REPBIO-81; No.

of Pages 8
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Available online at www.sciencedirect.com

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journal homepage: http://www.elsevier.com/locate/repbio

Original Research Article

The Bax/Bcl-2 apoptotic pathway is not responsible

for the increase in apoptosis in the RU486-treated
rat uterus during early pregnancy
Kathrine E. Theron a,*, Clement B. Penny b, Margot J. Hosie a
a

School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown
2193, South Africa
b
Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road,
Parktown 2193, South Africa

article info

abstract

Article history:

An increase in apoptotic activity has been observed in both the rabbit and the rat endometria

Received 23 November 2012

following treatment with RU486. The aim of this study was to assess whether Bax and Bcl-2

Accepted 23 September 2013

signaling, in response to RU486, could be crucial role players mediating apoptosis in the rat

Keywords:

antagonist, functioning to actively silence P4 receptor gene-associated transcription. Al-

uterus during early pregnancy. RU486 is a partial progesterone (P4) and estrogen receptor
Mifepristone

though an increase in apoptosis as a result of RU486 administration has been previously

RU486

reported in rabbits, the specic apoptotic factors and pathways involved in driving this

Apoptosis

process have not yet been established. Immunouorescent techniques were used to deter-

Bax

mine protein expression levels of both Bax and Bcl-2 in RU486-treated endometria at days

Bcl-2

4.5, 5.5 and 6.5 of pregnancy. The Bax/Bcl-2 index was used to determine the overall pro- or
anti-apoptotic setting at each day of pregnancy, following RU486 administration. Changes in
the Bax and Bcl-2 gene expression levels as a consequence of RU486 administration were
evaluated using RT-qPCR. Both the protein and gene expression analyses suggest that RU486
induces a change toward an overall anti-apoptotic signal within the Bax/Bcl-2 pathway.
These results suggest that the observed increase in apoptosis following RU486 administration is not driven by a shift in the Bax/Bcl-2 ratio toward cell death, when the P4 and estrogen
receptors are partially inactivated by RU486, but is possibly regulated by another apoptotic
pathway.
# 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and
Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban &
Partner Sp. z o.o. All rights reserved.

* Corresponding author. Present address: Department of Medical Sciences, Public Health and Health Promotion, School of Health Sciences,
University of Limpopo, Private Bag X1106, Sovenga 0727, South Africa. Tel.: +27 15 268 3362/4056; fax: +27 86 515 3340.
E-mail address: kathrine.theron@ul.ac.za (K.E. Theron).
1642-431X/$ see front matter # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of
Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
http://dx.doi.org/10.1016/j.repbio.2013.09.002
Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

1.

Introduction

RU486 or Mifepristone (marketed as MifegyneTM), an 11bdimethyl-amino-phenyl derivative of norethindrone, is a

potent progesterone (P4) and glucocorticoid receptor antagonist [1]. A receptor complex activated by RU486 results in an
inhibitory function in the C-terminal region of the hormonebinding domain, rendering the DNA-bound receptor transcriptionally inactive [2,3]. With P4 not being able to bind to its
receptor and produce its effects on the endometrium, the strict
ovarian hormonal control of the priming of the endometrium
for blastocyst implantation is disrupted, resulting in a
disruption of the plasma membrane transformation of the
uterine epithelial cells during early pregnancy. Initially RU486
was used for medical termination of intrauterine pregnancies,
however, it has been shown to be highly effective as an
emergency contraceptive method [47].
An important function of P4 during early pregnancy is the
regulation of apoptosis. In general, P4 has been shown to
protect against apoptosis, whereas withdrawal of P4 causes
apoptosis to occur. The apoptotic cell death that occurs
throughout the endometrium has been shown to be suppressed by P4 in ovariectomized animals [8]. Apoptosis is
regulated by various proteins, including the Bcl-2 protein
family. This family of proteins consists of both pro-apoptotic
(e.g. Bax, Bak, Bad, Bag and Bcl-xs) and anti-apoptotic (e.g. Bcl2, Bcl-xL, Mcl-1) proteins [9,10]. The ratio of Bcl-2/Bax
heterodimers to Bax/Bax homodimers determines whether
or not a cell will undergo apoptosis, where excess Bax will
promote cell death. In both the human [11] and rat [12]
endometrium, the shift in the Bax/Bcl-2 ratio has been
attributed to the cyclic changes in endometrial growth and
regression observed in the menstrual and estrous cycles,
respectively.
Although an increase in apoptosis as a result of RU486
administration has been previously reported in the rabbit
endometrium [13] as well as the rat endometrium [14], the
specic apoptotic factors and pathways involved in driving
this process have not yet been established. As the antiapoptotic factor Bcl-2 and the pro-apoptotic factor Bax have
been reported to be expressed in the endometrium during the
menstrual [15,16] and estrous cycles [12], as well as in the
decidua of the rat uterus during the post-implantation stage of
early pregnancy, it was queried here whether Bax and Bcl-2, in
response to RU486 could be crucial role players mediating
apoptosis.

