proteome of human endometrium: identification of differentially expressed proteins in proliferative...

RESEARCH ARTICLE

Proteome of human endometrium: Identification

of differentially expressed proteins in proliferative

and secretory phase endometrium

Priyanka Rai1, Venkatesh Kota1, Curam Sreenivasacharlu Sundaram1, Mamta Deendayal2

and Sisinthy Shivaji1

1 Centre for Cellular and Molecular Biology Uppal Road, Hyderabad, Andhra Pradesh, India2 Infertility Institute and Research Centre, Hyderabad, Andhra Pradesh, India

Received: May 13, 2009

Revised: September 11, 2009

Accepted: September 16, 2009

Purpose: To exploit the potential of proteomics to identify and study additional yet-uniden-

tified important proteins present in human endometrium.

Experimental design: The proteome of human endometrium would be established using

2-DE and MALDI and the data analyzed to identify differential protein expression in the

proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI.

Results: In the present work, 2-DE of human endometrium protein led to the resolution of

over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194

proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of

which 49 could be identified using MALDI. These differentially expressed proteins included

structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins

related to immunity, RNA biogenesis, protein biosynthesis and others. The differential

expressions of seven representative proteins in secretory and proliferative phase endome-

trium tissue were confirmed by immunoblot analysis.

Conclusion and clinical relevance: This study establishes the 2-D proteome of human

endometrium represented by 194 identified protein spots. The present data provides an

important clue towards determining the function of these proteins with respect to endo-

metrium related diseases.

Keywords:

2-DE / Endometrium / Menstrual cycle / Proliferative phase / Secretory phase

1 Introduction

Endometrium, the mucosal lining of the uterus, is func-

tionally involved in providing a suitable site for implantation

and development of a fertilized oocyte. This highly dynamic

tissue undergoes cyclic variation with every menstrual cycle

during the reproductive years. The menstrual cycle is divi-

ded into three distinct phases – menstrual, proliferative

(follicular) and secretory (luteal). During menstruation, the

entire functional layer (functionalis) of the endometrium is

shed with subsequent regeneration of the tissue from the

remaining basal layer (basalis) [1]. The proliferative phase

begins after menses and terminates at ovulation. During

this phase, under the influence of estrogen there is rapid

cellular proliferation of all cell types and new extracellular

matrix is laid down. Shortly after ovulation, in the secretory

phase the endometrium undergoes progesterone dependent

functional differentiation which provides a suitable

environment for embryo implantation. Characteristically,

Abbreviations: Gapdh, glyceraldehyde phosphate dehydrogen-

ase; GRP, glucose-regulated proteins; MVP, major vault protein;

TBST, Tween 0.1% v/v in TBS

Correspondence: Dr. Sisinthy Shivaji, Centre for Cellular and

Molecular Biology, Uppal Road, Hyderabad 500 007, Andhra

Pradesh, India

E-mail: [email protected]

Fax: 191-40-27160311

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

48 Proteomics Clin. Appl. 2010, 4, 48–59DOI 10.1002/prca.200900094

the glands become increasingly tortuous with considerable

secretory activity and the stromal cells begin a differentia-

tion process, termed as decidualization, a prerequisite

for successful implantation. The endometrium is

receptive to embryonic implantation in the mid-secretory

phase, approximately for a period of 5 days and this

process coincides with a peak in the levels of progesterone.

In the absence of pregnancy, a decline in progesterone

levels in the late secretory phase leads to endometrial

regression and menstruation [2, 3]. Any abrogation in

endometrial remodeling or endometrial physiology

would lead to diseases like endometriosis [4–10],

endometrial polyps, hyperplasia and endometrial cancer

[11–14].

Earlier studies using the conventional approach

of purifying and characterizing the proteins from the

endometrium in different physiological states has led to the

identification of a number of endometrium-specific proteins

and the functions of some of these proteins like sialic acid

binding protein (54 kDa glycoprotein) [15], glycodelin [16],

IGF-binding proteins, PP14 [17, 18] and CYP3A7, a cyto-

chrome P450 isoform, which metabolizes estrogens [19] in

human proliferative as well as secretory phase endometrium

has been established. These earlier studies were limited with

respect to the identification of only a few proteins. However,

the advent of genomics and proteomics has led to the

identification of several genes and proteins that are char-

acteristic of the different phases of the menstrual cycle and

some of the identified candidate proteins have been impli-

cated in uterine receptivity and oocyte implantation [20–23].

