Myeloid

ecotropic

viral

integration

site

-LRB-

MEIS

-RRB-

involvement

in

embryonic

implantation

BACKGROUND

The

homeobox

gene

controls

embryonic

uterine

development

and

adult

endometrial

receptivity

The

three-amino-acid

loop

extension

-LRB-

TALE

-RRB-

family

homeobox

genes

like

myeloid

ecotropic

viral

integration

site

-LRB-

MEIS

-RRB-

provide

enhanced

target

gene

activation

and

specificity

in

HOX-regulated

cellular

processes

by

acting

as

HOX

cofactors

Introduction

The

molecular

mechanisms

responsible

for

endometrial

development

and

receptivity

to

embryonic

implantation

are

poorly

understood

-LRB-

Cross

et

al.

Wang

and

Dey

-RRB-

In

the

last

few

years

therefore

a

large

number

of

high-throughput

expression

profiling

studies

have

been

performed

on

both

human

and

animal

model

tissues

to

identify

the

molecular

signaling

involved

in

the

endometrial

development

and

embryo-endometrium

interactions

leading

to

successful

embryonic

implantation

These

include

DNA-microarray

studies

-LRB-

reviewed

in

Horcajadas

et

al.

see

also

Chen

et

al.

Pan

et

al.

-RRB-

as

well

as

SAGE

-LRB-

serial

analysis

of

gene

expression

-RRB-

studies

-LRB-

Blomberg

et

al.

Ma

et

al.

-RRB-

In

addition

two

databases

have

been

set

up

that

contain

expression

data

on

genes

thought

to

be

involved

in

endometrium

development

and

function

and

endometrium.bcm.tmc.edu

HOX-class

homeobox

genes

are

leading

candidates

for

the

regulation

of

endometrium

differentiation

in

preparation

for

embryonic

implantation

-LRB-

Taylor

Eun

Kwon

and

Taylor

-RRB-

HOX

genes

assign

the

correct

identity

to

undifferentiated

body

segments

along

several

developmental

axes

-LRB-

Krumlauf

Carroll

Moens

and

Selleri

Wellik

Zakany

and

Duboule

-RRB-

They

are

however

not

only

typically

expressed

during

embryogenesis

but

also

persistently

active

in

both

the

mouse

and

human

female

reproductive

tract

-LRB-

Taylor

et

al.

Taylor

-RRB-

The

continued

expression

of

these

HOX

genes

allows

the

reproductive

tract

to

maintain

developmental

plasticity

and

to

differentiate

appropriately

during

each

menstrual

cycle

and

throughout

pregnancy

The

development

of

the

para-mesonephric

duct

in

the

embryo

is

dictated

in

part

by

the

abdominal-B

group

of

HOX

genes

-LRB-

Taylor

et

al.

Taylor

Eun

Kwon

and

Taylor

-RRB-

Specifically

is

responsible

for

proper

uterine

development

in

both

mouse

and

man

-LRB-

Taylor

et

al.

Block

et

al.

-RRB-

Female

null-mutant

mice

demonstrate

uterine

factor

infertility

-LRB-

Satokata

et

al.

-RRB-

These

mice

ovulate

normally

and

produce

normal

embryos

but

their

uteri

will

not

support

the

implantation

of

their

own

or

transplanted

wild-type

embryos

In

humans

expression

is

up-regulated

in

the

mid-luteal

phase

at

the

time

of

implantation

and

its

expression

is

driven

by

estrogen

and

progesterone

-LRB-

Taylor

et

al.

-RRB-

is

a

well-characterized

marker

of

endometrial

receptivity

which

exerts

pleiotropic

effects

on

multiple

aspects

of

adult

endometrial

development

such

as

stromal

decidualization

leukocyte

infiltration

and

pinopode

development

-LRB-

Bagot

et

al.

Daftary

and

Taylor

Qian

et

al.

-RRB-

Pinopodes

-LRB-

also

called

uterodomes

-RRB-

are

the

best-characterized

structural

markers

of

endometrial

receptivity

in

rodents

and

humans

-LRB-

Nikas

Nardo

et

al.

Nikas

and

Makrigiannakis

-RRB-

Defective

endometrial

expression

has

been

described

in

several

conditions

that

result

in

diminished

human

embryo

implantation

such

as

endometriosis

polycystic

ovarian

syndrome

and

hydrosalpinx

fluid

-LRB-

Taylor

et

al.

Daftary

and

Taylor

Cermik

et

al.

-RRB-

One

of

the

known

downstream

target

genes

of

in

endometrial

cells

is

-LRB-

Daftary

et

al.

-RRB-

HOX

genes

perform

their

function

by

acting

as

transcription

factors

They

bind

to

the

regulatory

regions

of

downstream

target

genes

through

their

homeobox

domain

and

thereby

activate

or

repress

transcription

HOX

proteins

bind

only

weakly

to

DNA

by

themselves

and/or

exhibit

a

high

degree

of

redundancy

in

binding

site

specificity

-LRB-

Pellerin

et

al.

-RRB-

It

has

become

increasingly

evident

that

HOX

proteins

usually

act

as

a

part

of

hetero-dimeric

-LRB-

van

Dijk

et

al.

Kurant

et

al.

-RRB-

or

hetero-trimeric

complexes

-LRB-

Jacobs

et

al.

Shen

et

al.

Schnabel

et

al.

-RRB-

The

TALE

-LRB-

three-amino-acid

loop

extension

-RRB-

family

of

homeodomain

proteins

represents

the

most

important

group

of

HOX

cofactors

The

unique

TALE

motif

within

their

homeodomain

allows

TALE

proteins

to

interact

with

other

homeobox-containing

proteins

-LRB-

Bürglin

Piper

et

al.

-RRB-

Unlike

most

other

homeobox

gene

families

TALE

genes

share

sequence

homology

outside

of

the

homeobox

domain

This

extensive

homology

appears

linked

to

their

function

Single

TALE

proteins

have

only

low

DNA-binding

activity

and

specificity

but

once

complexed

with

a

TALE

protein

from

another

subfamily

and

a

HOX-class

homeobox

protein

via

these

homologous

domains

they

show

powerful

and

specific

downstream

target

promoter

regulation

-LRB-

reviewed

in

Geerts

et

al.

-RRB-

Abd-B-like

HOX

transcription

factors

like

can

form

dimers

or

trimers

with

TALE

proteins

that

thereby

regulate

the

multiple

HOX

downstream

target

genes

necessary

for

the

implantation

process

The

myeloid

ecotropic

viral

integration

site

-LRB-

MEIS

-RRB-

genes

belong

to

the

TALE

homeobox

family

MEIS

proteins

are

not

just

involved

in

TALE

complex

DNA

binding

and

complex

stabilization

on

the

target

DNA

but

can

also

recruit

other

TALE

partner

proteins

into

the

nucleus

-LRB-

Rieckhof

et

al.

-RRB-

was

first

identified

as

a

major

integration

site

for

leukemogenic

virus

in

a

murine

leukemia

model

-LRB-

Moskow

et

al.

-RRB-

Also

in

human

leukemia

high

expression

of

was

found

in

bone

marrow

cells

of

acute

myelogenous

leukemia

-LRB-

AML

-RRB-

patients

where

it

was

always

co-expressed

with

-LRB-

Smith

et

al.

Lawrence

et

al.

-RRB-

expression

was

found

to

cause

transformation

of

normal

hematopoietic

cells

to

AML

cells

only

when

expressed

together

with

-LRB-

Kroon

et

al.