2.

Materials and methods

2.1.

Animals and treatment regime

Thirty-six adult, virgin female inbred Hooded Wistar rats (12

14 weeks of age and weighing
200 g) were used in this study.
Animals were maintained in temperature-controlled quarters
at 23 8C, with a 12-h lightdark cycle. Food and water were
freely available. The animals were divided into six groups and
the presence of a vaginal plug, as well as spermatozoa in the
vaginal smear taken the following morning, was used to

conrm successful mating. This was designated as day 0.5 of

pregnancy.
At day 3 of pregnancy, three groups of animals (n = 18) were
injected subcutaneously with 8 mg/kg (body weight) of RU486.
The three control groups (n = 18) were injected with vehicle
(ethanol/peanut oil 1:1) only. Uterine tissue was isolated from
euthanized animals 4.5, 5.5 and 6.5 days after mating. Fifteen
minutes prior to sacricing, the animals were anesthetized
with 0.3 ml of RompinKetamin (1:4) and a ventral midline
incision made. One ml of 1% (w/v) Pontamine Sky Blue dye
solution was injected into the inferior vena cava to aid in
visualizing tissue edema, an indicator of decidualization [17].
Uterine horns were removed and one was xed whole in 10%
(v/v) buffered formalin and then cut into implantation and
non-implantation sites. The other horn was placed into a 1%
(w/v) saline solution and the blastocysts were ushed out
using a syringe lled with 1% (w/v) saline solution. The uterine
horn was then cut into implantation and non-implantation
sites which were individually ash frozen in liquid nitrogen
and stored at 80 8C for further analysis. The implantation
sites were distinguished from the non-implantation sites by
prominent blue bands of the aforementioned Pontamine Sky
Blue dye solution. Prominent blue bands were, however, not
always evident in the 4.5 day pregnant rats. Areas in which the
blood vessels appeared aggregated were identied as implantation sites and areas in which little vessel aggregation was
visible were considered non-implantation sites. On average,
there were 56 implanted embryos per uterine horn in the
control pregnant groups and only 01 on average in the RU486treated groups, indicating that RU486 successfully inhibited
embryo implantation in the rat uterus.

2.2.

Immunouorescence

Parafn sections (5 mm) were deparafnized, rehydrated and

then immersed in Trisethylenediamine tetraacetic acid (EDTA;
Sigma, St Louis, MO, USA) buffer (pH 9.0) for retrieval of
antigenicity. Sections were heated in a microwave at medium
heat (720 W) for 10 min and allowed to cool in the buffer for
20 min. The sections were covered with 0.1% (v/v) Triton-X100
(Sigma) in 0.1% (w/v) BSA (Sigma)/PBS to permeabilize the cells.
Tissue sections were incubated overnight in a moist chamber at
4 8C with primary antibodies: rabbit anti-Bax or mouse anti-Bcl2 at 1:50 dilution (Santa Cruz Biotechnology, Dallas, TX, USA). On
the following day, the sections were incubated in a dark
chamber with goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 or donkey anti-mouse IgG secondary
antibody conjugated to Alexa Fluor 488 at 1:200 dilution
(Invitrogen, Grand Island, NY, USA) for 1 h at room temperature.
After each step the sections were rinsed with PBS (pH 7.6). For
negative controls, the primary antibody was omitted and
tissues were incubated with 0.1% (w/v) BSA/PBS. The labeled
tissues were viewed and photographed with an Olympus XM10
Camera mounted on an Olympus IX71 uorescence microscope
(Olympus, Center Valley, PA, USA) with appropriate lters.

