Effects of interferon tau on endometrial epithelial cells in caprine in vitro
Introduction
IFNT is a type 1 interferon secreted by trophoblast of conceptus working in a concentration-dependent style (Chethan et al., 2014). Recently, interferon tau (IFNT) is recognized as a signal of maternal recognition of pregnancy (Sandra et al., 2015, Kusama et al., 2016). However, molecular mechanism of IFNT on embryo attachment in caprine remained undefined. We supposed that caprine EECs might make response to different concentration of IFNT to regulate embryo attachment of caprine. In this study, caprine EEC cell line was dealt with 0, 1, 10 and 100 ng/ml of IFNT. EEC cell apoptotic rate and cell cycle were applied to estimate cell apoptosis. Besides, the protein level of pro-apoptotic proteins, Caspase-3 (Abe et al., 2006), FAS (Song et al., 2002) and PRβ (Wang et al., 2003), together with anti-apoptotic proteins, SP1 (Vizcaino et al., 2015), PTN (Park et al., 2008), ERα (Nakamura et al., 2012), and IGF1R (Chen et al., 2016) was measured to analyze the mechanism of cell apoptosis. Although there is not inevitable between these gene expression level and apoptotic status, the expression levels still provides reference for further research. To explore the effect of IFNT on JAK1-STAT3 pathway, mRNA level of JAK1 and STAT3 was determined. Except for apoptotic-related proteins, embryo attachment biomarkers were measured to assess the proper concentration of IFNT for embryo attachment. It is reported that PTEN (Chen et al., 2008) and HOXA10 (Benson et al., 1996, Xu et al., 2014) participate in the formation of normal endometrium. Both OPN and VEGF contribute to endometrial receptivity by involving in angiogenesis and vascular remodeling (Gong et al., 2015). Besides, an indispensable enzyme for arachidonic acid to synthesize to prostaglandins, COX-2, is necessary for embryo attachment (Reese et al., 1999, Zavan et al., 2016). LIF and PRL are confirmed to participate in endometrial preparation for attachment (Dimitriadis et al., 2000). AKT is a serine/threonine protein kinase that plays an essential role in endometrial receptivity (Veillette et al., 2013). It is reported that unexplained infertility is possibly due to reduced STAT3 phosphoration (Dimitriadis et al., 2007). Although JNK, MAPK, STAT3 (Dimitriadis et al., 2007) and AKT pathways (Nurse, 1990) are essential and related to endometrial receptivity, little is known about the effects of IFNT on the pathways in caprine.
Section snippets
Cell culture
EEC cell line was generously provided by Professor Yaping Jin in Northwest A & F University (Wang et al., 2015). EECs were cultured in DMEM/F12 (Gibco, Co., Ltd., Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Co., Ltd., Grand Island, NY, USA). EECs were incubated in a humidified atmosphere with 5% CO2 at 37 °C and dealt with 0, 1, 10 and 100 ng/ml of IFNT. We chose EECs dealt with 0 ng/ml of IFNT as control group.
PTN gene knock-down
Goat PTN siRNA (GenePharma, Shanghai, China) was used to
IFNT at 1 ng/ml and 10 ng/ml induces EEC apoptosis
As is shown in Fig. 1A obtained by flow cytometric analysis, the apoptotic rate was significantly increased in EECs dealt with 1 ng/ml and 10 ng/ml IFNT (P < 0.01). Expression of apoptosis-related proteins (FAS, Caspase-3, SP1, ERα, PRβ, IGF1R and PTN) was measured to analyze the mechanism of apoptosis regulation. Protein expression of activated-Caspase-3 (Act-Caspase-3) was decreased when Caspase-3 was significantly increased at 1 ng/ml of IFNT (P < 0.01, Fig. 1B). FAS and Progestogen Receptor
Discussion
IFNT, which is characterized as a signal of maternal recognition of pregnancy, induces apoptosis in a concentration-dependent style (Chethan et al., 2014, Sandra et al., 2015). In the present study, EEC treated with 0, 1, 10 and 100 ng/ml of IFNT enhanced apoptotic rate at 1 ng/ml and 10 ng/ml of IFNT in caprine EECs cultured in vitro (P < 0.01). Nevertheless, the protein level of activated-Caspase-3 declined in EECs at 1 ng/ml and 10 ng/ml of IFNT. We conjectured that the expression of
Conflict of interest
All of the authors have no conflicts of interest.
Acknowledgements
Thank Nature science foundation of shaanxi Province (2015JM3087), Shaanxi science and technology innovation project plan (2013KTZBO2-02-01), and Shaanxi science and technology innovation project plan (2015KTCQ03-08) for supporting this study. Thank Professor Yaping Jin in Northwest A & F University for generously offering caprine EEC cell line.
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2022, TheriogenologyCitation Excerpt :Goat EECs were seeded at 80% confluency in 6-well plates and incubated with a serum-free medium overnight. Then, the cells were treated with IFNT at the concentration of 1 ng/ml for 12h [17]. As IFNT was diluted with 0.5% BSA, EECs treated with 0.5% BSA only were used as the control.