-RRB-

Specifically

expression

is

vital

for

keeping

myeloid

cells

in

an

undifferentiated

proliferating

state

refractory

to

granulocyte-colony

stimulating

factor-induced

terminal

differentiation

-LRB-

Calvo

et

al.

-RRB-

Though

co-expression

and

interaction

of

with

Abd-B-like

HOX

transcription

factors

was

convincingly

demonstrated

in

leukemia

the

data

on

gene

expression

and

function

in

the

endometrium

were

still

scarce

In

the

current

study

we

performed

data-mining

on

gene

expression

sets

in

the

public

domain

to

investigate

the

expression

of

the

MEIS

genes

-LRB-

-RRB-

in

the

human

female

reproductive

tract

We

next

characterized

the

menstrual

cycle-specific

pattern

of

in

human

endometrium

To

block

or

over-express

maternal

expression

with

small

interfering

RNA

-LRB-

siRNA

-RRB-

or

over-expression

constructs

in

a

mouse

model

we

used

liposome-mediated

gene

transfection

in

the

murine

uterus

to

investigate

whether

maternal

expression

in

the

endometrium

contributes

to

implantation

We

conclude

from

these

experiments

that

manipulation

of

adult

expression

significantly

affected

fertility

in

the

mouse

Materials

and

Methods

Meis

gene

expression

in

normal

tissue

Affymetrix

data

for

a

set

of

different

normal

human

tissue

types

-LRB-

the

human

body

index

set

-RRB-

representing

a

total

of

tissue

samples

were

retrieved

from

public

gene

expression

omnibus

-LRB-

GEO

-RRB-

data

sets

on

the

National

Center

for

Biotechnology

Information

-LRB-

NCBI

-RRB-

website

-LRB-

Barrett

et

al.

-RRB-

CEL

data

from

the

Affymetrix

GeneChip

Human

Genome

Plus

array

data

sets

were

downloaded

and

intensity

values

and

the

accompanying

P-values

assigned

with

GeneChip

Operating

Software

-LRB-

GCOS

-RRB-

using

the

algorithm

-LRB-

Affymetrix

Santa

Barbara

CA

USA

-RRB-

Annotations

and

clinical

data

for

the

tissue

samples

analyzed

were

available

from

through

its

GEO

ID

Human

tissue

collection

Endometrial

tissue

was

collected

during

routine

endometrial

biopsy

from

women

with

a

normal

menstrual

cycle

who

were

eligible

for

IVF

and

embryo

transfer

because

of

-LRB-

partial

-RRB-

tubal

blockage

Informed

consent

was

obtained

from

all

patients

The

tissue

collection

met

with

the

conditions

described

in

the

Declaration

of

Helsinki

for

Medical

Research

involving

Human

Subjects

as

described

in

-LRB-

-RRB-

Also

approval

was

obtained

from

the

Tongji

Hospital

research

and

ethics

committee

One

half

of

the

tissue

sample

was

immediately

frozen

in

liquid

nitrogen

and

stored

at

°C

The

other

half

was

fixed

in

formalin

embedded

in

paraffin

sectioned

and

stained

with

hematoxylin

and

eosin

Menstrual

cycle

dating

was

determined

by

menstrual

history

and

confirmed

by

histological

examination

using

the

Noyes

criteria

-LRB-

Noyes

et

al.