2.2.1.

Image analysis

Quantication of IF sections was performed using a 40

objective lens. The image analysis software, analySIS Life
Science Research program FIVE Digital imaging Solutions

Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

(Olympus Soft Imaging Solutions; Olympus), was used to

analyze the intensity of the uorescence of the 40 micrographs. This was done by rst using the software to dene the
regions of interest (ROIs); namely, the epithelium, the
subepithelial stroma, the glands and the glandular stroma.
The mean intensity of each ROI was then analyzed using the
image analysis software.

2.2.2.

Statistical analysis

The values of the intensity of each ROI for Bax and Bcl-2
immunosignal are presented as mean
SEM. The O'Brien's
test was used to test whether the variances within each
treatment group for Bax and Bcl-2 were equal. If the variances
were found to be equal ( p < 0.05) then this test was followed by
a one-way analysis of variance. If the variances were not equal
( p > 0.05) then a Welch ANOVA was performed to test whether
there was a signicant difference between the means, whilst
allowing for the variances to be unequal (JMP statistics
program version 8; SAS Institution Inc., Cary, NC, USA). The
ANOVA and Welch ANOVA were followed by a TukeyKramer
post hoc analysis. Statistical analysis was performed using the
JMP statistics program (version 8: SAS Institution Inc., Cary,
NC, USA). Differences with p values less than 0.05 were
considered signicant.

2.2.3.

The Bax/Bcl-2 index

As different uorescent secondary antibodies were used for

localization of Bax and Bcl-2 here, reliable quantitative
analysis cannot be carried out due to the difference in the
uorescent intensities between the secondary antibodies.
Thus the Bax/Bcl-2 index reported within this section was
performed to show any trends in the change in the Bax/Bcl-2
ratio rather than to dene denite shifts in the Bax/Bcl-2 ratio
toward an anti- or pro-apoptotic environment. A decrease in
Bax levels and/or an increase in Bcl-2 levels results in a
decrease in the Bax/Bcl-2 index, suggesting a shift to a more
anti-apoptotic environment. Conversely, an increase in Bax
protein levels and/or a decrease in Bcl-2 protein levels results
in an increase in the Bax/Bcl-2 ratio, indicating a trend toward
a more pro-apoptotic milieu.

2.3.
Real-time quantitative reverse transcription
polymerase chain reaction
2.3.1.

RNA isolation and reverse transcription

Total RNA was isolated using the RNeasy Mini kit (Qiagen,
Valencia, CA, USA). The frozen tissues were homogenized and

lysed using a RLT buffer b-mercaptoethanol solution. The

purity and concentration of total RNA were determined by
NanoDrop 3300 uorospectrometer (Thermo Fisher Scientic,
Wilmington, DE, USA). Samples with A260/A280 ratio between
1.8 and 2.0 were used in the experiment. To determine the
integrity of the extracted RNA, the samples were electrophoresed to resolve the 18S and 28S RNA bands.
In all cases, 270 ng of RNA was treated with MultiScribe
Reverse Transcriptase (Applied Biosystems, Foster City, CA,
USA) and subjected to reverse transcription for 30 min at 48 8C
in 10 ml of reaction mixture containing RT-buffer (1 TaqMan
Buffer, 5.5 mM MgCl2; Applied Biosystems) 500 mM of each
dNTP, 2.5 mM of Oligo d(T)16, 0.4 U/ml of RNase Inhibitor and
1.25 U/ml of MultiScribe Reverse Transcriptase (Applied Biosystems). The reaction was terminated by heating for 5 min at
95 8C.

2.3.2.

