Steroid regulation of secreted phosphoprotein 1 (SPP1) expression in ovine endometrium

Secreted phosphoprotein 1 (SPP1) is an extracellular matrix glycoprotein that is highly expressed at the maternal–fetal interface and is a critical mediator of embryo implantation. The objectives of this study were to examine the spatial and temporal cyclical expression patterns and steroid regulation of SPP1 mRNA and protein in ovine endometrium, which may be further indicative of their functionality in embryo implantation. Uterine tissue was obtained following hysterectomy from ovariectomised ewes treated with ovarian steroids. In parallel, invitro culture of endometrial cells was used to investigate the effects of ovarian steroids on SPP1 expression in endometrial and luminal epithelial (LE) cells. A significant sustained mid-luteal phase increase in SPP1 mRNA in intercaruncular regions of the endometrium was observed, indicating that glandular epithelium is likely to be the primary source of SPP1 production. This increase in SPP1 was induced by progesterone treatment and was shown at the protein level by immunohistochemistry analysis. Similarly, treatment of stromal cells with 10ng mL−1 progesterone or in combination with 1ng mL−1 oestradiol significantly increased SPP1 expression (P<0.05). Collectively, expression levels of SPP1 are cycle-dependent and peak in the progesterone-dominant luteal phase. They are dependent on the interaction of uterine LE and stromal cells and may involve paracrine signalling by progesterone receptor-positive stromal cells.

Download Full-text

Regulation of the hyaluronan system in ovine endometrium by ovarian steroids

Reproduction ◽

10.1530/rep-13-0001 ◽

2013 ◽

Vol 145 (5) ◽

pp. 491-504 ◽

Cited By ~ 16

Author(s):

Kabir A Raheem ◽

Waleed F Marei ◽

Karen Mifsud ◽

Muhammad Khalid ◽

D Claire Wathes ◽

...

Keyword(s):

Molecular Weight ◽

Protein Expression ◽

Mrna Expression ◽

Differential Expression ◽

Luteal Phase ◽

Follicular Phase ◽

Steroid Treatment ◽

Ovarian Steroids ◽

Protein Levels ◽

Steroid Regulation

In this study, we investigated steroid regulation of the hyaluronan (HA) system in ovine endometrium including HA synthases (HAS), hyaluronidases, and HA receptor-CD44 using 30 adult Welsh Mountain ewes. Eight ewes were kept intact and synchronized to estrous (day 0). Intact ewes were killed on day 9 (luteal phase; LUT; n=5) and day 16 (follicular phase; FOL; n=3). The remaining ewes (n=22) were ovariectomized and then treated (i.m.) with vehicle (n=6) or progesterone (n=8) for 10 days, or estrogen and progesterone for 3 days followed by 7 days of progesterone alone (n=8). Estradiol and progesterone concentrations in plasma correlated with the stage of estrous or steroid treatment. Our results showed trends (P<0.1) and statistically significant effects (P<0.05, by t-test) indicating that LUT had lower HAS1 and HAS2 and higher HAS3 and CD44 mRNA expression compared with FOL. This was reflected in immunostaining of the corresponding HAS proteins. Similarly, in ovariectomized ewes, progesterone decreased HAS1 and HAS2 and increased HAS3 and CD44, whereas estradiol tended to increase HAS2 and decrease CD44. Sometimes, HAS mRNA expression did not follow the same trend observed in the intact animals or the protein expression. HA and its associated genes and receptors were regulated by the steroids. In conclusion, these results show that the level of HA production and the molecular weight of HA in the endometrium are regulated by ovarian steroids through differential expression of different HAS both at the gene and at the protein levels.

