Hum. Reprod. Advance Access originally published online on November 25, 2005
Human Reproduction 2006 21(3):618-623; doi:10.1093/humrep/dei404
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Activin receptor expression and induction of apoptosis in rat blastocysts in vitro
OBST Research Unit, Université catholique de Louvain, 1200 Brussels, Belgium
1 To whom correspondence should be addressed at: Cliniques Universitaires Saint-Luc, Service d’Obstétrique 1014, Avenue Hippocrate 10, B-1200 Bruxelles, Belgium. E-mail: debieve{at}obst.ucl.ac.be
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Abstract |
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BACKGROUND: Apoptosis, a process of normal embryonic development, is enhanced in blastocyst from diabetic rats. Nevertheless, glucose seems not to be the only factor involved. Activin A, a TGF-
family member, is also increased in maternal serum from diabetic pregnancy. METHODS: Flushing medium, blastocysts and uterine cells were obtained from 5 day old pregnant rats. The presence of activin A in flushing medium was investigated by western blotting. RT–PCR was used to test for the presence of activin A subunit mRNA in cultured uterine cells. Blastocysts were stained by immunohistochemistry for activin receptor types IIA and IIB, and chromatin degradation (apoptosis) was investigated by terminal transferase-mediated dUTP nick end labelling in blastocysts exposed in vitro to activin. RESULTS: In this study, we demonstrate the presence of activin A protein in fluid from rat uterine horns at day 5 of pregnancy, as well as the presence of activin A receptors type IIB in the trophectoderm and inner cell mass and activin A receptor type IIA in trophectoderm cells only. Activin A increases the chromatin degradation level in vitro. CONCLUSIONS: Activin A protein was found in fluid from uterine horns, and mRNA expression of A activin subunit in cultured uterine cells suggests probable secretion from decidual cells. Moreover, activin A increases specifically the apoptosis level in rat blastocyst in vitro.
Key words: activin/apoptosis/blastocyst/rat
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Maternal diabetes is a metabolic disorder with severe consequences on embryo development (Moley, 1999
). These could account for the clinical manifestations in diabetic pregnancy: increasing miscarriage rate, congenital malformations and obstetric complications (Miller et al., 1981; Kitzmiller et al., 1996). The effects induced by this metabolic disorder during the preimplantation period of pregnancy include a decrease in the number of cells per blastocyst and a concomitant increase of two apoptosis nuclear markers: chromatin degradation and nuclear fragmentation (Pampfer et al., 1997). The hypothesis is that insufficient numbers of cells in the blastocyst may contribute to increasing the risk of a subsequent development defect.
As diabetes is associated with a myriad of hormonal, metabolic and other alterations, identification of imbalanced factors responsible for developmental anomalies detected at the blastocyst stage may prove difficult. The influence of high glucose and tumour necrosis factor-
(TNF-) concentrations on blastocyst development in vitro and in vivo has been demonstrated (De Hertogh et al., 1991; Pampfer et al., 1997). However, both clinical and experimental studies suggest that these two factors may account only for a part of the embryonic deficiencies. Indeed, even if blood glucose level correction with insulin therapy decreases the congenital malformation rate, diabetic pregnancy is still a high risk pregnancy with macrosomia persistence in some cases and particularly placental type complications in others such as growth retardation and pre-eclampsia (Gabbe and Graves, 2003).
Activin A is a homodimeric protein belonging to the transforming growth factor- (TGF-) superfamily, composed of disulphide-linked A-subunit (A) (Massague, 1990). Activin A acts through type II ligand-binding receptors and type I signal-transducing receptor, which interact to form functional receptor dimers. There are two type II receptors known for activin, termed activin receptor type IIA (ActR-IIA) and activin receptor type IIB (ActR-IIB) (Hilden et al., 1994). Differential effects on development with type IIA and IIB receptors are reported (New et al., 1997).
Activin A protein has a variety of functions including effects on cell growth, differentiation, and apoptosis. These different actions depend on the cell system examined and the dose of the ligand (Ethier and Findlay, 2001).
Activin A is present in maternal serum throughout pregnancy, increasing progressively and reaching its highest concentration at term (Tong et al., 2003). Activin A appears to be an important factor in developmental processes. It is secreted both by decidua and the feto-placental unit and modulates embryo differentiation and growth in mammals (Roberts and Barth, 1994; Tuuri et al., 1994).