RT-qPCR

To assess the expression proles of the two apoptosis genes,

Bax and Bcl-2, a quantitative RT-PCR (RT-qPCR) method was
used. Sequences of primers for the target genes and b-actin as
a reference gene (Actb) were designed using Primer Express
2.0 (Applied Biosystems). Actb was chosen as the reference
gene based on its consistent and stable expression within the
uterus, as compared to other reference genes [18,19]. The
primer sequences, expected product sizes, and references
are presented in Table 1. Real Time qPCR was performed
using SYBR Green PCR Master Mix (Applied Biosystems) in
the Applied Biosystems 7500 Real-Time PCR System (Applied
Biosystems). Each reaction was done as a triplicate in a
volume of 10 ml and consisted of SYBR Green PCR Master
Mix (Applied Biosystems, Foster City, CA, USA), AmpErase
UNG (Applied Biosystems), primer set 200 nM each (Synthetic
DNA Laboratory, University of Cape Town, Cape Town, RSA)
and 3 ml of cDNA. The real-time qPCR was performed with
activation of UNG to prevent PCR product carryover (2 min at
50 8C), followed by an initial denaturation (10 min at 95 8C), 40
cycles of denaturation (15 s at 95 8C), annealing and extension (1 min at 60 8C). A non-template control was used as a
negative control in which cDNA in the reaction mix was
replaced by nuclease-free water. A melting curve was
generated (by ramping to 95 8C) for each sample to ensure
that a single product was amplied in each reaction. The
comparative quantication cycle (Cq) method was used to
quantify the abundance of Bax and Bcl-2 relative to that of
Actb (Applied Biosystems Sequence Detection Software
version 1.2.3 7500 System SDS Software). An average Cq

Table 1 Primer sequences used for RT-qPCR.

Genes

GenBank accession number

Actb

NM_031144

Primer sequences 50 30

Amplicon size (bp)

F: CCTAAGGCCAACCGTGAAAA
R: TGGTACGACCAGAGGCATACAG

112

Bax

NM_017059

F: AGTGTCTCAGGCGAATTGGC
R: CACGGAAGAAGACCTCTCGG

102

Bcl-2

NM_016993

F: ACTGAGTACCTGAACCGGCATC
R: GGAGAAATCAAACAGAGGTCGC

108

F: forward primer; R: reverse primer; Actb: b-actin, a reference gene.

Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

Fig. 1 Light (a) and transmission electron (b) micrographs representative of the observed increase in apoptotic activity in the
RU486-treated rat endometrium; L: lumen; LE: luminal epithelium; S: stroma; AB: apoptotic body [21].
Reproduced with the consent of the copyright's owner: KE Theron.

value for the reference gene Actb was obtained and this value
was used to normalize the data from each gene. An average
Cq value was obtained for both the implantation and nonimplantation sites at days 5.5 and 6.5 of the control pregnant
and the RU486-treated animals for the Bax and Bcl-2 genes.
The change in Cq (DCq) for each day of pregnancy was
obtained by normalizing the data using the reference gene
average Cq value. The equation used was: Cq (target)  Cq
(reference gene) = DCq [20]. The calibrator sample chosen was the
implantation sites at day 5.5 of pregnancy in the control
pregnant animals, this being the site and day of blastocyst
implantation. The DCq values were then calibrated using the
chosen calibration sample, using the equation, DCq (target)  DCq (calibrator) = DDCq.
The up-regulation of gene expression, as compared to
the calibrator, for each day of pregnancy was established
using the equation 2DDCq. Down-regulation of gene expression relative to the calibrator sample was established using
the inverse (2DDCq). A fold change greater than 2 was
considered signicant.

3.

Results

Following RU486 administration, there was a marked increase

in the number of apoptotic cells observed within the uterine
epithelium at all three days of pregnancy, as observed by light
(Fig. 1a) and electron microscopy ([14,21]; Fig. 1b). This led to
the investigation of the Bax/Bcl-2 index to determine whether
these two apoptotic factors were involved in the observed
increase in apoptosis as a result of RU486 administration.
Evaluation of the Bax/Bcl-2 index revealed that there was a
decrease in both the implantation and non-implantation sites
at all three days of pregnancy with regards to all endometrial
compartments (Table 2). The decrease in the Bax/Bcl-2 index
here was as a result of a decrease in Bax protein expression
throughout the endometrium, following RU486 administration
(Fig. 2 and Table 3). Bcl-2 protein expression was not
signicantly affected by RU486 administration (Fig. 3 and
Table 3). Both Bax and Bcl-2 protein expression was observed
in the cytoplasm of the cells within the endometrial

