cyclooxygenase-2, a critical enzyme in synthesis of proinflammatory prostaglandins, was coordinately increased. Overexpression of miR-199a-3p and miR-214 in cultured human myometrial cells inhibited cyclooxygenase-2 protein and blocked TNF-α-induced myometrial cell contractility, suggesting their physiological relevance. Notably, E(2) treatment of ovariectomized mice suppressed, whereas P(4) enhanced uterine miR-199a-3p/214 expression. Intriguingly, these opposing hormonal effects were mediated by ZEB1, which is induced by P(4), inhibited by E(2) and activates miR199a/214 transcription. Together, these findings identify miR-199a-3p/miR-214 as important regulators of myometrial contractility and provide new insight into strategies to prevent preterm birth.

Phosphorylation of h1 Calponin by PKC epsilon may contribute to facilitate the contraction of uterine myometrium in mice during pregnancy and labor

BackgroundThe timely onset of powerful uterine contractions during parturition occurs through thick and thin filament interactions, similar to other smooth muscle tissues. Calponin is one of the thin filament proteins. Phosphorylation of calponin induced by PKC-epsilon can promote the contraction of vascular smooth muscle. While the mechanism by which calponin regulates the contraction of pregnant myometrium has rarely been explored. Here, we explore whether PKC-epsilon/h1 calponin pathway contribute to regulation of myometrial contractility and development of parturition.MethodsWe detected the expression of h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon in the different stages of mice during pregnancy and in labor by the method of western blot and recorded the contraction activity of myometrium strips at the 19th day during pregnancy with different treatments by the organ bath experiments.ResultsThe level of the four proteins including h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice. The ratios of phosphorylated h1 calponin/h1 calponin and phosphorylated PKC-epsilon/PKC-epsilon were reached the peak after the onset of labor in myometrium in the mice. After the treatment of more than 10(9-) mol/L Psi-RACK (PKC-epsilon activator), the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1 calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-epsilon. Meanwhile, the change of the ratio of phosphorylated h1 calponin/h1 calponin was consistent with that of contraction force of mice myometrium strips.ConclusionsThese data suggest that in mice myometrium, phosphorylation of h1 calponin induced by the PKC-epsilon might facilitate the contraction of uterine in labor and regulate pregnant myometrial contractility.

MicroRNA-200a serves a key role in the decline of progesterone receptor function leading to term and preterm labor

During pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) activity, but labor is facilitated by a series of events that impair PR function. Previously, we discovered that miR-200 family members serve as progesterone (P(4))-modulated activators of contraction-associated genes in the pregnant uterus. In this study, we identified a unique role for miR-200a to enhance the local metabolism of P(4) in myometrium and, thus, decrease PR function during the progression toward labor. miR-200a exerts this action by direct repression of STAT5b, a transcriptional repressor of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. Notably, overexpression of miR-200a in cultured human myometrial cells (hTERT-HM) suppressed STAT5b and increased 20α-HSD mRNA levels. In uterine tissues of ovariectomized mice injected with P(4), miR-200 expression was significantly decreased, STAT5b expression was up-regulated, and 20α-HSD mRNA was decreased, but in 15 d postcoitum pregnant mice injected with the PR antagonist RU486, preterm labor was associated with increased miR-200a, decreased STAT5b, and enhanced 20α-HSD expression. Taken together, these findings implicate miR-200a as an important regulator of increased local P(4) metabolism in the pregnant uterus near term and provide insight into the importance of miR-200s in the decline in PR function leading to labor.

Cdh1 Is Essential for Uterine Gland Development and Adult Uterine Function.