Download Full-text

Ovarian steroids affect prostaglandin production in equine endometrial cells in vitro

Journal of Endocrinology ◽

10.1530/joe-13-0185 ◽

2014 ◽

Vol 220 (3) ◽

pp. 263-276 ◽

Cited By ~ 12

Author(s):

Anna Z Szóstek ◽

António M Galvão ◽

Graça M Ferreira-Dias ◽

Dariusz J Skarzynski

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Protein Expression ◽

Stromal Cells ◽

Positive Control ◽

Dependent Manner ◽

Ovarian Steroids ◽

Endometrial Cells ◽

Prostaglandin Endoperoxide Synthase

This study aimed to evaluate the influence of ovarian steroids on equine endometrial epithelial and stromal cells, specifically i) prostaglandin (PG) production in a time-dependent manner, ii) specific PG synthases mRNA transcription and protein expression, and iii) cell proliferation. After passage I, cells were exposed to vehicle, oxytocin (OT, positive control, 10−7M), progesterone (P4, 10−7M), 17β estradiol (E2, 10−9M), or P4+E2for 12, 24, 48, or 72 h. Following treatment, PG concentration was determined using the direct enzyme immunoassay (EIA) method. Alterations inPGsynthases mRNA transcriptions,PGsynthases protein expression, and cell proliferation in response to the treatments were determined after 24 h using real-time PCR, western blot, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide respectively. After 24 h, E2and P4+E2increased PGE2and PGF2αsecretion as well as specific prostaglandin-endoperoxide synthase-2 (PTGS2), PGE2synthases (PGES), and PGF2αsynthases (PGFS) expression in the epithelial cells (P<0.05). Additionally, E2and P4+E2increased PTGS2 expression in stromal cells after 24 h (P<0.05). In stromal cells, P4+E2increased PGE2production as well as PGES expression after 24 h (P<0.05). Both E2and P4+E2increased PGF2αproduction by stromal cells after 24 h (P<0.05). Ovarian steroids affected proliferation of stromal and epithelial cells during the 24-h incubation period (P<0.05). We provide evidence that ovarian steroids affect PG production in equine endometrial cells, upregulating PTGS2, PGES, and PGFS expression. Ovarian steroid-stimulated PG production could be an important mechanism occurring in the equine endometrium that is involved in the regulation of the estrous cycle and early pregnancy.

Download Full-text

Immunosuppressive Factor MNSFβ Regulates Cytokine Secretion by Mouse Lymphocytes and Is Involved in Interactions between the Mouse Embryo and Endometrial Cells In Vitro

ISRN Immunology ◽

10.5402/2011/186541 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Yaping He ◽

Zhaogui Sun ◽

Yan Shi ◽

Yahong Jiang ◽

Zhefu Jia ◽

...

Keyword(s):

Stromal Cells ◽

Embryo Implantation ◽

Cytokine Secretion ◽

Embryonic Cells ◽

Suppressor Factor ◽

Endometrial Stromal Cells ◽

Endometrial Cells ◽

Coculture Model ◽

Mouse Lymphocytes

Immune tolerance at the fetomaternal interface must be established during the processes of implantation and pregnancy. Monoclonal nonspecific suppressor factor beta (MNSFβ) is a secreted protein that possesses antigen-nonspecific immune-suppressive function. It was previously reported that intrauterine immunoneutralization of MNSFβ significantly inhibited embryo implantation in mice. In the present study, MNSFβ protein expression was up- or downregulated by overexpression or RNA interference, respectively, in HCC-94 cells and the culture supernatants used to determine effects of MNSFβ on the secretion of IL-4 and TNFα from mouse lymphocytes as detected by ELISA. A coculture model of mouse embryos and endometrial stromal cells was also utilized to determine the effects of a specific anti-MNSFβ antibody on hatching and growth of embryos in vitro. The results show that MNSFβ induced secretion of IL-4 and inhibited secretion of TNFα from mouse lymphocytes. Following immunoneutralization of MNSFβ protein in the HCC-94 supernatant, the stimulatory effect of MNSFβ on IL-4 secretion from mouse lymphocytes was reduced, while the inhibitory effect on secretion of TNFα was abrogated. Expression of MNSFβ was detected in both embryonic and endometrial stromal cells, and its immunoneutralization inhibited the hatching and spreading of embryos in an in vitro coculture model. These results indicated that MNSFβ may play critical roles during the peri-implantation process by regulating cytokine secretion of lymphocytes and by mediating the crosstalk between embryonic cells and endometrial stromal cells.

