Myometrial Tissue Research Articles

Secretion of estradiol-17β by porcine endometrium and myometrium during early pregnancy and luteolysis

Past studies of the source of estrogens secreted during maternal recognition of pregnancy in pigs have focused on embryonic rather than uterine origin of these steroids. The present study documents: (1) the expression of the gene CYP 17, encoding cytochrome P450 17alpha-hydroxylase/C(17-20) lyase and (2) the synthesis and secretion of estradiol-17 beta (E(2)) in endometrial and myometrial tissues in gilts. The expression of CYP 17 gene was shown in porcine endometrium and myometrium. Basal endometrial secretion of E(2) was higher in pregnant gilts than in cyclic gilts (days 14-16). The myometrium secreted more E(2) during the expected time of luteolysis compared to early pregnancy. Basal secretion of E(2) during pregnancy was higher from the endometrium than from the myometrium. Conversely, during luteolysis E(2) secretion was higher from the myometrium and lower from the endometrium. In pregnant and cyclic gilts (days 14-16), progesterone (P(4), 10(-5)M) in vitro significantly increased E(2) secretion regardless of reproductive status. Oxytocin (OT, 10(-7)M) had no influence on E(2) secretion and did not change the stimulatory effect of P(4) in both tissues examined. In conclusions: (1) the CYP 17 gene transcript is present in porcine endometrium and myometrium; (2) porcine endometrium and myometrium release E(2) in vitro; (3) the endometrium releases more E(2) than the myometrium during early pregnancy; (4) the myometrium releases E(2) mainly during luteolysis; (5) the endometrium and myometrium can increase E(2) release in vitro if substrate (P(4)) is provided during early pregnancy and luteolysis. These data suggest active estrogen production by the myometrium and endometrium as an alternative source for this signal for recognition of pregnancy in the pig.

Expression of Prothrombin and Protease Activated Receptors in Human Myometrium During Pregnancy and Labor1

As thrombin is proposed to be involved in stimulating myometrial contractility during labor and preterm labor, we aimed to investigate the expression of prothrombin (F7), the precursor of thrombin, its receptors, the protease-activated receptor (PAR) family (F2R, F2RL1, F2RL2, and F2RL3), and prothrombinase FGL2 in human myometrium during pregnancy and labor. Messenger RNA and protein were isolated from human pregnant laboring and nonlaboring myometrial tissue and from human primary myometrial smooth muscle cells. Semiquantitative RT-PCR, real-time fluorescence RT-PCR, Western blotting, and fluorescence microscopy were performed to determine the expression levels of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in the myometrial tissues and cells. The expression of mRNA and protein for these molecules is reported for the first time in human myometrium at term pregnancy, at labor, and in the nonpregnant state. Importantly, an increase in F2R and a significant increase in F2RL3 mRNA expression at labor were demonstrated. Statistically significant increases in F2R and F2RL3 protein expression was also detected in human myometrium at labor. Furthermore, FGL2 mRNA expression at labor, and FGL2 protein expression at term pregnancy and at labor was observed in this tissue for the first time. The expression of F7, FGL2, F2R, F2RL1, F2RL2, and F2RL3 in human myometrium reveals that all the machinery necessary for thrombin activation and cellular activity is present in the myometrium during pregnancy and labor. These data, in conjunction with the demonstrated increase in F2R and F2RL3 expression at labor, suggest a principal role for these molecules in the regulation of myometrial function at labor, including preterm labor.