Epithelial differentiation and growth are key processes in mammalian development, which are central to the formation of many organs. Adherens junctions play a role in the establishment of cell polarity and maintenance of the epithelial phenotype, and are essential for epithelial development including cell differentiation and proliferation. CDH1 (E-cadherin) belongs to the cadherin superfamily of cell adhesion molecules, and forms structures known as adherens junctions that mediate intercellular adhesion through dynamic interactions with CTNNB1 (beta-catenin). The objective of this study is to determine a biological role of Cdh1 on postnatal uterine morphogenesis and adult uterine function. Because global disruption of Cdh1 in mice causes early embryo death, we generated the mice with Cdh1 conditionally deleted in the uterus using the progesterone receptor Cre knockin (Pgr-Cre) model. First, histological analysis was conducted in the uteri of Pgr+/+Cdh1f/f (control) and PgrCre/+Cdh1f/f (mutant) mice. Examination of the 4-5-week old uteri of control and mutant mice showed ablation of uterine glands and abnormal organization of the luminal epithelium (LE) in Cdh1 mutant mice. We did not see any significant differences of uterine size and weight at 5-week old. Further, critical regulators for uterine development (CTNNB1, Wnt4, Wnt5a, Wnt7a, Hoxa10 and Hoxa11), and PGR and ESR1 were not significantly altered by loss of Cdh1 in the uterus, but the expression of Foxa2, a marker of glandular epithelium, was reduced in Cdh1 mutant mice. We also examined whether CDH1 expression was regulated by endocrine disruptors: diethylstilbestrol (DES) or progesterone (P4). Although each model has several different phenotypes, both can lead to multiple patterning defects in the female reproductive tract including loss of endometrial glands and gene expression (Wnt and Hox). The exposure of neonatal mice to either DES or P4 did not affect CDH1 expression in the epithelium even though both hormones disrupted uterine gland development. The uteri of the 6-month old Cdh1 mutant mice showed an increase in uterine diameter. Examination of the uterine histology revealed a larger uterine lumen, thinner stroma, and thicker myometrium in Cdh1 mutant mice. Interestingly, immunohistochemical analysis of cyclin D1 and Ki67 indicated no cell proliferation in the LE, but an increase of stromal cell proliferation in Cdh1 mutant mice. To determine whether Cdh1 has a role in pregnancy, Female control and mutant mice were mated to wild-type male mice for 6 months. We have confirmed the presence of vaginal plugs in both control and Cdh1 mutant mice. During the time of observation, the control mice exhibited normal fecundity, whereas the mutant mice were found to be infertile. Further, we determined effects of ovarian steroid hormone on CDH1 expression in the adult uterus. The ovariectomized mice were given an injection of sesame oil (vehicle control, estradiol-17beta (E), P4, or E+P, subcutaneously. However, CDH1 was not altered by ovarian steroids in the adult mouse uterus. These results suggest that Cdh1 is essential for uterine gland development and adult uterine function. The defective mechanisms of gland development or adult uterine function in Cdh1 mutant mice may be different from those seen with disruption by endocrine disruptors in neonate or with regulation by steroid hormones in adult. Supported by NIH HD058222. (platform)

Cell-specific conditional deletion of Pten in the uterus results in differential phenotypes

Objective Endometrial cancer (EMC) is the most common gynecological malignancy. The etiology and the cell types that are conducive to EMC are not completely understood, provoking further studies. Our objective was to determine whether deletion of Pten specifically in the uterine stroma and myometrium induces cancer or manifests different phenotypes. Methods Pten Amhr2(d/d) mice with conditional deletion of Pten in the mouse uterine stroma and myometrium, but not in the epithelium, were generated by mating floxed Pten mice and anti-Mullerian hormone type 2 receptor ( Amhr2)- Cre mice. The phenotypes were compared between Pten f/f and Pten Amhr2(d/d) uteri. Results We show that conditional deletion of Pten in the mouse uterine stroma and myometrium, but not in the epithelium, fails to generate EMC even at the age of 5 months. Surprisingly Pten deletion by Amhr2-Cre transformed a large number of myometrial cells into adipocytes with lipid accumulation, possibly a result of increased levels of SREBP1 and PPARγ which regulate adipose differentiation. Conclusions These results provide evidence that deletion of Pten specifically in the stroma and myometrium does not result in EMC in female mice examined up to 5 months of age but alters the myocytes to adipocytes and mimics histologic similarities with lipoleiomyomas in humans, raising the possibility of using this mouse model to further explore the cause of the disease.

