Variability of mRNA abundance of leukemia inhibitory factorgene (LIF) in porcine ovary, oviduct and uterus tissues

Katarzyna Ropka-Molik • Maria Oczkowicz •

Aurelia Mucha • Katarzyna Piorkowska •

Agata Piestrzynska-Kajtoch

Received: 4 December 2011 / Accepted: 16 April 2012 / Published online: 28 April 2012

� Springer Science+Business Media B.V. 2012

Abstract The leukemia inhibitory factor (LIF) gene

encodes a pleiotropic cytokine which is produced by the

endometrium and plays an important role in implantation

and early embryonic development. Because of its function,

LIF gene is considered as a candidate gene for litter size in

many mammalian species including pig. The aim of pres-

ent study was to evaluate the expression of LIF gene in the

porcine ovary, oviduct and two regions of uterus (corpus

uteri, cornu uteri) in prepubertal and pubertal gilts. In order

to precise estimation of LIF transcript abundance we

evaluated the stability of expression for several candidate

housekeeping genes in investigated tissues across different

breeds and different stage of oestrus cycle. The geNorm

analysis indicated that the most stable reference genes

across analyzed tissues were: OAZ1 and RPL27. The

analysis conducted separately for each tissue confirmed

that the most stable gene was OAZ1 in all tissues expect

oviduct (the most stable was RPL27 gene). In prepubertal

pigs, the highest level of the LIF expression was obtained

in both regions of uterus compare to ovary and oviduct

tissues (P \ 0.01). A similar trend in LIF expression pat-

tern was observed in follicular phase—the significantly

highest transcript level was obtained in cornu uteri, it was

about ninefold higher than in ovary (P \ 0.05). In luteal

stage the highest expression was in corpus uteri. In pig, the

high expression in luteal phases suggested that, LIF may be

mainly secreted in respond to the increased of progesterone

concentration and it can be connected with the preparation

of the uterus for implantation.

Keywords Ovary � Oviduct � Uterus � Pig � LIF �Housekeeping genes

Introduction

The real-time PCR is one of the most accurate, reproduc-

ible and fast quantification methods for gene expression

measurements. The results obtained by quantitative real-

time PCR may be affected by various factors of which the

most significant is selection of the appropriate house-

keeping gene (HK). HK genes are constitutively expressed

in all cells of an organism and the proteins which they

code, are generally involved in the basic functions neces-

sary for the sustenance or maintenance of the cell. The

genes used as endogenous controls should have a constant

level of expression in a given tissue type, regardless of

experimental conditions [1]. Furthermore, well-chosen

reference gene should have similarly transcript abundance

compare to target gene [2]. However, the mRNA abun-

dance of HK genes may vary between tissues or cells and

may change under certain conditions. Thus, the selection of

appropriate internal control gene is critical for gene

expression studies, especially for reliable interpretation of

results.

The leukemia inhibitory factor (LIF) gene encodes a

pleiotropic cytokine which is produced by the

K. Ropka-Molik (&) � M. Oczkowicz � K. Piorkowska

Laboratory of Genomics, National Research Institute of Animal

Production, Krakowska 1, 32-083 Balice, Poland

e-mail: kropka@izoo.krakow.pl

A. Mucha

Department of Animal Genetics and Breeding, National

Research Institute of Animal Production, Krakowska 1, 32-083

Balice, Poland

A. Piestrzynska-Kajtoch

Department of Cytogenetics and Molecular Genetics of Animals,

National Research Institute of Animal Production, Krakowska 1,

32-083 Balice, Poland

123

Mol Biol Rep (2012) 39:7965–7972

DOI 10.1007/s11033-012-1642-8

endometrium and plays an important role in implantation

and early embryonic development in mice [3] and human

[4]. LIF protein acts through the LIFRb/gp130 receptor and

it is an important regulator of mouse embryonic stem cell

self-renewal (STAT3 pathways), cell differentiation—

MAPK pathways, and cell survival—PI3 K pathways [5].

Furthermore, LIFRb receptors and gp130 proteins were

found in mouse [6] and human [7] blastocystes before

implantation, which suggested that LIF regulates preim-

plantation embryo development and endometrial

sensitivity.