-RRB-

The

early

and

late

proliferative

phase

and

the

early

mid

and

late

secretory

phase

were

defined

as

Days

and

of

the

menstrual

cycle

respectively

RNA

isolation

and

RT

PCR

Total

RNA

was

extracted

using

the

total

RNA

isolation

reagent

-LRB-

TRI

reagent

Molecular

Research

Center

Cincinnati

OH

USA

-RRB-

according

to

the

manufacturer

's

instructions

Four

microgram

total

RNA

from

each

sample

was

denatured

at

°C

for

min

and

chilled

rapidly

on

ice

The

RNA

was

then

reverse

transcribed

in

a

µl

reaction

mixture

containing

µl

RT

buffer

µl

mM

dNTP

µl

µg/µl

Oligo

-LRB-

-RRB-

µl

U/µl

ribonuclease

inhibitor

µl

U/µl

Moloney

murine

leukemia

virus

transcriptase

-LRB-

Promega

Madison

WI

USA

-RRB-

with

the

following

reaction

condition

°C

for

min

°C

for

min

in

a

Biometra

T

Gradient

Thermocyler

-LRB-

Biometra

Göttingen

Germany

-RRB-

After

first

strand

complementary

DNA

-LRB-

cDNA

-RRB-

synthesis

µl

of

cDNA

was

amplified

in

µl

of

PCR

mixture

containing

µl

PCR

buffer

µl

mM

µl

mM

dNTP

µl

-LRB-

U/µl

-RRB-

Taq

polymerase

-LRB-

Fermentas

Foster

City

CA

USA

-RRB-

and

µl

-LRB-

pmol/µl

-RRB-

of

each

primer

pair

The

primers

used

were

-LRB-

forward

reverse

-RRB-

for

human

-LRB-

-RRB-

for

mouse

-LRB-

-RRB-

for

mouse

integrin

β3

-LRB-

-RRB-

-LRB-

-RRB-

for

human

dehydrogenase

-LRB-

GAPDH

-RRB-

-LRB-

-RRB-

and

for

mouse

β-actin

-LRB-

-RRB-

The

PCR

reactions

were

performed

as

follows

°C

for

min

followed

by

a

number

of

cycles

of

°C

for

s

annealing

for

s

at

different

annealing

temperatures

and

°C

for

min

followed

by

incubation

at

°C

for

min

Annealing

temperatures

for

human

mouse

mouse

mouse

β-actin

and

human

GAPDH

were

°C

°C

°C

°C

and

°C

respectively

and

the

numbers

of

cycles

were

and

respectively

The

PCR

products

were

separated

on

agarose

gels

and

visualized

by

ethidium

bromide

-LRB-

EtBr

-RRB-

staining

under

UV

light

The

resulting

PCR

products

were

bp

for

human

-LSB-

representing

nucleotides

-LRB-

nt

-RRB-

of

RefSeq

-RRB-

bp

for

mouse

-LRB-

nt

of

-RRB-

bp

for

mouse

-LRB-

nt

of

-RRB-

bp

for

human

GAPDH

-LRB-

nt

of

-RRB-

and

bp

for

mouse

β-actin

-LRB-

nt

of

-RRB-

Mouse

primers

used

in

the

experiments

described

yielded

a

bp

PCR

product

containing

endogenous

mouse

as

well

as

human

-LRB-

nt

of

-RRB-

resulting

from

the

transgene

Human

or

mouse

and

mRNA

levels

were

calculated

as

a

ratio

of

the

densitometric

values

for

or

to

their

corresponding

GAPDH

or

β-actin

values

Care

was

taken

to

ensure

a

logarithmic

signal

for

the

PCR

products

measured

RT

PCR

reactions

performed

for

human

showed

differential

and

equal

GAPDH

expression

for

all

samples

when

RT

PCR

was

performed

for

or

cycles

For

all

three

cycle

numbers

used

and

GAPDH

signal

intensities

were

different

between

cycle

numbers

Similar

results

were

obtained

for

mouse

-LRB-

or

cycles

-RRB-

and

mouse

-LRB-

or

cycles

-RRB-

-LRB-

results

not

shown

-RRB-

In

situ

hybridization

Diethyl

pyrocarbonate-treated

Milli-Q

water

was

used

in

each

step

Sections

-LRB-

µm

thick

-RRB-

were

immersed

in

solution

for

min

to

eliminate

endogenous

peroxidase

activity

After

rinsing

in

mM

phosphate-buffered

saline

-LRB-

PBS

-RRB-

sections

were

treated

with

pepsin

-LSB-

-LRB-

w/v

-RRB-

in

citric

acid

-RSB-

at

°C

for

min

Thereafter

sections

were

fixed

in

freshly

prepared

-LRB-

w/v

-RRB-

paraformaldehyde

in

PBS

for

min

at

°C

rinsed

in

PBS

and

immersed

in

acetic

anhydride

mM

triethanolamine

and

-LRB-

w/v

-RRB-

NaCl

consecutively

each

for

min

at

room

temperature

The

probe

for

human

was

a

molar

mixture

of

single

stranded

DNA

oligonucleotides

complementary

to

the

-LRB-

nt

of

-RRB-

and

-LRB-

nt

of

-RRB-

sequences

labeled

with

digoxigenin

Sections

were

hybridized

overnight

at

°C

in

hybridization

buffer

containing

digoxigenin-labeled

oligonucleotide

probe

at

a

final

concentration

of

mg/l

After

hybridization

sections

were

washed

in

fresh

SSC

-LRB-

saline

sodium

citrate

-RRB-

at

°C

for

min

SSC

at

°C

for

min

and

SSC

at

°C

for

min

then

incubated

in

-LRB-

w/v

-RRB-

bovine

serum

albumin

for

min

at

room

temperature

and

in

biotin-anti-digoxigenin

at

°C

for

h

and

finally

washed

and

incubated

in

streptavidin

biotin

complex

-LRB-

SABC

-RRB-

for

min

at

°C

Probe

labeling

and

digoxigenin

biotin-anti-digoxigenin

and

SABC

incubations

were

performed

using

the

kit

-LRB-

Boster

Bio-technology

Wuhan

Hubei

China

-RRB-

The

brown

precipitate

generated

at

the

site

of

digoxigenin-probe

hybridization

by

reaction

of

the

anti-digoxigenin

antibody

bound

peroxidase

with

diaminobenzidine

tetrahydrochloride

-LRB-

DAB

-RRB-

-LRB-

Boster

Bio-technology

-RRB-

was

scored

as

presence

of

mRNA

Hematoxylin

was

used

for

counterstaining

A

total

of

samples

were

examined

The

staining

of

consecutive

cells

in

five

non-adjacent

microscopic

fields

was

evaluated

to

quantify

the

expression

of

Western

blot

analysis

Tissues

from

each

mouse

uterus

were

washed

twice

with

PBS

and

lysed

on

ice

in

lysis

buffer

-LSB-

mM

Tris

HCl

-LRB-

pH

-RRB-

mM

NaCl

-LRB-

w/v

-RRB-

sodium

dodecyl

sulfate

-LRB-

SDS

-RRB-

-LRB-

w/v

-RRB-

sodium

deoxycholate

-LRB-

v/v

-RRB-

-LRB-

w/v

-RRB-

sodium

azide

and

freshly

added

protease

inhibitors

-LRB-

phenylmethylsulphonyl

fluoride

to

µg/μl

and

aprotinin

to

µg/ml

-RRB-

-RSB-

Solid

cellular

debris

was

removed

by

centrifugation

at

for

min

Protein

concentration

was

measured

by

the

Coomassie

Brilliant

Blue

assay

Protein

samples

-LRB-

µg

each

-RRB-

were

subjected

to

SDS

polyacrylamide

gel

electrophoresis

and

transferred

onto

polyvinylidene

difluoride

membrane

using

a

Bio-Rad

electroblot

apparatus

-LRB-

Bio-Rad

Beijing

China

-RRB-

Non-specific

binding

sites

were

blocked

in

-LRB-

w/v

-RRB-

non-fat

dry

milk

in

PBS

containing

-LRB-

v/v

-RRB-

The

primary

goat

polyclonal

antibody

-LRB-

Santa

Cruz

CA

USA

-RRB-

was

used

at

and

incubated

at

°C

overnight

This

antibody

was

raised

against

a

C-terminal

epitope

of

human

and

recognizes

both

human

and

murine

since

their

protein

sequences

are

identical

in

that

region

-LRB-

also

see

below

section

constructs

-RRB-

As

secondary

antibody

rabbit

peroxidase-conjugated

anti-goat

immunoglobulin

-LRB-

Ig

-RRB-

G

-LRB-

Zhongshan

Biotechnology

Beijing

China

-RRB-

was

used

at

and

incubated

at

°C

for

h.

Rabbit

anti-actin

antibody

-LRB-

Zhongshan

Biotechnology

as

a

loading

control

-RRB-

was

used

at

and

incubated

at

°C

overnight

and

the

secondary

antibody

goat

anti-rabbit

IgG

-LRB-

Zhongshan

Biotechnology

-RRB-

was

used

at

and

incubated

at

°C

for

h.

Protein

bands

were

visualized

by

enhanced

chemiluminescence

-LRB-

Pierce

Biotechnology

Rockford

IL

USA

-RRB-

constructs

The

complete

human

coding

sequence

was

isolated

by

PCR

using

neuroblastoma

tumor

cDNA

as

a

template

with

forward

primer

and

reverse

primer

The

forward

primer

consisted

of

a

non-coding

flag

containing

an

EcoRI

recognition

site

-LRB-

underlined

-RRB-

and

the

ATG

initiation

codon

-LRB-

in

bold

capitals

-RRB-

followed

by

codons

-LRB-

in

capitals

-RRB-

representing

nt

of

the

human

NCBI

RefSeq

coding

sequence

The

reverse

primer

consisted

of

a

non-coding

flag

containing

a

SalI

recognition

site

-LRB-

underlined

-RRB-

and

the

TAG

stopcodon

-LRB-

in

bold

capitals

-RRB-

preceded

by

the

final

codons

-LRB-

in

capitals

-RRB-

representing

nt

of

the

coding

sequence

This

sequence

encoded

the

most

widely

expressed

splice

variant

-LRB-

see

also

Geerts

et

al.

and

the

locus

in

TranscriptView

at

-RRB-

The

derived

human

protein

-LRB-

NCBI

sequence

residues

-RRB-

was

identical

to

mouse

protein

-LRB-

NCBI

sequence

-RRB-

except

for

substitution

of

the

Ile

residue

at

position

in

the

human

sequence

by

an

analogous

Val

residue

at

position

in

the

mouse

sequence

The

bp

PCR

product

was

restricted

with

EcoRI

and

SalI

purified

by

agarose

gel

electrophoresis

using

Qiaex

II

gel

extraction

-LRB-

Qiagen

Venlo

The

Netherlands

-RRB-

and

ligated

into

EcoRI

SalI

restricted

pCI-neo

vector

-LRB-

Promega

Benelux

Leiden

The

Netherlands

-RRB-

backbone

The

sequence

was

excised

from

this

construct

using

EcoRI

and

the

polylinker

NotI

site

and

subcloned

into

the

mammalian

expression

vector

-LRB-

Invitrogen

Breda

The

Netherlands

-RRB-

restricted

with

EcoRI

and

NotI

resulting

in

The

construct

was

verified

by

sequencing

and

expression

of

protein

by

the

construct

was

demonstrated

by

western

blot

analysis

of

transiently

transfected

cells

and

stably

transfected

human

neuroblastoma

cells

-LRB-

Revet

et

al.

manuscript

in

preparation

see

also

Fig.

in

Geerts

et

al.