Download Full-text

Gonadotropin-releasing hormone and TGF-β activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00200.2004 ◽

2004 ◽

Vol 287 (5) ◽

pp. E991-E1001 ◽

Cited By ~ 31

Author(s):

Xiaoping Luo ◽

Li Ding ◽

Nasser Chegini

Keyword(s):

Stromal Cells ◽

Embryo Implantation ◽

Differential Regulation ◽

Gonadotropin Releasing Hormone ◽

Nuclear Accumulation ◽

Dependent Manner ◽

Releasing Hormone ◽

Endometrial Cells ◽

Fibronectin Expression ◽

Tgf Β1

Gonadotropin-releasing hormone analog (GnRHa) is used for medical management of endometriosis and premature luteinizing hormone surge during controlled ovarian stimulation. Human endometrium expresses GnRH receptors, and GnRHa alters the expression of transforming growth factor-β (TGF-β) and receptors in endometrial cells. Because the diverse biological actions of GnRHa and TGF-β are mediated in part through the MAPK pathway, we determined whether utilization of MAPK/ERK and transcriptional activation of immediate early genes c- fos and c- jun result in differential regulation of fibronectin, known as key regulator of embryo implantation and endometriosis progression. Using endometrial stromal cells (ESC) and the endometrial epithelial cell line HES, we demonstrated that GnRHa and TGF-β, in a dose-, time-, and cell-dependent manner, increased the level of phosphorylated ERK1/2 (pERK1/2). GnRH antagonist Antide also increased pERK1/2 induction in ESC and HES, whereas pretreatment reduced GnRHa-induced pERK2 in ESC but not in HES. Cotreatments with GnRHa plus TGF-β1 did not have an additive or an inhibitory effect on pERK1/2 induction compared with GnRHa or TGF-β1 action alone. TGF-β1 and GnRHa increased ERK1/2 nuclear accumulation and inversely regulated the expression of c- fos and c- jun and that of fibronectin in a cell-specific manner. Pretreatment with U-0126, a MEK1/2 inhibitor, blocked basal, as well as GnRHa- and TGF-β1-induced pERK1/2; however, it differentially affected c- fos, c- jun, and fibronectin expression. In conclusion, the results indicate that GnRHa and TGF-β signaling through MAPK/ERK results in differential regulation of fibronectin expression in endometrial cells, a molecular mechanism where short- and long-term GnRHa therapy and locally expressed TGF-β could influence embryo implantation and endometriosis implants, respectively.

Download Full-text

Altered retinoid signaling compromises decidualization in human endometriotic stromal cells

Reproduction ◽

10.1530/rep-16-0592 ◽

2017 ◽

Vol 154 (3) ◽

pp. 207-216 ◽

Cited By ~ 9

Author(s):

Mary Ellen Pavone ◽

Saurabh Malpani ◽

Matthew Dyson ◽

Serdar E Bulun

Keyword(s):

Stromal Cells ◽

Embryo Implantation ◽

Carrier Protein ◽

Normal Endometrium ◽

Endometrial Stromal Cells ◽

Paracrine Factors ◽

Endometrial Cells ◽

Retinoid Signaling ◽

The Media ◽

Metabolizing Enzyme

Decidualization alters multiple molecular pathways in endometrium to permit successful embryo implantation. We have reported that paracrine factors, including retinoids, secreted from progesterone-treated endometrial stromal cells, act on nearby epithelial cells to induce the estradiol metabolizing enzyme HSD17B2. This same induction is not seen in endometriotic stromal cells. We have also shown significant differences in retinoid uptake, metabolism and action in endometriotic tissue and stromal cells compared to normal endometrium. Here, we characterize retinoid signaling during decidualization in these cells. Endometrial and endometriotic cells were isolated, cultured and incubated and decidualized. Genes involved in retinoid metabolism and trafficking were examined using RT-PCR and Western blotting. Prolactin, a decidualization marker, was also examined. We found that both endometrial and endometriotic stromal cells express all intracellular proteins involved in retinoid uptake and metabolism. Decidualization significantly reduced the expression of the genes responsible for retinoid uptake and shuttling to the nucleus. However, expression of CRBP1, an intracellular carrier protein for retinol, increased, as did RBP4, a carrier protein for retinol in the blood, which can function in a paracrine manner. Secreted RBP4 was detected in the media from decidualized endometrial cells but not from endometriotic cells. We believe that retinoid trafficking in endometrial stromal cells during decidualization may shift to favor paracrine rather than intracrine signaling, which may enhance signaling to the adjacent epithelium. There is blunting of this signaling in endometriotic cells. These alterations in retinoid signaling may help explain the decidualization defects and deficient estradiol inactivation (via HSD17B2) seen in endometriosis.