As on date only two studies have so far been carried out on

the differential protein expression of endometrium both in

proliferative and secretory phases [22, 23]. One group has

analyzed both the proliferative and secretory phases of the

human endometrium by using ProICAT approach and

identified 119 proteins, among which only five of the

proteins showed consistent differential expression [22]. The

other group used DIGE and was able to identify 76 out of

196 differentially expressed proteins [23]. Thus the two

available studies do not provide a consistent profile of the

proteome of the human endometrium. In an attempt to a

get a more consistent profile of the human endometrium

2-D PAGE, followed by densitometry for relative quantifi-

cation, and subsequent identification of proteins by MS and/

or MS/MS analysis was carried out using human endome-

trium in the proliferative and secretory phase. A total

of 194 proteins were identified which includes structural

proteins, molecular chaperones and proteins related to

metabolism, immunity, protein biosynthesis and RNA

biogenesis. Further 57 proteins were differentially

expressed in proliferative and secretory phase and 49 of

these differential proteins were successfully identified by

MALDI MS and/or MS/MS. This combined study has

resulted in the identification of proteins which could give

us new insights into endometrial development and

remodeling.

2 Materials and methods

2.1 Human endometrial tissue samples

Endometrial tissue samples were obtained from women

admitted to Infertility Institute and Research Centre, Hyder-

abad, India, for diagnostic purposes, including infertility,

tubal re-enastomosis or pelvic pain. The tissue sample were

obtained from proliferative (days 7–10; n 5 12) and secretory

(days 20–24; n 5 12) phases of the menstrual cycle from

women with normal menstrual cycle, normal hormonal

profiles and free of uterine abnormalities. All tissue samples

were collected during routine surgical procedures. Written

consent was obtained for the sampling of eutopic endome-

trium for research purposes. Irregularly cycling, amenorrheic,

postmenopausal women and those who had received steroid

hormone therapy in the last three months were excluded from

the study. The samples were frozen in liquid nitrogen and

preserved at �801C until protein was extracted. The accurate

phasing of the endometrial samples was done by histo-

pathology section using standard procedure [24]. All experi-

ments were performed in accordance with the guidelines of

the Institutional Review Board of the Centre for Cellular and

Molecular Biology, Hyderabad, India.

2.2 Proteins extraction and 2-D PAGE

The frozen endometrial tissue samples were thawed, washed

with PBS and lysed with lysis buffer [containing 7 M urea, 2 M

thiourea, 2% NP40, 50 mM DTT, 0.5% pharmalytes 3–10, and

protease inhibitor cocktail (Roche, Mannheim, Germany)].

The suspension was then homogenized for approximately

5 min and kept at 41C for 2 h. Subsequently, the lysate was

centrifuged at 12 5000� g for 1 h at 41C and the solubilized

protein recovered carefully without disturbing the sediment.

The supernatant was precipitated using trichloroacetic acid/

acetone and resultant pellet was resuspended in lysis buffer

and protein was quantified by amido black method [25]. The

protein (200mg) was then loaded on to a commercially avail-

able IPG strip (7 cm, pH 4–7; Bio-Rad, Hercules, CA, USA) by

passive rehydration for 12 h. Later IEF was performed at

50mA/IPG strips at 8000 V for 20 000 Vh using a Protean IEF

cell (Bio-Rad). After the IEF run was completed strips were

equilibrated in buffer I (containing 6 M urea, 0.375 M Tris pH

8.8, 2% SDS, 20% glycerol and 2% DTT), followed by a second

incubation in buffer II which contained all the ingredients of

buffer I except that DTT was replaced with 2.5% iodoaceta-

mide. Each equilibration step was carried out for 20 min

under gentle agitation. Strips were then transferred onto a

10% SDS-PAGE gel (10� 10.5� 0.10 cm3) and embedded

into the gel with 1% agarose containing a trace amount of

bromophenol blue. SDS-PAGE was performed using vertical

gel electrophoresis system (GE Healthcare Bio-Sciences,

Piscataway, NJ, USA) at 20 Amp/gel. The gels were stained

with colloidal Coomassie stain [26].

Proteomics Clin. Appl. 2010, 4, 48–59 49


2.3 Image capture and analysis

To compare the 2-D gels of proliferative (n 5 6; in dupli-

cates) and secretory (n 5 6; in duplicates) phase endome-

trium, the stained gels were scanned using a Fluor-STM

Multi Imager (Bio-Rad) and transferred to PDQuest

Advanced 2D Analysis Software Version 8.0.1 software (Bio-

Rad). The semi automated routines available in this software

were used to detect and quantify protein spots as well as to

match the profiles across a gel series. Protein spots in the

gels were identified after normalization based on the local

regression model (LOESS). The protein spots from all the

gels in the same group were matched for reproducibility

analysis in our test system and the scatter plot tool was used

to show the correlation coefficient among the gels in each

group. For gel comparison, a statistical approach was

applied when determining differentially expressed proteins

using the PDQuest software. Mann–Whitney Unpaired

2-sample test was performed with 95% significance level to

determine which proteins were differentially expressed

between the proliferative and secretory phase endometrium

gels. A minimum of 1.2-fold change was considered for the

upregulated/downregulated proteins.

