Mol. Hum. Reprod. Advance Access originally published online on September 2, 2008
Molecular Human Reproduction 2008 14(10):589-594; doi:10.1093/molehr/gan050
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The role of T-lymphoma invasion and metastasis inducing protein 1 in early pregnancy in mice
Department of the Reproductive Physiology, College of Public Health, Chongqing Medical University, No.1, Xueyuan Road, Yuanjiagang, Yuzhong District, Chongqing 400016, PR China
1 Correspondence address: E-mail: zwz007cn{at}yahoo.com.cn
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Abstract |
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T-lymphoma invasion and metastasis inducing protein 1 (Tiam1) is involved in tumorigenesis processes, including cell migration, adhesion and invasion, proteolysis, cytoskeleton reorganization, cell morphological transformation and intracellular signaling. These processes are also critical for embryo implantation, although the role of Tiam1 during embryo implantation remains poorly understood. The aim of this study was to investigate the spatio-temporal expression of Tiam1 in endometria of mice comparing early pregnancy and non-pregnancy. Fluorescent quantitative-PCR and immunohistochemical analysis showed that the expression of Tiam1 mRNA and protein in endometria of pregnant mice was higher than that of non-pregnant mice, and gradually increased from Day 1 of pregnancy reaching a maximum level on Day 5 and then declining on Days 6 and 7. Immunohistochemistry showed that Tiam1 protein was present in luminal epithelium from Days 3–5 of pregnancy and in gland epithelium from Days 4 to 6 and enhanced significantly in stromal cells on Day 5, regarded as the ‘implantation window’ period. The number of implantation sites was greatly decreased by the intrauterine injection with anti-Tiam1 polyclonal antibody in the early morning of the Day 4 of pregnancy. These findings suggest that Tiam1 might play an important role in embryo implantation in mice.
Key words: embryo/endometrium/implantation/early pregnancy/Tiam1
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Introduction |
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Tiam1 was originally identified as an oncogene in 1994 based on its ability to induce invasiveness and metastasis in T lymphoma cells (Habets et al., 1994). Tiam1 maps to human 21q22.1 and mouse chromosome 16, and encodes a 1591 amino acid protein with several functional domains, including a Dbl homology domain (DH), a discs large homology region and two pleckstrin homology (PH) domains (Habets et al., 1994; Worthylake et al., 2000). Tiam1, as a specific guanine nucleotide exchange factor of Rac1, has been demonstrated to have multiple biological activities, such as increasing cellular migration, adhesion and invasion in some cells, and promoting cytoskeleton reorganization, transforming cell morphology and proteolysis (Minard et al., 2004; Uhlenbrock et al., 2004). The Tiaml–Rac1 signaling pathway also plays a role in the regulation of adhesion molecules, transcriptional activation and the cell cycle (Malliri et al., 2004; Mertens et al., 2005).
Embryo implantation is a complex physiological process regulated by a large number of genes and proteins (Enders, 2000), and depends on a series of key events including blastocyst migration, apposition and adhesion to the luminal epithelium, extensive degradation and remodeling of extracellular matrix (ECM), invasion of the maternal endometrium by the trophoblast cells of the developing blastocyst, and secretion of embryonic factors which elicit a cellular reaction in the maternal endometrium (Carson et al., 2000; Achache and Revel, 2006; Gentilini et al., 2007). Synchronization between the development of the blastocyst and the modifications of the maternal endometrium is crucial for successful embryo implantation (Carson et al., 2000; Enders, 2000).
Tiam1 plays a role in the migration, adhesion, proteolysis and invasion of tumor cells (Mertens et al., 2003; Lee et al., 2007); and these properties are shared by trophoblasts in embryo implantation (Janneau et al., 2002; Ferretti et al., 2007).Similarities between mice and human beings in the molecular mechanisms of embryo implantation suggest that a mouse model might provide some insights for embryo implantation in humans (Lee and DeMayo, 2004). However, little information is available in public databases regarding whether Tiam1 has a function in early pregnancy. We speculated that Tiam1 might be involved in the embryo implantation, so studies of Tiam1 spatio-temporal expression in the endometria of non-pregnant- and pregnant-mice, and a functional study of Tiam1 in the embryo implantation of mice, were performed. Here, we present experiments to test our hypothesis. The possible mechanisms in molecular regulation during embryo implantation are also discussed.
