Matrix metalloproteinases in endometrial breakdown and repair: functional significance in a mouse model

Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.     

Mimicking the events of menstruation in the murine uterus

Menstruation and endometrial regeneration occur during every normal reproductive cycle in women and some Old World primates. Many of the cellular and molecular events of menstruation have been identified by correlative or in vitro studies, but the lack of a convenient model for menstruation in a laboratory animal has restricted functional studies. In this study, a mouse model for menstruation first described by Finn in the 1980s has been modified for use in a commonly used inbred strain of mouse. A decidual stimulus was applied into the uterine lumen of appropriately primed mice and leukocyte numbers and apoptosis were examined over time following progesterone withdrawal. Endometrial tissue breakdown was initiated after 12-16 h, and by 24 h, the entire decidual zone had been shed. Re-epithelialization was nearly complete by 36 h and the endometrium was fully restored by 48 h. Leukocyte numbers increased significantly in the basal zone by 12 h after progesterone withdrawal, preceding stromal destruction. Stromal apoptosis was detected by TUNEL staining at 0 and 12 h but decreased by 16 h after progesterone withdrawal. This mouse model thus mimics many of the events of human menstruation and has the potential to assist in elucidation of the functional roles of a variety of factors thought to be important in both menstruation and endometrial repair.     

Identification of label-retaining perivascular cells in a mouse model of endometrial decidualization, breakdown, and repair

The human endometrium is incredibly dynamic, undergoing monthly cycles of growth and regression during a woman's reproductive life. Endometrial repair at the cessation of menstruation is critical for reestablishment of a functional endometrium receptive for embryo implantation; however, little is understood about the mechanisms behind this rapid and highly efficient process. This study utilized a functional mouse model of endometrial breakdown and repair to assess changes in endometrial vasculature that accompany these dynamic processes. Given that adult endometrial stem/progenitor cells identified in human and mouse endometrium are likely contributors to the remarkable regenerative capacity of endometrium, we also assessed label-retaining cells (LRC) as candidate stromal stem/progenitor cells and examined their relationship with endometrial vasculature. Newborn mouse pups were pulse-labeled with bromodeoxyuridine (BrdU) and chased for 5 wk before decidualization, endometrial breakdown, and repair were induced by hormonal manipulation. Mean vessel density did not change significantly throughout breakdown and repair; however, significantly elevated endothelial cell proliferation was observed in decidual tissue. Stromal LRC were identified throughout breakdown and repair, with significantly fewer observed during endometrial repair than before decidualization. A significantly higher percentage of LRC were associated with vasculature during repair than before decidualization, and a proportion were undergoing proliferation, indicative of their functional capacity. This study is the first to examine the endometrial vasculature and candidate stromal stem/progenitor cells in a functional mouse model of endometrial breakdown and repair and provides functional evidence suggesting that perivascular LRC may contribute to endometrial stromal expansion during the extensive remodeling associated with this process.     

Induction of overt menstruation in intact mice

The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply.     

CXCR4, regulated by HIF1A,promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45⁺ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.     

Antiprogestins as a model for progesterone withdrawal

The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The withdrawal of progesterone is the initiating factor for breakdown of the endometrium. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. By administration of the antiprogestin mifepristone, it is possible to mimic progesterone withdrawal and study local events in early pregnancy decidua that may play a role in the process of early pregnancy failure. Our data indicate that antagonism of progesterone action at the receptor level results in an up-regulation of key local inflammatory mediators, including NF-kappaB, interleukin-8 (IL-8), monocyte chemotactic peptide-1 (MCP-1), cyclooxygenase 2 (COX-2) and others in decidua. Bleeding induced by mifepristone in the mid-luteal phase of the cycle is associated with changes in the endometrium similar to those that precede spontaneous menstruation including up-regulation of COX-2 and down-regulation of PGDH. Administration of antagonists of progesterone provide an excellent model to study the mechanisms involved in spontaneous and induced abortion as well as providing information which may help devise strategies for treating breakthrough bleeding associated with hormonal contraception.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

小鼠月经生理模型的探讨

目的减少Finn CA于1984年首次报道的小鼠月经模型的观察时间点,以期为月经生理学研究提供一种较廉价且易操作的月经模型。方法应用成年雌性去势C57BL/6小鼠,给予续贯性激素处理,最末次激素处理后4～6h,实验组小鼠宫腔内注射花生油以诱导子宫内膜蜕膜化反应,对照组小鼠给予同样激素处理但无宫腔油剂注射。分别于油剂处理后31～35h(T3组)、56～70h(T4组)处死小鼠,称量子宫湿重,制作H&E组织切片,运用图像分析软件CAST2,计算全子宫横截面积(TUA)与子宫内膜横截面积(EA)。结果H&E染色子宫组织切片示在单纯雌激素作用下宫内膜呈单层立方上皮,核浆比较高,内膜基质疏松;雌孕激素联合处理后,分泌细胞易见,腺腔内可见分泌物。激素撤退后实验组T3观察到子宫内膜剥离,T4组示子宫内膜修复。对照组子宫内膜始终完整。子宫湿重在激素撤退后,实验组下降较慢。激素撤退后实验组T3的TUA继续上升而EA则维持原水平,T4组TUA与EA均明显下降。结论此模型在子宫内膜剥落期和早期修复期组织学特征与人类子宫内膜有一定的相似性。     