In pig, LIF gene is considered as a candidate gene for

litter size. Spotter et al. [8] showed that polymorphism in

third exon of porcine LIF gene is significantly associated

with the number of piglets born alive. In large white pigs,

Lin et al. [9] indicated that LIF influenced a total number

born and number born alive of piglets. In mice and human,

there are a lot of researches about expression profile and

protein level of LIF and their association with fertility.

However, only a few studies investigated the exact amount

of LIF mRNA in pig tissues in different stage of the oestrus

cycle. The precise analysis of expression patterns of leu-

kemia inhibitory factor in porcine reproductive tissues

might clarify the exact role of this protein in embryo

development and implantation in this species.

The aim of present study was to evaluate the expression of

leukemia inhibitory factor gene (LIF) in the porcine ovary,

oviduct and two regions of uterus (corpus uteri, cornu uteri)

in prepubertal and pubertal gilts. In order to precise estima-

tion of LIF transcript abundance in four investigated tissues,

we evaluated the stability of expression of eight candidate

HK genes across different breeds and different stage of

oestrus cycle. The two most stable genes were used as a

endogenous controls in quantitative real-time PCR.

Materials and methods

Animals

In present study we investigated two breeds of pig: polish

landrace (PL) and polish large white (PLW), which are

included in the national breeding program as a dam line.

Animals were maintained in the pig testing Stations of the

National Research Institute of Animal Production in Chor-

zelow and Pawłowice under the same housing and feeding

conditions. All gilts (with an average weight of 100 kg) were

fasted 48 h before the slaughter. Furthermore, pigs repre-

sented two different physiological stage of the oestrus cycle:

luteal or follicular phases or were a prepubertal gilts (divi-

sion resulting from the macroscopic analysis of the ovary).

Immediately after slaughter all tissues (ovary, oviduct

and two regions of uterus: corpus uteri, cornu uteri) were

collected in tubes with RNAlater solution (Ambion Inc.,

Austin, USA) and stored at -20 �C.

RNA isolation and reverse transcription

The total RNA from ovary and oviduct was isolated using

TRI-reagent (Sigma-Aldrich, Poznan, Poland) according to

the method described by Chomczynski [10]. Total RNA

form corpus uteri and cornu uteri was isolated with the use of

PARIS Kit—protein and RNA isolation system (Ambion

Inc., Austin, USA), according to the attached protocol. The

quality and quantity of extracted RNA was estimated using

NanoDrop 2000 (Thermo Scientific, Wilmington, USA), and

2 % agarose electrophoresis. One lg of total RNA was

reverse transcribed into cDNA using high capacity RNA-to-

cDNA Master Mix (Applied Biosystems, Warsaw, Poland).

The DNA contamination in RNA samples was checked by

real-time PCR with ‘‘No-RT’’ control.

Validation of HKGs using quantitative real-time PCR

The normalization of candidate gene stability was carried

out on 16 samples from each tissues (in total 64 samples).

To estimate the impact of breed on gene stability each

tissue was represented by 8 samples from PL and PLW.

We tested eight candidate HKGs previously recommended

as a endogenous controls for other tissues: ACTB—b-actin,

OAZ1—ornithine decarboxylase antizyme 1, RPL27—60S

ribosomal protein L27, RPS29—40S ribosomal protein S29,

RPS20—40S ribosomal protein S20, RPS13—40S ribosomal

protein S13, GAPDH—glyceraldehyde-3-phosphate dehy-

drogenase, HPRT—hypoxantine phsophoribosyltransferase.

Furthermore, to evaluate that gene studied were not affected

across the different stage of the oestrus cycle normalization

was performed on tissues derived from pigs in luteal (n = 8)

or follicular (n = 8) phases.

Relative quantification of the mRNA abundance of the

genes studied was performed with 7,500 Real-Time PCR

system using TaqMan� MGB probes labeled with FAM,

VIC or NED. Reaction for each samples was carried out in

three repeats in total volume 25 lL and according to the

TaqMan Universal PCR Master Mix protocol: 2 initial steps

at 50 �C for 2 min (UNG incubation) and 95 �C for 10 min

(AmpliTaq Gold activation), and 40 cycles of 95 �C for 15 s

(denaturation) and 1 min at 60 �C (annealing/extension).

