implantation-associated gene-1 (iag-1): a novel gene ... · implantation-associated gene-1 (iag-1):...

Implantation-associated gene-1 (Iag-1): a novel gene involvedin the early process of embryonic implantation in rat

Hong-Fei Xia1,2, Jing Sun1,2, Quan-Hong Sun1, Ying Yang1 and Jing-Pian Peng1,3

1State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080,

People’s Republic of China; 2Graduate University of the Chinese Academy of Sciences, Beijing 100080, People’s Republic of China

3Correspondence address. Tel: þ86-10-64807183; Fax: þ86-10-64807099; E-mail: [email protected]

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 initiallyderived from suppressive subtracted hybridization of a cDNA library of rat uterus, which was used to analyse differ-entially expressed genes between the preimplantation and implantation period. METHODS: The full-length cDNAsequence of Iag-1 was cloned from rat uterus on D5.5 of pregnancy by 50- and 30-RACE. The expression of Iag-1 inthe uterus of early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantationwas detected by northern blotting, in situ hybridization, western blotting and immunofluorescence. Endometrialstromal cells (ESCs) were isolated from rat uterus. The effect of Iag-1 on ESCs proliferation and apoptosis were deter-mined by MTT assay, TUNEL and Hoechst staining. Apoptosis-related proteins in ESCs were detected by westernblotting. RESULTS: Differential patterns of Iag-1 expression were detected in rat embryo and in the uterus duringthe peri-implantation period. Iag-1 was specifically localized in glandular epithelium and luminal epithelium. In con-trast, 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 introducedIag-1 inhibited the proliferation of in vitro-cultured ESCs. Significant apoptosis was also detected in the ESCs over-expressing Iag-1, along with the enhancement of p53 and Bax protein expression. CONCLUSIONS: Overexpression ofIag-1 can inhibit ESCs proliferation and induce ESCs apoptosis, and p53 and Bax may play an important role in theprocess of Iag-1-induced apoptosis.

Keywords: Iag-1; embryo implantation; uterus; rat

Introduction

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 infor-

mation about whole molecular events of embryo implantation.

To obtain a global view and identify novel pathways of implan-

tation, 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 implan-

tation (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 litera-

ture 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

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Human Reproduction Vol.23, No.7 pp. 1581–1593, 2008 doi:10.1093/humrep/dem401

Advance Access publication on February 21, 2008

at Pennsylvania State University on February 27, 2014

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technique of SSH and differential display of reverse transcrip-

tion–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 sub-

tracting 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 mechan-

ism of embryo implantation.

Materials and Methods

Experimental animals and protocols

Sexually mature, healthy female Sprague–Dawley rats (220–260 g

body weight) were purchased from Institute of Genetics and Develop-

ment 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 vasec-

tomized males and was confirmed by checking the vaginal plug. The

presence of a vaginal plug was considered as Day 1 of pseudopreg-

nancy. The uteri were collected from Days 4–7 of pseudopregnancy.

On Day 5 of pseudopregnancy, when the uterus was optimally sensi-

tized to deciduogenic stimulus, 100 ml 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-17b (0.5 mg/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 50 and 30-cDNA ends

50 and 30 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 infor-

mation for Iag-1. Briefly, 50 and 30-RACE ready cDNA was syn-

thesized using 1 mg 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

(50 RACE primer: 50-TGA CGA TCA TCC ACT CCA GGC G-30;

30-RACE primer: 50-TGG GTG AGC TCT TTG CCC TCA G-30)

based on the sequence of a cDNA fragment isolated from a SSH

library were used for the 50 and 30-RACE experiments. The PCR pro-

ducts were cloned into pMD18-T vector (TaKaRa). Searches for hom-

ology 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 mg) from each tissue were used as templates for reverse transcrip-

tion using Superscript III (Invitrogen). The primers used to amplify the

1353 bp coding sequence of Iag-1 cDNA were 50-ATG CCA GCC

AGA CAA CTC CAA ACC CT-30 (forward) and 50-TCA GAA

GAG GAC TCT CCC CAG CTC GAA C-30 (reverse).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified

as an internal control.

