Regulation of Embryonic Signal on Talin1 in Mouse Endometrium

Embryonic signals can affect the spatiotemporal-specific expression of the uterus to establish a successful pregnancy. Our previous study has found that talin1 underwent dynamic changes in the mouse endometrium during peri-implantation period. However, whether talin1 is affected by the embryo signals is not clear. In order to investigate the effect of embryonic signals, especially human chorionic gonadotropin (HCG) on talin1, we have designed mouse models of pseudopregnancy, delayed implantation and activation, and HCG treatment. Using these models, the expression of talin1 in the mouse endometrium was determined by immunohistochemistry and Western blotting. In the pseudopregnancy model, an increased expression of talin1 was found from day 3 to day 5, whereas the talin1 protein was decreased on day 5 in the normal pregnant mice. In the delayed implantation model, a strong cytoplasmic staining of talin1 was found, especially in stromal cells. However, after activation of the implantation, the expression of talin1 decreased ( P < .05). Furthermore, a significantly lower expression of talin1 was found at the implantation site when compared to the interimplantation sites ( P < .05). In the HCG treatment model, an intrauterine perfusion of 10u HCG significantly reduced the expression of talin1 in both stromal and epithelial cells in pseudopregnant mice ( P < .05), although further increase in the HCG concentration did not have additional effect on expression of talin1. Taken together, our data suggest that the presence of embryos can affect expression of talin1 in the mouse endometrium, and a certain concentration of HCG can regulate its expression.

Download Full-text

The Role of the FOXO1/β2-AR/p-NF-κB p65 Pathway in the Development of Endometrial Stromal Cells in Pregnant Mice under Restraint Stress

International Journal of Molecular Sciences ◽

10.3390/ijms22031478 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1478

Author(s):

Jiayin Lu ◽

Yaoxing Chen ◽

Zixu Wang ◽

Jing Cao ◽

Yulan Dong

Keyword(s):

Oxidative Stress ◽

Stromal Cells ◽

Restraint Stress ◽

Target Genes ◽

Mrna Levels ◽

Endometrial Stromal Cells ◽

Protein Levels ◽

Pregnant Mice

Restraint stress causes various maternal diseases during pregnancy. β2-Adrenergic receptor (β2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the β2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the β2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, β2-AR, and even the protein levels of FOXO1 and β2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when β2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the β2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.

Download Full-text

Delayed implantation induced by letrozole in mice

10.1101/2021.09.15.460438 ◽

2021 ◽

Author(s):

Fang Wang ◽

Shijie Li ◽

Lingshuai Meng ◽

Ye Kuang ◽

Zhonghua Liu ◽

...

Keyword(s):

Embryo Implantation ◽

Late Pregnancy ◽

High Dose ◽

Successful Pregnancy ◽

Short Delay ◽

Wild Mice ◽

Plasma Progesterone ◽

Delayed Implantation ◽

Gene Ablation ◽

At Will

Implantation timing is key for a successful pregnancy. Short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the delay on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneally administration of letrozole at 5 mg/kg body weight on the day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, high dose of estrogen (25 ng) caused compromised implantation. We also found that only 67% of the female mice could be pregnant normally and finally gave birth when the injection of estradiol (3 ng) was on day 5 of pregnancy (i.e., one day delay). Most of the failed pregnancies had impaired decidualization, decreased plasma progesterone levels and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily used to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.

Download Full-text

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese black bear, Ursus thibetanus japonicus, during pregnancy

Reproduction ◽

10.1530/rep.0.1210587 ◽

2001 ◽

pp. 587-594 ◽

Cited By ~ 20

Author(s):

T Tsubota ◽

S Taki ◽

K Nakayama ◽

JI Mason ◽

S Kominami ◽

...

Keyword(s):

Cytochrome P450 ◽

Corpus Luteum ◽

Fetal Development ◽

Black Bear ◽

Corpora Lutea ◽

Luteal Cells ◽

Ursus Thibetanus ◽

Delayed Implantation ◽

Post Implantation ◽

Implantation Period

The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the cholesterol side chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3betaHSD, P450c17 and P450arom. P450scc and 3betaHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3betaHSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.

Download Full-text

HB-EGF regulates Prss56 expression during mouse decidualization via EGFR/ERK/EGR2 signaling pathway

Journal of Endocrinology ◽

10.1530/joe-16-0636 ◽

2017 ◽

Vol 234 (3) ◽

pp. 247-254 ◽

Cited By ~ 7

Author(s):

Jie Liu ◽

Fei Gao ◽

Yue-Fang Liu ◽

Hai-Ting Dou ◽

Jia-Qi Yan ◽

...

