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.     

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.     

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     

人T细胞急性淋巴细胞白血病小鼠动物模型的建立

目的:通过建立一理想的动物模型来模拟T细胞急性淋巴细胞白血病的发病状态,为探索急性淋巴细胞白血病全新的治疗方法提供平台。方法:采用抗鼠-CD122抗体注射NOD/SCID小鼠进行预处理,通过尾静脉注射9例不同病例的白血病细胞,以及1株T-ALL细胞系。通过检测小鼠体内白血病细胞及脏器白血病细胞浸润情况,观察白血病细胞植入。将白血病细胞进行二次移植,观察移植稳定性。对白血病动物模型进行药物处理,观察小鼠生存期,模拟人体治疗反应。结果:有4例病例的细胞及T-ALL细胞株成功植入。流式细胞检测显示受体小鼠体内较多比例人CD45+细胞表达,免疫组化显示CD45+细胞浸润小鼠不同脏器,系列移植也获得成功。体内药物处理显示地塞米松能延长小鼠的生存期,与临床观察相一致。结论:成功建立经抗鼠CD122单抗预处理的人T细胞急性淋巴细胞白血病NOD/SCID小鼠模型,具有原发疾病的所有主要特征。     

Neutrophil depletion retards endometrial repair in a mouse model

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. This work was funded by the National Health and Medical Research Council of Australia (#143798, #241000) and by an Australian Postgraduate Scholarship to T.K.     

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.     

Progesterone inhibits rejection of xenogeneic transplants in the sheep uterus

OBJECTIVES: One of the proposed roles of progesterone is to prevent maternal immunological destruction of the allogeneic conceptus. Here, it was demonstrated that progesterone allows survival of a xenotransplant placed in the uterine lumen. METHODS: Ovariectomized ewes, surgically prepared to have ligatures around each uterine horn, were given daily subcutaneous injections of 50 mg progesterone or vehicle (sesame oil). After 30 days of treatment, mouse hybridoma cells were transplanted to one ligated uterine horn and phosphate-buffered saline was injected into the other horn. The uterus was flushed after an additional 14 days of treatment and hybridoma cells were identified by immunofluorescence. RESULTS: Overall, hybridoma cells were recovered from 4 of 5 progesterone-treated ewes and 1 of 5 vehicle-treated ewes. Immunohistochemical analysis of intercaruncular endometrium using antibodies towards CD8, gammadelta, and CD45R lymphocyte markers revealed that local presence of hybridoma cells caused a significant increase in CD8+ cells in all tissue compartments. While not significant, the numbers of CD8+ cells in the luminal and glandular epithelium were lower for progesterone-treated ewes. Progesterone tended to increase gammadelta T cell numbers in the glandular epithelium. CONCLUSIONS: Results demonstrate that xenograft rejection in the uterus is associated with an increase in CD8+ cells in the endometrium and that progesterone can inhibit uterine tissue graft rejection responses sufficiently to allow survival or delay rejection of xenograft tissue.     

Wild fulvous fruit bats (Rousettus leschenaulti) exhibit human-like menstrual cycle

We investigated the menstrual cycle of wild fulvous fruit bats (Rousettus leschenaulti), focusing on changes in the endometrial and ovarian structure and pituitary and steroid hormones. The menstrual cycle lasts for 33 days in bats studied in their natural habitat and in captivity. Vaginal bleeding was restricted to a single day (Day 1). A preovulatory follicle was found in the ovary on Day 18 when the levels of LH and FSH reached their maxima, accompanied by a thickened endometrium. On Day 24, serum levels of progesterone and estradiol-17 were also maximal, and uterine glands increased in size. After that, the levels of progesterone dropped precipitously, leading to menstrual bleeding. Both the morphologic and hormonal changes observed in fulvous fruit bats during the menstrual cycle resemble similar changes in humans. Fulvous fruit bats may be useful nonprimate laboratory models to study menstruation and menstrual dysfunction.     

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.     

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。     

Activated networking of platelet activating factor receptor and FAK/STAT1 induces malignant potential in BRCA1-mutant at-risk ovarian epithelium

Background

Recent data provide significant evidence to support the hypothesis that there are sub-populations of cells within solid tumors that have an increased tumor initiating potential relative to the total tumor population. CD133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages has been identified as a marker for tumor initiating cells in solid tumors of the brain, colon, pancreas, ovary and endometrium. Our objectives were to assess the relative level of CD133 expressing cells in primary human endometrial tumors, confirm their tumorigenic potential, and determine whether CD133 expression was epigenetically modified.

Methods

We assessed CD133 expression in primary human endometrial tumors by flow cytometry and analyzed the relative tumorigenicity of CD133+ and CD133- cells in an in vivo NOD/SCID mouse model. We assessed potential changes in CD133 expression over the course of serial transplantation by immunofluorescence and flow cytometry. We further examined CD133 promoter methylation and expression in normal endometrium and malignant tumors.