Thirty microgram of total RNA from uteri of pregnant and pseudo-

pregnant rats was subjected to electrophoresis in 1% denatured

agarose gel, and capillary method used to transferred to nylon mem-

brane (Hybond Nþ; 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 658C for 4 h. We used the same primer

pairs as in RT–PCR to synthisize the probe which was labelled with32P. Hybridizations were performed at 658C overnight in the hybridiz-

ation buffer containing specific radioactive probe. Membranes were

washed and exposed to autoradiography (Kodak BioMax MS film,

Eastman Kodak Co., Rochester, NY, USA) overnight at 2808C. 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 lin-

earized 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 Bio-

sciences), then the probes were incubated with 0.4 mmol/l NaHCO3

and 0.6 mmol/l Na2CO3 at 608C for 35 min to hydrolyse them into

small fragments (between 200 and 250 bp). Frozen sections (8 mm)

of uterus of pregnancy and pseudopregnancy rats were hybridized

with fluorescein-labelled antisense probes in 50% formamide buffer

at 558C for 16 h. The fluorescein-labelled cRNA probe was detected

using anti-fluorescein antibodies at a dilution of 1:500. Finally, the

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colour reaction was developed with BCIP/NBT solution overnight in

the dark at 48C. The sections were hybridized with sense probes as

negative controls. Samples were viewed with an Eclipse 80i micro-

scope (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 (50- GGC

GGA TCC ATG CCA GCC AGA CAA CTC -30) and reverse/HindIII(50-CGG AAG CTT GAC TCT CCC CAG CTC GAA C-30).

After being double digested with BamHI and HindIII, the PCR

product was cloned into the prokaryotic expression vector pET28A

(þ) (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-b-

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 man-

ufacturer’s protocol (Amersham Biosciences).

Female New Zealand rabbits purchased from the Institute of Gen-

etics of the Chinese Academy of Sciences with body weight �2 kg

were immunized s.c. with 400 mg 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 mg 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 2208C 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/b-mercaptoethanol sample buffer, and 50 mg samples were loaded

onto each lane of 15% polyacrylamide gels. The proteins were separ-

ated by electrophoresis, and the proteins in the gels were blotted onto

nitrocellulose (NC) membranes (Amersham Biosciences) by electro-

phoretic 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 48C.

The membranes were then incubated with rabbit anti-IAG-1 ployclo-

nal antibody, rabbit anti-p53 ployclonal antibody (sc-6243, SantCruz),

mouse anti-Bax monoclonal antibody (sc-7480, Santa Cruz Biotech-

nology Inc., Santa Cruz, CA, USA), mouse anti-Bcl-2 monoclonal

antibody (sc-7382, SantCruz), rabbit anti-caspase-3 ployclonal anti-

body (sc-7148, SantCruz), rabbit anti-Fas ployclonal antibody

(sc-716, SantCruz), rabbit anti-Fas-L ployclonal antibody (sc-834,

SantCruz) or rabbit anti-b-actin ployclonal antibody (sc-1616-R, Sant-

Cruz) for 2 h at 378C. 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 peroxi-

dase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG

(Jackson Immunoresearch Laboratories, Inc.). Detection by the chemi-

luminescence (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 mm) 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 48C 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

378C, 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 sup-

plemented with 6.5 mg/ml trypsin and 25 mg/ml pancreatin. The

tube was kept at 48C 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 � 105 cells/0.5 ml (6-well plates) at 378C in a humidi-

fied 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 pro-

cedure 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 con-

tains a neo gene that expresses a G418 resistant product. Thus the

transfected cells were selected with 500 mg/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 transfec-

tion. 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

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well) in DMEM/F-12 culture medium, and allowed to attach over-

night. 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 A490nm 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 adher-

ent 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 15–258C, 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 phospha-

tidylinositol (5 mg/ml) and coverslipped. Apoptotic cells were

counted in different optical fields (�400 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 mg/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 (�400 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 mg/ml. Cells were

incubated for 15 min in an atmosphere at 378C 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.

Results

Cloning and sequence analysis of rat Iag-1 cDNAand protein

According to the sequence of a cDNA fragment from our SSH

cDNA library of rat uterus, we performed 50- and 30-RACE,

which produced the cDNA sequence 2121 bp in full length

(Fig. 1; GenBank accession number DQ451016). 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 30 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.

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, XR_001601, AC135633), 75.78% hom-

ology to human (BC106065, NM_017909, AK000634) 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, NP_079619) and human

(80%, NP_060379, AAI19684). 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 trans-

membrane 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 phosphoryl-

ation site (380–387). DNAStar analysis shows that the isoelec-

tric point of IAG-1 is 8.651 and the charge at pH 7.0 is 10.048.