Primers and probes for OAZ1, RPL27, RPS29, RPS13,

RPS20 were designed using Primer Express 3.0 (Applied

Biosystems) (Table 1). Primers and probes for ACTB and

HPRT were purchase as assays from Applied Biosystems

and for GAPDH as described by Wang et al. [11]. Quan-

titative real-time PCR for presented genes was performed

in multiplex: ACTB and OAZ1; RPS29 and RPL27; HPRT

and GAPDH.

7966 Mol Biol Rep (2012) 39:7965–7972

123

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Mol Biol Rep (2012) 39:7965–7972 7967

123

The results were analyzed with the use of Sequence

Detection System 7500 software v. 2.0.1 (Applied Bio-

systems). The efficiency of real-time PCR reactions was

defined by using the standard curve method. The relative

transcript abundance was calculated as 1/E(Ct) [E = effi-

ciency (10[-1/slope]), Ct = cycle determined by the thresh-

old applied to the maximum amplification of the standard

curve]. In order to identification of the most stable refer-

ence genes and normalization factor (NF) we used the

geNorm software [12].

Quantification of LIF mRNA abundance

The estimation of LIF expression was performed on four

studied tissues derived from animals in luteal or follicular

stage of the oestrus cycle and in prepubertal gilts (number

of samples was shown in Fig. 1). Relative quantification of

the transcript level of the LIF (leukemia inhibitory factor)

gene was evaluate with the use of two most stable HKGs

(OAZ1, RPL27). Primers and probes for porcine LIF gene

were designed and synthesized by Applied Biosystems.

Reactions (in a total volume 25 lL) were carried out in

three repeats and in multiplex with two endogenous con-

trols. The NF was calculated based on the geometric mean

of the normalized quantity of the two endogenous genes.

Relative quantity of LIF mRNA was calculated according

to Pfaffl [13].

The Shapiro–Wilk test and the Kolmogorov–Smirnov

test were performed to test if variables examined are nor-

mally distributed. The Levene’s test was used to assess the

equality of variances. Statistical analysis was performed

using the ANOVA procedure (SAS Institute, Cary, NC, v.

8.02, 2001)1. All results were shown as a means ± SEM.

Results and discussion

In the present study, we evaluated the stability of expres-

sion of eight HK genes in porcine ovary, oviduct and two

regions of uterus. In order to find the most stable reference

gene across four tissues and different phase of oestrus cycle

we used geNorm software, which calculates average gene

expression stability (M) and the average pair-wise variation

(V). The genes with M below 1.5 are considered to have

more stable expression levels and are the most suitable to

use as reference control genes.

We indicated that the most stable reference genes across

analyzed tissues were: OAZ1 (M = 0.921), RPL27

(M = 0.970) and ACTB (M = 0.995). The highest M was

obtained for RPS13 (M = 1.858) and HPRT (M = 1.632)

genes (Fig. 2). The geNorm analysis conducted separately

for each tissue confirmed that the most stable gene was

OAZ1 in all tissues expect oviduct (the most stable was

RPL27 gene) and the least stable was RPS13 in ovary,

oviduct, corpus uteri and HPRT in cornu uteri (Table 2).

Furthermore, we confirmed that all analyzed genes had

expression at the same level across the PL and PLW breeds

and in different phase of oestrus cycle.

There are a lot of investigations conducted in porcine

reproductive tissues (ovary, oviduct, uterus) where GAP-

DH, ACTB or 18S rRNA genes are used as HK genes [14–

16]. On the other hand, a number of studies have provided

evidence that expression levels of these most commonly

used HK genes might vary under different experimental

conditions, between different tissues or developmental

stages [17, 18]. Jemiolo and Trappe [19] showed that in

human muscles transcript abundance of four HKGs

(GAPDH, ACTB, B2 M and 18S rRNA), differed signifi-

cantly before, during and after exercise. Additionally,

Rubie et al. [20] confirmed high variability of 21 endoge-

nous controls (particularly GAPDH) in cancers and normal

human tissues. Fu et al. [21] investigated the stability of 20

Fig. 1 Relative quantity of LIFgene in ovary, oviduct and

uterus (corpus uteri, cornuuteri) in prepubertal gilts and in

different phases of oestrus cycle

(n-number of samples in each

group; *P \ 0.05, **P \ 0.01)

1 SAS Enterprise Guide 4.1 (4.1.0.1000) Copyright � 2006 by SAS

Institute Inc.