Table 2 Effect of RU486 administration at day 3.0 of pregnancy on the Bax/Bcl-2 index (mean W SEM) in the rat
endometrium.
Time
Day of pregnancy
Untreated control
4.5
5.5
6.5

RU486-treated
4.5
5.5
6.5

Endometrial compartment
IS/NIS

Luminal epithelium

Subepithelial stroma

Glandular epithelium

Glandular stroma

IS
NIS
IS
NIS
IS
NIS

3.11
2.49
3.19
2.54
1.70
2.27

2.62
1.75
2.18
1.88
2.35
1.78

5.74
3.67
4.73
3.87
5.00
3.83

2.53
1.72
2.64
2.00
2.81
1.78

IS
NIS
IS
NIS
IS
NIS

2.18
2.17
1.29
1.39
1.40
1.74

1.79
1.71
0.83
1.14
1.32
1.36

3.55
3.88
1.48
1.83
3.10
2.53

1.78
1.94
1.23
1.33
1.79
1.56

IS: implantation site; NIS: non-implantation site.

Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

Fig. 2 Exemplary images of immunofluorescent localization of Bax (red) in the control pregnant (a) rat endometrium as
compared to the RU486-treated (b) endometrium; (c) negative control; scale bar = 20 mm; LE: luminal epithelium; S: stroma.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 3 Effect of RU486 administration at day 3.0 of pregnancy on Bax and Bcl-2 protein expression (mean W SEM) in the
rat endometrium.
Protein

Time
Day

Bax

Bcl-2

Site

Endometrial compartment
Luminal epithelium

Subepithelial stroma

Glandular epithelium

Untreated control
IS
4.5
NIS
5.5
IS
NIS
6.5
IS
NIS

Glandular stroma

116.92
1.32 d
91.49
1.18 f
140.83
1.63 b
132.44
1.53 c
167.92
1.46 a
83.21
1.54 gh

78.21
1.36 b
44.83
1.39 ef
75.79
1.26 b
60.19
1.65 c
138.38
1.13 a
49.45
1.20 de

182.52
1.65 b
96.82
1.59 de
180.71
1.58 b
151.23
1.30 c
195.44
1.29 a
102.52
1.17 d

64.76
1.48 c
43.30
1.47 e
73.01
1.40 b
54.26
1.57 d
90.79
1.43 a
42.10
1.18 e

RU486-treated
IS
4.5
NIS
5.5
IS
NIS
6.5
IS
NIS

77.59
1.24 hi
72.31
1.51 i
80.17
1.58 gh
87.22
1.40 fg
99.40
1.64 e
104.38
1.64 e

45.32
1.00 ef
38.85
1.37 f
40.15
1.51 f
48.67
1.39 de
56.72
1.38 c
54.08
1.78 cd

75.97
1.65 h
87.73
1.67 fg
62.81
1.53 i
75.55
1.56 h
92.73
1.33 ef
85.30
1.15 g

31.69
1.25 f
37.21
1.38 ef
32.06
1.39 f
37.67
1.57 ef
44.10
1.63 e
51.73
1.28 d

Untreated control
IS
4.5
NIS
5.5
IS
NIS
6.5
IS
NIS

37.55
1.20 ef
36.80
1.82 f
44.17
1.32 e
52.24
1.76 d
98.70
1.16 a
36.58
1.40 f

29.90
1.82 efg

25.61
1.81 fg
34.80
1.55 de
31.96
1.37 ef
58.94
1.19 a
27.73
1.25 efg

31.80
1.82 cde

26.37
1.48 ef
38.19
1.42 abc
39.11
1.79 ab
39.09
0.94 ab
26.78
1.18 ef

25.61
1.31 cd
25.11
1.48 cd
27.70
1.25 abc
27.14
1.26 abc
32.36
1.26 ab
23.64
1.26 cde