Download Full-text

225.Expression of chemokines and their receptors at the human maternal - embryonic interface

Reproduction Fertility and Development ◽

10.1071/srb04abs225 ◽

2004 ◽

Vol 16 (9) ◽

pp. 225 ◽

Cited By ~ 2

Author(s):

N. J. Hannan ◽

R. L. Jones ◽

L. A. Salamonsen

Keyword(s):

Cell Lines ◽

Expression Patterns ◽

Embryo Implantation ◽

Receptor Expression ◽

Trophoblast Invasion ◽

Epithelial Cell Line ◽

Embryonic Cells ◽

Trophoblast Cells ◽

Endometrial Cells ◽

Decidual Cells

Human embryo implantation is a complex process involving attachment of the developing blastocyst to the receptive endometrial epithelium, and subsequent trophoblast invasion through decidua. This is regulated by crosstalk between the maternal and embryonic cells, however little is known about the factors involved in enabling and directing trophoblast invasion. Chemokines are cytokines that regulate leukocyte chemotaxis via stimulation of adhesion molecules and cell migration. We have previously shown that two chemokines, fractalkine and MCP-3, are produced by endometrial epithelial and decidual cells, maximally around the time of implantation and early pregnancy (1, 2). We hypothesized that endometrially derived fractalkine and MCP-3 are important for the attachment/invasion of fetal trophoblast cells during implantation. To investigate this, expression of fractalkine, MCP-3 and their receptors (CX3CR1, CCR1, CCR2, CCR3 and CCR5) were assessed in cell types present at the maternal-embryonic interface. RNA, extracted from three trophoblast cell lines (JEG-3 and two trophoblast-choriocarcinoma hybrids), a human epithelial cell line (HES), primary endometrial epithelial cells, mid-secretory endometrium and placental tissue, was subjected to RT-PCR for the chemokines and receptors. Both chemokines were produced by endometrial and placental cells. Chemokine receptor expression was more variable, CX3CR1, CCR1, 2 and 3 were expressed by one or more of the trophoblast cells lines while CX3CR1, CCR1, 2 and 5 were expressed by endometrial cells. Marked differences in expression patterns in the different cell lines highlight the importance of studies to select those cell lines of most physiological relevance: in this case, one that most closely resembles early invasive trophoblasts. These data confirm that chemokines are produced by maternal and embryonic cells during implantation and the strong expression of their receptors on trophoblast cells supports a role for chemokines in embryo implantation. Further, these studies have characterized a number of trophoblast cells for future trophoblast migration and attachment assays. (1) Hannan, N., et al. JCEM (in press). (2) Jones, R., et al. JCEM (in press).

Download Full-text

Analysis of differences in the transcriptomic profiles of eutopic and ectopic endometriums in women with ovarian endometriosis

PeerJ ◽

10.7717/peerj.11045 ◽

2021 ◽

Vol 9 ◽

pp. e11045

Author(s):

Xiao Feng ◽

Lingbin Qi ◽

Xiaoyu Xu ◽

Yun Feng ◽

Xiaoming Gong ◽

...