Keyword(s):

Signaling Pathway ◽

Stromal Cells ◽

Embryo Implantation ◽

Implantation Site ◽

Mouse Uterus ◽

Delayed Implantation ◽

Initial Stage ◽

And Function ◽

Key Steps

Embryo implantation and decidualization are key steps for successful reproduction. Although numerous factors have been identified to be involved in embryo implantation and decidualization, the mechanisms underlying these processes are still unclear. Based on our preliminary data, Prss56, a trypsin-like serine protease, is strongly expressed at implantation site in mouse uterus. However, the expression, regulation and function of Prss56 during early pregnancy are still unknown. In mouse uterus, Prss56 is strongly expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy compared to inter-implantation site. Under delayed implantation, Prss56 expression is undetected. After delayed implantation is activated by estrogen, Prss56 is obviously induced at implantation site. Under artificial decidualization, Prss56 signal is seen at the primary decidual zone at the initial stage of artificial decidualization. When stromal cells are induced for in vitro decidualization, Prss56 expression is significantly elevated. Dtprp expression under in vitro decidualization is suppressed by Prss56 siRNA. In cultured stromal cells, HB-EGF markedly stimulates Prss56 expression through EGFR/ERK pathway. Based on promoter analysis, we also showed that Egr2 is involved in Prss56 regulation by HB-EGF. Collectively, Prss56 expression at implantation site is modulated by HB-EGF/EGFR/ERK signaling pathway and involved in mouse decidualization.

Download Full-text

Blastocyst-induced ATP release from luminal epithelial cells initiates decidualization through the P2Y2 receptor in mice

Science Signaling ◽

10.1126/scisignal.aba3396 ◽

2020 ◽

Vol 13 (646) ◽

pp. eaba3396

Author(s):

Xiao-Wei Gu ◽

Zi-Cong Chen ◽

Zhen-Shan Yang ◽

Yan Yang ◽

Ya-Ping Yan ◽

...

Keyword(s):

Epithelial Cells ◽

Stromal Cells ◽

Embryo Implantation ◽

Atp Release ◽

Sterile Inflammation ◽

Short Exposure ◽

Uterine Epithelial Cells ◽

High Concentrations ◽

Implantation Period ◽

Luminal Epithelial Cells

Embryo implantation involves a sterile inflammatory reaction that is required for the invasion of the blastocyst into the decidua. Adenosine triphosphate (ATP) released from stressed or injured cells acts as an important signaling molecule to regulate many key physiological events, including sterile inflammation. We found that the amount of ATP in the uterine luminal fluid of mice increased during the peri-implantation period, and this depended on the presence of an embryo. We further showed that the release of ATP from receptive epithelial cells was likely stimulated by lactate released from the blastocyst through connexin hemichannels. The ATP receptor P2y2 was present on uterine epithelial cells during the preimplantation period and increased in the stromal cells during the time at which decidualization began. Pharmacological inhibition of P2y2 compromised decidualization and implantation. ATP-P2y2 signaling stimulated the phosphorylation of Stat3 in uterine luminal epithelial cells and the expression of early growth response 1 (Egr1) and prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2), all of which are required for decidualization and/or implantation, in stromal cells. Short exposure to high concentrations of ATP promoted decidualization of primary stromal cells, but longer exposures or lower ATP concentrations did not. The expression of genes encoding ATP-degrading ectonucleotidases increased in the decidua during the peri-implantation period, suggesting that they may limit the duration of the ATP signal. Together, our results indicate that the blastocyst-induced release of ATP from uterine epithelial cells during the peri-implantation period may be important for the initiation of stromal cell decidualization.

Download Full-text

Mesenchymal Stromal Cells From Myelodysplastic Syndrome Patients Show Lower DICER1 and DROSHA Expression, as Well as Decreased Mir-155 and Mir-181a MicroRNA Expression, When Compared with Healthy Controls

Blood ◽

10.1182/blood.v116.21.397.397 ◽

2010 ◽

Vol 116 (21) ◽

pp. 397-397

Author(s):

Carlos Santamaría ◽

Olga López-VIllar ◽

Sandra Muntión ◽

Belén Blanco ◽

Soraya Carrancio ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Protein Expression ◽

Myelodysplastic Syndrome ◽

Western Blot ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Microrna Expression ◽