There are 59 strongly basic (þ) amino acids, 52 strongly acidic

(2) 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, pitu-

itary, stomach, liver, spleen, small intestine, large intestine,

kidney, skeletal muscle, uterus and ovary. There was no detect-

able 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).

Dynamics of Iag-1 in embryo and uterus during ratperi-implantation period

To study the role of Iag-1 in embryo implantation, we first exam-

ined 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 cyto-

plasm and in or above the nucleus (Fig. 3C and D). At

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Figure 1: Nucleotide sequences of Iag-1The longest open reading frame, from bases 102–1454 predicts a 450-amino acid sequence before a stop signal (*) is encountered. The numberingon the left indicates amino acid sequence and the numbering on the right indicates nucleic acid sequence


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blastocyst stage, strong signals were found in the trophecto-

derm and weak signals were found in the inner cell mass

(Fig. 3E).

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 epi-

thelium 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).

Western blotting analysis showed that the expression level

of IAG-1 (normalized to b-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).

Dynamics of Iag-1 in the uterus of pseudopregnant,experimentally induced decidualization and delayedimplantation

To further explore whether Iag-1 expression is associated with

the induction of implantation, we detected the effect of pseudo-

pregnancy, experimentally induced decidualization and

delayed implantation on Iag-1 expression.

The expression levels of Iag-1 mRNA and protein, respect-

ively, 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). Immuno-

fluorescence 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

Figure 3: Immunofluorescent localization of IAG-1 protein in preim-platation embryosGreen represents IAG-1 staining, and red indicates nuclear staining.Yellow colouration represents an overlap of green and red. (A)2-cell embryo; (B) 4-cell embryo; (C) 8-cell embryo; (D) morulastage; (E) blastocyst stage; (F) Negative control: primary antibodiesreplaced by rabbit preimmune serum

Figure 2: Tissue distribution of Iag-1 mRNA expressionTotal 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 for RT–PCR analysis

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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 cyto-

plasm, with weak staining in/above the nucleus (Supplemen-

tary 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

Figure 4: Changes in uterine Iag-1 mRNA during pregnancy and pseudopregnancy(A) Northern blotting analysis of uterine Iag-1 mRNA expression on the Days 4–8 of pregnancy. (B) Northern blotting analysis of uterine Iag-1mRNA expression on Days 4–7 pseudopregnancy. Hybridization was done with a 32P-labelled probe for Iag-1 and GAPDH. The black curverepresents the optical densities of the signals quantified by densitometric analysis and represented as Iag-1 intensity/Gapdh intensity to normalizefor gel loading and transfer. (C) In situ localization of Iag-1 mRNA in the uterus of early pregnancy. Uterine sections from Day 4 (a), Day 5.5 (b),Day 6 (c) of pregnancy were subjected to in situ hybridization using fluorescein-labelled cRNA antisense probes specific to Iag-1. The stain wasdeveloped with BCIP/NBT. (d) Represents a uterine section from Day 5 of pregnancy hybridized with a sense probe for Iag-1 as a negativecontrol. Red arrows indicate hybridization signal. The photographs are shown at �100 original magnification. m, myometrium; v, uterineblood vessels; mg, maternal gland; s, stroma; le, luminal epithelium


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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.

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 flu-

orescence 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).

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).

Discussion

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

Figure 5: The expression pattern of IAG-1 protein in rat uterusIAG-1 protein expression in the uterus of early pregnant rats (A), pseudopregnant rats (B), artificial decidualization (C) and delayed implantation(D) was detected by western blotting. Total protein was separated by SDS–PAGE, then transferred to NC membranes. The membrances wereprobed with anti-IAG-1 antibody, then incubated with anti-rabbit IgG-HRP, and developed using the ECL reagent. The bands were analysedusing Quantity One analyzing system (Bio-Rad Laboratory Inc.). The black curve and histogram all represent the optical densities of thesignals quantified by densitometric analysis and are represented as IAG-1 intensity/b-actin intensity. *indicates significant differences (P ,0.05) from all other samples