Identification of novel antigens in blood vessels in rectovaginal endometriosis

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Differential expression of microRNA species in human uterine leiomyoma versus normal myometrium

To determine whether microRNAs (miRNAs) are differentially expressed in human leiomyoma versus matched myometrial tissue. Microarray with real-time polymerase chain reaction (PCR) validation. Academic medical center. Premenopausal subjects (n = 15), who were undergoing hysterectomies for leiomyoma-related symptoms. None. Statistically differential expression of miRNAs in leiomyoma versus myometrium. Forty-six miRNA species were differentially expressed in leiomyoma versus normal myometrium. Of these, 19 were overexpressed and 27 were underexpressed in leiomyomas. The changes ranged from 1.2-fold to 11.8-fold. These findings were confirmed using real-time reverse transcriptase PCR for selected miRNAs (miRNAs 21, 34a, 125b, 139, and 323). Our findings indicate that miRNAs are differentially expressed between human leiomyoma and matched myometrium. Given this differential expression, miRNAs may play a role in the pathogenesis of uterine leiomyoma and may serve as future therapeutic targets for the treatment of these tumors.

Glycosaminoglycan expression in versican variants is tightly regulated by TGF-β3 in leiomyoma cells

Leiomyomas exhibit a large degree of extracellular matrix (ECM) disorganization. Versican, a hyaluronan-chondroitin sulfate binding glycosaminoglycan (GAG), forms an integral part of the ECM and serves as a repository for growth factors important for leiomyoma proliferation. Versican has four splice variants which express different amounts of GAGs. Versican variant, V0, contains the largest amount of GAGs. Using myometrial and leiomyoma tissue and immortalized cell cultures, we hypothesized that V0 expression would be regulated by TGF-β3, which has been implicated in numerous fibrotic diseases. Laboratory study. After IRB approval, we obtained spontaneous uterine leiomyoma and matched myometrium from hysterectomy specimens. Primary cell cultures were generated and immortalized using recombinant retrovirus containing the E6/E7 open reading frames of HPV type-16. Cells were grown to 80% confluence, serum-starved for 48 hours, and then treated for 24 hours with various concentrations of TGF-β3 and Anti-TGF-β3. V0 expression was analyzed by end point RT-PCR and quantitative RT-PCR in both tissue and immortalized cell cultures. V0 was upregulated in leiomyoma vs. myometrial tissue (mean 3.61, Wilcoxon signed rank P<0.05) and cell culture (mean 2.96, P<0.05). With the addition of TGF-β3, myometrial cells demonstrated increased relative fold expression of V0 compared to untreated myometrial expression (1.65, 34.5, and 11.4, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). Leiomyoma cells exhibited a less pronounced stimulatory effect of TGF-β3 compared to untreated myometrial cells (2.48, 12.9, and 2.62, P<0.05 at 0.1 ng/ml, 1.0 ng/ml, and 10 ng/ml of TGF-β3). The addition of Anti-TGF-β3 decreased the relative fold expression of leiomyomas cells compared to untreated leiomyomas cells (0.27, 0.20, and 0.02 at 0.1 ug/ml, 1.0 ug/ml, and 10 ug/ml of Anti-TGF-β3). The GAG-rich V0 is overexpressed in leiomyoma vs. myometrial tissue and immortalized cell culture. The addition of TGF-β3 stimulated the expression of V0 in both myometrial and leiomyoma cells, while Anti-TGF-β3 decreased the expression of V0 in leiomyomas cells. Differing levels of GAG composition and amount in leiomyomas contribute to the disorganized ECM by obstructing the tight packing of collagen fibrils. Our data suggest that the larger GAG-rich V0 affects ECM composition and is important to the leiomyoma phenotype.

Demonstration of Multipotent Stem Cells in the Adult Human Endometrium by In Vitro Chondrogenesis

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported. Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)-beta2 or TGF-beta 3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.

Uterine Stretch Regulates Temporal and Spatial Expression of Fibronectin Protein and Its Alpha 5 Integrin Receptor in Myometrium of Unilaterally Pregnant Rats1