Results

As determined by flow cytometric analysis, the percentage of CD133+ cells in primary human endometrial cancer samples ranged from 5.7% to 27.4%. In addition, we confirmed the tumor initiating potential of CD133+ and CD133^- cell fractions in NOD/SCID mice. Interestingly, the percentage of CD133+ cells in human endometrial tumor xenografts, as evidenced by immunofluorescence, increased with serial transplantation although this trend was not consistently detected by flow cytometry. We also determined that the relative levels of CD133 increased in endometrial cancer cell lines following treatment with 5-aza-2'-deoxycytidine suggesting a role for methylation in the regulation of CD133. To support this finding, we demonstrated that regions of the CD133 promoter were hypomethylated in malignant endometrial tissue relative to benign control endometrial tissue. Lastly, we determined that methylation of the CD133 promoter decreases over serial transplantation of an endometrial tumor xenograft.

Conclusions

These findings support the hypotheses that CD133 expression in endometrial cancer may be epigenetically regulated and that cell fractions enriched for CD133+ cells may well contribute to endometrial cancer tumorigenicity, pathology and recurrence.     

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.     

The effects of danazol, mefenamic acid, norethisterone and a progesterone-impregnated coil on endometrial prostaglandin concentrations in women with menorrhagia

The effects of four medical treatments have been assessed on menstrual blood loss (MBL) and endometrial prostaglandin (PG) concentrations in 30 women with objectively confirmed menorrhagia. Patients were randomly treated with danazol, 200 mg daily (n = 6), mefenamic acid, 500 mg three times daily during menses (n = 8), norethisterone, 5 mg twice daily from day 15-25 of the cycle (n = 8) or a progesterone-impregnated coil releasing 65 micrograms progesterone daily (n = 8). Endometrial biopsies were obtained in the mid-luteal phase before and after treatment in 23 cases, and assayed for PG content using radioimmunoassay. Treatment with norethisterone had no effect on either MBL or the concentration of PGs in the endometrium. MBL was significantly reduced after treatment with mefenamic acid (P = 0.05, n = 6) and the progesterone coil (P less than 0.05, n = 6), and was reduced in each of 4 cases treated with danazol in whom endometrial biopsies were available. Although there was no consistent change in endometrial PG concentrations in either the mefenamic acid or danazol groups, the lower MBL after insertion of the progesterone coil was associated with a reduced endometrial content of PGE, PGF2 alpha and "total" PG (6oxo PGF1 alpha + PGE + PGF2 alpha)-P = 0.05. Whereas the cyclooxygenase inhibitor mefenamic acid is likely to exert its effect on endometrial PGs at the time of menstruation itself, the continuous administration of progesterone throughout the menstrual cycle could result in both an impairment in estrogen receptor generation leading to reduced estrogen-mediated cyclooxygenase activity, and an increase in endometrial PG metabolism.     

Characterization of intraepithelial lymphocytes in human endometrium

Intraepithelial lymphocytes (IELs) were characterized and quantitated in normal non-pregnant endometrium and in early pregnancy decidua using H & E and phloxine tartrazine stains and a panel of monoclonal antibodies in an indirect immunoperoxidase technique. The relative numbers of granulated and non-granulated IELs varied according to menstrual cycle stage and in early pregnancy all IELs appeared to be granulated. There was a higher surface:gland ratio for IELs in proliferative endometrium compared with late secretory phase and early pregnancy endometrium. In proliferative endometrium most IELs were T cells, predominantly of the CD8 + subset. In first trimester decidua, higher numbers of CD56 + cells were observed, in keeping with the increased proportion of granulated IELs. IEL populations in human endometrium vary according to menstrual cycle stage and endometrial IELs appear to show phenotypic differences compared with IELs in the human gastrointestinal tract.     

Protein synthesis and secretion by the human endometrium during the menstrual cycle and the effect of progesterone in vitro

To identify markers of endometrial differentiation specimens of endometrium from the menstrual cycle were incubated in vitro with [35S]methionine, in the absence or presence of progesterone, and protein synthesis and secretion were studied by fluorographic analysis of one dimensional SDS/gradient polyacrylamide gels. Changes were demonstrated in the rate of synthesis and secretion of a number of endometrial proteins (EP) during the cycle and in response to progesterone. Endometrial proteins were classified into three groups: Group I-synthesized and secreted throughout the menstrual cycle and unaffected by progesterone exposure; Group II-synthesis and secretion associated with histological type of endometrium and unaffected by progesterone exposure, e.g. EP 13 (Mr 33,000) with proliferative, EP 15 (Mr 28,000) with secretory and EP 14 (Mr 32,000) with late secretory endometrium; Group III-synthesis and secretion regulated by progesterone exposure irrespective of source of endometrium, e.g. EP 9 (Mr 54,000) and 11 (Mr 45,000). The Group II proteins EP 14 and 15 were also the major secretory protein products of endometrium from first and second trimester pregnancy respectively, the native forms referred to as pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG). We conclude that EP 15 (alpha 2-PEG) represents a human analogue of uteroglobin.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.     

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem-like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b⁺CD146⁺ cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from the menstrual endometrium underwent significantly more rounds of self-renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self-renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium.