Hum. Reprod. Advance Access originally published online on February 21, 2008
Human Reproduction 2008 23(7):1581-1593; doi:10.1093/humrep/dem401
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Implantation-associated gene-1 (Iag-1): a novel gene involved in the early process of embryonic implantation in rat
1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China 2 Graduate University of the Chinese Academy of Sciences, Beijing 100080, People's Republic of China
3 Correspondence address. Tel: +86-10-64807183; Fax: +86-10-64807099; E-mail: pengjp{at}ioz.ac.cn
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Abstract |
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BACKGROUND: In order to study the novel genes related to rat embryonic implantation, a novel implantation-associated gene, Iag-1, was identified and characterized from rat uterus of early pregnancy. Iag-1 was initially derived from suppressive subtracted hybridization of a cDNA library of rat uterus, which was used to analyse differentially expressed genes between the preimplantation and implantation period.
METHODS: The full-length cDNA sequence of Iag-1 was cloned from rat uterus on D5.5 of pregnancy by 5'- and 3'-RACE. The expression of Iag-1 in the uterus of early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantation was detected by northern blotting, in situ hybridization, western blotting and immunofluorescence. Endometrial stromal cells (ESCs) were isolated from rat uterus. The effect of Iag-1 on ESCs proliferation and apoptosis were determined by MTT assay, TUNEL and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting.
RESULTS: Differential patterns of Iag-1 expression were detected in rat embryo and in the uterus during the peri-implantation period. Iag-1 was specifically localized in glandular epithelium and luminal epithelium. In contrast, the expression of Iag-1 was not significantly altered in uterus of pseudopregnancy and artificial decidualization, but was significantly increased in the uterus after activation of delayed implantation. Stable expression of introduced Iag-1 inhibited the proliferation of in vitro-cultured ESCs. Significant apoptosis was also detected in the ESCs overexpressing Iag-1, along with the enhancement of p53 and Bax protein expression.
CONCLUSIONS: Overexpression of Iag-1 can inhibit ESCs proliferation and induce ESCs apoptosis, and p53 and Bax may play an important role in the process of Iag-1-induced apoptosis.
Key words: Iag-1/embryo implantation/uterus/rat
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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Implantation is a highly coordinated sequence of events that begins with the attachment of an embryo to the uterine luminal epithelium and ultimately results in the formation of the placenta. Implantation of the embryo to the uterine wall is regulated by various factors, e.g. hormones (Defeo, 1963
; Venners et al., 2006), cytokines (Sun et al., 2005; Xia et al., 2006), growth factors (Koyama et al., 2006; Wang et al., 2006) and matrix metalloproteinases (Riley et al., 1999; Li et al., 2002). Although a growing list of molecules are known to be involved in the implantation process, specific mechanisms associated with the onset of uterine receptivity remain to be determined.
High-throughput techniques make it possible to get information about whole molecular events of embryo implantation. To obtain a global view and identify novel pathways of implantation, high-throughput screening methods [e.g. microarray analysis, suppressive subtracted hybridization (SSH) and serial analysis of gene expression (SAGE)] have been used to identify differentially regulated genes from uterus of human and mouse during the window of receptivity in different models. In human, natural cycles (Carson et al., 2002; Kao et al., 2002; Borthwick et al., 2003; Riesewijk et al., 2003; Mirkin et al., 2005), controlled ovarian stimulated cycles (Mirkin et al., 2004; Horcajadas et al., 2005; Simon et al., 2005) and endometriosis (Lebovic et al., 2000, 2002; Eyster et al., 2002; Arimoto et al., 2003; Kao et al., 2003; Matsuzaki et al., 2004, 2005) have been used to study the genomics of the endometrium in the window of implantation. In mouse, normal pregnancy (Nie et al., 2000; Yoshioka et al., 2000; Yoon et al., 2004; Chen et al., 2006; Ma et al., 2006), pseudopregnancy (Campbell et al., 2006; Pan et al., 2006) and delayed implantation (Reese et al., 2001; Lee et al., 2003) have been used to analyse the genomics of the endometrium during implantation.
However, up to now, the available knowledge in the literature about differentially regulated genes of rat endometrium in the window of implantation is mainly from the reports of Simmons. Simmons et al. (1999, 2002, 2004) used the technique of SSH and differential display of reverse transcription–PCR to detect differential expression genes of rat uterus of pseudopregnancy and delayed implantation, and found that glucose-regulated protein 78 (GRP78), uterine sensitization-associated gene-1 (USAG-1) and vitamin D3 up-regulated protein 1 (Vdup1) were concerned with establishment of the receptive state of endometrium.
To further explore the differential expressed genes of rat uterus in the window of implantation and the mechanism of embryo implantation, we constructed a SSH library by subtracting mRNAs of Day 5.5 of pregnancy rat uterus from that of Day 4 of pregnancy rat uterus, and identified various novel cDNA sequences from the library (data not shown). In this paper, we report the cloning, characterization and function of a novel gene, which we have termed Iag-1, from this subtracted cDNA library. Characterization of novel genes expressed in uterus may provide new insights into the mechanism of embryo implantation.
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Experimental animals and protocols
Sexually mature, healthy female Sprague–Dawley rats (220–260 g body weight) were purchased from Institute of Genetics and Development Biology, Chinese Academy of Sciences. Rats were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. All animal procedures were approved by the Institutional Animals Care and Use Committee of the Institute of Zoology. Female rats were caged overnight with male Spargue–Dwaley rats, and the presence of a vaginal plug or sperm was considered as Day 1 of pregnancy (g.d.1). Uteri were excised from g.d.4 to g.d.8 rat and fixed with 4% paraformaldehyde solution (Sigma Chemical Co., St. Louis, MO, USA) for in situ hybridization or frozen in liquid nitrogen for RNA and protein analysis.
Pseudopregnancy was induced by caging adult females with vasectomized males and was confirmed by checking the vaginal plug. The presence of a vaginal plug was considered as Day 1 of pseudopregnancy. The uteri were collected from Days 4–7 of pseudopregnancy. On Day 5 of pseudopregnancy, when the uterus was optimally sensitized to deciduogenic stimulus, 100 µl sesame oil was infused into the lumen of one of the uterine horns to induce artificial decidualization. The contralateral uterine horn, which was not infused with oil, served as a control. At Day 7 and 8 of pseudopregnancy, the rats were killed, and the uterine horns were isolated.
To induce delayed implantation, the pregnant rats on g.d.4 were ovariectomized at 0830–0900 h. Progesterone (3 mg/rat, s.c.; Sigma) was injected to maintain delayed implantation from Days 5–7. The progesterone-primed delayed-implantation rats were treated with estradiol-17β (0.5 µg/rat; Sigma) to terminate the delayed implantation. The rats were killed by stunning and cervical dislocation was used to collect uteri at 24 h after estrogen treatment. The implantation sites were also identified by i.v. injection of Chicago blue solution (Sigma). Delayed implantation was confirmed by flushing the blastocysts from the uterus.
To get preimplantation embryos at different developmental stages, we flushed the oviduct or uterus at 0900 and 1400 h on g.d.2–g.d.5 in Sprague–Dawley rats. All embryos were then fixed for 30 min in freshly prepared 4% paraformaldehyde (Sigma) for indirect immunofluorescence.
Rapid amplification of 5' and 3'-cDNA ends
5' and 3' rapid amplification of cDNA ends (RACE), using the SMART RACE cDNA Amplification Kit (Clontech Laboratories Inc., Palo Alto, CA, USA), was used to obtain the full sequence information for Iag-1. Briefly, 5' and 3'-RACE ready cDNA was synthesized using 1 µg total RNA from uterus tissue of g.d.5 rat by reverse transcriptase according to the manufacturer’s protocol. Universal primer mix provided in the kit and gene-specific primers (5' RACE primer: 5'-TGA CGA TCA TCC ACT CCA GGC G-3'; 3'-RACE primer: 5'-TGG GTG AGC TCT TTG CCC TCA G-3') based on the sequence of a cDNA fragment isolated from a SSH library were used for the 5' and 3'-RACE experiments. The PCR products were cloned into pMD18-T vector (TaKaRa). Searches for homology to Genbank database sequences, potential structural motifs and open reading frame determination were done using the databases available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) and the Prosite database of protein families and domains (http://ca.expasy.org/prosite/).
RT–PCR and northern blotting analysis
Total RNAs from adult rat tissues including hypothalamus, pituitary, lung, heart, stomach, liver, spleen, small intestine, large intestine, kidney, skeletal muscle, uterus and ovary were extracted with TRIzol reagent (Invitrogen Life Technologies Inc., USA) according to the manufacture’s instructions for RT–PCR analysis. Total RNAs (2 µg) from each tissue were used as templates for reverse transcription using Superscript III (Invitrogen). The primers used to amplify the 1353 bp coding sequence of Iag-1 cDNA were 5'-ATG CCA GCC AGA CAA CTC CAA ACC CT-3' (forward) and 5'-TCA GAA GAG GAC TCT CCC CAG CTC GAA C-3' (reverse). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control.
Thirty microgram of total RNA from uteri of pregnant and pseudopregnant rats was subjected to electrophoresis in 1% denatured agarose gel, and capillary method used to transferred to nylon membrane (Hybond N+; Amersham Pharmacia Biotech, St Albans, Herts, UK). The membranes were prehybridized in prehybridization solution (0.2 mmol/l sodium phosphate, 0.1 mmol/l EDTA, 7% SDS, 1% BSA, 15% formamide) at 65°C for 4 h. We used the same primer pairs as in RT–PCR to synthisize the probe which was labelled with 32P. Hybridizations were performed at 65°C overnight in the hybridization buffer containing specific radioactive probe. Membranes were washed and exposed to autoradiography (Kodak BioMax MS film, Eastman Kodak Co., Rochester, NY, USA) overnight at –80°C. All experiments were repeated at least three times. The bands were analysed using Quantity One software (Bio-Rad, Hercules, CA, USA).
In situ hybridization
The whole coding sequence of Iag-1 cDNA was amplified from the uterus of g.d.5.5 rat by the above RT–PCR primers and cloned into pGEM-T plasmid (Promega, Madison, WI, USA). The cloned Iag-1 fragment was further verified by sequencing. These plasmids were linearized with appropriate enzymes for labelling. Fluorescein-labelled antisense or sense cRNA probes were transcribed in vitro using an RNA Colour Kit (T7 for sense, SP6 for antisense; Amersham Biosciences), then the probes were incubated with 0.4 mmol/l NaHCO3 and 0.6 mmol/l Na2CO3 at 60°C for 35 min to hydrolyse them into small fragments (between 200 and 250 bp). Frozen sections (8 µm) of uterus of pregnancy and pseudopregnancy rats were hybridized with fluorescein-labelled antisense probes in 50% formamide buffer at 55°C for 16 h. The fluorescein-labelled cRNA probe was detected using anti-fluorescein antibodies at a dilution of 1:500. Finally, the colour reaction was developed with BCIP/NBT solution overnight in the dark at 4°C. The sections were hybridized with sense probes as negative controls. Samples were viewed with an Eclipse 80i microscope (Nikon, Japan).
Recombinant protein expression and antibody production
The peptide used for raising antibody was derived from the full length of Iag-1. The primer pairs for cloning were: forward/BamHI (5'- GGC GGA TCC ATG CCA GCC AGA CAA CTC -3') and reverse/HindIII(5'-CGG AAG CTT GAC TCT CCC CAG CTC GAA C-3'). After being double digested with BamHI and HindIII, the PCR product was cloned into the prokaryotic expression vector pET28A (+) (Novagen, Madison, WI, USA) in frame with the C-terminal His 6-tagged fusion protein and the construct was verified by DNA sequencing. The recombinant construct was transformed into E. coli strain BL21 (DE3) and induced with 1.0 mmol/l isopropyl-thio-β-D-galactopyranoside at 0.4 optical density (at A600), and cells were harvested 4 h later. Tagged recombinant protein was purified using His-binding resin column HiTrap Chelating HP according to the manufacturer’s protocol (Amersham Biosciences).
Female New Zealand rabbits purchased from the Institute of Genetics of the Chinese Academy of Sciences with body weight 
2 kg were immunized s.c. with 400 µg of affinity-purified recombinant IAG-1 protein emulsified in Freunds complete adjuvant (Sigma), and were boosted three times at intervals of 2 weeks with 400 µg of protein emulsified in Freunds incomplete adjuvant (Sigma). Ten days after the last booster, the animals were bled and the serum was collected. Also the serum prior to immunization was collected, and all the serum was stored at –20°C for later use. Antibody titre was detected by enzyme-linked immunosorbent assay. Antibody speciality was detected by western blotting.
Western blotting analysis
Proteins obtained from uterine lysates were boiled in SDS/β-mercaptoethanol sample buffer, and 50 µg samples were loaded onto each lane of 15% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were blotted onto nitrocellulose (NC) membranes (Amersham Biosciences) by electrophoretic transfer in 25 mmol/l of Tris and 192 mmol/l of glycine buffer at pH 8.3. The membranes were blocked in 5% skimmed dry milk in TBST (TBS containing 0.1% Tween-20) overnight at 4°C. The membranes were then incubated with rabbit anti-IAG-1 ployclonal antibody, rabbit anti-p53 ployclonal antibody (sc-6243, SantCruz), mouse anti-Bax monoclonal antibody (sc-7480, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse anti-Bcl-2 monoclonal antibody (sc-7382, SantCruz), rabbit anti-caspase-3 ployclonal antibody (sc-7148, SantCruz), rabbit anti-Fas ployclonal antibody (sc-716, SantCruz), rabbit anti-Fas-L ployclonal antibody (sc-834, SantCruz) or rabbit anti-β-actin ployclonal antibody (sc-1616-R, SantCruz) for 2 h at 37°C. Rabbit anti-IAG-1 ployclonal antibody was diluted 1:5000 and other antibodies were diluted 1:200. The specific protein–antibody complex was detected by using horse-radish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc.). Detection by the chemiluminescence (ECL) reaction was carried using the ECL kit (Pierce Biotechnology Inc., Rockford, IL, USA). All experiments were repeated at least three times. The bands were analysed using Quantity One analyzing system (Bio-Rad).
Immunofluorescence
After fixation in 4% paraformaldehyde, sections (8 µm) from uteri of pregnant and pseudopregnant rats or embryos were washed three times in PBS and permeabilized for 20 min in PBS containing 0.1% Triton X-100 (Sigma) at room temperature. After rinsing several times in PBS, sections or embryos were incubated in 5% BSA for 45 min at room temperature to block non-specific binding of the antibodies, then incubated with rabbit anti-IAG-1 antibody diluted 1:1000 in PBST at 4°C overnight. After rinsing in PBS, sections or embryos were incubated in fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:300, Jackson Immunoresearch Laboratories) for 1 h at 37°C, then rinsed in PBS. Nuclei were stained with 0.01 mg/ml of propidium iodide (Sigma) for 10 min. Sections or embryos were viewed under a laser-scanning confocal microscope (ZEISS LSM 510 META, Germany). To evaluate the specificity of the antibodies, negative control staining was performed by substituting normal rabbit serum for the primary antibody.
Preparation of ESCs and transfection of pCR3.1–Iag-1 plasmid into ESCs
Endometrial stromal cells (ESCs) were enzymatically isolated from the rat uterus according to the method described by McCormack and Glasser (1980) with slight modifications. Uteri were removed from g.d.5 rat and washed in sterile PBS. After trimming off the fatty and connective tissues, the uteri were sliced longitudinally. All sliced uteri were put together into a 15-ml tube containing 10 ml PBS supplemented with 6.5 mg/ml trypsin and 25 mg/ml pancreatin. The tube was kept at 4°C for 60 min and at room temperature for another 45 min. As the enzyme-containing PBS was carefully poured away, Dulbecco’s modified Eagle’s medium (DMEM)/F-12 culture medium containing 10% fetal bovine serum, 1% non-essential amino acids, 100 IU/ml penicillin and 10 mg/ml streptomycin was added to stop the activity of the trypsin. The medium was replaced with PBS 5 min afterward. The tissues were then gently shaken for 30 s. Uterine tissues were removed and the filtrate was centrifuged at 1000g for 5 min. Then the cell pellet was resuspended in DMEM/F-12 medium. The isolated endometrial cells were plated at a density of 2 x 105 cells/0.5 ml (6-well plates) at 37°C in a humidified 5% CO2 incubator.
The whole coding sequence of Iag-1 cDNA were amplified by above RT–PCR primers and cloned into pMD18-T (TaKaRa). After PstI–BamHI restriction enzyme digestion, products were separated and purified on agarose gel and inserted into the eukaryotic expression vector pCR3.1. Sequences were confirmed by sequencing. The procedure of the transfection experiment, G418 screening and apoptosis detection was from the method described by Kumar et al. (2004) and Kobayashi et al. (1998). In brief, pCR3.1–Iag-1 were transfected into ESCs with the lipofectamine 2000 (Invitrogen) essentially as described as the manufacture’s manuals. The transfected cells survive in culture medium containing G418 antibiotic as pCR3.1 contains a neo gene that expresses a G418 resistant product. Thus the transfected cells were selected with 500 µg/ml Geneticin G418 (determined by the killcurve assay) in the culture medium to select the cells that express resistance to this marker two days after transfection. Three weeks later, the cells of control and treatment groups were cultured in the serum-free medium. After 48 h culture, cell apoptosis was detected by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) and Hoechst staining. Total protein was extracted from the cells to detect the expression of Iag-1 three weeks after Geneticin G418 screening.
Cell proliferation assay
Cell proliferation was estimated with an MTS assay using the Cell Titer 96 Aqueous One Solution cell proliferation assay (Promega). ESCs were seeded in 96-well plates at low density (5000 cells per well) in DMEM/F-12 culture medium, and allowed to attach overnight. After 48 h, Cell Titer 96 Aqueous One Solution was added to each well, the plates were incubated for 4 h, then the absorbance was recorded at A490 nm with a 96-well plate reader (Bio-Rad, 3550). Each measurement was made in triplicate, and the results were described as a ratio of transfected pCR3.1–Iag-1 or as transfected pCR3.1 versus untreated ESCs.
Detection of apoptosis assay by TUNEL and Hoechst staining
TUNEL and Hoechst staining analysis was performed as previously described (Sun et al., 2006).
Briefly, in situ detection of apoptotic cells was performed on adherent cells cultured on chamber slides by using an in situ cell death detection kit, Fluorescein (Roche, Mannheim, Germany). Air-dried cell samples were fixed with a freshly prepared fixation solution for 1 h at 15–25°C, and then incubated in permeabilization solution for 2 min on ice, and the TUNEL procedure was conducted according to the manufacturer’s instructions. For the correlation of TUNEL with nuclear morphology, cultures were counterstained with phosphatidylinositol (5 µg/ml) and coverslipped. Apoptotic cells were counted in different optical fields (x400 magnification) selected in a random manner, and at least 500 cells were counted for each sample. To confirm the specificity of TUNEL, cultures were treated with 1 µg/ml DNase I (Sigma) at room temperature for 10 min to create positive controls. TdT was omitted from the labelling reaction mixture in negative controls. Samples were viewed at excitation 488 nm/emission 512 nm by fluorescence microscopy (ZEISS LSM 510 META). At least 500 cells for each sample were evaluated for apoptosis in different optical fields (x400 magnification) randomly selected. The results were expressed as the ratio of TUNEL-positive ESCs to total ESCs. Each treatment was repeated three times.
The fragmented nuclei were stained with Hoechst 33342 (Sigma Chemical Co.). Hoechst 33342 was diluted with PBS and added to the medium to the final concentration of 10 µg/ml. Cells were incubated for 15 min in an atmosphere at 37°C and visualized with a fluorescence microscope (ZEISS LSM 510 META).
Statistical analysis
All values are reported as the mean ± SEM. Statistical analysis was done by one-way ANOVA. When significant effects of treatments were indicated, the Student–Newman–Keuls multi-range test was employed among the groups using SPSS version 13.0. A value of P < 0.05 was considered statistically significant.
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Results |
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Cloning and sequence analysis of rat Iag-1 cDNA and protein
According to the sequence of a cDNA fragment from our SSH cDNA library of rat uterus, we performed 5'- and 3'-RACE, which produced the cDNA sequence 2121 bp in full length (Fig. 1; GenBank accession number DQ451016 [GenBank] ). A search of nucleotide databases from NCBI revealed 93.42% homology to mouse (over the open reading frame), 81.15% homology to human and 81.08% homology to rhesus monkey Iag-1. In the 3' untranslated region, a typical putative polyadenylation signal, AATAAA, was found. A search of GenBank contig maps assigned Iag-1 to rat chromosome 1. Iag-1 is spliced by 12 exons and 11 introns.
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The Iag-1 cDNA contains a complete open reading frame of 1353 bp (from bases 102–1454), predicting a protein of 450 amino acids. The predicted molecular mass was
51.6 kDa. The IAG-1 protein sequence was aligned with deposited sequences using DNAman 5.5 displaying 92.89% homology to mouse (AC121964
[GenBank]
, XR_001601, AC135633
[GenBank]
), 75.78% homology to human (BC106065
[GenBank]
, NM_017909
[GenBank]
, AK000634
[GenBank]
) and 75.11% homology to rhesus monkey, IAG-1 (XM_001098025). The homology domain mainly localized in the C-terminal. Using similar, the protein sequence of IAG-1 was also homologous to CG11679-PA Gene (98%, XP_001058471, XP_001054801) in rat and required for meiotic nuclear division protein 1 homolog (Rmnd1) in mouse (92%, Q8CI78, AAH27299
[GenBank]
, NP_079619
[GenBank]
) and human (80%, NP_060379
[GenBank]
, AAI19684
[GenBank]
). They have the same putative conserved domain DUF155 which is uncharacterized ACR, YagE family COG1723 (function unknown). Prosite, SignalP software in ExPASY server analysis revealed that IAG-1 protein is a non-secretory protein and has two typical transmembrane domains (304–323 and 429–450). Psort and Motifscan software in GenomNet server both reveal that the IAG-1 protein contains 10 protein kinase C phosphorylation sites (16–18; 29–31; 71–73; 104–106; 109–111; 218–220; 244–246; 313–315; 346–348; 397–399), nine casein kinase II phosphorylation sites (34–37; 166–169; 179–182; 218–221; 275–278; 292–295; 299–302; 332–335; 346–349), four N-glycosylation sites (77–80; 273–276; 365–368; 395–398), two N-myristoylation sites (103–108; 214–219), one amidation site (339–342) and one tyrosine kinase phosphorylation site (380–387). DNAStar analysis shows that the isoelectric point of IAG-1 is 8.651 and the charge at pH 7.0 is 10.048. There are 59 strongly basic (+) amino acids, 52 strongly acidic (–) amino acids, 159 hydrophobic amino acids and 116 polar amino acids in the sequence of IAG-1 protein.
Tissue distribution of Iag-1 mRNA expression
Total RNA from multiple tissues of normal rat were extracted and reverse-transcribed to perform PCR. From the results of RT–PCR, Iag-1 gene was expressed in hypothalamus, pituitary, stomach, liver, spleen, small intestine, large intestine, kidney, skeletal muscle, uterus and ovary. There was no detectable signal in lung and heart. The expression level of Iag-1 mRNA in liver was higher than in other tissues, and expression in normal rat uterus was lower (Fig. 2).
