Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells.

The present study was undertaken to investigate the role of estrogen and progesterone in the expression of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in human endometrial stromal cells (ESC). ESC were incubated with estradiol (10(-8) mol/l), medroxyprogesterone acetate (MPA, 10(-6) mol/l), or estradiol + MPA for 18 days. MPA significantly increased Cu,Zn-SOD and Mn-SOD mRNA levels and enzyme activities as well as the mRNA level of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker for decidualization. Estradiol only augmented the effects of MPA on Cu,Zn-SOD activity and IGFBP-1 mRNA level, and estradiol alone had no effect. To study the withdrawal of estrogen and progesterone (EP withdrawal), ESC that had been treated with estradiol + MPA for 12 days were washed and then incubated with or without estradiol + MPA for a further 11 days. Cu,Zn-SOD mRNA levels and activities declined after EP withdrawal, while they were gradually increased by the continuous treatment with estradiol + MPA. In contrast, Mn-SOD mRNA levels and activities were not affected by EP withdrawal. IGFBP-1 mRNA levels were significantly increased 4 days after EP withdrawal and decreased thereafter, whereas they were gradually increased by the continuous treatment with estradiol + MPA. In conclusion, Cu,Zn-SOD, Mn-SOD and IGFBP-1 are differently regulated by estrogen and progesterone in human ESC. The decrease in Cu,Zn-SOD after the ovarian steroid withdrawal may be involved in endometrial breakdown.     

Progesterone induces the fibulin-1 expression in human endometrial stromal cells

BACKGROUND: By using microarray analysis with human endometrial stromal cells (ESCs), we previously reported that the mRNA for fibulin-1, an extracellular matrix as well as a plasma glycoprotein, is up-regulated by progesterone. In the present study, we tried to clarify the spatial and temporal regulation mechanism of fibulin-1 in the human endometrium. METHODS AND RESULTS: Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with 6 alpha-methyl-17 alpha-hydroxy-progesterone acetate (MPA) or 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). MPA stimulated the fibulin-1 mRNA expression in a dose-dependent manner, and a progesterone antagonist, RU-486, inhibited the stimulatory effect almost completely. By contrast, beta-estradiol alone did not increase the fibulin-1 mRNA expression. CONCLUSIONS: These results suggest that fiblin-1 is an important molecule that mediates progesterone action in human ESC differentiation towards implantation.     

Altered apoptosis and proliferation in endometrial stromal cells of women with adenomyosis

BACKGROUND: The eutopic endometrium in a woman suffering from adenomyosis is known to be biologically different from that of healthy women. The aim of this study was to examine the apoptosis and proliferation of eutopic endometrium from women with adenomyosis. METHODS: We enrolled 23 women with adenomyosis (study group) and 21 without (control group). Eutopic endometrium was obtained and separated into single endometrial stromal cells (ESCs). ESCs were treated in vitro with hydrogen peroxide (H(2)O(2)) to examine their apoptosis using a fluorescence-activated cell sorter. Cells were also treated with estradiol (E(2)), medroxyprogesterone acetate, interleukin (IL)-6, lipopolysaccharide and interferon-gamma (IFN-gamma) to test their proliferation using a non-radioactive cell proliferation assay. RESULTS: The percentage of annexin V ( + )/7-amino-actinomycin D ( + ) ESCs was much lower in women with adenomyosis after 24 h culture with and without H(2)O(2) treatment when compared with the control group. ESCs of adenomyosis proliferated more rapidly than those of the control group, whether they were cultured alone or were treated with E(2), MPA, IL-6 or IFN-gamma. The immunocytochemical Ki-67 labelling index was much more prominent in adenomyotic ESCs than that of the control group (7.7% versus 1.1%, P < 0.001). CONCLUSIONS: Altered apoptosis and proliferation of eutopic endometrium possibly elucidate some aspects of the pathophysiology of adenomyosis. A high Ki-67 labelling index in immunocytochemistry might be a potential indicator in predicting the occurrence of adenomyosis.     

Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells.