Altered contribution of RhoA/Rho kinase signaling in contractile activity of myometrium in leptin receptor-deficient mice

In late gestation, enhanced myometrial contractility is mediated in part through increased Rho/Rho kinase. Since leptin, which is elevated in pregnancy and obesity, can directly depress myometrial function, we hypothesized that in leptin receptor-deficient mice, myometrial contractility would be greater in late pregnancy due to increased Rho/Rho kinase activity. To test this, we correlated RhoA and Rho kinase expression to contractility in myometrium from nonpregnant (NP) and late-pregnant (P18) heterozygous leptin receptor-deficient mice (db/+) vs. wild-type (WT) mice. In NP mice, KCl-induced contractions were similar between WT and db/+ myometrium. However, the Rho kinase-dependent component of the contractions was greater in db/+ mice, along with an increased expression of Rho kinase. KCl-induced contractions increased in strength in myometrium from P18 WT and db/+ compared with NP. Although the contribution of Rho kinase to contractions was unchanged in P18 WT mice, it was decreased in P18 db/+ mice. The decrease in Rho kinase-dependent contractions in P18 db/+ mice coincided with reduced RhoA and Rho kinase expression relative to NP db/+. Addition of high-fat-induced abnormal glucose utilization prevented changes in Rho kinase function. We conclude that abnormal leptin signaling increases expression and function of Rho kinase to maintain contractile function in NP myometrium and that during pregnancy the contribution of RhoA and Rho kinase expression to myometrial function is reduced despite an increase in myometrial contractility. Thus, other signaling mechanisms appear to compensate when leptin signaling is reduced to maintain contractile function during pregnancy.

Neuromedin B and Its Receptor Induce Labor Onset and Are Associated with the RELA (NFKB P65)/IL6 Pathway in Pregnant Mice1

Although the neuromedin B receptor (NMBR), a bombesin receptor family member, has been implicated in thermoregulation and in stimulation of both urogenital and gastrointestinal smooth muscle contraction, its underlying role in labor onset and its associated molecular mechanisms remain poorly understood. We examined the relationship between temporal and spatial NMBR expression in the myometrium of pregnant mice and potential mechanistic pathways leading to labor onset. Resultant data indicate that NMBR expression peaked at term and before parturition. Maternal exposure to the NMBR agonist neuromedin B (NMB) shortened the gestational age of pups, an effect that was also observed after oxytocin administration. Both RELA (NFKB P65) DNA-binding activity and interleukin 6 (Il6) mRNA expression were greatest during parturition and after maternal exposure to the highest NMB concentration administered (150 μg/kg). Furthermore, a significant correlation was observed among NMBR mRNA expression, RELA DNA-binding activity, and Il6 mRNA expression. These data demonstrate that NMB and its receptor can induce the onset of labor via a RELA/IL6-mediated pathway.

SK3 channel expression during pregnancy is regulated through estrogen and Sp factor-mediated transcriptional control of theKCNN3gene