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Dynamics of Iag-1 in embryo and uterus during rat peri-implantation period
To study the role of Iag-1 in embryo implantation, we first examined its temporal and spatial distribution in both the embryo and the uterus during the peri-implantation period. In order to get IAG-1 antibody, the recombinant IAG-1 protein was expressed and purified (Supplementary Material, Fig. S1A). Antiserum was obtained from the immunized rabbit using the purified IAG-1 recombinant protein. Antibody speciality was detected by western blotting (Supplementary Material, Fig. S1B).
Immunofluorescence results showed that IAG-1 protein was mainly localized in the cell membranes of 2- and 4-cell embryos (Fig. 3A and B). At the 8-cell embryo and morula stages, we detected a wide distribution of IAG-1 in the cytoplasm and in or above the nucleus (Fig. 3C and D). At blastocyst stage, strong signals were found in the trophectoderm and weak signals were found in the inner cell mass (Fig. 3E).
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Iag-1 mRNA and protein also showed unique expression patterns in the uterus during the peri-implantation period. Northern blotting analysis showed the expression level of Iag-1 mRNA is higher in implantation period (g.d.5–g.d.6) than in the preimplantation (g.d.4), and post-implantation period (g.d.7–g.d.8) (Fig. 4A). In situ hybridization results showed that Iag-1 mRNA had slight staining in myometrium on the Day 4 of pregnancy (Fig. 4Ba). On the Day 5.5 and 6 of pregnancy, strong signals were found in glandular epithelium and luminal epithelium, and weaker signals were found in stroma (Fig. 4Bb and c). No staining was found in uterine sections from Day 5 of pregnancy hybridized with a sense probe for Iag-1 as negative control (Fig. 4Bd).
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Western blotting analysis showed that the expression level of IAG-1 (normalized to β-actin protein levels) was increased gradually in early pregnancy. IAG-1 protein level significantly increased in the implantation period (g.d.5–g.d.6) compared with preimplantation period (g.d.4), and this high level was maintained until g.d.8 (Fig. 5A). Immunofluorescence results showed that weak immunoreaction was found in myometrium on g.d.4 (Fig. 6A). In the implantation period (g.d.5–g.d.6), strong signals were detected in glandular epithelium, luminal epithelium and myometrium, and very weak signals were found in stroma (Fig. 6B and C). There was no immunoreaction in uterus sections from Day 5 of pregnancy incubated with normal rabbit serum as the negative control (Fig. 6F).
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Dynamics of Iag-1 in the uterus of pseudopregnant, experimentally induced decidualization and delayed implantation
To further explore whether Iag-1 expression is associated with the induction of implantation, we detected the effect of pseudopregnancy, experimentally induced decidualization and delayed implantation on Iag-1 expression.
The expression levels of Iag-1 mRNA and protein, respectively, detected by northern blotting and western blotting were not significantly altered between Days 4–7 pseudopregnancy (Figs 4C and 5B).
The expression level of IAG-1 protein detected by western blotting was not significantly altered in experimentally induced decidualization uterus and non-stimulated uterus on the Day 7 and 8 of pseudopregnancy (Fig. 5C). Immunofluorescence results showed that immunoreaction was found in glandular epithelium and luminal epithelium in uterus not infused with oil on the Day 7 and 8 of pseudopregnancy (Fig. 6D). In oil-infused uterus, IAG-1 protein was mainly localized in myometrium and glandular epithelium (Fig. 6E).
A low level of IAG-1 protein was detected in uterus under delayed implantation by western blotting. The protein level of IAG-1 was dramatically increased after implantation was activated with estrogen treatment (Fig. 5D).
The effects of Iag-1 on ESCs proliferation and apoptosis
ESCs were isolated from the g.d.5.5 rat uterus. The cells were incubated with rabbit anti-IAG-1 antibody prepared by our laboratory (Supplementary Material, Fig. S1). Spontaneous IAG-1 protein was detected in ESCs, mainly located in cytoplasm, with weak staining in/above the nucleus (Supplementary Material, Fig. S2Aa). ESCs were transfected with pCR3.1–Iag-1, and the expression of Iag-1 was detected after Geneticin G418 screening three weeks. As shown in Supplementary Material, Fig. S2Ab, the correct expression of pCR3.1–Iag-1 protein was identified with the specific antibody by Immunofluorescence and western blotting. Strong signal was found in the cytoplasm and in or above the nucleus. There was no immunoreaction in ESCs incubated with normal rabbit serum as negative control (Supplementary Material, Fig. S2Ac). The protein level of IAG-1 was higher in ESCs expressing pCR3.1–Iag-1 than in cells expressing pCR3.1 or in normal ESCs (Supplementary Material, Fig. S2B).