2.4 In-gel digestion and protein identification by

MALDI

The protein spots were manually cut out from gels and

processed for MALDI. The excised protein spots were

destained for 1 h using 50% v/v ACN and 100 mM

NH4HCO3 and then briefly washed with 100% ACN,

vacuum-dried in a SpeedVac concentrator (Labconco

Corporation, Kansas City, MO, USA) and then incubated in

15mL of trypsin solution (10mg/mL trypsin in 10 mM

NH4HCO3) for 16 h at 371C. The tryptic peptides were

spotted on the MALDI plate and dried prior to the addition

of 1 mL of 5 mg/mL of CHCA in 50% ACN. Protein spots

were then identified by MALDI MS and/or MS/MS using

MALDI TOF/TOF 4800 Proteomics Analyzer (Applied

Biosystems, Foster city, CA, USA). Peptide mass calibration

was performed with external mass standard (Calmix 5;

Applied Biosystems). Spectra were analyzed using in-house

GPS Explorer (TM) software, version 3.5 with fixed carb-

amidomethylcysteine and variable methionine oxidation.

Database used was Homo sapiens, NCBI 2008. Proteins that

identified by MALDI MS and/or MS/MS with significant

score have been tabulated. The results were also manually

validated based on protein pI and molecular weight.

2.5 Immunoblotting

Endometrium proteins from proliferative phase (n 5 6) and

secretory phase (n 5 6) were resolved by 1-D PAGE and then

electrotransferred onto an NC membrane at 100 V for 1 h

using the wet transfer system (Hoefer Scientific Instru-

ments, San Francisco, CA, USA). Subsequently, the

membrane was stained with 0.1% ponceau S, to check for

equal loading of the proteins. Membranes were then blocked

with 5% w/v non-fat milk in TBST (Tween 0.1% v/v in TBS)

for 1 h at room temperature, washed and incubated with the

primary antibody prepared in TBST containing BSA 1% w/v

for 1 h. Primary antibodies were diluted as follows: (i) HSP

27 (Stressgen, Ann Arbor, Michigan, USA) 1:1000; (ii)

glucose-regulated protein 94 (GRP 94) (Abcam, Cambridge,

UK),1:5000; (iii) Vinculin (Millipore, Billerica, MA, USA),

1:1000; (iv) Lamin B1 (Santa Cruz, CA, USA), 1:1000; (v)

GRP 78 (Santa Cruz, CA, USA), 1:1000; (vi) ERp57 (Milli-

pore), 1:1000; (viii) major vault protein 37 (MVP-37)

(generous gift of George L. Scheffer, VU Medical Center,

Amsterdam, the Netherlands), 1:1000 and (ix) glycer-

aldehyde phosphate dehydrogenase (Gapdh), loading

control (Millipore), 1:5000. After the incubation period, the

membranes were washed three times (10 min each wash

with TBST) and incubated in the appropriate secondary

antibody prepared in TBST containing BSA 1% w/v for 1 h

at room temperature. The secondary antibody used was

conjugated to HRP (Sigma) and was used at a concentration

of 1:10 000. The blots were developed using the ECL kit

(Amersham, Buckinghamshire, UK). Exposed films were

scanned with Fluor-STM Multi-Imager (Bio-Rad Labora-

tories), and band of interest were quantified using Gene-

Tools version 3.06.04 from SynGene (Cambridge, UK).

3 Results

3.1 Proteome of human endometrium

To establish the proteome of the human endometrium,

proteins were resolved on 2-D PAGE and protein spots were

identified using MALDI MS and/or MS/MS. The total

proteins of human endometrium tissue following 2-D

PAGE was resolved into a number of proteins in the

molecular weight range of 10–110 kDa and pI 4–7 (Fig. 1). A

total of 215 consistently appearing protein spots were

excised from the CBB stained 2-D gels of proliferative (n 5 3;

in duplicates) and secretory (n 5 3; in duplicates) phase

endometrium. Out of 215 spots, 194 protein spots were

unambiguously identified by MS and/or MS/MS

(Summarized in Table 1 and Supporting Information

Tables 1–3) and categorized based on their functional

properties as shown in Fig 2. The most abundant group

corresponds to proteins that are structural in function

(39%). The molecular chaperones constituted 20% of the

proteins and the other important categories of proteins

identified are those involved in signal transduction (10%),

metabolism (9%), immunity (7%), protein biosynthesis,

RNA biogenesis and nuclear proteins. In addition there are

proteins which are of diverse function and are categorized as

others.