The adaptive growth of the uterus during pregnancy is a critical event that involves increased synthesis of extracellular matrix (ECM) proteins and dynamic remodeling of smooth muscle cell (SMC)-ECM interactions. We have previously found a dramatic increase in the expression of the mRNAs that encode fibronectin (FN) and its alpha5-integrin receptor (ITGA5) in pregnant rat myometrium near to term. Since the myometrium at term is exposed to considerable mechanical stretching of the uterine wall by the growing fetus(es), the objective of the present study was to examine its role in the regulation of FN and ITGA5 expression at late gestation and during labor. Using myometrial tissues from unilaterally pregnant rats, we investigated the temporal changes in Itga5 gene expression in gravid and empty uterine horns by Northern blotting and real-time PCR, in combination with immunoblotting and immunofluorescence analyses of the temporal/spatial distributions of the FN and ITGA5 proteins. In addition, we studied the effects of early progesterone (P4) withdrawal on Itga5 mRNA levels and ITGA5 protein detection. At all time-points examined, the Itga5 mRNA levels were increased in the gravid uterine horn, compared to the empty horn (P < 0.05). Immunoblot analysis confirmed higher ITGA5 and FN protein levels in the myometrium, associated with gravidity (P < 0.05). Immunodetection of ITGA5 was consistently high in the longitudinal muscle layer, increased with gestational age in the circular muscle layer of the gravid horn, and remained low in the empty horn. ITGA5 and FN immunostaining in the gravid horn exhibited a continuous layer of variable thickness associated directly with the surfaces of individual SMCs. In contrast to the effects of stretch, P4 does not appear to regulate ITGA5 expression. We speculate that the reinforcement of the FN-ITGA5 interaction: 1) contributes to myometrial hypertrophy and remodeling during late pregnancy; and 2) facilitates force transduction during the contractions of labor by anchoring hypertrophied SMCs to the uterine ECM.

Differential expression of the metalloproteinase MMP3 and the 5 integrin subunit in human myometrium at labour

Extensive tissue remodelling occurs in the human myometrium before, during and after parturition. The aim of this study was to investigate the expression of two tissue remodelling molecules, matrix metalloproteinase 3 (MMP3) and alpha5 integrin (ITGA5) subunit in human myometrium, during pregnancy and labour. mRNA and protein were isolated from human pregnant labouring and non-labouring myometrial tissue, and also from human primary uterine smooth muscle cells (SMCs). Semi-quantitative RT-PCR, real-time fluorescence RT-PCR and western blotting were subsequently performed to determine the expression levels of MMP3 and ITGA5 in the myometrial tissues during pregnancy and labour, and in the primary uterine SMCs. The expression of MMP3 and ITGA5 mRNA and protein are reported for the first time during pregnancy and labour in human myometrium. Furthermore, a significant increase in expression of MMP3 mRNA (41-fold, P = 0.001), and a significant decrease in ITGA5 mRNA expression (4-fold, P < 0.001) at labour, were observed. Protein expression of these two molecules was also altered at labour, pro-MMP3 protein expression significantly increased while ITGA5 protein expression decreased, with labour onset. Expression of these molecules was also observed in primary cultured human uterine SMCs. The differential expression of these two tissue remodelling molecules at labour and their detection in uterine SMCs highlights their potential importance in myometrial function during pregnancy, labour and post-partum.

Up-Regulation of Myometrial RHO Effector Proteins (PKN1 and DIAPH1) and CPI-17 (PPP1R14A) Phosphorylation in Human Pregnancy Is Associated with Increased GTP-RHOA in Spontaneous Preterm Labor1