Several studies have demonstrated that diabetic pregnant women have an elevated serum activin A concentration. It decreases after insulin therapy reaching the range of healthy women at the same gestational age (Petraglia et al., 1995; Gallinelli et al., 1996). Even if activin A is known to stimulate insulin secretion (Florio et al., 2000), the influence of elevated concentration of activin A in diabetic pregnancy remains unknown.
In the present study, we used different cellular and molecular techniques in order to determine if the preimplantation stage embryo is exposed to activin A in uterine lumen and if activin A has a deleterious effect on blastocyst development.
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Materials and methods |
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Embryo collection and culture
All investigations were performed in accordance with the Guide for Care and Use of Laboratory Animals (National Academy of Sciences, 1996).
Sexually mature female Wistar rats from our breeding centre were mated overnight with males and examined the next morning (day 1 of pregnancy) to attest the presence of a vaginal plug. On day 5, pregnant rats were anaesthetized and the uterine horns were flushed with phosphate-buffered saline (PBS) to recover the blastocysts. The embryos were washed and incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 h. The basal culture medium was Ham’s F-10 (Life Technologies Inc., Paisley, UK) supplemented with 0.1% bovine serum albumin (BSA), 100 IU/ml penicillin and 100 mg/ml streptomycin. In some experiments, the incubation medium was supplemented with bovine activin A at 100 ng/ml (Innogenetics, Gent, Belgium). The chosen concentration of activin A was based on previous published work on rat oocytes (Zhao et al., 2000). According to the manufacturer’s protocol and the study of Zhao et al. (2000) this activin is an active form. The specificity of activin A action was confirmed using a neutralizing rabbit anti-bovine activin A antibody at 10 µg/ml (Innogenetics). The non-toxicity of this concentration was checked in experiments with concentration gradient using the Tarkowski method (Tarkowski, 1966) and was the highest concentration without toxicity.
The flushing media were harvested, avoiding any blood contamination, briefly centrifuged at 3000 g for 5 min to avoid contamination with cellular debris, concentrated 10 times using centrifugal filter and stored at –20°C.
Ovaries from four different rats were recovered and resuspended in TRI-Reagent (Sigma Chemicals) to isolate total RNA and proteins.
Protein concentrations were determined (DC Protein assay; BioRad, Hercules, CA, USA) with BSA as standard.
Uterine cells
Primary cultures of uterine luminal cells were prepared from the uterus of pregnant rat on day 5 by a method described elsewhere (Gu and Gibori, 1995) and used here with minor modifications. Briefly, uterine horns were opened lengthwise and pooled in 5 ml of Ca2+/Mg2+-free Hanks’ balanced salt solution (HBSS) (Life Technologies) at 4°C before transfer into 5 ml of HBSS containing 0.25% pancreatin (Life Technologies) and 0.5% trypsin (Sigma Chemicals, St Louis, MO, USA). Enzymatic digestion was continued for 1 h at 4°C followed by 1 h at 20°C. The enzyme solution was carefully removed and replaced with 5 ml of HBSS. Uterine cells were released from the tissues by mild vortexing and separated from single cells by repeated sedimentation at unit gravity. The remaining uterine tissues were further digested a second time with 70 IU/ml deoxyribonuclease (Sigma Chemicals) in Ca2+/Mg2+-free HBSS for a total of 40 min at 37°C and vortexed vigorously to release a mixture of uterine cells. The cells obtained after sequential enzymatic treatment were pooled and pelleted at 500 g for 10 min, resuspended in culture medium composed of Dulbecco’s minimal essential medium (DMEM) (Life Technologies) mixed 1:1 with Ham’s F-12 medium (Life Technologies) completed with 15 mmol/l HEPES (Sigma chemicals), 1 g/l bovine serum alubmin (BSA) (Sigma Chemicals), 100 IU/ml of penicillin and streptomycin (Life Technologies), and 5% of FBS (Life technologies) and plated at one uterine-horn equivalent per 100 µl.
In some experiments, the uterine cells were resuspended in TRI-Reagent (Sigma Chemicals) to isolate total RNA. In others, the uterine cells were cultured in previously described culture medium for 3 days to perform immunochemistry experiments. Experiments were repeated three times.