-RRB-

The

siRNA

expression

constructs

were

prepared

as

follows

oligos

and

targeting

the

TCAAGAACTGGATAACTTG

sequence

in

mRNA

-LRB-

nt

in

the

human

RefSeq

and

in

the

murine

RefSeq

-RRB-

or

and

targeting

the

CTTGATGATTCAAGCCATA

sequence

in

mRNA

-LRB-

nt

in

and

in

-RRB-

were

annealed

and

ligated

into

pTER

restricted

with

BglII

and

HindIII

as

described

-LRB-

van

de

Wetering

et

al.

-RRB-

to

create

and

respectively

A

non-targeting

pTER

construct

contained

a

similar

sequence

that

had

no

homology

to

the

human

or

murine

genome

and

was

used

as

a

negative

control

The

constructs

were

verified

by

sequencing

and

siRNA-mediated

protein

and

mRNA

knockdown

by

both

constructs

was

demonstrated

-LRB-

Revet

et

al.

manuscript

in

preparation

see

also

Fig.

in

Geerts

et

al.

-RRB-

The

and

constructs

have

complete

homology

to

human

as

well

as

to

murine

and

target

all

annotated

splice

variants

-LRB-

i.e.

isoforms

A-D

see

also

Geerts

et

al.

-RRB-

DNA/liposome

preparation

Plasmid

DNA

-LRB-

µl

-RRB-

was

mixed

with

µl

Opti-MEM

reduced

serum

medium

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

added

to

µl

liposome

-LSB-

a

-LRB-

w/w

-RRB-

formulation

of

-LSB-

-LRB-

sperminecarboxamido

-RRB-

ethyl

-RSB-

propanaminium

trifluoroacetate

-LRB-

DOPSA

-RRB-

and

dioleoylphosphatidyl

ethanolamine

-LRB-

DOPE

-RRB-

-LRB-

Invitrogen

-RRB-

-RSB-

in

µl

OptiMEM

and

incubated

for

min

at

room

temperature

Final

concentrations

were

µg/ml

DNA

and

µg/ml

liposome

In

vivo

gene

transfection

Nulliparous

female

and

male

Kunming

mice

of

reproductive

age

were

supplied

by

Center

of

Experimental

Animals

Tongji

Medical

College

-LRB-

Wuhan

China

-RRB-

Female

mice

were

mated

and

examined

every

h

until

detection

of

a

vaginal

plug

which

was

designated

Day

of

the

pregnancy

The

mice

were

anesthetized

h

following

plug

detection

with

butaylone

given

by

i.p.

injection

A

laparotomy

was

performed

to

expose

the

uterus

and

µl

of

the

DNA/liposome

complex

was

injected

into

the

base

of

each

uterine

horn

using

a

needle

as

described

-LRB-

Bagot

et

al.

Hsieh

et

al.

-RRB-

The

incision

was

closed

in

two

layers

-LRB-

peritoneal

and

cutaneous

-RRB-

with

vicryl

suture

The

mice

were

euthanized

by

cervical

vertebrae

dislocation

under

anesthesia

before

excision

of

the

uteri

For

the

experiments

studying

the

effect

of

expression

manipulation

on

expression

litter

size

and

pinopode

formation

the

group

sizes

were

five

mice

each

transfected

with

empty

siRNA

construct

or

non-targeting

pTER

respectively

-LRB-

mice

in

total

-RRB-

For

the

experiments

studying

the

effect

of

expression

manipulation

on

expression

mice

each

were

transfected

with

empty

siRNA

construct

or

non-targeting

pTER

respectively

-LRB-

mice

in

total

-RRB-

The

numbers

of

mice

available

for

expression

analysis

in

Fig.

were

and

respectively

-LRB-

the

remainder

were

not

pregnant

in

spite

of

the

detection

of

the

vaginal

plug

or

died

of

post-surgical

complications

-RRB-

The

presented

work

conformed

to

the

Guiding

principles

in

the

Care

and

Use

of

Animals

as

described

in

DHEW

publication

No

-LRB-

NIH

-RRB-

Revised

Office

of

Science

and

Health

Reports

DRR/NIH

Bethesda

MD

USA

was

approved

by

the

ethical

committee

of

Tongji

Medical

College

and

was

also

covered

by

Chinese

animal

husbandry

legislation

Scanning

electron

microscropy

Uteri

were

dissected

from

mice

h

after

vaginal

plug

detection

The

specimens

were

dehydrated

in

an

acetone

series

dried

in

a

critical

point

drier

using

carbon

dioxide

mounted

on

the

specimen

holder

coated

with

gold

and

examined

under

a

Stereoscan

Scanning

Electron

Microscope

-LRB-

Hitachi

Tokyo

Japan

-RRB-

From

each

biopsy

six

to

eight

tissues

fragments

measuring

mm

in

thickness

and

mm

in

length

were

evaluated

A

minimum

of

an

aggregated

of

well-preserved

epithelial

surface

was

analyzed

to

increase

the

likelihood

that

the

observations

were

representative

by

negating

the

influence

of

local

differences

in

endometrial

morphology

The

evaluations

were

performed

with

coded

tissue

and

read

blindly

by

an

experienced

investigator

according

to

Nikas

and

Makrigiannakis

-LRB-

-RRB-

Immunohistochemistry

The

expression

of

mouse

protein

in

the

uterus

was

evaluated

by

immunohistochemistry

using

a

goat

polyclonal

antibody

to

-LRB-

Santa

Cruz

-RRB-

The

full-thickness

biopsies

were

obtained

at

the

time

of

histological

evaluation

from

each

uterine

horn

The

specimens

were

embedded

in

paraffin

and

serial

µm

sections

obtained

The

sections

were

deparaffinized

in

xylene

and

ethanol

Endogenous

peroxidase

was

blocked

with

at

room

temperature

for

min

After

blocking

for

min

with

normal

horse

serum

in

PBS

the

sections

were

incubated

overnight

at

°C

with

primary

antibody

-LRB-

-RRB-

in

PBS

The

sections

were

then

incubated

with

biotinylated

rabbit

anti-goat

IgG

-LRB-

Zhongshan

Biotechnology

-RRB-

as

a

secondary

antibody

at

and

incubated

at

°C

for

min

subsequently

with

avidin

and

biotinylated

peroxidase

at

room

temperature

for

min

and

finally

with

DAB

-LRB-

mg/ml

-RRB-

at

room

temperature

for

min

Hematoxylin

was

used

for

counterstaining

The

staining

of

consecutive

cells

in

five

non-adjacent

microscopic

fields

was

evaluated

Immunoreactivity

of

was

scored

as

follows

of

cells

stained

but

of

cells

stained

but

of

cells

stained

and

of

cells

stained

Overall

intensity

was

scored

as

weak

-LRB-

-RRB-

moderate

-LRB-

-RRB-

or

strong

-LRB-

-RRB-

The

sum

of

cell

stained

scores

and

intensity

scores

was

calculated

to

quantify

the

expression

of

Statistical

analysis

All

numerical

results

are

expressed

as

the

mean

value

SD

except

for

the

Affymetrix

expression

values

of

mRNAs

in

normal

human

tissue

which

are

represented

as

mean

value

SEM

The

data

of

mRNA

in

endometrium

during

the

menstrual

cycle

were

analyzed

by

analysis

of

variance

Comparisons

between

the

expression

of

in

each

group

of

murine

tissue

in

each

group

of

murine

tissue

and

litter

size

of

each

group

of

mice

were

performed

using

two-tailed

unpaired

t-tests

The

differences

of

pinopode

development

between

each

group

of

murine

tissue

were

analyzed

using

a

chi-square

test

P

was

considered

significant

in

all

tests

The

Statistical

Package

for

the

Social

Sciences

software

package

for

Windows

-LRB-

Version

-RRB-

was

used

for

all

statistical

analyses

Results

Expression

of

and

genes

in

the

normal

human

uterus

Affymetrix

data

for

a

set

of

different

normal

human

tissue

types

-LRB-

the

human

body

index

set

-RRB-

representing

a

total

of

tissue

samples

were

retrieved

from

the

NCBI

website

As

shown

in

Fig.