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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 epi-

thelium. 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 dis-

tribution 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 inva-

sion. 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 preimplanta-

tion period, showing that it may participate in uterine myome-

trium tissue-remodelling processes, to prepare for receiving

embryos. In the implantation period, luminal epithelium,

stroma and myometrium were the major sites of IAG-1

Figure 6: Immunofluorescent localization of IAG-1 protein in rat uterus of early pregnancy and artificial decidualizationUterus sections from Day 4 (A), Day 5.5 (B) and Day 6 (C) of pregnancy were subjected to immunofluorescence using anti-IAG-1 antibody.Artificial decidualization was induced by infusing 100 ml sesame oil into the lumen of one of the uterine horns on the Day 5 of pseudopregnancy(stimulated). The contralateral uterine horn, which was not infused with oil, served as a control (non-stimulated). At Day 8 of pseudopregnancy,the rats were killed, and IAG-1 in uterus sections from non-stimulated (D) and stimulated (E) were also detected by immunofluorescence. Greenrepresents IAG-1 staining, and red indicates nuclear staining. Yellow colouration represents an overlap of green and red. To evaluate the speci-ficity of the antibodies, negative control staining was performed by substituting normal rabbit serum for the primary antibody (F). White arrowsindicate immunoreactivity. m, myometrium; mg; maternal gland; s, stroma; le, luminal epithelium. The scale bar indicated a distance of 200 mM

Figure 7: The effects of Iag-1 on ESCs proliferationESCs were isolated from rat uterus on g.d.5. ESCs expressing pCR3.1or pCR3.1–Iag-1 were cultured for 2 days, and cell proliferation wasdetermined using an MTS assay. The results were expressed at SI(ratio of A490nm with ESCs expressing pCR3.1–Iag-1 or pCR3.1versus untreated ESCs). *indicates significant differences (P ,0.05). SI, stimulation index


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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, immuno-

reaction 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 obser-

vation showed that Iag-1 may mainly be synthesized by the

glandular epithelial cells after the initiation of implantation.

Figure 8: The effects of Iag-1 on ESCs apoptosisESCs were transfected with pCR3.1 and pCR3.1–Iag-1, and the apoptosis was detected after Geneticin G418 screening for three weeks.(A) TUNEL staining (a–e) and Hoechst staining (f–h) of ESCs. (a) Normal ESCs after treatment with DNAse I (positive control), and (b)without terminal deoxynucleotide transferase (negative control); (c) Normal ESCs without any treatment; (d) ESCs expressing pCR3.1; (e)ESCs expressing pCR3.1–Iag-1. (f) ESCs without any treatment; (g) ESCs expressing pCR3.1; (h) ESCs of expressing pCR3.1–Iag-1 werestained. Yellow/green cells were TUNEL (þ) cells. (B) Histogram of ratio of TUNEL-positive ESCs. The results were expressed as ratio ofTUNEL-positive ESCs versus total ESCs. **indicates significant differences (P , 0.01)

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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 blasto-

cysts 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 decid-

ualization. In addition, in implantation-delayed mice, IAG-1

protein levels increased dramatically following activation of

the delayed implantation 12 h by treatment with estradiol-17b,

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 prolifer-

ation 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 apop-

tosis, 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 deter-

mined by TUNEL and Hoechst staining. The results showed

that 10 and 11.8% of the cells in the control groups were under-

going 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 fragmenta-

tion appeared in ESCs of the treatment group in contrast with

ESCs of the control groups. These results showed that exogen-

ous 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 apop-

tosis 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). Apopto-

sis 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 pro-

teins 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 sig-

nificantly different among the normal ESCs, the ESCs expres-

sing 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 simul-

taneously 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 decidualiza-

tion 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

Figure 9: Western blotting analysis of the expression of apoptosis-related protein in ESCsTotal protein was separated by SDS–PAGE, then transferred to NCmembranes. The membranes were probed with p53, Bax, Bcl-2,caspase-3, Fas or Fas-L antibody, then incubated with anti-rabbit ormouse IgG-HRP, and developed using the ECL reagent. The bandswere analysed using the Quantity One analyzing system (Bio-RadLaboratory Inc.). The expression of b-actin served as an internalcontrol


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implantation. In addition, high levels of Iag-1 may trigger

apoptosis of ESCs from the uterus of g.d.5 rat via Bax enhance-

ment. 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 pro-

moting 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.

Supplementary material

Supplementary material is available at Humrep Journal online.

Acknowledgements

We would like to thank Dr Li Xu and Shu-Qun Shi for their sincerehelp on protein purification.

Funding

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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Submitted on September 10, 2007; resubmitted on November 9, 2007; acceptedon November 21, 2007


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