50 P. Rai et al. Proteomics Clin. Appl. 2010, 4, 48–59


3.2 Differential protein expression of endometrium

tissue from secretory and proliferative phase

To identify proteins associated with proliferative and secre-

tory phase eutopic endometrium, the 2-D proteome of

endometrium tissue from proliferative (days 7–10; n 5 6)

and secretory (days 20–24; n 5 6) phase in the molecular

weight range of 10–110 kDa and pI range of 4–7 were

compared and analyzed using PDQuest software. The

reproducibility of 2-D gels was analyzed by the scatter plots

generated in the same software. The correlation coefficient

of 40.8 in secretory phase group gels and 40.8 in prolif-

erative phase group gels indicated good reproducibility of

the gels in each group. These gels were then used to

synthesize a master gel. The synthesized master gel

contained 215 discovered spots and was chosen for

comparison with the proteomes of the proliferative and

secretory phase endometrium. Those protein spots that

showed significant changes of more than 71.2-folds

(po0.05) across all individuals in the group were considered

as differentially expressed. Fifty-seven protein spots were

identified as differentially expressed on comparison of

secretory and proliferative phase eutopic endometrium.

Among these a total of 17 protein spots showed a significant

increase, whereas 38 protein spots showed a significant

decrease in expression in secretory phase and two protein

spots were unique to proliferative phase. Out of a total of 57

differential protein spots, 49 protein spots were successfully

identified by MALDI MS and/or MS/MS (Table 1 and

Supporting Information Table 1). Table 1 gives the identity

of the differentially expressed proteins and characteristics

such as its pI, mass, peptides matched, sequence coverage

and the fold difference compared to proliferative phase

endometrium.

3.3 Validations of differential proteins by

immunoblotting

To validate the differential protein expression in secretory

and proliferative phase endometrium tissue identified by

PDQuest analysis, the proteins were separated by 1-D SDS-

PAGE and immunoblotting was performed with the

respective antibody using a total of 12 samples representing

six from each phase of the menstrual cycle. Figure 3 shows

immunoblot and quantitation results of GRP 96, GRP 78,

HSP27, ERp57, vinculin, major vault protein and lamin B1

of three individual samples from each phase. The results

from the remaining six samples were also identical (data not

shown). Gapdh was used as internal loading control. The

band intensities were normalized with internal control and

were compared as secretory versus proliferative. The level of

significance for the differences in expression was deter-

mined by the Student’s t-test (po0.05). The results of

HSP27 and vinculin revealed that there was about 1.5-fold

and 1.4-fold upregulation respectively in the secretory phase

endometrium compared to the proliferative phase, while

GRP 94, GRP 78, ERp57 and lamin B1 were about 1.4- to

1.7-fold downregulated in the secretory phase. These results

were consistent with the results of the seven differentially

expressed proteins in 2-DE experiments.

4 Discussion

Human endometrium undergoes remarkable histological

and structural changes throughout the menstrual cycle, in

preparation for embryonic implantation and subsequent

shedding and regeneration in non-conception cycles [1–3].

Disturbance to this normal endometrial remodeling process

could lead to a diseased condition such as endometriosis

[4–10], endometrial polyps, hyperplasia and endometrial

cancer [11–14]. Therefore, there is a need to establish the

proteome of human endometrium as well to study the

differential protein expression between proliferative and

secretory phase of the cycle.

In the present study, 194 proteins in the proteome of

human endometrium were identified out of which 57

proteins were differentially expressed when the proteomes

of the endometrium in the proliferative and secretory phase

were compared (Fig. 1; Table 1 and Supporting Information

Tables 1–3). Among these differentially expressed proteins,

17 proteins showed a significant increase while 38 proteins

showed a decrease in expression in secretory phase of the

cycle. The change in the expression levels of five repre-

Figure 1. 2-DE map of Human endometrial tissue proteins. The

first dimension was performed by IEF on pH 4–7 IPG strips, the

second dimension on 10% SDS-PAGE gels and the proteins were

visualized by CBB G250. The indicated spots (1–194) were

excised from the gel and identified by MALDI MS and/or MSMS

as listed in Table 1.



Tab

le1.

Lis

to

fd

iffe

ren

tiall

yexp

ress

ed

pro

tein

s(s

ecr

eto

ryvers

us

pro

life

rati

ve

ph

ase

)in

hu

man

en

do

metr

ium

iden

tifi

ed

by

MA

LD

I

S.