RU486-treated
IS
4.5
NIS
5.5
IS
NIS
6.5
IS
NIS

35.53
1.38 f
33.37
1.32 f
62.02
1.55 c
62.85
1.69 c
71.12
1.43 b
59.95
1.42 c

25.36
2.09 fg
22.70
1.16 g
48.54
1.35 b
42.76
1.51 bc
43.03
1.62 bc
39.82
1.26 cd

21.41
1.18 f
22.60
1.67 f
42.31
1.41 a
41.30
1.41 a
29.95
1.36 de
33.70
1.72 bcd

17.82
1.40 e
19.22
1.34 de
26.09
1.38 bcd
28.24
1.45 abc
24.64
1.65 cde
33.24
1.90 a

IS: implantation site; NIS: non-implantation site; means in the same column without the same superscript (ai) are considered signicantly
different ( p < 0.05).

compartments (Figs. 2 and 3). These data suggest that RU486

induces a change toward an overall anti-apoptotic signal in the
Bax/Bcl-2 pathway.
RT-qPCR evaluation of Bax and Bcl-2 gene expression
supported the results of the protein expression patterns.
Overall, RU486 treatment decreased Bax gene expression in
both the implantation and non-implantation sites of the rat
uterus at days 5.5 and 6.5 of pregnancy; whereas it was
observed to have no effect on or increase Bcl-2 gene expression

in both the implantation and non-implantation sites of the rat

uterus (Table 4).

4.

Discussion

Observation of large protrusions on the surface of the luminal

epithelial cells of the endometrium in the RU486-treated
animals in a prior ultrastructural study, led to the formulation

Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

Fig. 3 Exemplary images of immunofluorescent localization of Bcl-2 (green) in the control pregnant (a) rat endometrium as
compared to the RU486-treated (b) endometrium; (c) negative control; scale bar = 20 mm; LE: luminal epithelium; S: stroma.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 4 Bax and Bcl-2 gene expressions in the implantation (IS) and non-implantation (NIS) sites at days 5.5 and 6.5 of
pregnancy in the uteri of control pregnant and RU486-treated rats.
Gene

Bax

Day of pregnancy

5.5
6.5

Bcl-2

5.5
6.5

IS/NIS

Group
Untreated control

RU486-treated

Fold change
SEM

Fold change
SEM

ISa
NIS
IS
NIS

1
0.32
1.09
0.35
1.77
0.45
2.27
0.73

27.28
0.20
27.86
0.15
1.68
0.19
3.36
0.11

ISa
NIS
IS
NIS

1
0.57
2.50
0.30
1.93
0.35
2.14
0.36

1.14
0.17
3.29
1.05
2.64
0.10
6.82
0.23

Calibrator sample (for more details see Section 2); a fold change of 2 or more denotes a significant difference in gene expression; IS:
implantation site; NIS: non-implantation site.

of the hypothesis that these blebs were forming due to

increased apoptosis [14]. Further light microscopy and
transmission electron microscopy (TEM) analysis conrmed
an increase in apoptotic activity within the rat endometrium
as a result of RU486 administration at day 4.0 of pregnancy [21].
In addition, Rotello et al. [13] previously noted an increase in
apoptotic activity following RU486 administration in the rabbit
endometrium.
Reports that a shift in the Bax/Bcl-2 ratio toward a proapoptotic environment is responsible for the increase in
apoptosis observed during the estrous cycle, as a consequence
of either estrogen withdrawal or increased P4 levels [12], led
the investigators in the current study to assess whether the
Bax/Bcl-2 apoptotic pathway is responsible for this observed
increase in apoptosis in the RU486-treated rat endometrium.
In assessing the Bax/Bcl-2 index here, the results suggest that
there is an increased anti-apoptotic stimulus within the
RU486-treated animals. While this posits the maintenance
of a pro-cell survival Bax/Bcl-2 ratio, following from our
previous morphological studies [14] and the observations of
the current study, it is evident that RU486 treatment
substantially increases apoptotic activity, in agreement with
the prior study in the rabbit endometrium [13]. Altogether,
these data indicate that the Bax/Bcl-2 signaling pathway is not
necessarily responsible for the increased apoptosis observed
in the RU486-treated animals within this study.