Granulocytes and vascularization regulate uterine bleeding and tissue remodeling in a mouse menstruation model

Menstruation-associated disorders negatively interfere with the quality of life of many women. However, mechanisms underlying pathogenesis of menstrual disorders remain poorly investigated up to date. Among others, this is based on a lack of appropriate pre-clinical animal models. We here employ a mouse menstruation model induced by priming mice with gonadal hormones and application of a physical stimulus into the uterus followed by progesterone removal. As in women, these events are accompanied by menstrual-like bleeding and tissue remodeling processes, i.e. disintegration of decidualized endometrium, as well as subsequent repair. We demonstrate that the onset of bleeding coincides with strong upregulation of inflammatory mediators and massive granulocyte influx into the uterus. Uterine granulocytes play a central role in regulating local tissue remodeling since depletion of these cells results in dysregulated expression of matrix modifying enzymes. As described here for the first time, uterine blood loss can be quantified by help of tampon-like cotton pads. Using this novel technique, we reveal that blood loss is strongly reduced upon inhibition of endometrial vascularization and thus, is a key regulator of menstrual bleeding. Taken together, we here identify angiogenesis and infiltrating granulocytes as critical determinants of uterine bleeding and tissue remodeling in a mouse menstruation model. Importantly, our study provides a technical and scientific basis allowing quantification of uterine blood loss in mice and thus, assessment of therapeutic intervention, proving great potential for future use in basic research and drug discovery.     

Regulation and function of LEFTY-A/EBAF in the human endometrium. mRNA expression during the menstrual cycle,control by progesterone,and effect on matrix metalloprotineases

The human endometrium is a unique tissue that is periodically shed during menstruation. Although overall triggered by ovarian steroids withdrawal, menstrual induction of matrix metalloproteinases (MMPs) and resulting tissue breakdown are focal responses, pointing to additional local modulators. LEFTY-A, a novel member of the transforming growth factor-beta family identified originally as an endometrial bleeding-associated factor (EBAF), is a candidate for this local control. We measured LEFTY-A and beta-ACTIN mRNA concentration during the menstrual cycle in vivo and found that their ratio was dramatically ( approximately 100-fold) increased at the perimenstrual phase. A similar increase was seen when proliferative explants were cultured for 24 h in the absence of ovarian steroids; this was followed by spontaneous production of proMMP-1, -3, and -9. Both responses were inhibited by progesterone. Moreover, addition of recombinant LEFTY-A to proliferative explants was sufficient to stimulate the expression of proMMP-3 and -7; this response was also blocked by ovarian steroids. Collectively, these data indicate that LEFTY-A may provide a crucial signal for endometrial breakdown and bleeding by triggering expression of several MMPs. Progesterone appears to exert a dual block, upstream by inhibiting LEFTY-A expression and downstream by suppressing its stimulatory effect on MMPs.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Transforming growth factor-beta1 attenuates expression of both the progesterone receptor and Dickkopf in differentiated human endometrial stromal cells

TGFbeta1 is thought to be intimately involved in cyclic tissue remodeling and inflammatory events associated with menstruation. Menstruation is initiated by progesterone withdrawal; however, the underlying mechanisms are not well understood. In the present study, we have tested the hypothesis that locally produced TGFbeta1 may influence expression of progesterone receptor (PR) or the Wnt antagonist Dickkopf-1 (DKK) with consequential impact on regulation of menstruation. Endometrial stromal cells (ESC) were isolated from endometrial biopsy samples collected from patients undergoing gynecological procedures for benign indications. Treatment of differentiated ESC with TGFbeta1 (10 ng/ml) significantly inhibited the expression of mRNAs encoding PR and DKK. TGFbeta1 also attenuated the protein expression of PR and secretion of DKK proteins in culture supernatants. Neutralization of endogenous TGFbeta1 signaling abolished the TGFbeta1-induced effects, significantly increased expression of PR, and increased DKK protein release levels to that of differentiated ESCs, confirming the specificity of the TGFbeta1 effect. Additionally, in vitro decidualization of ESCs significantly augmented DKK protein release. Moreover, although TGFbeta1 was capable of signaling via the Sma- and mothers against decapentaplegic (MAD)-related protein (SMAD) pathway, the inhibitory effect on DKK was SMAD independent. Conversely, the inhibitory effect of TGFbeta1 on PR was dependent on SMAD signal transduction. In conclusion, these results suggest that local TGFbeta1 signaling can potentiate progesterone withdrawal by suppressing expression of PR and may coordinate tissue remodeling associated with menstruation by inducing Wnt-signaling via inhibition of DKK, which we found to be up-regulated as a consequence of decidualization of ESCs.     