No

.S

po

tn

o.a

)P

rote

inn

am

eA

ccess

ion

no

.b)

pI/

mass

ID meth

od

MO

WS

Esc

ore

c) /s

eq

cov

(%)

Pep

tid

em

atc

hed

Fo

ldd

iffe

ren

ced

)

Str

uctu

ral

pro

tein

s

12

a2

typ

eV

Ico

llag

en

iso

form

2C

2p

recu

rso

rg

i|115527062

5.8

5/1

09709

MS

127/1

922

1.8

25

Co

llag

en

,ty

pe

VI,a

1p

recu

rso

rg

i|87196339

5.2

6/1

09602

MS

/MS

187/3

025

�1.7

39

Vin

culi

nis

ofo

rmm

eta

-VC

Lg

i|7669550

5.5

0/1

24292

MS

100/1

622

1.9

421

Gels

olin

iso

form

bg

i|38044288

5.5

8/8

0876

MS

/MS

209/3

225

1.5

584

Kera

tin

8,

iso

form

CR

A_a

gi|119617057

5.4

1/5

7829

MS

/MS

173/4

322

2.0

6110

VIM

gi|47115317

5.0

9/5

3604

MS

/MS

172/5

120

�1.7

7118

Dyn

act

insu

bu

nit

2g

i|22096346

5.1

0/4

4318

MS

/MS

183/5

019

�2.0

8121

Vim

en

tin

gi|5030431

4.9

4/4

7062

MS

/MS

137/4

517

�2.2

9122

Vim

en

tin

gi|62414289

5.0

6/5

3676

MS

/MS

295/6

831

�2.0

10

131

Act

in,b

gi|14250401

5.2

3/4

0116

MS

/MS

161/4

817

�2.1

11

162

Tro

po

myo

sin

3is

ofo

rm2

iso

form

10

gi|73961081

4.7

7/2

7386

MS

84/4

812

2.5

12

184

Gam

ma-a

ctin

gi|178045

5.2

0/2

8478

MS

/MS

85/4

010

2.5

13

187

F-a

ctin

cap

pin

gp

rote

inb

sub

un

itg

i|4826659

5.6

9/3

0952

MS

71/3

410

1.8

Mo

lecu

lar

ch

ap

ero

nes

14

17

Heat

sho

ckp

rote

in90

kDab,

mem

ber

1g

i|4507677

4.7

6/9

2696

MS

/MS

203/2

928

�4.8

15

18

Tu

mo

rre

ject

ion

an

tig

en

(gp

96)