RU486 has previously been reported to block estrogen

effects on the endometrium as this anti-progestin is also able
to bind to ERs [2224]. If a shift in the Bax/Bcl-2 ratio to a proapoptotic environment was under the inuence of estrogen
withdrawal, then a change in the Bax/Bcl-2 ratio as a result of
RU486 administration would be expected. This, however, is not
evident in the present study. As RU486 partially blocks P4
binding and subsequent P4 action on the endometrium [2,3], it
is feasible that the decrease in the Bax/Bcl-2 ratio and
subsequent anti-apoptotic environment is directly related to
the decrease in P4 signaling in the RU486-treated endometrium. These results further imply that Bax and Bcl-2 are likely
not responsible for the RU486-induced increase in apoptosis
observed during early pregnancy.
In a study in ovariectomized mice, an increase in the
apoptotic index was observed within the endometrium one to
two days post-ovariectomy, without hormonal replacement
[25]. These results indicate that ovariectomy induces apoptosis within the uterus, resulting in rapid involution [25]. In the
current study, apoptotic activity within the endometrium of
the pregnant rat was raised after RU486 administration, as
evident from the ultrastructural and light microscopy observations. This being in agreement with the study in ovariectomized mice [25]. This correlation between the ovariectomized
endometrium and the RU486-treated endometrium supports
the hypothesis that RU486 results in a more hormonally

Please cite this article in press as: Theron KE, et al. The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the
RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002

REPBIO-81; No. of Pages 8

reproductive biology xxx (2013) xxxxxx

under-stimulated uterus rather than one displaying unopposed estrogen action [14]. Burroughs et al. [26] reported
maximal apoptosis in all cell types when P4 and estrogen
levels were both low. However, the results in the current study
suggest that this increase in apoptosis is not driven by a shift in
the Bax/Bcl-2 ratio toward cell death, when the P4 and estrogen
receptors are partially inactivated by RU486, but is possibly
regulated by another apoptotic pathway.
Glucocorticoids are potent inducers of apoptosis in many
cell types [27]. Glucocorticoid signaling increases the expression of the pro-apoptotic proteins Bax/Bak and down-regulates
anti-apoptotic protein such Bcl-2 or Bcl-xL [27]. The blocking of
the glucocorticoid receptors by RU486 binding may account for
the shift in the Bax/Bcl-2 ratio toward a more anti-apoptotic
environment, however, this does not account for the increase
in observed apoptotic activity within the RU486-treated rat
endometrium. Sato et al. [25] demonstrated increased levels of
TNF-a, and Fas and Fas-ligand (FasL) 24 h after ovariectomy
within the endometrium. Both TNF-a and Fas and FasL have
been reported to be involved in the up-regulation of apoptosis
[28,29]. This led to the formulation of the hypothesis that the
increase in apoptotic cell death observed in the ovariectomized mouse uterus is mediated by the Fas and TNF-a
pathways [25].
In an endometrial cell line, P4 was shown to decrease
apoptosis by increasing the ratio of Bcl-XL to Bcl-XS, which
favors cell survival, whereas RU486 was shown to inhibit this
effect by increasing the ratio of the Bcl-X splice variants, Bcl-XS
to Bcl-XL, toward cell death. Bcl-XL inhibits cell death, whereas
Bcl-XS promotes apoptosis [30,31]. Future studies investigating
the expression of the Bcl-X splice variants, Bcl-XL and Bcl-XS,
as well as the Fas and TNF-a pathways may ascertain whether
these pathways could be responsible for the drastic increase in
apoptotic activity observed in the RU486-treated rat uterus.

Conicts of interest
None of the authors have any conicts of interest to disclose.

Acknowledgements
This work was supported by a THUTHUKA grant from the
National Research Foundation (NRF), South Africa awarded to
MJH. KET was supported by a Grant-holder Scholarship and an
Innovation Postdoctoral Fellowship from the NRF.

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RU486-treated rat uterus during early pregnancy. Reprod Biol (2013), http://dx.doi.org/10.1016/j.repbio.2013.09.002