was

highly

expressed

in

both

uterine

tissues

present

in

the

data

set

myometrium

and

endometrium

In

fact

the

expression

in

these

two

tissues

was

the

highest

observed

in

all

tissue

types

investigated

In

contrast

expression

was

high

in

myometrium

although

several

other

tissues

show

even

higher

expression

but

only

moderate

in

endometrium

Finally

the

gene

showed

overall

much

lower

expression

-LRB-

-RRB-

than

the

and

genes

The

high

expression

of

compared

with

and

in

endometrium

prompted

us

to

investigate

the

expression

and

function

of

the

gene

in

the

reproductive

cycle

Figure

Expression

of

mRNAs

in

normal

human

tissue

Visual

representation

of

and

expression

in

the

human

body

index

Affymetrix

data

set

in

the

public

domain

samples

representing

different

normal

human

tissues

For

reasons

of

presentation

only

tissues

consisting

of

five

or

more

different

samples

were

shown

tissues

representing

samples

expression

in

endometrium

and

myometrium

was

higher

than

in

any

of

the

non-shown

sets

Average

MEIS

expression

in

all

tissues

was

and

For

comparison

average

expression

of

dehydrogenase

-LRB-

GAPDH

-RRB-

and

β-actin

in

this

data

set

was

and

respectively

Average

MEIS

gene

expression

in

endometrium

-LRB-

samples

-RRB-

was

and

Average

MEIS

expression

in

myometrium

-LRB-

samples

-RRB-

was

and

The

average

expression

over

the

different

samples

per

tissue

is

shown

The

error

bars

represent

the

SEM

Expression

of

mRNA

during

the

human

reproductive

cycle

expression

has

been

previously

demonstrated

in

epithelial

cells

of

the

developing

female

reproductive

tract

-LRB-

Dintilhac

et

al.

-RRB-

To

investigate

the

expression

of

in

cyclic

development

of

the

endometrium

the

menstrual

cycle

stage-specific

expression

was

determined

by

RT

PCR

Representative

EtBr

stained

agarose

gel

pictures

of

RT

PCR

are

shown

in

Fig.

mRNA

expression

was

evident

throughout

the

menstrual

cycle

Figure

mRNA

expression

in

human

endometrium

during

the

menstrual

cycle

RT

PCR

analysis

of

expression

A

representative

agarose

gel

stained

with

ethidium

bromide

-LRB-

EtBr

-RRB-

picture

is

shown

was

expressed

in

all

stages

of

the

human

menstrual

cycle

investigated

the

first

half

of

the

proliferative

phase

-LRB-

-RRB-

the

second

half

of

the

proliferative

phase

-LRB-

-RRB-

and

the

first

third

of

the

secretory

phase

-LRB-

-RRB-

the

mid-secretory

phase

-LRB-

-RRB-

and

the

late

secretory

phase

-LRB-

-RRB-

The

sample

numbers

for

the

and

groups

were

and

respectively

M

size

markers

No

PCR

product

was

observed

in

control

samples

that

did

not

contain

RT

enzyme

during

RT

synthesis

or

template

DNA

during

PCR

-LRB-

not

shown

-RRB-

RT

PCR

analysis

was

repeated

at

least

three

times

per

sample

expression

was

analyzed

by

densitometry

and

normalized

to

GAPDH

in

endometrial

samples

The

densitometric

analysis

was

repeated

three

times

with

similar

results

The

data

were

analyzed

using

analysis

of

variance

The

average

ratio

in

each

group

was

and

There

was

no

statistical

difference

within

the

proliferative

phase

-LRB-

P

-RRB-

but

the

difference

between

the

proliferative

phase

and

the

secretory

phase

was

significant

-LRB-

P

-RRB-

To

assess

potential

regulation

of

expression

during

the

reproductive

cycle

densitometric

analysis

was

performed

on

all

samples

and

the

average

abundance

of

during

each

stage

of

the

menstrual

cycle

normalized

to

GAPDH

calculated

No

difference

was

apparent

between

the

levels

of

expression

in

the

segments

of

the

proliferative

phase

In

contrast

expression

at

the

mid

secretory

phase

was

elevated

relative

to

other

stages

-LRB-

see

legend

to

Fig.

-RRB-

These

results

were

a

first

indication

that

expression

varied

during

the

menstrual

cycle

markedly

increasing

at

the

mid

secretory

phase

-LRB-

-RRB-

which

corresponded

to

the

time

of

implantation

mRNA

is

expressed

in

the

human

endometrial

stroma

and

glands

To

investigate

the

site

of

expression

in

the

human

endometrium

and

as

a

support

of

the

expression

detected

with

RT

PCR

described

above

in

situ

hybridization

was

performed

Fig.

and

b

shows

high-power

views

of

expression

in

proliferative

and

secretory

endometrium

was

expressed

in

both

glandular

and

stromal

cells

This

supports

the

results

of

RT

PCR

in

Fig.

and

further

suggests

that

may

play

a

role

in

embryonic

implantation

Figure

mRNA

expression

in

human

endometrial

tissue

In

situ

hybridization

analysis

of

expression

in

human

endometrial

samples

Views

of

two

representative

samples

demonstrate

expression

in

both

the

glandular

and

stromal

cells

of

-LRB-

a

-RRB-

proliferative

phase

and

-LRB-

b

-RRB-

secretory

phase

endometrium

mRNA

is

stained

brown

No

staining

was

observed

when

the

probe

was

omitted

-LRB-

not

shown

-RRB-

Scale

bar

µm

Each

sample

was

analyzed

at

least

in

triplicate

Down-regulation

of

expression

affects

murine

embryo

implantation

rate

To

assess

whether

an

up-regulation

of

expression

during

embryo

implantation

was

essential

to

this

process

the

expression

of

maternal

was

down-regulated

in

a

mouse

model

by

transfection

in

vivo

as

described

by

-LRB-

Bagot

et

al.

Hsieh

et

al.

-RRB-

A

first

indication

of

decreased

expression

in

the

uterus

was

obtained

by

RT

PCR

and

this

was

confirmed

by

western

blot

on

Day

after

transfection

Representative

results

for

mRNA

are

shown

in

Fig.

and

b.