Western blot
Fifteen microlitres of concentrated flushing medium were separated on 10% polyacrylamide gels by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride microporous membranes (Millipore). The WesternBreeze Chemiluminescent Immunodetection Kit (for mouse primary antibody detection) (Invitrogen) was used for immunodetection. The primary antibody was mouse anti-activin A (R&D systems) at 1 µg/ml. Rat ovary served as a positive control. A total of 10 µg protein was loaded on each lane.
Blastocyst mRNA extraction
mRNA was isolated from pools of 60 blastocysts after 24 h culture using the Micro-FastTrack mRNA isolation system (K1520–02; Invitrogen; Carlsbad, CA, USA) according to the manufacturer’s instructions.
RT–PCR
The RNA was used to synthesize single-stranded cDNA using oligo(dT)18 primers and RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas; Hanover, MD, USA).
The synthesized cDNA was used as template for PCR amplification. The quality of the obtained cDNA was checked by GAPDH (glyceraldehyde phosphate dehydrogenase) PCR amplification (data not shown). Synthetic oligonucleotides used for PCR primers are listed in Table I based on the published sequences. The PCR reactions were performed in 50 µl PCR mixture containing 1xPCR buffer, 0.2 mmol/l of each dNTP, 25 pmol of each primer, 1 mmol/l MgCl2, 2.5 IU Taq DNA polymerase (Fermentas) and cDNA template. Negative controls with no cDNA template in PCR reaction and positive control using rat ovary cDNA were used (data not shown).
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Uterine cell immunochemistry
Uterine cell cultures were analysed at 72 h after plating. Cells were rinsed with PBS at 4°C and fixed for 10 min at 4°C in 100% methanol, rinsed again with PBS. Preparations were preincubated in PBS containing 0.5% BSA, 2.5% normal goat serum and 0.1% saponin for 15 min at 4°C, then incubated with mouse anti-activin A (R&D systems) at 25 µg/ml in the same solution overnight at 4°C. The primary antibody was replaced with normal IgG in control slides. Cells were rinsed for 5 min six times in PBS containing 0.1% saponin, followed by a 1 h incubation at room temperature with goat anti-mouse IgG–fluorescein isothiocyanate (FITC) secondary antibody (Vector Laboratories, Burlingame, CA, USA) at 15.3 µg/ml. Cell nuclei were counterstained with TOPRO-3 iodide (Molecular Probes Inc., Eugene, OR, USA) in washing solution for 5 min at room temperature, followed by six rinses. Slides were mounted using VectaShield mounting medium (Vector) examined using confocal microscopy (Zeiss, Germany).
Blastocyst immunocytochemistry
After removal of their zona pellucida in Tyrode acid solution, blastocysts were transferred onto Concanavalin A-coated coverslips. The embryos were fixed in 1.7% paraformaldehyde in PBS, permeabilized in 1% Triton X-100 in PBS and transferred in the primary antibody solution for overnight incubation at 4°C. The primary antibody was either goat anti-ActR-IIA at 0.5 mg/ml (Santa Cruz Biotechnology) or goat anti-ActR-IIB at 0.5 mg/ml (Santa Cruz Biotechnology) in PBS with 1% Tween-50 (PBS-T) and 3% BSA. Negative control reactions consisted of replacing the primary antibody with normal goat IgG at similar concentrations. Blastocysts were then washed three times for 15 min each in PBS-T and transferred into a solution of secondary antibody for 60 min incubation at 37°C. The secondary antibody was rabbit anti-goat IgG-FITC (Vector) at 1.7 mg/ml. Blastocysts were incubated for 15 min in a solution of TOPRO-3 iodide (Molecular Probes Inc.) to counterstain their nuclei. Mounting was performed in Vectashield medium (Vector) before examination by laser-scanning confocal microscopy (Zeiss, Germany). Stacks of digital sections were acquired along the z-axis (z-series) at an interval of 5 µm throughout the embryo. Whole embryos were reconstituted onto a single image by a vertical projection of z-series. Each blastocyst was scanned in two channels to detect the nuclei staining (TOPRO-3 iodide) and ActR-IIA and ActR-IIB (IgG-FITC). Each experiment was repeated four times, using a total of >25 blastocysts in each experimental group.