7968 Mol Biol Rep (2012) 39:7965–7972

123

reference genes in human ovarian tissues under different

conditions. The authors indicated that genes belonging to

the family of ribosomal protein (60S)—RPL4 and RPL20

have the most stable expression and are recommended to

the normalization of quantitative real-time PCR in ovary.

In mice, Koudjo et al. [22] confirmed that GAPDH, and

beta-actin genes are expressed in all tissues with significant

difference in their expression levels. Furthermore, they

chose several HK genes i.a. eEF-2, RPL37 and RPL38

which were the most stable in panel of tissues including

ovary and uterus. Likewise, our analysis confirmed that in

porcine reproductive tissues the combination of the OAZ1

and RPL27 should be recommended as a much more

suitable compared to GAPDH gene. In recent studies

conducted by Kim et al. [23] RPL7 (ribosomal protein L7)

was used as the endogenous control to normalize the

activated leukocyte cell adhesion molecule (ALCAM)

transcript amount in the porcine endometrium.

One of the most economically important traits in pig

production is liter size. There are a lot of researches about

the impact of various candidate genes on porcine repro-

ductive traits. Some of genes, for example the estrogen

receptor (ESR) [24, 25], the follicle-stimulating hormone

beta (FSHb) [26], the prolactin receptor (PRLR) [27], the

retinol-binding protein 4 (RBP4) [28] and osteopontin

(OPN) gene [29] proved to be significantly associated with

such traits as number of piglets born, live born and still

born piglets.

The leukemia inhibitory factor (LIF) is also considered

as a candidate gene for liter size in many mammalian

species including human, mouse and pig. Steward et al. [4]

indicated that mice females lacking a functional LIF gene

are fertile, but their blastocysts fail to implant and do not

develop. On the other hand, LIF overexpression is lethal

and leads to the absence of differentiated mesoderm [30].

Moreover, the presence of LIFRb receptors in preimplan-

tation blastocysts and enhancement of their development

after addition of exogenous LIF in in vitro culture indicated

that this protein is critical in preimplantation development

[31].

In pig, there is the greatest embryos mortality during

implantation process (around 30 day of the gestation) and

thus this period is essential for liter size [32]. Previous

studies indicated that the expression of LIF gene is

observed in the uterus of mice, human, rabbit and cow

around implantation [33, 34]. In present study, concerning

the prepubertal pigs we obtained the highest level of the

LIF expression in both regions of uterus compare to ovary

and oviduct tissues (P \ 0.01). The lowest abundance of

LIF mRNA was observed in oviduct—approximately sev-

enfold lower than in cornu uteri. A similar trend in LIF

expression pattern was observed in follicular phase—the

Table 2 Analysis of gene expression stability measure M for genes analyzed conducted separately for each tissue (geNorm software)

Tissue\gene ACTB OAZ1 RPL27 RPS29 RPS20 RPS13 GAPDH HPRT

Uterus (cornu uteri) 0.769 0.756 0.833 0.791 1.496 1.018 1.018 1.666

Uterus (corpus uteri) 0.638 0.529 0.575 0.584 0.612 1.101 0.660 0.771

Oviduct 0.897 0.796 0.714 0.800 0.723 1.621 0.966 1.285

Ovary 0.883 0.820 0.861 0.882 0.829 2.338 1.540 1.968

In each tissues, the lowest M values were highlighted in green, the highest were highlighted in red

Fig. 2 Average expression

stability values of remaining

control genes (geNorm

software)

Mol Biol Rep (2012) 39:7965–7972 7969

123

highest transcript level was obtained in uterus (cornu

uteri), and it was about ninefold higher than in ovary

(P \ 0.05). In luteal stage, the highest expression was in

corpus uteri (Fig. 1). Yang et al. [34] showed that LIF gene

is highly expressed in the mouse uterus around the time of

ovulation. Another studies conducted in human tissues

confirmed that levels of LIF mRNA and protein increased

during ovulation and on day 4 of pregnancy [35, 36]. The

transcript of LIF gene is present in human uterus at the high

level in the luteal stage of the menstrual cycle [37], and in

infertile women degreasing of secretion of LIF protein in

this phase were observed, what confirms its importance in

implantation process. Chen et al. [37] showed presence of

leukemia inhibitor factor in human uterus in the follicular

and late-luteal phases, but at very low levels. Similarly, in

marmoset, LIF protein was detected in endometrium during

the early luteal phase, reached maximum level during the

mild-luteal phase and decreased at the end of luteal stage.