Keyword(s):

Malignant Transformation ◽

Expression Patterns ◽

Embryo Implantation ◽

Differential Expression Analysis ◽

Enrichment Analysis ◽

Gene Module ◽

Ovarian Endometriosis ◽

Endometrial Cells ◽

Go Enrichment ◽

And Migration

Background Endometriosis is a common gynecological disease among women in their reproductive years. Although much effort has been made, the pathogenesis of this disease and the detailed differences between eutopic endometrial cells and ectopic endometrial cells are still unclear. Methods In this study, eutopic and ectopic endometrial cells were collected from patients with and without endometriosis and RNA sequencing was performed. The gene expression patterns and differentially expressed genes (DEGs) in eutopic and ectopic endometrial cells, as well as control endometrial cells, were analyzed using a weighted gene co-expression network analysis (WGCNA) and the DESeq2 package. The functions of significant genes were detected using Gene ontology (GO) enrichment analysis, and qRT-PCR validation was performed. Results The results indicated that eight gene modules were found among these three groups. They also indicated that the gene module, which is highly related to eutopic endometrial cells, was mainly enriched in cell adhesion, embryo implantation, etc., while the gene module related to ectopic endometrial cells was mainly enriched in cell migration, etc. The results of differential expression analysis were generally consistent with the WGCNA results through identified significant DEGs between different groups. These DEGs may play an important role in the occurrence of endometriosis, including the infertility associated gene ARNTL and PIWIL2, tissue remodeling gene MMP11, cell survival and migration gene FLT1, inflammatory response gene GNLY, the tumor suppressor genes PLCD1, etc. Further analysis suggested the function of adhesion is stronger in ectopic endometrial cells than in eutopic endometrial cells, while the ectopic endometrium may have a higher potential risk of malignant transformation than eutopic endometrium. Conclusions Overall, these data provide a reference for understanding the pathogenesis of endometriosis and its relationship with malignant transformation.

Download Full-text

Regulation of homeobox A10 expression in the primate endometrium by progesterone and embryonic stimuli

Reproduction ◽

10.1530/rep-07-0234 ◽

2007 ◽

Vol 134 (3) ◽

pp. 513-523 ◽

Cited By ~ 39

Author(s):

G B Godbole ◽

D N Modi ◽

C P Puri

Keyword(s):

Growth Factor ◽

Luteal Phase ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Macaca Radiata ◽

Endometrial Cells ◽

Preimplantation Stage ◽

Bonnet Monkey ◽

Embryonic Signaling

Homeobox A10 (HOXA10), a member of abdominal B subclass of homeobox genes, is responsible for uterine homeosis during development. Intriguingly, in the adult murine uterus, HOXA10 has been demonstrated to play important roles in receptivity, embryo implantation, and decidualization. However, the roles of HOXA10 in the primate endometrium are not known. To gain insights into the roles of HOXA10 in the primate endometrium, its expression was studied in the endometria of bonnet monkey (Macaca radiata) in the receptive phase and also in the endometria of monkeys treated with antiprogestin onapristone (ZK98.299) or in conception cycle where the presence of preimplantation stage blastocyst was verified. In addition, the mRNA expression of HOXA11 and insulin-like growth factor-binding protein 1 (IGFBP1) was evaluated by real-time PCR in these animals.The results revealed that HOXA10 in the luteal phase primate endometrium is differentially expressed in the functionalis and the basalis zones, which is modulated in vivo by progesterone and also by the signals from the incoming embryo suggesting the involvement of HOXA10 in the process of establishment of pregnancy in primates. In addition, the results also demonstrated that the expression of IGFBP1 but not HOXA11 is coregulated with HOXA10 in the endometria of these animals. The pattern of changes in the expression of HOXA10 in response to the two stimuli suggests that endometrial receptivity and implantation not only requires a synchrony of maternal and embryonic signaling on endometrial cells in the primates but there also exists a controlled differential response among the cells of various uterine compartments.

Download Full-text

249.Activin A upregulates endometrial metalloproteases: potential mechanisms for promotion of decidualisation and implantation

Reproduction Fertility and Development ◽

10.1071/srb04abs249 ◽

2004 ◽

Vol 16 (9) ◽

pp. 249

Author(s):

R. L. Jones ◽

P. Paiva ◽

T. J. Kaitu'u ◽

L. A. Salamonsen

Keyword(s):