Determination of chromatin degradation
Incidence of chromatin degradation was analysed by terminal transferase-mediated dUTP nick end labelling (TUNEL) coupled with bisbenzimide staining (HO staining).
Zona pellucida-freed blastocysts were fixed in 4% paraformaldehyde in PBS, exposed to 0.3% hydrogen peroxide in methanol and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate. Blastocysts were then prestained in 10 mg/ml of bisbenzimide. After rinsing in PBS, the embryos were incubated with 50 IU/ml of terminal deoxynucleotidyl transferase and 15 mmol/l of fluorescein-deoxyuridine 5-triphosphate (dUTP; Roche Molecular Biochemicals), and then exposed to a sheep anti-fluorescein antibody conjugated with peroxidase. Exposure of the embryos to UV light allowed visualizing and counting the total number of bisbenzimide nuclei. Observation under visible light allowed for the counting of TUNEL-positive chromatin degradation. For each blastocyst, the index of chromatin degradation was calculated as percentage of the total cell number. Each experiment was repeated four times, resulting in a total of >30 blastocysts in each experimental group.
Statistical analysis
Results were presented as means ± SEM. One-way ANOVA coupled to Scheffe’s F-test was used to identify statistically significant differences between the different culture groups.
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Results |
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Activin A protein expression in fluid of rat uterine horns
In order to determine if rat blastocysts on day 5 in uterine horns were exposed to activin A, we performed western blot with an activin A-specific monoclonal antibody on flushing medium concentrated 10 times before analysis. Positive control of reaction was performed on rat ovary (Figure 1). As the antibody used recognized all forms of activin A, we observed a large number of activin A precursors, particularly 110 and 70 kDa proteins. They represent a homodimer composed of two pro
A subunits and a heterodimer composed of one pro-A and one A subunits respectively. The same pattern was found in rat ovary and in the flushing medium from uterine horns. The mature activin A protein (28 kDa) was clearly observed in rat ovaries, but only slightly in uterine fluids, probably because of a low concentration.
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Activin A subunit mRNA expression and activin A protein expression in rat uterine cells
The mRNA expression of A subunit in rat uterine cells from day 5 pregnant rats was investigated by RT–PCR. As shown in Figure 2, rat uterine cells expressed the activin A subunit on day 5 of gestation. Amplification of cDNA preparations from freshly collected uterine cells using rat A subunit-specific primers generated an amplicon with the predicted size of 565 base pair (bp). The confirmation of activin A protein expression by rat uterine cells was obtained by immunochemistry experiments using activin A antibody (Figure 3).
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Activin receptor expression in blastocysts
The expressions of the two types of activin receptor II in rat blastocyst were investigated by RT–PCR. As shown on Figure 4, the two types of activin receptor II, ActR-IIA and ActRII-B, were expressed in rat blastocysts collected on day 5 and cultured for 24 h in vitro. Amplification of cDNA preparations from rat blastocysts using rat ActRII-A- and ActRII-B-specific primers generated amplicons with the predicted size of 462 and 300 bp respectively.
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The localizations of activin receptor II were investigated by immunocytochemistry after incubation for 24 h in control culture medium (Figure 5). The confocal staining patterns shown in this figure are representative of all blastocysts analysed. For ActRII-B, positive immunostaining was detected in all cells of the embryos, indicating that expression of ActRII-B is evenly distributed in the two cell lineages of blastocyst, the inner cell mass and the trophectoderm. In contrast, the ActRII-A immunosignal was present only in trophectoderm cells. Control experiment performed with no immune goat IgG instead of primary antibody confirmed that non-specific background signals were negligible.
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Apoptotic effect of activin A on blastocysts
Rat blastocysts were incubated for 24 h in presence or absence of activin A and stained to detect cells showing signs of chromatin degradation (TUNEL staining) (Figure 6).
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Compared with control blastocysts, the chromatin degradation index was significantly increased in blastocysts exposed to activin A (P 
0.05) although there was no difference in the number of cells per blastocyst (Figure 7A and C).
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The deleterious impact of activin A on blastocyst development was found to be significantly decreased when the embryos were co-treated with activin A and neutralizing activin A antibody (P 0.05) (Figure 7B and D).