1g

i|61656607

4.7

7/9

2567

MS

/MS

241/3

332

�1.7

16

34

GR

P78

pre

curs

or

gi|386758

5.0

3/7

2185

MS

/MS

258/4

128

�2.0

17

42

Heat

sho

ck70

kDa

pro

tein

9g

i|12653415

5.9

7/7

3890

MS

/MS

186/3

924

�2.5

18

43

Heat

sho

ck70

kDa

pro

tein

9B

pre

curs

or

gi|62897075

5.9

7/7

3890

MS

/MS

238/4

027

�2.1

19

44

HS

P70-2

gi|4529892

5.4

8/7

0267

MS

/MS

227/4

426

1.2

20

46

Heat

sho

ck70

kDa

pro

tein

8is

ofo

rm1

gi|5729877

5.3

7/7

1082

MS

/MS

236/4

429

�1.8

21

58

Mit

och

on

dri

al

heat

sho

ck60

kDa

pro

tein

1g

i|189502784

5.8

3/6

0813

MS

/MS

190/4

125

�1.9

22

64

Mit

och

on

dri

al

heat

sho

ck60

kDa

pro

tein

1g

i|189502784

5.8

3/6

0813

MS

/MS

202/4

925

�4.3

23

69

Tco

mp

lex

pro

tein

1su

bu

nit

ep

silo

ng

i|194373691

5.3

7/5

1968

MS

/MS

103/3

315

�2.1

24

72

Ch

ap

ero

nin

con

tain

ing

TC

P1,

sub

un

it8

(th

eta

)g

i|62896539

5.4

2/6

0183

MS

/MS

119/2

817

�2.4

25

82

Pro

tein

dis

ulfi

de

iso

mera

sefa

mily

A,

mem

ber

3g

i|119597640

6.7

8/5

4468

MS

/MS

244/4

825

�1.5

26

116

Pro

tein

dis

ulfi

de

iso

mera

se-r

ela

ted

pro

tein

5g

i|1710248

4.9

5/4

6512

MS

/MS

157/5

017

1.2

27

181

Heat

sho

ckp

rote

inb-

1g

i|4504517

5.9

8/2

2826

MS

/MS

119/5

512

3.3

28

182

Heat

sho

ckp

rote

inb-

1g

i|4504517

5.9

8/2

2826

MS

/MS

134/5

413

1.5

29

192

Heat

sho

ckp

rote

inb-

1g

i|4504517

5.9

8/2

2826

MS

/MS

134/4

913

1.7

Imm

un

ity-r

ela

ted

pro

tein

s

30

15

Fib

rin

og

en

gam

ma

gi|223170

5.5

4/4

6823

MS

/MS

225/5

321

�2.5

31

16

Fib

rin

og

en

gam

ma

chain

,is

ofo

rmC

RA

_og

i|119625326

5.5

4/4

7971

MS

/MS

205/6

020

�2.5

32

35

a-1-B

-gly

cop

rote

ing

i|69990

5.6

5/5

2479

MS

/MS

141/3

617

�3.2

33

161

AN

XA

5g

i|49168528

4.9

4/3

5840

MS

/MS

273/7

124

1.7

34

169

AN

XA

5g

i|49168528

4.9

4/3

5840

MS

/MS

203/5

220

�1.7

Meta

bo

lic

pro

tein

s

35

36

NA

DH

deh

yd

rog

en

ase

(ub

iqu

ino

ne)

Fe-S

pro

tein

1,

75

kDa

gi|21411235

5.8

/80415

MS

/MS

201/3

625

�2.3

36

90

Mit

och

on

dri

al

Ald

eh

yd

eD

eh

yd

rog

en

ase

gi|6137677

5.7

/54394

MS

/MS

157/3

719

�1.8



Tab

le1.

Co

nti

nu

ed

S.

No

.S

po

tn

o.a

)P

rote

inn

am

eA

ccess

ion

no

.b)

pI/

mass

ID meth

od

MO

WS

Esc

ore

c) /s

eq

cov

(%)

Pep

tid

em

atc

hed

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sentative downregulated proteins, GRP 78, GRP 94, ERp57,

lamin B1 and MVP and two representative upregulated

proteins namely, HSP27 and vinculin were further

confirmed using immunoblot analysis (Fig. 3), which

showed similar results as obtained by using PDQuest soft-

ware. In the two earlier studies on the differentially

expressed proteins in secretory versus proliferative phase

endometrium, one group had identified 119 proteins by

ICAT and online MS/MS. Out of 119 proteins, only five of

the proteins showed consistent differential expression [22].

The other group had identified 196 differentially expressed

proteins but only 76 could be identified using DIGE and

MS. They found 39 proteins were downregulated and 157

were upregulated in secretory phase [23]. Among the 49

differential protein spots identified in this study, 20 proteins

were consistent with the earlier two reports, thus implying

the identification of additional differentially expressed

proteins in the proliferative and secretory phase of the

human endometrium. The reasons for the observed discre-

pancy in the results between the earlier and the present

study could be possibly because of the different techniques

used such as ICAT and DIGE used by the earlier workers

and may also be due to the experimental conditions such as

the gel size, the use of gradient gel and the tissue used in

terms of days of the menstrual cycle. In addition, in our

study it was interesting to observe that more proteins are

downregulated than upregulated in the secretory phase.

Similar observation have been noted previously by others

using DNA microarray [21, 27]. Downregulation of proteins

in the secretory phase could be because these may be

estradiol dependent proteins and require estradiol action

which in the secretory phase is low [3]. Therefore proteins

that were upregulated by estradiol (E2) in the proliferative

Figure 3. Immunobloting

analysis of proliferative (Pro)

and secretory (Sec) phase

endometrium tissue protein

lysates. Panels A-G shows

representative immunoblots of

GRP 78, GRP 94, lamin B1,

ERp57, HSP27, vinculin and

MVP, respectively. Panels A0 to

G0 shows the graphical repre-

sentation of the immunoblots

A–F, which was obtained by

densitometry analysis. The

semiquantitative data of A to G

immunoblots was normalized

by relative intensity of Gapdh.�Asterisk indicates significant

difference (po0.05, as deter-

mined by Student’s t-test)

between the two phases.

Experiments were performed

with endometrial tissues from

12 different individuals repre-

senting six each in the prolif-

erative and secretory phase,

respectively.

Figure 2. Functional distribution of the proteins isolated from the

2-DE gels and identified by MS and/or MS/MS in human endo-

metrial tissue proteins.



phase are now downregulated due to the loss of E2 action

[28, 29]. In addition, direct downregulation by progesterone

and multiple progestomedins during the implantation

window may result in more downregulated proteins

compared with upregulated proteins. The proteins identified

could be categorized into eleven groups based on their

functions and are briefly discussed below.