Healthy Controls ◽

Lower Expression

Abstract Abstract 397 Mesenchymal stromal cells (MSC) are closely related to the regulation of hematopoietic stem cell niche. Recently, Raaijmakers et al (Nature, 2010), published that deletion of Dicer1, a RNase III enzyme involved in microRNA biogenesis, in murine MSC-derived osteoprogenitors triggered peripherical blood cytopenias, myelodysplasia and subsequent AML, showing that molecular alterations in bone marrow microenvironment could result in clonal impaired haematopoiesis. Here, we have investigated whether MSC from myelodysplastic syndrome (MDS) patients show differences in DICER1 and DROSHA, another RNA III endonuclease, in comparison to healthy MSC. In addition, we have analyzed several hematopoietic-related microRNAs in these same samples. Bone marrow MSC from MDS patients (n=35; 10 5q- syndrome, 4 RA, 5 RARS, 10 RCMD, 3 RAEB, 2 MDS-U and 1 hypocellular MDS) and healthy donors (HD, n=20) were isolated and in vitro expanded following standard procedures until the third passage. Additionally, paired mononuclear cells (MNC) from 13 MDS and 8 HD were obtained. Total RNA was isolated using TRIzol reagent (Invitrogen). DICER1 and DROSHA relative gene expressions were assessed by quantitative PCR (Q-PCR) using commercial TaqMan® assay (Applied Biosystems®) with GAPDH as control gene. DICER1 and DROSHA (Abcam) protein expression were evaluated in whole cell lysates by western blot, using calnexin (Stressgen) as control. Several microRNAs with known role in hematopoiesis and immune system regulation were analyzed in 25 MDS and 12 HD by Q-PCR using commercial TaqMan® MicroRNA assay (Applied Biosystems®) with RNU43 as control microRNA. MSC from MDS showed significant lower DICER1 (0.0035±0.0020 vs. 0.0076±0.0092; p=0.044) and DROSHA (0.0070±0.0028 vs. 0.0135±0.0176; p=0.019) gene expression levels than healthy controls. Moreover, MSC from MDS showed lower protein expression of both DICER1 and DROSHA by western blot analysis, confirming Q-PCR findings. By contrast, no difference in either DICER1 (0.0197±0.0151 vs. 0.0173±0.0112; p=0.9) or DROSHA (0.0089±0.0023 vs. 0.0067±0.0037; p=0.09) gene expression were observed between MNC from MDS and HD. As far as microRNA expression, we observed a lower expression of mir-155 (0.63±0.92 vs. 0.94±0.49; p=0.007) and mir-181a (1.30±0.95 vs. 2.02±1.05; p=0.041) in MSC from MDS in comparison to healthy controls. Mir-155 and mir-181a are involved in T-cell and B-cell differentiation, while mir-155 are also related to erythroid and megakarycytic differentiation. We conclude that MSC from MDS patients show lower expression of DICER and DROSHA, two relevant RNA-III endonucleases involved in the microRNA biogenesis, confirming recent findings in murine models. Moreover, the expression of some microRNA is impaired in these cells, raising the possibility that these microenvironmental alterations could be involved in the MDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Lenalidomide Differentially Modifies the Genomic Profile and miRNA Expression of Mesenchymal Stromal Cells From Patients with 5q- Syndrome,

Blood ◽

10.1182/blood.v118.21.3810.3810 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3810-3810

Author(s):