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Discussion |
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Decidualization of uterine endometrial cells is an important process, taking part in blastocyst implantation in mammals. In rats, the endometrium behaves as an endocrine organ expressing prolactin-like-related proteins, follistatin,
2 macroglobulin and activin (Gu et al., 1995). Activin has been shown to be involved in the decidualization process, as well as in early implantation (Jones et al., 2002). This study confirms the mRNA expression of the A subunit in rat uterine cells from day 5 pregnant rats, as well as the activin A protein. Moreover, we demonstrate that activin A protein is present in uterine fluid from day 5 pregnant rat uterine horns. A large number of activin A precursors were observed in western blot analysis, but also a weak band of the mature activin A protein. These different bands in western blot analysis could correspond to different molecular forms resulting from post-transcriptional modifications within the proregion of the precursor A chain (Vejda et al., 2002). Nevertheless, the bioactivity of all these precursors is not well described. Data suggest that the presence of at least one mature chain is sufficient to confer bioactivity (Mason et al., 1996). A contamination from maternal blood was avoided during horn dissection, but the maternal origin is improbable since rat serum activin A is detected only from day 12 of gestation (Draper et al., 1997). There is substantial evidence that the blastocyst, at the time of implantation, is in an environment containing maternally derived activin A, as suggested for humans by Petraglia et al. (1998). It is important to note that previous studies about rat decidual activin expression were carried out from day 9 pseudopregnant rats with induced decidualization of uterine endometrium by scratching the antimesometrial surface (Gu and Gibori, 1995; Gu et al., 1995). Our study suggests that activin A is expressed and secreted in vivo in the maternal decidualization process as soon as day 5.
There is now evidence that activins are important regulators of the early developmental process in vertebrates with the mesoderm induction and placental formation (Feijen et al., 1994; Roberts et al., 1996). In the present study, activin receptor ligand binding IIB and IIA show different distribution patterns in the blastocyst: ActR-IIB is evenly distributed in the inner cell mass and the trophectoderm, whereas ActR-IIA signal is restricted to the trophectoderm cell lineage. A different function of activin in trophectoderm and inner cell mass could therefore be suspected. This is also in accordance with other reports in early rat embryo development where ActR-IIB is the predominant receptor involved in growth and differentiation of epithelial and endothelial cell types, whereas in adult tissue, ActR-IIA is the predominant type of receptor detected (Roberts and Barth, 1994; Manova et al., 1995). Moreover, ActR-IIA has recently been implicated in mediation of the effect of bone morphogenic proteins, probably also involved in the implantation process (Yamashita et al., 1995; Chen et al., 1997). This is in accordance with expression of ActR-IIA being restricted to the trophectoderm cells.
The first step of these experiments demonstrates that activin A is secreted in fluid from rat uterine horns, probably by decidual cells. Moreover, the rat blastocyst in the peri-implantation period is able to respond to this activin A present in its environment. The second step of the experiments demonstrates that activin A, this well-known apoptosis inducer in many cell lines (Chen et al., 2000), is able to induce apoptosis in the blastocyst. This effect is specific to the activin action, since the use of a blocking antibody reduces this apoptosis rate. The whole activin A activity was probably not suppressed by the neutralizing antibody, but increasing the antibody amount was itself toxic for the embryo.
Apoptosis, a process of normal embryonic development (Jacobson et al., 1997), is enhanced in blastocyst exposed to high glucose concentration, as present in diabetic conditions (Pampfer et al., 1997). Other factors, such as TNF-, oxidative stress and bcl-2, were associated with the regulation of apoptosis in blastocyst (Pampfer et al., 2001; Leunda-Casi et al., 2002). Activin A in maternal serum is increased in diabetic pregnancy (Petraglia et al., 1995; Gallinelli et al., 1996). Moreover, activin A is associated with human pancreatic insulin production (Florio et al., 2000). This TGF- family member could thus be involved in apoptosis induction in diabetes. Nevertheless, further experiments investigating the activin production by uterine cells at the peri-implantation stage in diabetes and the mechanisms of induction of apoptosis through activin A should be addressed.
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Acknowledgements |
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The authors are grateful to Prof. P.Courtoy for providing access to confocal microscopy (grant 9.4531.94, FNRS). The research was supported by the ‘Fonds Spéciaux de Recherche’ from the ‘Université Catholique de Louvain’, Brussels, Belgium.
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Submitted on May 24, 2005; resubmitted on August 9, 2005; accepted on October 18, 2005.
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