Fibronectin is a major component of decidual basement membrane. In the present study, we have investigated the effect of progestin on the synthesis and secretion of fibronectin in human endometrial stromal cells. Stromal cells were isolated during the menstrual cycle and cultured in RPMI-1640 with 2% fetal calf serum supplemented with progesterone or medroxyprogesterone acetate (MPA) in a long-term culture system. Indirect immunofluorescent staining showed that fibronectin was uniformly distributed in the intracellular and extracellular regions of stromal cells treated with MPA for 14 days. The biosynthesis and secretion of this protein and the accumulation of cellular fibronectin mRNA were studied after various culture periods. Cells were pulse-labelled with [35S]methionine to determine the amount of newly synthesized fibronectin secreted into the culture medium. A monoclonal antibody (Mab) identified human fibronectin on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), showing a predominant band (Mr 230-250 kDa) which migrated with authentic fibronectin run in parallel. In six endometrial specimens, the amount of radioactivity incorporated as [35S]fibronectin was increased by progestin. Maximal stimulation occurred after 6 days treatment with MPA. Culture beyond 16 days reduced the rate of synthesis and secretion to 40% of the maximum. The effect of progestin was dose dependent with 0.02, 0.2 and 1 microM progesterone, producing 2.0, 3.8 and 11-fold increases respectively, over the control. Medroxyprogesterone acetate was more effective than progesterone, the maximal response (10-fold increase) being achieved at 0.02 microM MPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of insulin-like growth factor-binding protein-related protein 1 in decidualization of human endometrial stromal cells

Uterine decidualization is crucial for successful implantation and the establishment of pregnancy. In the present study, the expression of insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1) in the human uterus and endometrial stromal cells (ESCs) and its physiological significance in decidualization were examined. IGFBP-rP1 protein was localized in the glandular epithelium and stromal cells, and blood vessels in the endometrium. Cultured stromal cells expressed IGFBP-rP1 and secreted it into the medium. IGFBP-rP1 was localized mostly in the cytoplasm near the nucleus. Knocking down the endogenous IGFBP-rP1 expression in stromal cells, by a small interfering (si)RNA, diminished the expression of prolactin and IGFBP-1 which serve as decidual markers. These results suggest that IGFBP-rP1 may play a role in decidualization of ESCs.     

Immunohistochemical localization of superoxide dismutase in the human ovary.

To elucidate the physiological function of superoxide dismutase (SOD) in the ovary, we examined the immunohistochemical distribution of CuZn-SOD in the human ovary. We also measured the CuZn-SOD concentration in human follicular fluid by enzyme-linked immunosorbent assay (ELISA). The germinal epithelium and tunica albuginea showed no or weak immunoreactivity. No or weak staining activity was also observed in the non-antral follicle. Once the follicle began to form the antral cavity, theca interna cells began to show intensive immunostaining of SOD, as compared with no staining in the granulosa and theca externa cells. In the gestational corpus luteum, theca and granulosa lutein cells showed intensive and moderate staining activity, respectively. The concentration of CuZn-SOD was 0.222 +/- 0.186 ng/mg protein (mean +/- SD) in the preovulatory follicular fluid. In the present study, the immunohistochemical distribution of SOD was confirmed in the human ovary for the first time. Taking into consideration the fact that SOD catalyses the dismutation reaction of superoxide anion radicals, the present results suggest that theca interna cells play an important role in the protection of the developing oocyte from oxygen radicals by acting as a blood-follicular barrier during follicle maturation.     

Induction of superoxide dismutase by decidualization in human endometrial stromal cells