Sandra Muntión ◽

Carlos Santamaría ◽

Beatriz Roson ◽

Carlos Romo ◽

Olga López-Villar ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profile ◽

Mesenchymal Stromal Cells ◽

Expression Profile ◽

Stromal Cells ◽

Microrna Expression ◽

Advisory Committees ◽

Healthy Donors ◽

Lower Expression

Abstract Abstract 3810 Mesenchymal stromal cells (MSC) are a non-hematopoietic BM cell population considered to be not only the osteoblastic progenitors, but also a key component of the hematopoietic microenvironment. Raaijmakers et al (Nature, 2010) have recently shown that deletion of Dicer1 in MSC-derived osteoprogenitors as well as its target gene SBDS resulted in myelodysplasia (MDS) in a murine model. We have previously confirmed these results in human MSC from MDS patients (ASH 2010, # 397). In a previous paper (Leukemia, 2009) we showed that MSC from 5q- syndrome patients were different from MSC from other types of MDS and could be involved in their development. We have hypothesized that lenalidomide, the standard treatment of 5q- patients could act not only on hematopoietic progenitors but also on the BM microenvironment. For this purpose BM-MSC from healthy donors (HD) (n=7) and 5q- syndrome patients (n=5) were expanded in vitro and treated with 50 uM lenalidomide or its solvent (DMSO) as control. RNA was obtained from MSC and DICER1, DROSHA and SBDS relative gene expression was assessed by real-time PCR using TaqMan® assay as well as several microRNAs with known role in hematopoiesis and immune system regulation. In addition, MSC gene expression profile was studied. Labeled samples were hybridized to affymetrix of oligonucleotide HU 1.OST arrays in 5q- patients (n=4) and compared with MSCs from HD (n=3). For this purpose the ratio lenalidomide-treated sample and its paired DMSO control was calculated and markers with a fold change >1.5 were selected for hierarchical clustering analysis (HCA). MSCs from 5q-syndrome showed lower expression of DICER1 when compared with those from HD (.35 x10−3 vs.20 x10−3 p=0.03) but this expression was recovered when 5q-MSCs were treated with Lenalidomide (0.32 x10−3 p= 0.34). By contrast, no differences in DROSHA expression were observed. In addition, 5q-MSC showed SBDS lower expression than HD-MSC and in both groups the expression increased when they were treated with lenalidomide fig1). When microRNAs were analyzed, we observed a lower microRNA expression in lenalidomide-treated MSC from healthy donors when was compared to paired non-treated cells, especially for miRNA-155 (p=0.028), miRNA-222 (p=0.028),and miRNA-181a (p=0.075; Table 1). By contrast, lenalidomide-treated MSC from MDS showed a trend towards higher microRNA expression in comparison to paired non-treated MSC.Table 1.HD-MSC DMSO vs LENA5q-MSC DMSO vs LENAmiRNA 1460.50 vs 0.30p=0.2490.07 vs 0.10p=0.7miRNA 1500.004 vs 0.0065p=0.60.001 vs 0.006p=0.07miRNA 1550.90 vs 0.58p=0.0280.80 vs 0.96p=0.7miRNA 181a2.47 vs 1,83p=0.0751.66 vs 2.32p=0.07miRNA 22286.2 vs 68.0p=0.02843.2 vs 56.2p=0.07 When the gene expression profile was carried out based in 421 selected probes including 306 known genes, MSC-treated cells from 5q- were separated from HD MSC by HCA (Fig2). We can conclude that Lenalidomide not only acts on HPC from 5q- patients but also on microenvironment by modifying the expression of DICER-1 and SBDS as well as the expression of some microRNAs and genes. Disclosures: San Miguel: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees. del Cañizo:Celgene Corp.: Spanishn Adviory committee.

Download Full-text

Changes in thymidine phosphorylase expression in response to neoadjuvant chemotherapy for primary breast cancer

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.655 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 655-655

Author(s):

F. Puglisi ◽

M. Mansutti ◽

A. Minisini ◽

S. Russo ◽

G. Cardellino ◽

...

Keyword(s):

Breast Cancer ◽

Neoadjuvant Chemotherapy ◽

Tumor Cells ◽

Anticancer Agents ◽

Stromal Cells ◽

Primary Breast Cancer ◽

Histological Evaluation ◽

Cytoplasmic Staining ◽

Primary Mouse ◽

Response To Neoadjuvant Chemotherapy

655 Background: Thymidine phosphorilase (TP) is a key enzyme involved in nucleoside metabolism. Recently, it has been hypothesized that TP modulation could enhance the therapeutic activity of TP-targeting chemotherapy such as capecitabine. In addition, some evidence exists that anticancer agents could upregulate TP. The present study analyzed TP immunohistochemical expression in response to neoadjuvant chemotherapy for primary breast cancer. Methods: Fifty-five women with operable breast cancer (T ≥ 2 cm, N0–1, M0) were treated with anthracycline-based (all cases) and anthracycline/taxane-based (n= 40 cases) neoadjuvant chemotherapy. Tumor samples from diagnostic large core biopsy (n=55) and from surgery (n=53) were available for histological evaluation and immunohistochemical analysis of TP. Immunohistochemistry was performed at a single central laboratory using a primary mouse anti-TP monoclonal antibody (Roche molecular biochemicals).TP expression was evaluated on tumor cells (nuclear and cytoplasmic staining) and on stromal cells. The intensity of cytoplasmic immunoreactivity was scored as 0, 1, 2 or 3 denoting negative, weak, moderate and strong staining, respectively. Results: An increase in TP cytoplasmic expression was observed in 35.89% (95% CI: 0.21–0.52%) of tumor samples after neoadjuvant chemotherapy. In particular, increases in cytoplasmic TP score were more common after taxane-containing regimens (40.74%, 95% CI: 0.22–0.61%) than after regimens without taxanes (25%, 95% CI: 0.05–0.57%). No significant changes of TP expression were found in nuclei of tumor cells after neoadjuvant chemotherapy. Similarly, no significant changes of TP expression were observed in stromal cells. There was no significant association between clinical or pathological response rate and TP changes in both tumor and stromal cells. Conclusions: This study provides further evidence that, at least in breast cancer, TP is upregulated after anthracycline and/or taxane-containing chemotherapy. According to these results, a strong rationale exists in combining TP-inducing and TP-targeting anticancer agents. No significant financial relationships to disclose.

Download Full-text