Overexpression of the small-conductance calcium-activated K(+) channel 3 (SK3) in transgenic mice compromises parturition, suggesting that the SK3 channel plays a role in pregnancy. In wild-type mouse myometrium, expression of SK3 transcript and protein is significantly reduced during pregnancy, but the mechanism(s) responsible for this attenuation of channel expression is unknown. The promoter region of the SK3-encoding mouse KCNN3 gene contains two binding sites for specificity protein (Sp) transcription factors, two of which are expressed in the uterus: Sp1, which enhances gene transcription in response to estrogen; and Sp3, which competes for the same binding motif as Sp1 and can repress gene expression. We investigated the hypothesis that Sp1 and Sp3 regulate SK3 channel expression during pregnancy. In mouse myometrium, Sp1 expression was reduced during late gestation, whereas Sp3 expression levels were constant throughout pregnancy. Using a reporter system, we found that Sp1 overexpression resulted in a significant increase in SK3 promoter activation and that Sp3 cotransfection reduced promoter activation to basal levels. These findings indicate that Sp3 outcompetes Sp1 to decrease SK3 transcription. To determine whether high levels of estrogen in vivo can affect the regulation of SK3 protein levels by Sp factors, ovariectomized mice were implanted with a 17β-estradiol or placebo pellet for 3 wk; estrogen-treated mice had reduced uterine SK3 protein expression compared with placebo-treated counterparts. In human myometrial cells overexpressing Sp1, estrogen treatment stimulated expression of the SK3 transcript. Overall, our findings indicate that Sp1 and Sp3 compete to regulate SK3 channel expression during pregnancy in response to stimulation by estrogen.

The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)-injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT-PCR/qRT-PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down-regulated in myometrium from LPS-treated-mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.

Efeitos da hiperprolactinemia sobre o útero de camundongos no proestro

To evaluate the effect of hyperprolactinemia induced by metoclopramide on the endometrium and myometrium of female mice in the proestrus phase. 24 female mice were randomly divided in two groups: CtrG/control and ExpG/treated with metoclopramide (6.7 mg/g daily). After 50 days, the animals were sacrificed in the proestrus phase, and the blood was collected to determine the levels of estradiol, progesterone and prolactin. The uterine horns were removed, fixed in 10% formaldehyde and processed before being included in paraffin. Slices of 4 microm were stained by hematoxylin and eosin (H/E). In the morphological analysis, a Carl Zeiss light microscope, with objectives varying from 4 to 400 X was used for each histological slice characterization. In the morphometrical analysis, the superficial epithelium, the lamina propria and the myometrium thickness were evaluated, with the help of an image analyzer (AxionVision - Carl Zeiss) attached to the light microscope (Carl Zeiss). The statistical analysis was done by ANOVA, followed by the Wilcoxon test. P-value was considered as significant, when <0.05. Our findings have shown an increase in the seric levels of prolactin (295.6+/-38.0 ng/mL) and significant decrease in the progesterone levels (11.3+/-0.9 ng/mL) in the ExpG, as compared to the CtrG (45.5+/-5.2 ng/mL and 18.2+/-1.6 ng/mL, respectively; p<0.001). Concerning the seric level of estradiol, significant differences between the groups were not obtained (ExpG=119.1+/-12.3 pg/mL and CtrG=122.7+/-8.4 pg/mL; p=0.418). The morphological study has shown that the uterus from the ExpG presented the endometrium with more developed superficial epithelium and lamina propria, as compared to the CtrG, the same happening with the myometrium. The thickness morphometrical values of the luminal epithelium (8.0+/-1.1 microm) and endometrium (116.2+/-21.1x10(2) microm) from the CtrG were lower than the ones from the ExpG (10.2+/-0.8 microm and 163.2+/-23.3x10(2) microm, respectively) with p<0.05. Nevertheless, data obtained in the myometrium have not shown significant differences between the groups (CtrG=152.2+/-25.2x10(2) microm and ExpG=140.8+/-18.0x10(2) microm). Data have shown that hyperprolactinemia induced by metoclopramide determines endometrial proliferation and interferes with the ovarian function, mainly in the progesterone production.

Loss of functional K+ channels encoded by ether‐à‐go‐go‐related genes in mouse myometrium prior to labour onset

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1-3 (KCNH1-3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 microM), E4031 (1 microM) and Be-KM1 (100 nM) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K(+) currents with distinctive 'hooked' kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K(+) channels as a precursor to late pregnancy physiological activity.

A Dominant Loss-of-Function GJA1 (Cx43) Mutant Impairs Parturition in the Mouse1

Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1(Jrt)) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1(Jrt)/+), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1(Jrt) mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition.