The present study was undertaken to investigate the effect of decidualization on superoxide dismutase (SOD) expression in human endometrial stromal cells (ESC). To induce decidualization, isolated ESC were incubated with medroxyprogesterone acetate (MPA, 10(-6) mol/l) and oestradiol (10(-8) mol/l) for 23 days. Insulin-like growth factor-binding protein-1 (IGFBP-1) was used as a marker of decidualization. SOD mRNA in ESC was significantly increased on day 12 of the hormone treatment (P < 0.01), which was concomitant with the onset of IGFBP-1 mRNA expression, and further increased until day 23 of the treatment in a manner similar to the change in IGFBP-1 expression. To examine the synergistic effect of human chorionic gonadotrophin (HCG) with MPA and oestradiol on SOD and IGFBP-1 expression, ESC were incubated with HCG in the presence or absence of MPA and oestradiol. HCG had no synergistic effect on SOD and IGFBP-1 expression. SOD activities in the decidualized endometrial tissue obtained from patients given oestradiol and progesterone for 7-10 days were significantly higher than those in the non-decidualized endometrial tissue from patients without the hormone treatment (P < 0.01). In conclusion, SOD expression in ESC was induced by MPA and oestradiol accompanied by decidualization, suggesting that SOD may play important roles in decidualization of ESC.     

Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.     

Molecular interactions during pregnancy: A mouse monoclonal antibody, S2n8, detects a 140 kDa protein on the surface of human endometrial stromal cells and decidual cells

We developed a mouse monoclonal antibody, S2n8, by immunizingmice i.p. with human decidual cells collected in the first trimesterof pregnancy. By indirect immunofluorescence staining of frozensections, S2n8 was found to react with decidual cells and endometrialstromal cells throughout the menstrual cycle, but not with endometrialglandular cells or with the endometrial surface epithelium.Judging from the fluorescence intensity, the antigen expressionon stromal cells was weak in the proliferative phase, and becamestronger in the secretory phase. Decidual cells in the firsttrimester of pregnancy and decidual cells at term showed strongexpression of this antigen. Indirect immunofluorescence stainingof enzymatically dispersed decidual tissue revealed that theS2n8 antigen was expressed on the decidual cell surface. Flowcytometric analysis of 12 freshly prepared stromal cell-enrichedcell suspensions showed that 74.8–94.2% (mean ±SD 86.1 ± 6.6%) of the cells carried the antigen. Theexpression of S2n8 antigen on cultured stromal cells was enhancedby the addition of oestradiol and/or progesterone. The antigenicmolecule was purified by immunoaffinity chromatography fromdecidua collected in the first trimester of pregnancy, and themolecular weight was estimated to be 140 kDa. These findingsindicate that the S2n8 antigen is a useful cell surface markerfor stromal cells/decidual cells and is associated with theirdifferentiation.     

Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells

BACKGROUND: Decidualization of endometrial stromal cells is essential for successful implantation and pregnancy. Prostaglandins (PG) have been shown to be required for the initiation and maintenance of decidualization in animal models. The transport of PG across the plasma membrane is mediated by carriers such as prostaglandin transporter (PGT). Our recent data have shown the expression of human PGT (hPGT) in the endometrium during the menstrual cycle. The objective of the present study was to characterize hPGT in decidualized stromal cells. METHODS AND RESULTS: Human endometrial stromal cells were treated with a combination of cAMP and medroxyprogesterone acetate to induce decidualization. Decidualization was confirmed by morphological differentiation and increased secretion of prolactin. A large increase in hPGT mRNA level, as measured by real-time PCR analysis, was observed in decidual cells compared with control. Similarly, a 2-fold up-regulation of hPGT and 3-12-fold increase in PG biosynthetic enzymes were obtained at the protein level. Decidual cells exhibited a higher isotopic PGE2 uptake and greater intracellular PG levels than control. CONCLUSIONS: The higher uptake of PG by decidual cells is highly likely to be mediated via hPGT. PGT is a newly identified regulator of PG action at the cellular level and likely contributes to the regulation of PG action in female reproductive processes.     

Uterus and endometrium: Inhibition of human endometrial stromal cell proliferation by interleukin 6

We investigated the ability of interleukin 6 (IL-6) to modulatehuman endometrial stromal cell growth in vitro Stromal cellproliferation in response to treatment with varying concentrationsof IL-6 was determined. Endometrial tissue was obtained from10 normally cycling women during the secretory phase of theirmenstrual cycle. Treatment with IL-6 resulted in a dose- andcell-density-dependent inhibition of endometrial stromal cellproliferation in vitro. The maximal inhibition was observedwith 200 pg/ml of IL-6 and at a concentration of 10⁵ cells/well.During in-vitro culture, stromal cells produced low amountsof IL-6 and demonstrated the presence of IL-6 receptor. Thesedata demonstrate that IL-6 acts as a growth-regulatory signalfor human endometrial stromal cells. We postulate that IL-6may contribute to the maintenance of homeostasis in normal endometriumand that perturbation of IL-6 mediated responses may play arole in disorders of the endometrium such as endometrial cancerand endometriosis.     