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Material and Methods |
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Animals
Six- to eight-week-old NIH mice (23–28 g) were purchased from Laboratory Animal Center of Chongqing Medical University in China. According to vaginal smear (Nelson et al., 1982), the oestrus mice were chosen as non-pregnant group in this study (Day 0 means non-pregnancy). Two female mice were housed with one male mouse from 6:00 p.m. to 6:00 a.m. and then checked for the presence of vaginal plug. Females with vaginal plug were separated and examined for the presence of sperm in a vaginal smear. The day of vaginal plug with sperm present was considered as the Day 1 of pregnancy. Pregnant mice were divided into six groups (Days 2, 3, 4, 5, 6 and 7 of pregnancy), 40 mice in each group. Mice in each group were sacrificed at 8:00–9:00 a.m. Twenty mice uterus in each group were collected and stored in liquid nitrogen for fluorescent quantitative (FQ)-PCR and the other 20 mice uterus were fixed in 4% paraformaldehyde for immunohistochemistry. Another 80 pregnant mice were randomly divided into four groups to undergo intrauterine injection for functional tests of Tiam1, 20 mice in each group. All experimental protocols were in accordance with the policy of the Ethical Committee, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences.
Reagents
The primers for Tiam1 and β-actin were designed and synthesized by Invitrogen Biotechnology Co. (Shanghai, China). SYBR Green 1 Cat (No. SS-7583) was purchased from Shanghai OPE Technology Development Co. (Shanghai, China). Trizol reagent, M-MLV reverse transcriptase, Taq DNA polymerase, Oligo(dT)18, 250 mM MgCl2 and RNAase inhibitor were all from TAKARA Biotechnology Co., Ltd (Dalian, China). Ten millimolar dNTP (Cat No. D0056) was from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Tiam1 (C-16) polyclonal antibody was from Santa Cruz Biotechnology, Inc (CA, USA). HistostainTM-Plus Kits (SP-9001) and DAB color reagent Kit were all purchased from ZSGB-BIO, Inc (Beijing, China). The standard preparation of β-actin was from Shanghai Yingjun Biological Technology & Services Co., Ltd (Shanghai, China).
FQ-PCR of Tiam1 mRNA in mice endometria
RNA extraction
Total RNA was extracted from the frozen mice endometrial tissues using Trizol reagent according to the manufacturer's instructions and then stored at –20°C. Quantification and purity assessment were performed by optical density measurement at 260 and 280 nm. Integrity of the total RNA was identified by agarose gel electrophoresis.
FQ-PCR
The reverse transcription of cDNA was done from 5 µg of total RNA in a 20 µl reaction. Briefly, the RNA 5 µl (1 µg/µl), 1 µl oligo(dT) (500 ng/µl), 4 µl of 5 x First Strand Buffer (250 mM Tris–HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), dNTPs (10 mmol/l) 1 µl, 0.5 µl RNAase inhibitor (40 U/µl), 1 µl M-MLV reverse transcriptase (200 U/µl) and an adequate volume of diethylpyrocarbonate treated water were mixed to make up a total volume of 20 µl and then heated at 70°C for 5 min, followed by an incubation at 42°C for 60 min and heated at 70°C for another 15 min.
The sequences of the primers of Tiam1 and β-actin are given in Table I. A blast search was performed to check the specificity of these primers’ sequences. Quantification of the transcripts was carried out in the ABI Prism® 7000 HT. Reactions were prepared with a 96-well MicroAmp optical plate by the addition of 24 µl PCR master mixture consisting of 5 µl 5 x Taq (HS) buffer, 0.75 µl 10 mM dNTP, 0.3 µl 250 mM MgCl2, 0.25 µl 5 U/µl Taq (HS), 0.7 µl primers (10 pM), 1 µl 1 x SYBR Green 1 and 14 µl ddH2O and 2 µl cDNA. The PCR conditions were set up as follows: initial denaturation at 94°C for 3 min, 40 cycles at 93°C for 15 s (denaturation) and at 60°C for 45 s (annealing and extension), followed by a final extension at 40°C for 10 min.
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Target gene copy values were derived from a standard curve generated by plotting the Ct values for 10-fold serial dilutions of 100–105 copies of the β-actin standard preparation. Each run contained at least three β-actin controls to establish the baseline emission intensity of the quenched reporter dye. The gene expression rate was obtained by normalizing the amount of Tiam1 cDNA with that of β-actin.
Immunohistochemistry study of Tiam1 protein expression in mice endometria
Immunohistochemistry was performed with DAB color reagent Kit, according to the manufacturer's instructions. Whole mice uterus tissues were fixed with PBS containing 4% paraformaldehyde for 24 h and embedded in paraffin. A total of 5 µm sections were taken. Endogenous peroxidase activity was quenched by incubation with 3% H2O2 for 15 min and followed by a rinse in PBS. Antigen retrieval was achieved by heating 10 mmol/l of citrate buffer (pH 6) in a microwave at 92–98°C for 15 min followed by incubation at room temperature for 15 min, and finally rinsing in PBS. Some sections were incubated at 37°C for 2 h with rat anti-mouse Tiam1 polyclonal antibodies (treatment group), and the others incubated with a substitution of preimmune sera for primary antibodies (negative control). After incubation with biotinylated goat anti-Rat IgG, the chromogenic reaction was developed with DAB for 3–5 min and terminated by rinsing in water. Slides were counterstained with hematoxylin before permanent mounting and then evaluated under a light microscope. Tiam1 protein localization was analyzed and quantified using Chengdu Jinpan Media Mix Image Analysis Software 1.0 version (Chengdu, China).