RHO GTP-binding proteins are important regulators of actin-myosin interactions in uterine smooth muscle cells. Active (GTP-bound) RHOA binds to RHO-associated protein kinase (ROCK1), which inhibits the myosin-binding subunit (PPP1R12A) of myosin light chain phosphatase, leading to calcium-independent increases in myosin light chain phosphorylation and tension, which are termed "calcium sensitization." The RHO effector protein kinase N (PKN1) also increases calcium sensitization by phosphorylating the protein kinase C (PRKCB)-dependent protein CPI-17 (PPP1R14A) to inhibit the PPP1c subunit of myosin phosphatase. Moreover, other RHO proteins, such as RHOB, RHOD, and their effectors (DIAPH1 and DIAPH2), may modulate PKN1/ ROCK1 signaling to effect changes in myosin phosphatase activity and myosin light chain phosphorylation. The increases in contractile activity observed in term and preterm labor may be due to an increase in RHO activity and/or changes in RHO-related proteins. We found that the RHOA and RHOB mRNA levels in the myometrium were increased in pregnancy, although the expression levels of the RHOA and RHOB proteins did not change with pregnancy or labor. GTP-bound RHOA was increased in pregnancy, and this increase was significant in spontaneous preterm labor myometrium. PKN1 expression and PPP1R14A phosphorylation were dramatically increased in the pregnant myometrium. We also observed increases in DIAPH1 expression in spontaneous term and preterm labor myometrial tissues. The present study shows that human pregnancy is characterized by increases in PKN1 expression and PPP1R14A phosphorylation in the myometrium. Moreover, increases in GTP-bound RHOA and DIAPH1 expression may contribute to the increase in uterine activity in idiopathic preterm labor.

The regulation of uterine relaxation

The regulation of uterine relaxation is poorly understood but research in myometrial tissue and other types of smooth muscle has defined a number of receptors, ion channels and regulatory proteins that are likely to be involved. Some of these proteins are substrates for protein kinases, especially cyclic nucleotide dependent kinases. More research is necessary to identify the key molecules involved in the maintenance of uterine quiescence in pregnancy. The use of tocolytics in preterm labour remains controversial; there is a need to identify better pharmacological targets to provoke safe and selective uterine relaxation and improve neonatal outcome.

Increased expression of platelet-derived growth factor C messenger ribonucleic acid in uterine leiomyomata

This study was intended to investigate the possible roles of the platelet-derived growth factor (PDGF) gene family's involvement in the pathogenesis of uterine leiomyomata. We examined the differential gene expressions of PDGF-A, -B, -C, -D, PDGF receptor alpha (PDGFR-alpha), and receptor beta (PDGFR-beta) between uterine leiomyomata and the adjacent normal myometrium. Expression of PDGF-C in leiomyomata was significantly higher (approximately 2.4-fold) than in the adjacent normal myometrium, whereas there were no significant differences in the expressions of PDGF-A, -B, -D, PDGFR-alpha, or PDGFR-beta between leiomyomata and the adjacent myometrial tissues.

Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis

To investigate types of nerve fibers in endometrium and myometrium in women with endometriosis. Laboratory study using human tissue. University-based laboratory. Women with and without endometriosis undergoing hysterectomy. Histologic sections of contiguous endometrial and myometrial tissues were prepared from hysterectomies performed on women with and without endometriosis. Types and density of nerve fibers in endometrium and myometrium in women with and without endometriosis were determined using a series of specific markers for neuronal structure and function: PGP9.5, NF, SP, CGRP, TH, VAChT, VIP, and NPY. Nerve fibers stained with PGP9.5 and NF in endometrium and myometrium were significantly increased in women with endometriosis compared with women without endometriosis. Nerve fibers in the functional layer of endometrium in women with endometriosis were likely to be sensory C, a mixture of sensory A delta, sensory C, and adrenergic fibers in the basal layer of the endometrium, a mixture of sensory A delta, sensory C, adrenergic and cholinergic fibers in the myometrium. Increased nerve fiber density in endometrium and myometrium, and sensory C fibers and adrenergic nerve fibers in the endometrium in women with endometriosis may play an important role in the mechanisms of pain generation in this condition.