Epithelial Cells ◽

Stromal Cells ◽

Transforming Growth Factor ◽

Embryo Implantation ◽

Activin A ◽

Matrix Metalloproteases ◽

Trophoblast Invasion ◽

Endometrial Cells ◽

Mmp Inhibitor

Activin and inhibin subunits are co-expressed by human endometrial epithelial and decidualised stromal cells. Activin A is a potent stimulator of decidualisation in vitro, but the mechanisms are unknown. Matrix metalloproteases (MMPs) are known to be important during decidualisation, as administration of a broad spectrum MMP inhibitor in the rat results in reduced decidualisation. Transforming Growth Factor(TGF)-βs are closely related to activins and inhibit MMP production in endometrial epithelial cells. We hypothesised that activins regulate MMP production during decidualisation and/or trophoblast invasion. Epithelial and stromal cells were isolated from human endometrium and treated for 24 h with activin, inhibin, activin/inhibin, and follistatin. Media were collected and subjected to gelatin and caesin zymography. In epithelial cells, activin A stimulated the expression of latent forms of MMPs-1, -2, -7 and -9, and increased formation of active forms of MMPs-2 and -7. Cotreatment with inhibin prevented this stimulation, whilst inhibin alone completely inhibited MMP production. Treatment with follistatin treatment reduced MMP levels. Similar regulation was seen in stromal cells for MMPs-1, -2 and -9. These data show that activin stimulates the production and activation of MMPs in both endometrial cells, and that inhibin is a potent inhibitor. It is interesting that activin is acting in an opposing manner to TGF-β, indicating that these two closely related proteins have divergent signalling pathways in endometrial cells. Further, this is the first demonstration of a role for inhibin in regulating MMPs and indeed for inhibin action in the endometrium. These findings are of potential importance in understanding regulation of MMPs in the peri-implantation endometrium. Activin is the predominant dimer produced by decidual and epithelial cells, where it may be promoting decidualisation though enhancing MMP production and activation. Furthermore, activin secretion by invasive cytotrophoblasts may stimulate focal decidual MMP production promoting their invasion during embryo implantation.

Download Full-text

Decreased mixed lineage leukemia 1 is involved in endometriosis-related infertility

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0193 ◽

2021 ◽

Vol 66 (1) ◽

pp. 45-57

Author(s):

Xue Wen ◽

Yao Xiong ◽

Huimin Liu ◽

Ting Geng ◽

Ling Jin ◽

...

Keyword(s):

Early Pregnancy ◽

Stromal Cells ◽

Expression Patterns ◽

Embryo Implantation ◽

Hypoxia Inducible Factor ◽

Gene Promoter ◽

Mixed Lineage Leukemia ◽

Normal Endometrium ◽

Endometrial Stromal Cells

The aberrant histone methylation patterns contribute to the pathogenesis of endometriosis (EM). Mixed lineage leukemia 1 (MLL1), a histone methyltransferase, is crucial for gene expression by catalyzing the trimethylation of histone 3 lysine 4 (H3K4me3) in gene promoter. This study aimed to explore whether MLL1 is involved in EM-related infertility. The expressions of MLL1 and H3K4me3 were analyzed in the eutopic endometria from EM women with infertility (n = 22) and the normal endometria from EM-free women (n = 22). Mouse EM model was established. The MLL1 and H3K4me3 expression patterns in mice endometria of early pregnancy were also investigated. Immortalized human endometrial stromal cells (iESCs) were cultured and underwent in vitro decidualization. The chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to find the target gene of MLL1 during decidual process. Results showed that both MLL1 and H3K4me3 decreased in the eutopic endometrium from EM patients compared to that in the normal endometrium. During early pregnancy and the decidual process, MLL1 and H3K4me3 were significantly upregulated in stromal cells. ChIP-seq and ChIP-qPCR found that the cytochrome c oxidase subunit 4I 2 (COX4I2) was directly targeted by MLL1. The dominance of COX4I2-containing enzyme induced the expression of hypoxia-inducible factor-2α (HIF-2α), whose expression in the peri-implantation endometrium is essential for embryo implantation. Further results showed that MLL1 was directly regulated by progesterone (P4) – P4 receptors (PRs). Our study proved that MLL1 was involved in EM-related infertility, which may provide a novel approach to treat the nonreceptive endometrium in EM patients.

Download Full-text