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Regular Article |
a Physiology of Human Reproduction Research Unit,
b Cellular Biology Unit,
c Medical Biochemistry Unit, and
d Veterinary Research Unit, Université Catholique de Louvain, 1200 Brussels and 1348 Louvain-la-Neuve, Belgium
ABSTRACT
Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 µM] against caspase-3 and aurin [1 µM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.
apoptosis, implantation/early development
INTRODUCTION
Programmed cell death, or apoptosis, is a common feature of mammalian development [1]. A few studies have shown that apoptosis already occurs in preimplantation embryos. Several cells in blastocysts are eliminated by apoptosis during the normal course of embryo development in mice, rats, and humans [2, 3]. It has been hypothesized that the function of this eliminative process is to allow the removal of redundant or defective cells from the embryo before further differentiation of the prefetal stem cells during gastrulation.
The identity of the cellular mechanism implicated in the regulation and execution of apoptosis in blastocysts remains to be investigated. Recent reports have shown that several members of two major families of apoptotic genes, the Bcl-2 family and the caspase family, were expressed at that development stage [4, 5]. Bcl-2-related gene products are known to act at a distal step in the apoptotic pathway and may either suppress (Bcl-2, Bcl-XL) or promote (Bax, Bad) the induction of apoptosis. Caspases are cytoplasmic cysteine proteases, which are usually present in zymogen forms and become converted into active enzymes by proteolytic processing [6].
Prior observations have shown that the incidence of apoptotic cells can be modulated in blastocysts when maintained under specific conditions. For instance, adding transforming growth factor-
(TGF-) to the culture medium of the mouse two-cell embryos significantly reduced the frequency of apoptotic cells at the blastocyst stage, to levels close to those detected in blastocysts developing in utero [7]. On the opposite, supplementing the culture medium with high concentrations of glucose significantly increased apoptosis in mouse and rat blastocysts compared with embryos incubated in control medium [8, 9]. Collectively, these data suggest that external signals can influence the proportion of cells that are eliminated from blastocysts. Thus, under certain pathological conditions, an imbalance between proapoptotic and antiapoptotic signals may lead to inappropriate cell allocation in the different cell lineages that are differentiating within the implanting embryo. Our laboratory and others have shown in various animal models that maternal preconceptional diabetes could decrease the number of cells found in the inner cell mass (ICM) lineage in mouse and rat blastocysts [3]. This cell deficiency in the cellular population responsible for forming the fetal components was correlated with a higher proportion of cells showing signs of chromatin degradation and nuclear fragmentation [8, 9]. It is hypothesized that insufficient allocation of cells to the ICM may contribute to increasing the risk of subsequent developmental deficiencies, thus providing an explanation for the high recurrence of fetal complication in preconceptional diabetic pregnancies.
In the present paper, we determined whether two proapoptotic effectors, caspase-3 and caspase-activated deoxyribonuclease (CAD), are expressed in rat blastocysts and we examined the role of these effectors in the induction of cell death by high glucose. CAD exists as an inactive complex bound with inhibitor of CAD (ICAD) in the cyto-plasm of living cells. Activated caspase-3 cleaves ICAD to release CAD, which then enters the nucleus to degrade DNA [10].
MATERIALS AND METHODS
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 male and female Wistar rats from our breeding center were mated overnight and examined the next morning (Day 1 of pregnancy) to control the presence of a vaginal plug. On Day 5, pregnant rats were anesthetized and the uterine horns were flushed with culture medium to recover the blastocysts. The embryos were incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 h. The basal culture medium was Hams F-10 (07490088; Life Technologies Inc., Paisley, UK) supplemented with 0.1% bovine serum albumin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Glucose concentration in the culture medium was either 6 mM or 28 mM and the osmolarity was adjusted with NaCl to 285–300 mOs. In some experiments, the incubation medium was supplemented with an inhibitor for either caspase-3 or CAD. DEVD-CHO (235423; Calbiochem, La Jolla, CA) was used to inhibit the activity of caspase-3-like proteases. Aurin (R9502; Sigma Chemicals, St. Louis, MO) was used to inhibit CAD. Aurin stock solution was prepared in methanol at 500 µM, whereas DEVD-CHO inhibitor was prepared in dimethyl sulphoxide (DMSO) at 1 mM. In each experiment, the final concentration of the inhibitor was adjusted to 1 µM for aurin and to 10 µM for DEVD-CHO by diluting with the culture medium.