The Onset of Labor Alters Corticotropin-Releasing Hormone Type 1 Receptor Variant Expression in Human Myometrium: Putative Role of Interleukin-1β

CRH targets the human myometrium during pregnancy. The efficiency of CRH actions is determined by expression of functional receptors (CRH-R), which are dynamically regulated. Studies in myometrial tissue biopsies using quantitative RT-PCR demonstrated that the onset of labor, term or preterm, is associated with a significant 2- to 3-fold increase in CRH-R1 mRNA levels. Detailed analysis of myometrial CRH-R1 mRNA variants showed a decline of the pro-CRH-R1 mRNA encoding the CRH-R1beta variant during labor and increased mRNA levels of CRH-R1d mRNA. Studies in myometrial cells identified IL-1beta as an important regulator of myometrial CRH-R1 gene expression because prolonged treatment of myometrial cells with IL-1beta (1 ng/ml) for 18 h induced expression of CRH-R1 mRNA levels by 1.5- to 2-fold but significantly attenuated CRH-R1beta mRNA expression by 70%. In contrast, IL-1beta had no effect on CRH-R1d mRNA expression. Studies using specific inhibitors suggest that ERK1/2, p38 MAPK, and downstream nuclear translocation of nuclear factor-kappaB mediate IL-1beta effects on myometrial CRH-R1 gene. However, the increased CRH-R1 mRNA expression was associated with a dampening of the receptor efficacy to activate the adenylyl cyclase/cAMP signaling cascade. Thus, our findings suggest that IL-1beta is an important regulator of CRH-R1 expression and functional activity, and this interaction might play a role in the transition of the uterus from quiescence to active contractions necessary for the onset of parturition.

Separate and synergistic effect of progesterone and estradiol on induction of annexin 2 and its interaction protein p11 in pregnant sheep myometrium

To search for myometrial candidate genes regulated by progesterone, we isolated annexin 2 cDNA by subtractive hybridization and cloning. We also examined the effect of estradiol and/or progesterone, individually or combined, on expressions of annexin 2 and its ligand protein, p11 in pregnant sheep intrauterine tissues. Annexin 2 is a Ca(2+)-dependent phospholipid-binding protein which interacts with p11 to form a bivalent heterotetramer. The heterotetramer was indicated in the production of prostaglandins through the regulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Thus, annexin 2 and p11 could be the important players in Ca(2+) signaling and prostaglandin production in uterine smooth muscles. Twenty-two ewes were treated with vehicle (n = 6), or 5 mg estradiol administered intramuscularly twice a day for 2 days (n = 6), or 100 mg progesterone administered intramuscularly twice a day for 14 days (n = 5), or estradiol plus progesterone with 100 mg progesterone administered intramuscularly twice a day for 10 days (n = 5) and then with vehicle for 2 days followed by estradiol for 2 days (5 mg administered intramuscularly twice a day). At 121 days of gestation age (dGA), endometrium, myometrium, placenta, and cervix were obtained under halothane anesthesia. Subtractive hybridization was used to isolate differentially expressed mRNAs from myometrial tissues treated with progesterone, which was confirmed by western blot at protein level. Annexin 2 and p11 interaction was validated by immunoprecipitation and their expressions in intrauterine tissues were determined by western blot analysis. Data were analyzed by ANOVA. A cDNA clone from myometrial-subtracted library representing differentially expressed mRNA in the pregnant sheep myometrium with progesterone treatment was identified as annexin 2 by sequence analysis and BLASTN search. Annexin 2 was present in both cytosolic and membrane-associated fractions of intrauterine tissues. In contrast, p11 was detectable only in myometrial cytosolic fraction. Both annexin 2 and p11 significantly increased in myometrial cytosolic fraction after progesterone or progesterone plus estradiol treatment. Furthermore, estradiol and progesterone combined had a more profound effect on induction of annexin 2 than progesterone alone. Annexin 2 was immunoprecipitated specifically by p11 antibody from the myometrium. This study indicates that progesterone may play an important role in the control of myometrial contractility by modifying protein expression associated with Ca2+ and prostaglandin signaling systems during pregnancy. Enhanced expression of myometrial annexin 2 in progesterone plus estradiol group supports our hypothesis that estrogen's stimulation is optimized by progesterone's priming in the pregnant sheep uterus.

Morphological disintegration of biological fluids and tissues and the imbalance of macro- and microelements

The contents of and interrelations between macro- and microelements were determined in human myometrial tissue and myomas. Benign growth of the myometrium was found to be associated with changes in its elemental composition. Especially illustrative are the changes in the correlation between zinc and copper (and, to a lesser degree, other elements).