Spontaneous Endometrial Hyperplasia in the Uteri of IL-2 Receptor Beta-Chain Transgenic Mice

We found frequent and spontaneous proliferation of glandular epithelium and dilated cysts in the uteri of interleukin-2 receptor (IL2R) beta-chain transgenic (Tg2Rbeta) mice. The aim of this study was to examine the involvement of IL2R beta-chain in the pathogenesis of endometrial hyperplasia (EH). Mouse uteri and serum were collected from Tg2Rbeta and normal littermates (NL), which were classified into three groups according to age. The incidence of EH increased in an age-dependent manner in both types of mice. However, in old age, Tg2Rbeta mice showed more serious phenotypes of EH than NL. Immunohistochemical analysis revealed specific localization of IL2R beta-chain in the glandular epithelial cells, with a correlation to the degree of EH, not only in the Tg2Rbeta uteri but also in the NL uteri. Immunoreactions of CD3 and CD25 were detected in the uteri of Tg2Rbeta but were weak in the uteri of NL, and CD25-positive cells were distributed in the endometrial stroma and myometrium in the Tg2Rbeta mice. These findings suggest that the IL2R beta-chain induces growth potential for glandular epithelial cells and an immune-privileged condition mediated by CD25+regulatory-T cells.

The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

BackgroundUterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium.MethodsFunctional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots.ResultsThe Kv channel blocker 4-aminopyridine (4-AP) caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar) but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were found in isolated myometrium from both nonpregnant and term-pregnant mice, and one of these proteins – Kv4.3 – was found to disappear in term-pregnant tissues.ConclusionThese findings suggest a role for Kv channels in the regulation of uterine contractility, and that changes in the expression and/or function of specific Kv channels may account for the functional changes seen in pregnant myometrium.

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca(2+) releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.

Adult Mouse Myometrial Label-Retaining Cells Divide in Response to Gonadotropin Stimulation

Conditional deletion of beta-catenin in the Müllerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in beta-catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5-bromo-2'-deoxyuridine (BrdU) and chased for up to 14 weeks. Myometrial label-retaining cells (LRCs) were observed in the myometrium and stroma throughout the chase period. After 12 weeks, phenotypic analysis of the LRCs by immunofluorescence demonstrated that the majority of LRCs colocalized with alpha-smooth muscle actin, estrogen receptor-alpha, and beta-catenin. Flow cytometry of myometrial cells identified a myometrial Hoechst 33342 effluxing "side population" that expresses MISRII-Cre-driven YFP. Functional response of LRCs was investigated by human chorionic gonadotropin stimulation of week 12 chase mice and demonstrated sequential proliferation of LRCs in the endometrial stroma, followed by the myometrium. These results suggest that conventional myometrial regeneration and repair is executed by hormonally responsive stem or progenitor cells derived from the Müllerian duct mesenchyme. Disclosure of potential conflicts of interest is found at the end of this article.

Oxysterol Nuclear Receptor LXRβ Regulates Cholesterol Homeostasis and Contractile Function in Mouse Uterus

The uterus is an organ where lipid distribution plays a critical role for its function. Here we show that nuclear receptor for oxysterols LXRbeta prevents accumulation of cholesteryl esters in mouse myometrium by controlling expression of genes involved in cholesterol efflux and storage (abca1 and abcg1). Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased in wild-type mice, whereas under basal conditions, lxralpha;beta(-/-) mice exhibited a marked decrease in abcg1 accumulation. This change resulted in a phenotype of cholesteryl ester accumulation. Besides, a defect of contractile activity induced by oxytocin or PGF2alpha was observed in mice lacking LXRbeta. These results imply that LXRbeta provides a safety valve to limit cholesteryl ester levels as a basal protective mechanism in the uterus against cholesterol accumulation and is necessary for a correct induction of contractions.