Tiam1 function study during embryo implantation
To determine if Tiam1 has a role in embryonic implantation, mice were given an intrauterine injection of anti-Tiam1 polyclonal antibodies at 8:00–9:00 a.m. on Day 4 of pregnancy before implantation. Eighty pregnant mice were randomly divided into four groups, A, B, C and D, 20 mice in each group. Group A was control group. Groups B, C and D were treated as described below: two uterus horns of each mouse were injected with sterile saline (5 µl) in Group B; two uterus horns of each mouse were injected with the anti-Tiam1 polyclonal antibodies (5 µl) in Group C; the left uterus horn of each mouse was injected with the anti-Tiam1 polyclonal antibodies (5 µl) and the right one was injected with sterile saline (5 µl) in Group D. Mice were sacrificed at 8:00–9:00 a.m. on Day 9 of pregnancy and then the number of implantation sites was recorded.
Statistical analyses
One-way analysis of variance and SNK-q test were used to evaluate the FQ–PCR data. The general linear model was used to analyze the disparity in different locations within same time points and different time points within same locations. The experimental data of functional study were analyzed using variance analysis, SNK-q test and the Wilcoxon matched pairs signed rank test. The data were displayed as mean ± SD. Difference was considered as significance at P < 0.05.
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FQ-PCR analysis of Tiam1 mRNA in mice endometria
The standard curve for FQ-PCR is shown in Fig. 1A. The amplification curve displays the highly amplifying efficiency of PCRs in Fig. 1B. Melting curves of PCR products could be used to monitor non-specific PCR products formed in the PCR (shown in Fig. 1C). The FQ-PCR results showed that the Tiam1 mRNA in endometria of pregnant mice was higher than that in non-pregnant mice, P < 0.05. The expression gradually increased as pregnant days and reached its maximum level on Day 5 of pregnancy and then decreased on Days 6 and 7. The relative amounts of Tiam1 mRNA (Tiam1/β-actin mRNA) in mice endometria were as follows: 0.03 ± 0.004 on Day 0, 0.11 ± 0.03 on Day 2, 0.27 ± 0.04 on Day 3, 0.38 ± 0.05 on Day 4, 0.49 ± 0.10 on Day 5, 0.34 ± 0.10 on Day 6 and 0.24±0.07 on Day 7, showing that there were no significant differences in the expression of Tiam1 mRNA in comparison of Days 3 and 7, and in comparison of Days 4 and 6, respectively (P > 0.05). The significant differences compared with Day 0 are shown in Fig 1D.
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Immunohistochemical analysis of Tiam1 protein expression in mice endometria
The immunolocalization of Tiam1 protein in mice endometria during early pregnancy is shown in Fig. 2A. Yellowish-brown stain was determined as positive. The image analysis results of Tiam1 protein expression localization are shown in Fig. 2B and Table II. The expression profile of Tiam1 protein is similar to its mRNA. Tiam1 located mainly in luminal epithelium from Day 3 to 6 of pregnancy, and in glandular epithelium from Day 4 to 7 and in stromal cells on Days 4, 5 and 6 of pregnancy. The strongest signals of the Tiam1 protein were detected in the luminal epithelium, glandular epithelium and stromal cells on Day 5 of pregnancy. Only faint signals of Tiam1 protein were detected in the luminal epithelium, glandular epithelium and stromal cells of non-pregnant mice endometrium. Control sections substituting preimmune sera for the primary antibodies did not produce any positive immunostaining signals for Tiam1 protein.
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Functional analysis of Tiam1 during embryo implantation
The results of uterus horn injection are shown in Fig. 3. The total number of implanted embryos in Group C (2.9+0.72) was fewer than that in Group A (14.25 + 0.97) and Group B (13.95 + 0.83) (P < 0.05) (shown in Fig. 3E). In Group D, the number of implantation sites in the left uterine horn (1.5 + 0.69) was obviously decreased compared with that in the right uterine horn (7.5 + 0.67) (P < 0.05, shown in Fig. 3F). There were no significant differences in the number of the implanted embryo between Groups A and B (P > 0.05), but significant differences were found comparing Group A versus Group C and Group B versus Group C, and the left uterus versus the right ones in Group E (P < 0.05), respectively. These data showed that the total number of implanted embryos after intrauterine injection with anti-Tiam1 antibodies in mice was fewer than that after no injection or injection of sterile saline.