Furthermore, LIF transcript and protein were absent during

follicular phase [38].

In our research, the comparison of LIF transcript

abundance between different phases of oestrus cycle

showed that the highest expression was in luteal phase in

ovary, oviduct and corpus uteri (Fig. 3). In cornu uteri

expression of LIF gene was at a similar level in follicular

and luteal stage and lower in prepubertal pigs (Fig. 4). Our

results indicated that the highest level of LIF mRNA was in

porcine uterus in luteal stage and these results are consis-

tent with previous reports conducted in others mammalian

species. Opposite to results obtained in human, we indi-

cated presence of the expression of LIF gene in follicular

phase in uterus and other analyzed tissues. In pig, Anegon

et al. [39] showed that LIF protein is produced by endo-

metrium during pre-implantation period and the oestrous

cycle. According to the authors, LIF activity in porcine

uterus luminal fluid increased at day 7 and 13 of the oestrus

cycle, however, LIF transcript was detected in endome-

trium only at day 11 i.e. during luteal phase.

The hormonal regulation of leukemia inhibitory factor in

endometrium has not been exactly understood. In mice,

secretion of LIF in endometrium tissue is regulated by

estrogens. Opposite, in rabbits, LIF level is up-regulated by

progesterone without any dependence with maternal

estrogens concentration. On the other hand, in sheep, Vo-

giagis et al. [40] observed that expression of LIF transcript

was relatively constant throughout the oestrus cycle,

decreased during early pregnancy (days 12–14) and was the

highest on day 16–20 of pregnancy. The lack of precisely

association between peak of LIF expression and implan-

tation period, authors explained by probable regulation of

LIF by both estradiol and progesterone in ovine endome-

trium. In porcine endometrium, LIF mRNA level increased

between 10 and 15 days of pregnancy. The exact mecha-

nism of this up-regulation has not been completely

understood, but it may be caused by estrogens produced by

conceptus, similarly as ovarian estrogens influence in mice

[41].

In conclusion, our results confirmed that LIF gene,

which plays an important role in embryo implantation is

highly expressed in swine uterus compare to ovary and

oviduct. In analyzed tissues, transcript abundance of LIF

gene was the highest in luteal phase of oestrus cycle

compare to follicular phase or in prepubertal gilts. These

founding indicated that abundance of LIF transcript in

porcine endometrium is under maternal control, irrespec-

tively of signals from the conceptus. The high expression in

luteal phases of oestrus cycle (statistically significant in

oviduct and corpus uteri) suggested that LIF is mainly

secreted in respond to the increased of progesterone con-

centration. In this phase, the high concentration of LIF may

be connected with the preparation of the uterus for

implantation. On the other hand, we can exclude influence

of estrogens on the level of leukemia inhibitory factor in

analyzed tissues due to the observed expression in follic-

ular phase.

The results obtained confirmed that the LIF gene also

can be considered as a candidate gene for litter size in pig.

The accurate estimation of LIF expression level in different

phase of oestrus cycle or its comparison between primipara

and multiparous sows would be helpful in better

Fig. 4 The comparison of LIF expression levels between different

phases of oestrus cycle in uterus (corpus uteri, cornu uteri)(**P \ 0.01)

Fig. 3 The comparison of LIF expression levels between different

phases of oestrus cycle in ovary and oviduct tissues (**P \ 0.01)

7970 Mol Biol Rep (2012) 39:7965–7972

123

understanding of LIF protein function and should be further

investigated.

Acknowledgments The study was supported by the Polish Ministry

of Science and Higher Education (Project No. NN311220938).

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