4.1 Structural proteins

This is the most abundant group in human endometrium

proteome and this is not surprising since the endometrium is

a heterogeneous tissue which comprises not only epithelial

cells, but also supportive stroma cells, blood and vessels which

all contribute a heterogeneous array of structural proteins.

Dynactin subunit 2 is one of the structural proteins that is

upregulated in the proliferative phase of the menstrual cycle,

which is characterized by rapid cellular proliferation. It is

known that dynactin is involved in cell proliferation [30, 31].

Two proteins are upregulated during secretory phase of the

menstrual cycle and it includes vinculin and F-actin capping

protein b–subunit, whereas two other proteins vimentin and

actin are downregulated. It is of interest to note that the

upregulated proteins, vinculin and F-actin capping protein

b-subunit are known to be involved in cell adhesion, in

maintaining cell morphology, cell proliferation and regulate

cell migration [32–36] implying that these processes have a

role in proliferative-to-secretory phase transition in endome-

trium. It is also not surprising that vimentin and actin are

downregulated in secretory phase of endometrium when the

endometrium undergoes structural and functional differ-

entiation and reorganization to provide a suitable environ-

ment for embryo implantation.

4.2 Molecular chaperones

The next most abundant group of proteins in the human

endometrium are the molecular chaperones which includes

several members of the HSP90, HSP70, HSP60 and HSP27

including the HSP homologous GRPs. These findings show

that human endometrium has a full complement of HSPs.

These proteins are estrogen-regulated and are known to be

involved in the correct folding and processing of proteins

[37] and in the signal transduction of various steroid

receptors, including the estradiol receptor and growth factor

receptors [38] and other tyrosine kinases [39]. Estrogen has

been shown to regulate uterine mRNA levels of HSP90 [28,

40, 41], GRP 94 [41], HSP70 [41, 42] and therefore it is not

surprising that twelve proteins of this category were upre-

gulated in proliferative phase (which includes GRP 96, GRP

78, mitochondrial heat shock 60 kD protein 1, isoforms of

HSP70, T-complex protein 1 subunit epsilon, and protein

disulfide isomerase family A, member 3). The role of HSPs

as molecular chaperones and their interaction with steroid

receptors and contribution to proliferation and anti-apopto-

sis [43] supports our findings. Isoforms of heat shock

protein b-1 (HSP27) that are known to be involved in stress

resistance and actin organization [44] and protein disulfide

isomerase related protein 5 that catalyzes the rearrangement

of -S-S- bonds during protein folding [45] were upregulated

in secretory phase. Protein disulfide isomerase mRNAs are

expressed in the endometrium and may play a role in acti-

vating immunoglobulin binding factor [46].

4.3 Immunity related proteins

Several proteins which are related to immunity have been

identified. This group includes isoforms of MHC class I

antigen, HLA-A a1 and a2 domains, fibrinogen g and

isoforms of annexin V. One of the isoform of annexin V

(spot no. 161) was found to be upregulated in secretory

phase. This protein has been implicated in ion-channel

regulation [47, 48] and as an inhibitor of protein kinase C

[49, 50], phospholipase A [51] and blood coagulation [52].

Interestingly, we observed that the other isoform of

annexin V (spot no. 169) was downregulated in secretory

phase. This result suggests that from proliferative to secre-

tory phase transition, annexin V might be undergoing post

translational modifications [53]. However, at this stage we

could not explain the precise basis of these isoformic

variations in expression. The proteins which are down-

regulated in secretory phase include isoforms of fibrinogen

g. These proteins play an important role in blood coagula-

tion. In addition, various cleavage products of fibrinogen

and fibrin regulate cell adhesion and spreading, and display

vasoconstrictor and chemotactic activities [54, 55]. The

upregulation of annexin V and downregulation of fibrino-

gen g during secretory phase underscores the importance of

maintaining an environment for anticoagulation during

implantation.

4.4 Metabolic Proteins

Majority of the proteins identified in this group are involved in

energy production. Metabolic proteins like NADH dehy-

drogenase (ubiquinone) Fe-S protein 1 and mitochondrial

aldehyde dehydrogenase were found to be downregulated in

secretory phase. Apart from their likely role in glucose meta-

bolism (via oxidative phosphorylation and glycolysis), they are

also involved in oxygen sensing, apoptosis, cell cycle regula-

tion, and immune recognition and response [56]. However,

the exact function with respect to endometrium is not clear.