Reverse Transcription and Polymerase Chain Reaction
Total RNA was isolated from blastocysts immediately after collection or following culture for 24 h. Reverse-transcription was performed using oligo(dT)15 primers (814270; Roche Molecular Biochemicals, Mannheim, Germany) and Expand reverse transcriptase (1785826; Roche Molecular Biochemicals).
For caspase-3, total cDNA was then amplified with rat caspase-3-specific primers based on the available rat cDNA sequence (GenBank accession number U34685; Table 1).
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For CAD, a first pair of primers (CAD1/CAD2) was designed, based on a preliminary comparison of the mouse and human CAD sequences (GenBank accession numbers AB009377 and AB013918, respectively) that identified highly conserved regions (Table 1). The amplicon generated by amplifying cDNA with this first primer pair was cloned and sequenced. Following a comparison between this partial rat cDNA sequence and mouse and human sequences, we then designed a second pair of primers (CAD3/CAD4) that were specific for rat CAD and used them to amplify total cDNA for rat blastocysts (Table 1).
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% paraformaldhehyde 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 rabbit anti-mouse caspase-3 at 0.5 µg/ml (AHZ0052; Biosource, www.biosource.com) or goat anti-human CAD at 0.5 µg/ml (SC-8261; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS with 1% Tween-50 (PBS-T) and 3% BSA. Negative control reactions consisted of replacing the primary antibody with either normal rabbit immunoglobulin G (IgG) or normal goat IgG at similar concentrations, or omitting the primary antibody step from the protocol. Blastocysts were then washed three times for 15 min each in PBS-T and transferred into a solution of secondary antibody for a 60-min incubation at 37°C. The secondary antibody was either goat anti-rabbit IgG-fluorescein isothiocyanate (FITC; F-1262; Sigma Chemicals) at 5.5 µg/ml for caspase-3 or rabbit anti-goat IgG-FITC (FI-5000, Vector, Burlingame, CA) at 1.7 µg/ml for CAD. Blastocysts were incubated for 15 min in a solution of TOPRO-3 iodide (T3605; Molecular Probes Inc., Eugene, OR) at 1 µM to counterstain their nuclei. Mounting was performed in Vectashield medium (H-1000; Vector) before examination by laser-scanning confocal microscopy. Each blastocyst was scanned in two channels, red to detect the nuclei staining (TOPRO-3 iodide) and green to detect caspase-3 or CAD (IgG-FITC). Each experiment was repeated four times, using a total of more than 25 blastocysts in each experimental group.
Nuclei Counting
The method of Tarkowski [11] was used to evaluate the number of nuclei in the blastocysts. This technique used a hypotonic treatment (trisodium citrate 0.9%) to disrupt the cell membrane followed by the addition of a few drops of freshly prepared fixative mixture (acetic acid/ethanol: volume/volume). The nuclei were stained with a 4% Giemsa solution (Merck, Darmstadt, Germany) in sodium phosphate buffer (pH 6.8, 0.005 mol/L) and counted under a microscope.
Determination of Chromatin Degradation and Nuclear Fragmentation
Incidence of chromatin degradation and nuclear fragmentation were analyzed by TUNEL coupled with bisbenzimide-staining (HO-staining) [12].
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 µg/ml of bisbenzimide. After rinsing in PBS, the embryos were incubated with 50 U/ml of terminal deoxynucleotidyl transferase and 15 µM of fluorescein-deoxyuridine 5-triphosphate (dUTP; 1684817; Roche Molecular Biochemicals), and then exposed to a sheep anti-fluorescein antibody conjugated with peroxidase. TUNEL-staining was developed in a solution of diaminobenzidine and nickel chloride. Index of cells with signs of nuclear bisbenzimide-stained fragmentation and TUNEL-positive chromatin degradation were expressed in individual blastocysts as percentages of the total number of cells counted per blastocyst. Each experiment was repeated four times, resulting in a total of more than 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.