THG113.31, a specific PGF2alpha receptor antagonist, induces human myometrial relaxation and BKCa channel activation

BackgroundPGF2alpha exerts a significant contractile effect on myometrium and is central to human labour. THG113.31, a specific non-competitive PGF2alpha receptor (FP) antagonist, exerts an inhibitory effect on myometrial contractility. The BKCa channel is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate potential BKCa channel involvement in the response of human myometrium to THG113.31.MethodsSingle and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes, were investigated in the presence and absence of THG113.31. Functional studies investigated the effects of THG113.31 on isolated spontaneous myometrial contractions, in the presence and absence of the BKCa channel blocker, iberiotoxin.ResultsSingle channel recordings identified the BKCa channel as a target of THG113.31. THG113.31 significantly increased the open state probability of these channels [control 0.023+/-0.006; 10 microM THG113.31 0.087+/-0.012 (P = 0.009); and 50 microM THG113.31 0.1356+/-0.018 (P = 0.001)]. In addition, THG113.31 increased whole-cell BKCa currents over a range of membrane potentials, and this effect was reversed by 100 nanoM IbTX. Isometric tension studies demonstrated that THG113.31 exerted a significant concentration-dependent relaxant effect on human myometrial tissue and pre-incubation of strips with IbTX abolished this effect on spontaneously occurring contractions.ConclusionThese data suggests that activation of the BKCa channel may contribute, at least partially, to the uterorelaxant effect of THG113.31.

Uterine leiomyomas express a molecular pattern that lowers retinoic acid exposure

To analyze expression of the retinoic acid signaling pathway genes that are involved in retinol metabolism, transport, transcriptional activation, and transcriptional products in spontaneous human leiomyomas. Laboratory study of human leiomyoma and patient-matched myometrial tissue. Eight women undergoing hysterectomy for symptomatic leiomyomas. Confirmation of an altered retinoic acid pathway analyzed by microarray, real time reverse transcription-polymerase chain reaction, Western blot, immunohistochemistry, and high-performance liquid chromatography (HPLC). Gene and protein expression. Regardless of patient demographics and leiomyoma location and size, we found decreased expression of the major genes involved in retinoic acid pathway including alcohol dehydrogenase-1 (-3.97- +/- 0.03-fold), aldehyde dehydrogenase-1 (-3.1- +/- 0.07-fold), cellular retinol binding protein-1 (-2.62- +/- 0.04-fold), and cellular retinoic acid binding protein-1 (-2.42- +/- 0.20-fold). Cytochrome P450 (CYP 26A1), which is responsible for retinoic acid metabolism, was highly up-regulated in leiomyomas (+5.4- +/- 0.53-fold). Nuclear receptors demonstrated a complex pattern of under-expression (RARalpha, RARbeta, RXRalpha, RXRgamma) and over-expression (RARgamma, RXRbeta) at both the mRNA and protein level. Differences in protein amounts were confirmed by Western blot. Finally, a reduced amount of cellular ATRA and 9-cis retinoic acid was confirmed by HPLC in leiomyomas compared with myometrial tissues. Molecular alterations in the retinoic acid pathway of leiomyomata result in a decrease in retinoic acid exposure.

Uterine innervation in fibroids: A qualitative study

SummaryThis study describes the innervation of uterine fibroids in a retrospective survey of archived material. A total of 24 uteri containing fibroids were identified; four nulliparous uteri with single large fundal fibroids (200 – 320 g) and 20 multiparous uteri with one, or more, fibroids (80 – 230 g). Tissue blocks from the uterine isthmus and the fibroids, were identified, sectioned and stained for nerves with anti-S100 using a standard immunohistochemical regimen. Normal uterine innervation includes concentrations of nerves in the subserosal layer and at the endometrial-myometrial interface with sparse neurovascular bundles distributed throughout the myometrial stroma. Nulliparous uteri with single large fibroids demonstrated relatively normal patterns of innervation with nerves distributed throughout the stroma of the fibroid. In multiparous uteri with fibroids, there were no nerves detectable in the substance of the fibroids. Increased numbers of nerve fibres were observed in the pseudo-capsule of some fibroids and may reflect compression of normal myometrial tissue and their contained nerves, or, a minor degree of nerve fibre proliferation. This study demonstrates that fibroids in multiparous uteri do not contain nerves. Single, large nulliparous fibroids situated at the uterine fundus appear to contain relatively normal patterns of innervation.