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Discussion |
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The first 5 days of pregnancy is a critical time for the embryo implantation in mice and Day 5 of pregnancy coincides with the implantation window (Dey et al., 2004). During this period, high expression of Tiam1 mRNA and protein in endometria of the early pregnant mice with a peak on Day 5 of pregnancy were observed, and the total number of implanted embryos was dramatically decreased by anti-Tiam1 intrauterine administration of antibodies on Day 4. All of these findings indicate that Tiam1 might be involved in early pregnancy, but more investigations are necessary to validate this hypothesis.
Several reports showed that Tiam1 can alter organization of the actin cytoskeleton and regulate formation and maintenance of tight junctions (Mertens et al., 2003, 2005). The temporal and cell-specific expression of adhesion junction proteins in the uterus results in molecular signaling, which is important for blastocyst attachment and subsequent invasion (Mertens et al., 2005). Tiam1, localized to adhesion junctions in a number of epithelial cell lines, regulates formation and maintenance of cell–cell adhesions, and its over-expression leads to increasing E-cadherin-based adhesions via regulation of the actin cytoskeleton (Malliri and Collard, 2003; Malliri et al., 2004). The PH domain of Tiam1, mediates its association with sub-membrane region of cells via protein–protein or protein–lipid interactions (Ceccarelli et al., 2007), is required for membrane localization and Rac-dependent membrane ruffling (Carson et al., 2000). The Dbl DH of Tiam1 plays a critical role in cellular transformation, cytoskeletal reorganization (Habets et al., 1994; Michiels et al., 1997; Uhlenbrock et al., 2004). Tiam1 is extensively involved in processes of tumor cells migration and adhesion (Schmitz et al., 2000; Hamelers et al., 2005). The expression of Tiam1 mRNA and protein in our study gradually increased during Days 1–5 of pregnancy and reached the maximum level at the time of the ‘implantation window’. On Days 2 and 3 of pregnancy before the ‘implantation window’ Tiam1 was significantly increased in luminal epithelium compared with that in stroma. We hypothesized that Tiam1 might be involved in apposition and adhesion of blastocyst by altering organization of actin cytoskeleton, forming and maintaining tight junctions and increasing E-cadherin-based adhesions.
Many secretions of endometrial glands are essential for uterine receptivity and successful implantation (Gray et al., 2001; Diedrich et al., 2007). As Tiam1 was highly expressed in the glandular epithelium in mice around the ‘implantation window’, it might play a role in secretory function during early pregnancy. Furthermore, matrix-degradation and proteolysis has an undisputed role in the early stages of embryo implantation, particularly with respect to invasion of the trophoblast (Chen et al., 2001). The invasive period of rodents is on Days 4–10 of pregnancy when endometrial stroma undergoes extensive remodeling in response to blastocyst implantation and the loss of the luminal epithelium is evident at this stage (Enders, 2000; Kennedy et al., 2007). Accumulated evidence showed that Tiam1 was involved in the proteolysis, cell morphological transformation and intracellular signaling (Mertens et al., 2003; Fleming et al., 2004), and tumor cell invasion (Malliri et al., 2006). Tiam1 protein expression was intensively enhanced in the stromal cells on Day 4 of pregnancy and reached the highest value on Day 5, the day of the ‘implantation window’ and remained elevated on Day 6. These findings suggest that Tiam1 might be involved in trophoblast invasion and ECM degradation at the invasive stage of implantation.
In conclusion, we report here the spatio-temporal expression profile of Tiam1 in mice endometria during early pregnancy. The high levels of Tiam1 in mice endometria around the ‘implantation window’ raises the possibility that Tiam1 could be involved in early pregnancy, especially, embryonic implantation. However, there was still a few implanted embryos when Tiam1 signal was blocked, suggesting that Tiam1 might not be the sole regulator during embryonic implantation of mice. Further investigation is necessary to explore and define the function and molecular mechanism of Tiam1 during embryo implantation in mice and determine whether this molecule may also be involved in implantation (and infertility) in humans.
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Acknowledgements |
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We thank Profs Mao-Sheng Yang, Yi Tan and Xue-Mei Lian in Chongqing Medical University for their helpful and constructive comments on the preparation of this manuscript, Dr Qiong Shi at the Medicine Laboratory Center, Chongqing Medical University for her assistance in primer design, Dr Nian-Qing Lv at Jiangsu Family Planning Research Institute for his excellent assistance in the revised manuscript. We apologize for unintended omission of any relevant references.
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Submitted on May 9, 2008; resubmitted on August 28, 2008; accepted on August 28, 2008.
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