Immunohistochemical study on tissue transglutaminase and copper-zinc superoxide dismutase in human myocardium: Its relevance to apoptosis detected by the nick end labelling method

The influence of free radicals on apoptosis was studied in the human heart; 45 autopsy cases were examined by the nick end labelling method (NELM) that detects DNA fragmentation. Immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and tissue transglutaminase (tTG) induced frequently during apoptosis were also studied. Positive immunoreaction for tTG was detected in mucinous degeneration of myocardial cells; these same cells were also positive for CuZn-SOD but negative for NELM. Myocardial cells showing basophilic alterations after haematoxylin and eosin staining were also positive for CuZn-SOD but negative for the other markers examined. Positive nuclear reaction by NELM was only observed in myocardial cells showing contraction band necrosis or irregularly shaped nuclei surrounding recent or long-standing infarcted foci. In these the other two markers were negative. Since mucinous degeneration lacks the distinguishing morphological features of apoptosis, immunoreactive tTG in this lesion may not imply that the cells are undergoing apoptosis. tTG can be induced in non-apoptotic conditions and may not be involved in apoptosis induced by infarction. Histological disassociation between CuZn-SOD expression and apoptosis suggests the possibility of a cytoprotective role played by endogenous CuZn-SOD against free radical generation in the human heart.     

Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

BACKGROUND: Human endometrium is a highly regenerative tissue. We hypothesizedthat the source of endometrial stromal and vascular regenerationis a resident stromal stem/progenitor cell population. Putativehuman endometrial stromal stem/progenitor cells have been identifiedusing clonal assays, a retrospective functional stem cell assay.Therefore, the aim of this study was to screen potential stemcell markers for the prospective isolation of human endometrialstromal stem/progenitor cells and to determine their capacityto identify colony-forming stromal cells. METHODS: Single-cell suspensions of human endometrial stromal cells weresorted using fluorescence-activated cell sorting into positiveand negative populations based on STRO-1, CD133, CD90 or CD146expression for clonal assays. All markers were immunolocalizedin human endometrium. RESULTS: Small populations (2–9%) of human endometrial stromalcells expressed each of the markers. Only CD146⁺ cells wereenriched for colony-forming cells, and CD90^hi cells showed atrend for greater enrichment compared with CD90^lo cells. STRO-1and CD146 were localized to perivascular cells of the endometrium.CD90 was strongly expressed by functionalis stroma and perivascularcells, but only weakly expressed in the basalis stroma. CD133was expressed by epithelial cells of the endometrium, ratherthan by stroma or perivascular cells. CONCLUSIONS: This study identified CD146 as a marker of colony-forming humanendometrial stromal cells supporting the concept that humanendometrium contains a population of candidate stromal stem/progenitorcells.     

Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins

Decidual cells arise by proliferation and differentiation ofendometrial stromal cells of the uterus after appropriate stimulationby ovarian hormones. Previously we have shown that progestinand relaxin stimulate the secretion of several decidual-cell-specificsecretory proteins in a long-term primary cell culture system.We now report the effects of progestin and relaxin on the morphologyof stromal cells in association with the production rate oftwo major decidual secretory proteins, prolaction and insulin-likegrowth factor binding protein-1 (IGFBP-1). Stromal cells werecultured in RPMI 1640 and 2% fetal bovine serum for 22 daysunder control conditions (no hormone), with relaxin or medroxyprogesteroneacetate (MPA), or MPA plus relaxin. Cells treated with MPA aloneor MPA plus porcine relaxin grew to a high density with manyareas of heaping while control cells and cells grown in mediumcontaining relaxin alone formed discontinuous layers. The cytoplasmwas distinguished by aggregates of rough endoplasmic reticulumand secretory granules. Surfaces of cells treated with MPA plusrelaxin had clusters of short blunt processes containing secretorygranules. The processes were rarely seen in cells exposed toMPA alone and completely absent in control cells or cells exposedto relaxin alone. Intercellular space was greatly widened incells treated with MPA alone or MPA plus relaxin. There weremany extended gap junctions in MPA and relaxin-treated cellsin contrast to controls. The production rates of prolactin andIGFBP-1 measured by radioimmunoassay and enzyme-linked immunosorbentassay respectively increased from 0.004 to 0.7 µg forprolactin and 0.01 to 44 µg for IGFBP-1 per 10⁶ cells/dayafter 20 days incubation with MPA and relaxin, in agreementwith the previous findings. The parallel biochemical eventsand morphological changes are similar to those of decidualizationof endometrium from the secretory phase to the early gestationin vivo.     

Oxidative stress and overexpression of manganese superoxide dismutase in patients with Alzheimer''s disease

Substantial evidence supports the hypothesis that oxygen free radicals are involved in various neurodegenerative disorders. To assess the presence of oxidative stress in Alzheimer's disease (AD) we examined the activity of the enzyme copper-zinc superoxide dismutase (CuZnSOD) in red blood cells, the levels of the mitochondrial inducible enzyme manganese superoxide dismutase (MnSOD) mRNA in lymphocytes, and the total radical-trapping antioxidant capacity (TRAP) in plasma of AD patients and in a group of age-matched non-demented controls. We found that CuZnSOD activity (P<0.01 vs. controls) was significantly increased as well as the MnSOD mRNA levels while the total antioxidant status (P<0.001 vs. controls) was decreased in AD patients. These findings support the role of oxidative alterations in the pathogenetic mechanism underlying AD neurodegeneration.     

Activity and electrophoretic multiplicity of molecular forms of superoxide dismutase in human blood cells

The level of superoxide dismutase (SOD) activity calculated relative to protein in the various human blood cells falls in the following order: platelet > erythrocyte > lymphocyte > granulocyte. During electrophoresis in homogeneous polyacrylamide gel of homogenates of granulocytes, lymphocytes, and platelets three zones of SOD activity were identified. Two fractions of the enzyme, disappearing after treatment with cyanide, were found in lysates of erythrocytes after removal of the hemoglobin. Of the two SOD fractions of platelets, lymphocytes, and granulocytes migrating rapidly toward the anode, the first corresponds in its ability to be inhibited by cyanide or organic solvents to the cytosol isozyme, the second to the mitochondrial isozyme. The third cathode fraction was not identified. The functional role of SOD in specialized blood cells and the cause of heterogeneity of the enzyme are discussed.Department of Hyperbaric Oxygenation, All-Union Scientific-Research Institute of Clinical and Experimental Surgery, Moscow. Department of Biochemistry, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 166–168, August, 1977.     

Increased interleukin-6 messenger RNA expression in macrophage-cocultured endometrial stromal cells in adenomyosis

PROBLEM: To determine the effects of macrophage on endometrial stromal cells (ESCs) in women with adenomyosis. METHOD OF STUDY: Eutopic endometrium was obtained and separated into single ESC in 10 women with adenomyosis (study group) and 11 without adenomyosis (control group). ESCs were then cultured alone or with macrophage for 24 hr. RESULTS: Immunohistochemistry identified the presence of interleukin-6 (IL-6), IL-8, and IL-10 in ESCs. Real-time quantitative PCR revealed that the IL-6 mRNA was significantly expressed in macrophage-cocultured ESCs in adenomyosis than that in the controls, but was not different in ESCs cultured alone between the two groups. The levels of IL-8 and IL-10 mRNA were similar in ESCs either cultured alone or with macrophage between women with and without adenomyosis. CONCLUSION: IL-6 mRNA was significantly expressed in ESCs after in vitro coculture with macrophage in adenomyosis. This aberrant behavior of ESCs might play a role in the formation of ectopic endometrial implants in adenomyosis.