Densitometric

analysis

was

performed

on

all

samples

and

the

average

abundance

of

mRNA

in

each

group

normalized

to

β-actin

mRNA

was

calculated

Levels

of

mRNA

and

protein

-LRB-

Fig.

and

c

-RRB-

were

significantly

decreased

in

endometrium

transfected

with

siRNA

compared

with

the

control

Figure

expression

manipulation

in

murine

uterine

tissue

-LRB-

a

-RRB-

RT

PCR

analysis

of

expression

in

murine

endometria

transfected

with

small

interfering

RNA

-LRB-

siRNA

-RRB-

constructs

or

respective

controls

-LRB-

five

animals

in

each

group

-RRB-

Shown

is

a

representative

EtBr

picture

-LRB-

b

-RRB-

mRNA

expression

was

analyzed

by

densitometry

and

normalized

to

β-actin

as

described

in

Fig.

except

that

data

were

analyzed

using

a

two-tailed

unpaired

t-test

No

PCR

product

was

observed

in

control

samples

that

did

not

contain

RT

enzyme

during

RT

synthesis

or

template

DNA

during

PCR

-LRB-

not

shown

-RRB-

RT

PCR

and

quantification

was

repeated

at

least

three

times

with

similar

results

Average

ratios

in

endometrium

transfected

with

-LRB-

U

-RRB-

and

control

DNA

-LRB-

Uc

-RRB-

were

and

respectively

There

was

a

significant

increase

in

mRNA

in

the

up-regulation

group

-LRB-

U

-RRB-

compared

with

the

control

-LRB-

Uc

-RRB-

-LRB-

P

-RRB-

Average

level

in

endometrium

transfected

with

siRNA

-LRB-

D

-RRB-

and

control

pTER

constructs

-LRB-

Dc

-RRB-

were

and

respectively

Levels

of

mRNA

were

significantly

decreased

in

siRNA-transfected

samples

-LRB-

D

-RRB-

compared

with

the

control

samples

-LRB-

Dc

-RRB-

-LRB-

P

-RRB-

-LRB-

c

-RRB-

Western

analysis

performed

on

murine

endometria

transfected

with

siRNA

or

respective

controls

-LRB-

five

animals

in

each

group

-RRB-

A

representative

western

blot

image

is

shown

-LRB-

d

-RRB-

protein

expression

was

analyzed

by

densitometry

and

normalized

to

β-actin

values

as

described

in

Fig.

No

band

was

detected

on

western

blot

in

the

absence

of

primary

antiserum

-LRB-

not

shown

-RRB-

Western

blot

analysis

and

quantification

was

repeated

at

least

three

times

with

similar

results

The

average

ratio

in

each

group

was

U

Uc

D

and

Dc

There

was

a

significant

increase

in

protein

in

endometria

transfected

with

-LRB-

U

-RRB-

compared

with

controls

-LRB-

Uc

-RRB-

-LRB-

P

-RRB-

Endometria

transfected

with

siRNA

constructs

-LRB-

D

-RRB-

demonstrated

a

significant

decrease

in

protein

compared

with

pTER

controls

-LRB-

Dc

-RRB-

-LRB-

P

-RRB-

At

Day

of

pregnancy

mice

were

killed

and

the

uteri

removed

and

examined

for

normal

histology

and

for

the

number

of

implantation

sites

per

uterus

as

a

measure

of

litter

size

The

endometrium

appeared

histologically

normal

and

demonstrated

adequate

decidualization

Also

the

total

number

of

pregnant

mice

did

not

vary

significantly

between

siRNA-treated

mice

and

the

control

Pups

of

mice

transfected

with

siRNA

were

born

following

a

normal

length

gestation

of

days

were

of

normal

size

and

demonstrated

no

morphological

abnormalities

However

the

average

number

of

implantation

sites

per

uterus

was

in

the

mice

transfected

with

the

control

pTER

plasmid

and

in

the

mice

transfected

with

a

siRNA

construct

-LRB-

Fig.

-RRB-

Decreased

expression

of

therefore

resulted

in

a

significant

decrease

in

litter

size

-LRB-

see

legend

to

Fig.

-RRB-

We

concluded

that

the

naturally

occurring

up-regulation

of

in

the

reproductive

cycle

contributes

to

normal

embryo

implantation

Figure

Effect

of

expression

on

mouse

litter

size

Litter

size

as

determined

by

counting

the

number

of

implantation

sites

per

uterus

examined

at

Day

of

pregnancy

Mice

were

transfected

day

after

detection

of

a

vaginal

plug

with

control

pTER

or

empty

-LRB-

a

-RRB-

Litter

size

was

reduced

by

in

the

group

of

mice

transfected

with

siRNA

compared

with

pTER

controls

This

difference

was

statistically

significant

according

to

a

two-tailed

unpaired

t-test

-LRB-

P

-RRB-

-LRB-

b

-RRB-

Litter

size

at

Day

of

gestation

was

not

affected

by

transfection

with

compared

with

controls

-LRB-

P

-RRB-

The

experiment

was

repeated

three

times

with

similar

results

To

evaluate

the

effect

of

up-regulation

of

expression

in

the

adult

murine

uterus

mice

were

transfected

with

the

expression

construct

or

with

empty

plasmid

-LRB-

as

a

negative

control

-RRB-

A

first

indication

of

increased

expression

in

the

uterus

was

obtained

by

RT

PCR

and

this

was

confirmed

by

western

blot

Levels

of

mRNA

and

protein

were

significantly

increased

in

endometrium

transfected

with

the

expression

construct

compared

with

the

control

-LRB-

Fig.

and

d

-RRB-

As

in

the

experiment

described

above

uterine

histology

was

not

affected

by

the

transfection

and

the

number

of

mice

pregnant

and

the

weight

and

morphology

of

the

pups

did

not

differ

between

the

groups

The

average

number

of

implantation

sites

was

in

the

treated

mice

and

in

the

control

mice

-LRB-

Fig.

-RRB-

In

contrast

to

down-regulation

up-regulation

of

expression

by

treatment

did

not

significantly

alter

litter

size

Effect

of

on

murine

pinopode

formation

For

further

clues

as

to

a

possible

function

for

in

murine

implantation

uterine

epithelial

morphology

was

investigated

As

described

above

no

uterine

histological

changes

could

be

detected

at

the

light

microscopy

level

in

mice

transfected

with

DNA

constructs

To

evaluate

the

effect

of

on

epithelial

morphology

the

uteri

of

these

mice

were

examined

with

scanning

electron

microscopy

for

evidence

of

altered

formation

of

pinopodes

Mice

which

were

transfected

in

vivo

as

described

above

were

killed

days

after

vaginal

plug

detection

the

uteri

removed

and

prepared

for

electron

microscopy

analysis

Fig.

and

b

shows

a

typical

section

of

endometrium

from

a

mouse

and

an

empty

control

mouse

there

was

a

significant

increase

in

the

development

of

pinopodes

in

the

mouse

transfected

with

over-expression

plasmid

Fig.

and

d

shows

a

typical

section

of

endometrium

from

and

control

pTER-treated

mouse

uteri

of

the

sections

from

siRNA-treated

mice

contained

normal

pinopodes

The

secretory

cells

were

covered

with

dense

microvilli

instead

of

with

pinopodes

and

were

slightly

bulging

In

contrast

all

sections

from

control

animals

contained

a

normal

number

of

pinopodes

Apparently

decreasing

expression

had

a

negative

effect

on

efficient

pinopode

maturation

Absence

of

a

normal

amount

of

functioning

pinopods

in

mice

transfected

with

siRNA

could

be

a

mechanism

contributing

to

a

less

efficient

embryonic

implantation

rate

and

diminished

litter

size

in

these

animals

Figure

Effect

of

expression

on

murine

pinopode

formation

Pinopode

development

in

transfected

mouse

uteri

examined

with

scanning

electron

microscopy

-LRB-

a

-RRB-

Transfection

with

demonstrated

fully

developed

pinopodes

on

most

-LRB-

-RRB-

secretory

cells

-LRB-

b

-RRB-

Transfection

with

empty

plasmid

as

a

negative

control

showed

a

wide

distribution

of

developing

pinopodes

covering

of

the

endometrial

luminal

surface

However

there

was

a

much

lower

occurrence

-LRB-

-RRB-

of

fully

developed

pinopodes

in

this

group

-LRB-

c

-RRB-

Transfection

with

siRNA

plasmid

resulted

in

the

majority

of

the

endometrial

surface

expressing

few

to

no

pinopodes

Instead

the

epithelial

cells

were

covered

with

short

and

thick

microvilli

-LRB-

d

-RRB-

Transfection

with

non-targeting

pTER

plasmid

as

a

control

resulted

in

developing

pinopodes

on

about

half

of

the

secretory

cells

Scale

bar

µm

The

experiment

was

repeated

three

times

The

data

were

analyzed

using

a

chi-square

test

Effect

of

on

expression

in

the

murine

uterus

Maternal

has

earlier

been

shown

to

be

involved

in

pinopode

formation

in

the

mouse

uterus

-LRB-

Bagot

et

al.