Comprehensive analysis of leukocytes, vascularization and matrix metalloproteinases in human menstrual xenograft model

In our previous study, menstrual-like changes in mouse were provoked through the pharmacologic withdrawal of progesterone with mifepristone following induction of decidualization. However, mouse is not a natural menstruation animal, and the menstruation model using external stimuli may not truly reflect the occurrence and development of the human menstrual process. Therefore, we established a model of menstruation based on human endometrial xenotransplantation. In this model, human endometrial tissues were transplanted subcutaneously into SCID mice that were ovarectomized and supplemented with estrogen and progestogen by silastic implants with a scheme imitating the endocrinological milieu of human menstrual cycle. Morphology, hormone levels, and expression of vimentin and cytokeratin markers were evaluated to confirm the menstrual-like changes in this model. With 28 days of hormone treatment, transplanted human endometrium survived and underwent proliferation, differentiation and disintegration, similar to human endometrium in vivo. Human CD45+ cells showed a peak of increase 28 days post-transplantation. Three days after progesterone withdrawal, mouse CD45+ cells increased rapidly in number and were significantly greater than human CD45+ cell counts. Mouse CD31+ blood vascular-like structures were detected in both transplanted and host tissues. After progesterone withdrawal, the expression levels of matrix metalloproteinases (MMP) 1, 2, and 9 were increased. In summary, we successfully established a human endometrial xenotransplantation model in SCID mice, based on the results of menstrual-like changes in which MMP-1, 2 and 9 are involved. We showed that leukocytes are originated from in situ proliferation in human xenografts and involved in the occurrence of menstruation. This model will help to further understand the occurrence, growth, and differentiation of the endometrium and the underlying mechanisms of menstruation.     

Extracellular matrix dynamics in scar-free endometrial repair: perspectives from mouse in vivo and human in vitro studies

Repair of the postmenstrual endometrium presents a unique opportunity to examine nonscarring repair in an adult tissue. We aimed to characterize and determine the importance of extracellular matrix (ECM) dynamics in cell migration during endometrial repair. Utilizing an in vivo mouse model of postmenstrual repair and an in vitro model of human endometrial re-epithelialization, we determined the dynamic changes in expression of ECM and related factors in both models by array analysis of repairing areas. We also validated expression of integrins, growth factors, protease inhibitors, basement membrane, and adhesion molecules in vitro and in both mouse and human repairing endometrium by quantitative RT-PCR and immunohistochemical studies. Finally, we determined the functional importance of integrin-fibronectin interactions and matrix metalloprotease (MMP)-facilitated cell movement during re-epithelialization and propose a model for cell locomotion during postmenstrual repair. These data demonstrated the dynamic expression and functional importance of ECM interactions in endometrial repair, which may be important for scar-free repair.     

Active Role of the Predecidual-Like Zone in Endometrial Shedding in a Mouse Menstrual-Like Model

Cyclic shedding of the endometrium is unique to menstruating species. The status of the decidua in mouse menstrual-like models seems to differ from that of the predecidua in humans before endometrial breakdown. The aim of this study was to determine how this difference in decidual status is related to endometrial breakdown. A mouse menstruallike model was generated by pharmacological progesterone withdrawal. Histomorphological analysis and reticular fiber staining were used to evaluate endometrial status. In situ zymography was used to determine the localization of active collagenase and gelatinase. The functional endometrial layer containing the mature decidual-like zone (MDZ) and predecidual-like zone (PZ) underwent breakdown. The reticular fibers underwent disruption and fragmentation and became loose or disappeared at 12 h in the PZ, where active collagenase and gelatinase were limited. The reticular fibers were visibly reduced at 24 h in the MDZ, where active collagenase was detected. A few reticular fibers remained; however, the functional layer had sloughed into the lumen of the uterus. The results showed that reticular fibers of the PZ are actively degraded during endometrial shedding.Key words: mouse menstrual-like model, predecidual-like zone, reticular fiber, gelatinase, collagenase     

Expression of nodal signalling components in cycling human endometrium and in endometrial cancer

Background  

The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development.