Tissue prothrombinase activity in myometrium from timed-pregnant rats.

Fgl2 and thrombin potentially play roles during inflammation-induced preterm delivery. These studies sought to demonstrate functional prothrombinase enzyme activity in rat uterus, consistent with the expression of Fgl2 in this tissue. Myometrial and other tissue obtained from non-pregnant and timed-pregnant rats was homogenized in Tris-buffered saline. Prothrombinase activity was determined based on the kinetic metabolism of a chromogenic thrombin substrate. Homogenates were incubated with prothrombin followed by the addition of the thrombin substrate. Thrombin activity was determined by comparing tissue activity to a standard curve generated using 0.01 to 0.04 units of active thrombin. Western blot studies were also performed to confirm tissue prothrombinase activity in myometrial homogenates. Prothrombin was incubated with tissue homogenates; the reactions were then terminated with sodium dodecyl sulfate (SDS) loading buffer. Prothrombinase activity in myometrial tissue was observed to be 0.047 to 0.077 U thrombin/10 min/microg protein. Heat denaturation and calcium removal eliminated prothrombinase activity, whereas the addition of Factor V enhanced activity. The Western blots confirmed the presence of prothrombin, the anticipated prethrombin fragments, and thrombin. Consistent with the enzyme studies, the thrombin band formed upon incubation with myometrial homogenates from pregnant and nonpregnant rats. In contrast, the thrombin band was not apparent with removal of calcium, heat denaturation and treatment with serine protease inhibitors. In summary, these studies have confirmed functionally active prothrombinase activity in rat myometrium, supporting the hypothesis that Fgl2 is expressed in this tissue.

Accumulation of Retinoid X Receptor-α in Uterine Leiomyomas Is Associated with a Delayed Ligand-Dependent Proteasome-Mediated Degradation and an Alteration of Its Transcriptional Activity

An alteration of the retinoid pathway can influence the development of uterine leiomyomas in animal models, and retinoids have shown efficacy in inhibiting the growth of this benign tumor both in vitro and in vivo. However, the underlying mechanisms and biological implications are unclear. The present study was based on the demonstration of an accumulation of full-length retinoid X receptor alpha (RXRalpha) in leiomyomas that was not associated with a modification of its gene expression. This accumulation was shown to increase the transcription of the RXR-responsive gene cellular retinoic acid binding protein II (CRABP-II) and to be linked to the cellular redistribution of the receptor and to its retarded degradation via the ubiquitin/proteasome pathway. Accordingly, treatment with a specific proteasome inhibitor but not with protease inhibitors strongly inhibited the degradation of full-length RXRalpha in cells deriving from both myometrium and leiomyoma, but the formation of RXRalpha/ubiquitin conjugates was differentially regulated between the two cell types. Moreover, full-length RXRalpha accumulated in leiomyomas was abnormally phosphorylated at serine/threonine residues relative to myometrial tissue. The ligand to RXRalpha, 9-cis-retinoic acid, induced the receptor breakdown in smooth muscle cells deriving from both normal and tumor tissue, whereas a MAPK-specific inhibitor was able to reduce RXRalpha levels only in leiomyoma cells. These results suggest that switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation in leiomyomas may be responsible for the accumulation of the receptor and the consequent dysregulation of retinoic acid target genes. The ability of retinoids to modify this molecular alteration may be the rationale for their use in the treatment of leiomyomas.