-RRB-

in

a

manner

resembling

the

effect

of

on

pinopode

formation

observed

in

this

study

To

investigate

a

possible

connection

between

and

function

in

murine

uterine

development

and

embryonic

implantation

we

evaluated

the

effect

of

expression

manipulation

on

expression

To

obtain

a

first

indication

of

differential

mRNA

expression

peri-implantation

uteri

from

mice

treated

with

or

empty

-LRB-

as

a

negative

control

-RRB-

or

with

or

non-targeting

pTER

-LRB-

as

a

negative

control

-RRB-

were

analyzed

by

RT

PCR

Fig.

shows

a

representative

EtBr

gel

picture

of

the

RT

PCR

Densitometric

analysis

was

performed

on

all

samples

and

the

average

abundance

of

in

each

group

normalized

to

β-actin

was

calculated

Levels

of

mRNA

appeared

significantly

decreased

in

endometrium

transfected

with

siRNA

compared

with

the

control

In

contrast

levels

of

mRNA

increased

in

endometrium

transfected

with

the

expression

construct

compared

with

the

control

-LRB-

see

legend

to

Fig.

-RRB-

Figure

Effect

of

expression

manipulation

on

murine

uterine

integrin

β3

-LRB-

-RRB-

expression

-LRB-

a

-RRB-

RT

PCR

analysis

of

expression

in

murine

endometria

transfected

with

-LRB-

U

-RRB-

empty

-LRB-

Uc

-RRB-

siRNA

-LRB-

D

-RRB-

or

control

pTER

constructs

-LRB-

Dc

-RRB-

A

representative

agarose

EtBr

picture

is

shown

No

PCR

product

was

observed

in

control

samples

that

did

not

contain

RT

enzyme

during

RT

synthesis

or

template

DNA

during

PCR

-LRB-

not

shown

-RRB-

RT

PCR

was

repeated

three

times

with

similar

results

expression

was

analyzed

by

densitometry

and

normalized

to

β-actin

as

described

in

Fig.

Average

ratio

in

each

group

was

U

Uc

D

and

Dc

There

was

a

significant

increase

in

mRNA

in

endometrium

transfected

with

compared

with

endometrium

transfected

with

empty

DNA

-LRB-

P

-RRB-

Levels

of

mRNA

in

endometrium

transfected

with

siRNA

were

significantly

decreased

when

compared

with

endometrium

transfected

with

control

pTER

-LRB-

P

-RRB-

-LRB-

b

-RRB-

Immunohistochemistry

of

protein

expression

in

the

endometrium

of

mice

transfected

with

either

or

siRNA

plasmid

Shown

are

representative

images

of

each

group

uterine

sections

from

mice

transfected

with

-LRB-

U

-RRB-

with

empty

vector

-LRB-

Uc

-RRB-

with

siRNA

-LRB-

D

-RRB-

or

with

non-targeting

pTER

vector

-LRB-

Dc

-RRB-

No

staining

was

observed

in

a

negative

control

without

antibody

as

the

primary

antiserum

-LRB-

not

shown

-RRB-

Scale

bar

µm

Statistical

analysis

of

immunoreactivity

was

performed

as

described

in

the

Materials

and

Methods

section

The

data

were

analyzed

using

a

two-tailed

unpaired

t-test

The

immunoreactivity

analysis

was

repeated

three

times

with

similar

results

The

average

immunoreactivity

scores

in

each

group

were

U

Uc

D

and

Dc

Uterine

sections

from

mice

showed

significantly

increased

glandular

and

stromal

expression

compared

with

control

mice

treated

with

empty

vector

-LRB-

P

-RRB-

Uterine

sections

from

siRNA-transfected

mice

showed

significantly

reduced

glandular

and

stromal

expression

compared

with

control

mice

treated

with

non-targeting

pTER

vector

-LRB-

P

-RRB-

To

support

the

results

of

the

RT

PCR

described

above

the

effect

of

expression

on

protein

levels

was

determined

by

immunohistochemistry

on

transfected

uterine

tissue

of

each

group

Representative

pictures

are

shown

in

Fig.

expression

levels

appeared

higher

in

mice

transfected

with

expression

construct

compared

with

the

control

In

contrast

expression

was

lower

in

mice

transfected

with

siRNA

expression

constructs

than

in

those

transfected

with

the

non-targeting

siRNA

construct

Statistic

analysis

of

the

immunoreactivity

was

performed

on

the

samples

and

the

average

abundance

of

in

each

group

was

calculated

Levels

of

expression

were

significantly

decreased

in

endometrium

transfected

with

siRNA

compared

with

the

control

In

contrast

protein

levels

were

increased

in

endometrium

transfected

with

the

expression

construct

compared

with

the

control

-LRB-

see

legend

to

Fig.

-RRB-

This

was

consistent

with

the

results

of

the

RT

PCR

Together

these

results

showed

that

in

a

murine

uterine

receptivity

model

the

expression

of

the

known

target

could

be

regulated

by

the

manipulation

of

expression

suggesting

action

through

co-operation

or

possibly

interaction

with

Discussion

is

essential

for

normal

embryonic

uterine

development

In

the

developing

mouse

embryo

altered

expression

as

a

result

of

targeted

mutation

-LRB-

Satokata

et

al.

-RRB-

or

exposure

to

diethylstilbestrol

leads

to

abnormal

uterine

development

-LRB-

Block

et

al.

-RRB-

expression

persists

in

the

adult

uterus

and

is

important

for

implantation

of

the

embryo

null-mutant

mice

or

mice

in

which

has

been

blocked

using

antisense

oligonucleotides

demonstrate

decreased

litter

size

due

to

failure

of

implantation

-LRB-

Benson

et

al.

Bagot

et

al.

-RRB-

HOX

genes

function

as

transcription

factors

and

either

activate

or

repress

target

genes

The

pleiotropic

effects

of

on

endometrial

differentiation

and

function

are

likely

modulated

through

the

regulation

of

multiple

downstream

targets

that

are

necessary

for

the

implantation

process

-LRB-

Daftary

et

al.

Troy

et

al.

-RRB-

has

been

demonstrated

to

be

involved

in

the

regulation

of

pinopode

development

and

expression

of

other

genes

involved

in

endometrial

development

such

as

and

-LRB-

Bagot

et

al.

Daftary

et

al.

Troy

et

al.