Cell proliferation and viability were determined using the MTS assay. As shown in Fig. 7, the lower level of proliferative activity was seen in ESCs expressing pCR3.1–Iag-1 (P < 0.05) was observed compared with that of normal ESCs and ESCs only expressing pCR3.1.
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Apoptosis in ESCs was determined by TUNEL and Hoechst staining. In situ TUNEL labelling showed that TUNEL-positive cells constituted
10–15% of the total cell count in normal ESCs and ESCs expressing pCR3.1. The TUNEL-positive ESCs were markedly increased in ESCs expressing pCR3.1–Iag-1, to 53.8% of total cell number. Hoechst staining by fluorescence microscopy analysed morphological indicators of apoptosis such as cell shrinkage, nuclear segmentation, and chromatin condensation. Similar to TUNEL staining, Hoechst staining showed that ESCs expressing pCR3.1–Iag-1 had more cell shrinkage and nuclear segmentation compared with normal ESCs or ESCs expressing pCR3.1 (Fig. 8).
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Effect of Iag-1 on apoptosis-related genes
Apoptosis-related proteins were detected by western blotting. Results showed that the p53, Bax and caspase-3 protein levels were enhanced and the Bcl-2 protein level was reduced in ESCs expressing pCR3.1–Iag-1. Fas and Fas-L were not significantly different among normal ESCs, the ESCs expressing pCR3.1 and the ESCs expressing pCR3.1–Iag-1 (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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Iag-1 was initially derived by the SSH cDNA library, which was used to analyse differentially expressed genes between the preimplantation and implantation periods. Herein we describe the cloning, partial characterization and function of Iag-1. Iag-1 may be an evolutionarily conserved gene among mammals, as homologues were found in mouse, human, rhesus monkey, orangutan, dog and cattle. At the time of submission, this gene had yet to be characterized or described in all the available literature, therefore based on its expression pattern and characterization, it was designated as implantation-associated gene-1 (Iag-1).
Based on our data of the RT–PCR analysis in the multiple tissues, we found that Iag-1 mRNA was transcribed in many tissues, but not heart and lung. We showed that Iag-1 expression had tissue-specificity. It was not clear whether that tissue-specificity was relative to Iag-1 function.
The process of implantation involves a preimplantation embryo developing to the blastocyst stage and the blastocyst hatching from the zona pellucida to establish a reciprocal interaction between the trophectoderm and uterine luminal epithelium. To study the role of Iag-1 in embryo implantation, we first examined its temporal and spatial distribution in both the embryo and the uterus during the peri-implantation period.
Iag-1 was regulated differentially in both the embryo and the uterus during the window of implantation. At 2- and 4-cell embryos, IAG-1 protein mainly localized in cell membrane. At 8-cell embryos and morula stages, we detected a wide distribution of IAG-1 in the cytoplasm and in/or above the nucleus. At the blastocyst stage, strong signals were found in trophectoderm and weak signals were found in the inner cell mass. The change of distribution of IAG-1 suggests that it may be involved in blastocyst formation and trophoblast invasion. The expression of uterine IAG-1 in the implantation period was higher than in preimplantation period. IAG-1 immunoreaction was found in myometrium in the preimplantation period, showing that it may participate in uterine myometrium tissue-remodelling processes, to prepare for receiving embryos. In the implantation period, luminal epithelium, stroma and myometrium were the major sites of IAG-1 expression. It suggestes that IAG-1 may play an important role in the process of embryo attachment and uterine tissue-remodelling. In the post-implantation period, immunoreaction was more intense in the glandular epithelium. It has been well-documented that an increase in protein synthesis and secretion from the glands occurs after the initiation of implantation and is maintained throughout early pregnancy to provide nutrition to the embryo (Weitlauf, 1994). This observation showed that Iag-1 may mainly be synthesized by the glandular epithelial cells after the initiation of implantation. Iag-1‘s function at the time of uterine receptivity remains unclear, and it could have actions on the luminal epithelium or stroma to participate in the acquisition of the receptive phase or it could act on the embryo itself.
To further explore whether Iag-1 expression is associated with the induction of implantation, we detected the effect of pseudopregnancy, experimentally induced decidualization and delayed implantation on Iag-1 expression. Implanting blastocysts seemed to be major contributors to the regulation of Iag-1, because its expression showed no apparent differences in uterus of Day 4–7 of pseudopregnancy and artificial decidualization. In addition, in implantation-delayed mice, IAG-1 protein levels increased dramatically following activation of the delayed implantation 12 h by treatment with estradiol-17β, whereas the same change could not be detected in uteri without blastocysts. These results further emphasize the importance of activated blastocysts in regulating the dynamics of Iag-1 in the uterus during the window of implantation.
