reproductive biology 13 (2013) 290–297

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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 specific apoptotic factors and pathways involved in driving this

Apoptosis

process have not yet been established. Immunofluorescent 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: [email protected] (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

reproductive biology 13 (2013) 290–297

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 [4–7]. 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 specific 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 light–dark 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

291

confirm 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 sacrificing, the animals were anesthetized with 0.3 ml of Rompin–Ketamin (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 fixed 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 flushed out using a syringe filled with 1% (w/v) saline solution. The uterine horn was then cut into implantation and non-implantation sites which were individually flash 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 identified as implantation sites and areas in which little vessel aggregation was visible were considered non-implantation sites. On average, there were 5–6 implanted embryos per uterine horn in the control pregnant groups and only 0–1 on average in the RU486treated groups, indicating that RU486 successfully inhibited embryo implantation in the rat uterus.

2.2.

Immunofluorescence

Paraffin sections (5 mm) were deparaffinized, rehydrated and then immersed in Tris–ethylenediamine 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 fluorescence microscope (Olympus, Center Valley, PA, USA) with appropriate filters.

2.2.1.

Image analysis

Quantification of IF sections was performed using a 40 objective lens. The image analysis software, analySIS Life Science Research program FIVE – Digital imaging Solutions

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(Olympus Soft Imaging Solutions; Olympus), was used to analyze the intensity of the fluorescence of the 40 micrographs. This was done by first using the software to define 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 significant 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 Tukey–Kramer 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 significant.

2.2.3.

The Bax/Bcl-2 index

As different fluorescent 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 fluorescent 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 define definite 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 fluorospectrometer (Thermo Fisher Scientific, 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 profiles 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 amplified in each reaction. The comparative quantification 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.

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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 significant.

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 significantly 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.

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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 (a–i) are considered significantly 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

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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

a

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 confirmed 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 [22–24]. If a shift in the Bax/Bcl-2 ratio to a proapoptotic environment was under the influence 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

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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.

Conflicts of interest None of the authors have any conflicts 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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Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the RU486-treated rat uterus during early pregnancy.

An increase in apoptotic activity has been observed in both the rabbit and the rat endometria following treatment with RU486. The aim of this study wa...
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