-RRB-

HOX

proteins

require

co-operation

with

other

proteins

to

bind

to

their

target

DNA

in

particular

aids

HOX

proteins

like

to

gain

this

specificity

physically

interacts

with

HOX

proteins

by

forming

heterodimeric

binding

complexes

on

a

DNA

target

containing

a

site

-LRB-

TGACAG

-RRB-

and

an

Abd-B-like

Hox

site

-LRB-

TTTTACGAC

-RRB-

-LRB-

Shen

et

al.

-RRB-

Studies

demonstrated

that

gene

and

genes

are

co-expressed

throughout

Müllerian

duct

differentiation

-LRB-

Dintilhac

et

al.

-RRB-

which

suggests

that

might

play

a

role

in

embryonic

female

genital

tract

development

We

have

performed

data-mining

on

Affymetrix

gene

expression

database

in

the

public

domain

showing

that

was

highly

expressed

in

human

endometrium

as

well

as

in

myometrium

-LRB-

Fig.

-RRB-

implying

it

may

play

a

role

in

reproductive

function

in

the

adult

human

uterus

We

therefore

investigated

the

expression

pattern

of

the

gene

at

different

stages

of

the

human

menstrual

cycle

demonstrating

that

appeared

to

be

expressed

in

the

endometrium

at

different

levels

depending

on

the

menstrual

cycle

stage

mRNA

levels

appeared

significantly

increased

in

the

mid

secretory

phase

closely

resembling

the

expression

pattern

of

This

observation

pointed

to

the

possibility

that

is

an

active

cofactor

of

in

the

human

endometrium

On

the

basis

of

the

results

described

above

we

postulated

that

and

might

have

closely

related

roles

in

vertebrate

reproduction

and

in

the

regulation

of

endometrial

cell

proliferation

or

differentiation

null-mutant

mice

die

between

embryonic

days

and

because

of

internal

hemorrhage

liver

hypoplasia

and

anemia

-LRB-

Azcoitia

et

al.

-RRB-

The

embryo

lethal

phenotype

has

precluded

investigation

of

the

function

of

in

the

adult

reproductive

tract

of

mutant

mice

Therefore

to

investigate

the

role

of

in

adult

reproductive

function

in

a

mouse

model

we

altered

the

expression

of

the

gene

in

the

adult

murine

endometrium

When

mouse

endometrium

was

transfected

with

siRNA

at

the

time

of

implantation

the

number

of

embryos

that

could

implant

in

the

uterus

was

reduced

by

This

significant

reduction

in

implantation

sites

demonstrated

that

in

normal

embryonic

development

optimal

implantation

benefits

from

maternal

expression

We

also

investigate

the

effect

of

on

pinopodes

Pinopode

formation

is

directly

regulated

by

-LRB-

Bagot

et

al.

-RRB-

In

this

study

we

examined

whether

the

expression

of

pinopodes

could

be

also

affected

by

altering

expression

levels

We

demonstrated

that

blocking

expression

reduced

pinopode

numbers

and

reduced

their

ability

to

differentiate

into

fully

mature

forms

In

contrast

supernumerary

pinopodes

could

be

induced

by

over-expression

of

To

elucidate

potential

mechanisms

by

which

might

control

endometrial

function

and

receptivity

to

the

implanting

blastocyst

we

tested

the

effect

of

altering

expression

on

expression

The

gene

has

been

identified

as

the

first

known

target

of

gene

regulation

in

the

endometrium

-LRB-

Daftary

et

al.

-RRB-

As

a

bridging

molecule

between

the

endometrium

and

trophoblast

-LRB-

Kiefer

et

al.

Van

Dijk

et

al.

Sueoka

et

al.

-RRB-

plays

an

important

role

in

embryonic

implantation

We

found

that

a

blockade

of

expression

with

siRNA

was

associated

with

a

decrease

in

mRNA

mRNA

levels

significantly

increased

after

up-regulation

of

expression

with

cDNA

It

appeared

likely

that

was

not

regulated

by

monomer

but

by

a

heterodimeric

or

heterotrimeric

transcription

complex

containing

The

interaction

of

Abd-B-like

HOX

transcription

factors

and

at

the

HOX

site

in

the

target

gene

promoter

has

been

demonstrated

in

myeloid

differentiation

The

co-activation

of

and

is

a

common

event

in

AML

-LRB-

Lawrence

et

al.

Camós

et

al.

-RRB-

but

co-regulation

of

and

and

also

of

might

play

a

role

in

pathogenesis

of

acute

lymphocytic

and

acute

myeloid

leukemias

associated

with

fusions

-LRB-

Rozovskaia

et

al.

Ferrando

et

al.

Dik

et

al.

-RRB-

In

addition

Pineault

et

al.

-LRB-

-RRB-

demonstrated

that

enhances

the

effect

of

a

rearrangement

releasing

HOX

expression

necessary

for

leukemia

induction

Finally

binding

of

to

could

be

demonstrated

in

vitro

-LRB-

Williams

et

al.

-RRB-

-LRB-

also

known

as

or

-RRB-

is

a

transcriptional

target

of

in

differentiating

myelomonocytic

cells

reporter

expression

is

enhanced

after

transfection

with

and

especially

when

was

co-transfected

-LRB-

Bromleigh

and

Freedman

-RRB-

It

is

likely

that

a

similar

regulation

occurs

with

and

cofactor

in

the

endometrium

Recently

Taylor

et

al.

have

confirmed

that

this

potential

protein

protein

interaction

does

occur

in

endometrial

cells

Ablation

of

the

PBX

cofactor

half-site

results

in

a

loss

of

binding

to

the

probe

suggesting

the

importance

of

the

protein

protein

interaction

in

endometrium

for

high

affinity

binding

to

its

target

genes

-LRB-

Sarno

et

al.

-RRB-

In

conclusion

this

study

shows

for

the

first

time

that

expression

contributes

to

endometrial

receptivity

in

mouse

can

function

in

cancer

as

an

inhibitor

of

differentiation

and

have

a

positive

influence

on

cellular

proliferation

-LRB-

see

Introduction

-RRB-

In

addition

the

MEIS

genes

are

potent

enhancers

of

early

cell

proliferation

during

development

-LRB-

reviewed

in

Geerts

et

al.

-RRB-

could

have

a

similar

proliferative

function

during

endometrial

development

The

expression

pattern

of

its

contribution

to

pinopode

formation

in

mouse

and

its

effect

on

expression

suggest

that

the

effects

of

on

endometrial

function

occur

through

affecting

the

affinity

and

specificity

of

DNA

interactions

leading

to

the

regulation

of

downstream

targets

of

The

identification

of

as

a

potential

co-operating

gene

could

lead

to

elucidation

of

a

partial

network

in

endometrial

development

but

could

also

establish

in

a

signaling

network

in

its

own

right

Author

's

Role

B.X.

conception

and

design

of

experiments

acquisition

of

data

analysis

and

interpretation

of

data

writing

and

revising

of

manuscript

final

approval

of

manuscript

to

be

published

D.G.

design

of

experiments

acquisition

analysis

and

interpretation

of

data

writing

and

revising

of

the

manuscript

final

approval

of

manuscript

to

be

published

K.Q.

conception

and

design

of

experiments

revision

of

manuscript

final

approval

of

manuscript

to

be

published

H.Z.

conception

and

design

of

experiments

final

approval

of

manuscript

to

be

published

G.Z.

interpretation

of

data

revision

of

manuscript

final

approval

of

manuscript

to

be

published

Funding

Dutch

Cancer

Society

-LRB-

KWF

Kankerbestrijding

-RRB-

-LRB-

to

D.G.

-RRB-

The

Program

of

China

-LRB-

No.

-RRB-

O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O B O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O 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