Embryo implantation is regulated not only by cell proliferation and differentiation, but also by programmed cell death, or apoptosis. During the initial steps of implantation in rodents, the uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. Apoptosis occurs in specific uterine cells during blastocyst implantation and decidualization (Schlafke et al., 1985; Parr et al., 1987; Welsh and Enders, 1991).
In order to study the effect of exogenous Iag-1 on ESC apoptosis, the transfection of pCR3.1–Iag-1 into ESCs was performed. The expression of Iag-1 in ESCs was detected by immunofluorescence and western blotting. The weak expression of endogenous Iag-1 was detected in the cytoplast and nuclei of ESCs in the control groups and intense signals were detected in the cytoplast and nuclei of ESCs after pCR3.1–Iag-1 transfection into ESCs. The results indicated that pCR3.1–Iag-1 was expressed in ESCs. However, we are not clear about the reason for the intense signals detected in the nuclei of ESCs. The levels of apoptosis in ESCs were determined by TUNEL and Hoechst staining. The results showed that 10 and 11.8% of the cells in the control groups were undergoing apoptosis, however, the levels of apoptosis in treated cells were 53.8%, which indicated that Iag-1 significantly enhanced ESCs apoptosis three weeks after pCR3.1–Iag-1 transfection into ESCs and G418 selection. DNA fragmentation appeared in ESCs of the treatment group in contrast with ESCs of the control groups. These results showed that exogenous Iag-1 can induce the apoptosis of ESCs. During the course of the G418 selection, we did not detect the effect of Iag-1 on ESCs apoptosis which was only examined after the three weeks selection. We speculate that the sensitivity of the ESCs to apoptosis is increased when cultured in the serum-free medium.
Many reports indicate that members of the Bcl-2 family are mediators of cell survival and apoptosis (Oltvai et al., 1993; Reed, 1994; Kernohan et al., 1996). Bcl-2 must bind to Bax protein to function and Bcl-2/Bax heterodimers or Bax/Bax homodimers are active cell death regulators (Yin et al., 1994). The p53 gene is classified as a tumour-suppressor gene, and its product plays a role in triggering apoptosis (Levine, 1997). Fas (CD95) and Fas ligand (Fas-L) belong to the tumour necrosis factor receptor family (Suda et al., 1993; Nagata, 1999). Fas is a cell-surface glycoprotein that transmits apoptotic signals from the cell surface to the cytoplasm, while Fas-L functions as an essential effector molecule triggering apoptotic reactions (Suda et al., 1993; Nagata, 1999). Apoptosis depends on activation of downstream executioner caspases, and activated caspase-3 cleaves various cellular proteins to cause the morphologic changes of cells (Nicholson and Thornberry, 1997; Grutter, 2000).
To investigate the possible mechanisms by which Iag-1 induces apoptosis, the above-mentioned apoptosis-related proteins were detected by western blotting. The investigation showed that the p53, Bax and caspase-3 protein levels were enhanced, and the Bcl-2 protein level was reduced in the ESCs expressing pCR3.1–Iag-1. Fas and Fas-L were not significantly different among the normal ESCs, the ESCs expressing control vector and the ESCs expressing pCR3.1–Iag-1. Many reports indicate that the bcl-2 and bax genes each contain p53-responsive elements (Miyashita et al., 1994a; Miyashita and Reed, 1995). The p53 protein triggers apoptosis by down-regulating the expression of bcl-2, while simultaneously up-regulating the expression of bax (Miyashita et al., 1994b; Selvakumaran et al., 1994). Overexpression of bcl-2 inhibits the activation of caspase-3 protease and subsequent cell death following various apoptotic stimuli (Chinnaiyan et al., 1996; Shimizu et al., 1996). All these facts suggest that p53 and Bax/Bcl-2 may play an important role in the process of Iag-1-induced apoptosis.
In conclusion, Iag-1 was detected at differentially levels in rat embryo and uterus during the peri-implantation period. Results obtained from pseudopregnant, artificial decidualization and implantation-delayed rats imply an important role for Iag-1 in implanting blastocysts and for the temporal and spatial changes of Iag-1 in the uterus during the window of implantation. In addition, high levels of Iag-1 may trigger apoptosis of ESCs from the uterus of g.d.5 rat via Bax enhancement. These functions generated by Iag-1 are associated with p53. Collectively, these results suggested Iag-1 may be involved in the process of embryo implantation, possibly promoting the development of preimplantation embryos and differentiation of the uterus during this process. Also, this study may provide new insights into the molecular mechanism of embryo implantation.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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Supplementary material is available at Humrep Journal online.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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This work was supported by grants from the National Basic Research Program of China (No. 2006CB504006; 2006CB944007) and the Natural Science Foundation of China (No. 30770246).
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We would like to thank Dr Li Xu and Shu-Qun Shi for their sincere help on protein purification.
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Submitted on September 10, 2007; resubmitted on November 9, 2007; accepted on November 21, 2007.
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