4.5 Signal transduction

Proteins which are involved in cell cycle control, cell

proliferation and anti apoptosis are downregulated in



secretory phase endometrium and this group includes 14-3-

3 protein subunit epsilon, MVP and RAB11B [57–63]. In

addition Rab family proteins, which play an essential role in

membrane transport, mitosis and cytokinesis [64, 65] are

found to be differentially expressed in secretory and prolif-

erative phase endometrium. For instance Rab11, which is

downregulated in secretory phase, has been implicated in

cytokinesis [64] where as Rab6A, which is upregulated in

secretory phase is required for the metaphase/anaphase

transition [65]. Annexin IV, which promotes membrane

fusion, involved in exocytosis, anti-apoptosis and signal

transduction [66] is found to be upregulated in secretory

phase.

4.6 RNA biogenesis, protein biosynthesis and

nuclear proteins

Proteins involved in RNA biogenesis such as heterogeneous

nuclear ribonucleoprotein C, and B23 nucleophosmin are

found to be downregulated in secretory phase of endome-

trium. These heterogeneous nuclear ribonucleoprotein

family members are regulated by estrogen and may bind to

other nuclear receptors [67–69] and have been linked to a

variety of cellular processes, including mRNA translation,

transcription, RNA processing, RNA shuttling and stabili-

zation, chromatin remodeling, cell survival, cell prolifera-

tion, differentiation, apoptosis, and cell cycle regulation [70,

71]. The B23 nucleophosmin is another protein which is

found to be downregulated in secretory phase endome-

trium. B23 nucleophosmin has several potentially important

roles in regulating cell function and signaling [72, 73]. The

expression level of B23 nucleophosmin is shown to be

induced by estrogen in vascular smooth muscle cells [74]

and MCF-7 breast cancer cells [75]. FP1047, which is a

translation elongation factor and which belongs to the EF-1-

b/EF-1-g family, is found to be downregulated in secretory

phase. Lamin B1 and B2, which play a role in nuclear

architecture, DNA replication and gene expression are

essential for cell proliferation [76–78] are found to be

downregulated in secretory phase.

4.7 Conclusions

In the present study, we have reported the proteomic identi-

fication of 194 proteins present in human endometrial

tissue samples. We have applied proteomic techniques to

study protein expression profiling between secretory

and proliferative phase endometrium. A comparative analysis

of the human endometrium tissue proteome by 2-D in

combination with MS and/or MS/MS enabled us to identify

49 proteins that showed significant changes in expression

levels. Furthermore, we have confirmed the downregulation of

GRP 78, GRP 94, ERp57, lamin B1 and MVP and upregula-

tion of HSP27 and Vinculin in secretory phase human

endometrium by immunoblot. In this study we have identified

additional 137 protein spots in 2-D of human endometrium

that had previously escaped identification. This study estab-

lishes the 2-D proteome of human endometrium represented

by 194 identified protein spots. However, more proteins could

be present in this kind of tissue, which could not be identified

due to problems related to acquiring sufficient amount of

tissue. Further, since the tissue is limited in quantity it is a

further hindrance to running bigger length gels. One could

pool matched tissues and run bigger gels but one knows that

this may not be the best approach since matched tissue do

exhibit variations between individual patients. Therefore, it

was decided to use small gels. The reported proteomic iden-

tification of proteins in the present work now provides the

basis for subsequent studies on the physiological and patho-

logical aspects of endometrium in aberrant condition such as

endometriosis. Following this study, we are currently investi-

gating both eutopic endometrial proteins in women with and

without endometriosis, during both the proliferative and

secretory phases of the menstrual cycle.

Priyanka Rai and Venkatesh Kota are the recipients of CSIRfellowship from Government of India. We thank Dr. ArchanaB. Siva, Dr. Satish S. Bhande and Y. Kameshwari for theirvaluable suggestions and technical help. We sincerely thankDr. George L. Scheffer for the anti-MVP-37 antibody.

The authors have declared no conflict of interest.

Clinical Relevance

Endometrium, a heterogeneous tissue lining the uterus, is

highly dynamic and exhibits marked cyclical changes

during the menstrual cycle. Any abrogation in endometrial

physiology would lead to diseases like endometriosis,

endometrial polyps, hyperplasia and endometrial cancer.

Therefore, there is a need to understand the molecular

basis of function of the endometrium per se. In the present

study, we have established the proteome of human

endometrium by 2-DE and MALDI, which comprises of

194 identified protein spots. A comparative analysis of

proliferative and secretory phase human endometrium

was also carried out, which enabled us to identify 49

out of 57 differentially expressed protein spots. This study

has led to the identification of proteins which are

characteristic of the two phases of the menstrual cycle

and instigates further investigations into global changes in

protein expression in disorders of the endometrium.

Finally, this study may also set the stage to develop a

screen for candidate proteins in patients with infertility

and for targeted drug discovery for enhancing (or

inhibiting) implantation for infertility treatment (or

contraception).



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