RESULTS
Apoptotic Effect of High Glucose on Blastocysts
Rat blastocysts were incubated for 24 h in either 6 mM and 28 mM D-glucose and stained to detect cells showing signs of nuclear fragmentation (bisbenzimide staining) or chromatin degradation (TUNEL staining; Fig. 1). In these blastocysts, the two nuclear events were found to occur in different cells. The combination of chromatin degradation and nuclear fragmentation within the same cell nucleus was extremely rare (below 1%) regardless of the concentration of glucose added to the medium.
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At the initiation of the culture, the mean cell number per blastocyst was 32.6 ± 4.5. After 24 h of incubation in control medium (6 mM D-glucose), the mean cell number per blastocyst was 64.3 ± 2.1. Compared with these control blastocysts, a 23% cell deficiency was counted in embryos incubated in the presence of 28 mM D-glucose (50.0 ± 2.8, P 
0.01). The nuclear fragmentation index was significantly increased in blastocysts exposed to high D-glucose compared with control embryos (0.88% ± 0.32% versus 3.73% ± 0.87% in control and high glucose groups, respectively, P 0.05). The chromatin degradation index was also significantly increased in blastocysts exposed to high D-glucose compared with control embryos (2.23% ± 0.51% versus 4.93% ± 0.67% in control and high glucose groups, respectively, P 0.05).
Caspase-3 Expression in Blastocysts
The presence of caspase-3 transcripts in blastocysts was investigated by reverse transcription-polymerase chain reaction (RT-PCR; Fig. 2). Amplification of cDNA preparations from freshly collected blastocysts and from blastocysts cultured for 24 h using rat caspase-3-specific primers generated an amplicon with the predicted size of 393 base pair (bp). Positive control reactions were performed on rat spleen cDNA and negative control reactions were carried out without cDNA input. These results showed that the expression of caspase-3 mRNA was active on Day 5 and was maintained for at least 24 h in vitro.
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The presence of the precursor, the active form of caspase-3, or both, were investigated by immunocytochemistry after incubation for 24 h in control culture medium (Fig. 3). Positive immunostaining was detected in all cells of the embryos, indicating that expression of caspase-3 is evenly distributed in the two cell lineages. Nuclear counterstaining showed that the caspase-3 immuno-signal was principally present in the cytoplasm of the embryonic cells. Control experiments performed without primary antibody or with nonimmune rabbit IgG instead of primary antibody confirmed that both control of fluorescence and nonspecific background signals were negligible.
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Inhibition of Caspase-3-Like Activity
To study the importance of caspase-3-like activity in the apoptotic effect induced by high glucose in blastocysts, DEVD-CHO, a cell-permeable peptide inhibitor was used.
Preliminary experiments were conducted to investigate the possible embryotoxicity of DEVD-CHO. Rat blastocysts were incubated for 24 h in 6 mM glucose in the presence of increasing concentrations (1 µM to 10 µM) of the compound or in 6 mM glucose in the presence of DMSO (the vehicle for DEVD-CHO) and then analyzed for their morphology and average cell number per embryo according to the method of Tarkowski (data not shown). The concentration of 10 µM was selected for further experiments based on the absence of embryotoxicity and because higher concentrations may diminish its specificity against caspase-3.
An experiment was then performed to test the consequence of DEVD-CHO-mediated inhibition of caspase-3-like activity on the effect of high glucose in blastocyst development. Blastocysts were incubated for 24 h in either 6 mM or 28 mM D-glucose in the presence or absence of DEVD-CHO (10 µM) and then examined for the frequencies of nuclear fragmentation and chromatin degradation (Fig. 4).
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Addition of DEVD-CHO to high glucose resulted in a significant decrease in the chromatin degradation index compared with high glucose alone (P < 0.01). No difference in the chromatin degradation index was found between blastocysts cultured in control culture medium and high glucose combined with DEVD-CHO, indicating that the inhibitor had completely inhibited the effect of glucose on chromatin degradation.
The identification and counting of cells showing signs of nuclear fragmentation were made difficult by the fact that the inhibitor per se induced the formation of a large number of highly condensed or scattered nuclear bodies in the blastocysts. This unexpected effect was independent of the concentration of glucose added in the culture medium and had not been detected in the preliminary observations aimed at verifying the absence of embryotoxicity. Most nuclear bodies were contained in cytoplasmic vesicles and delimited by cell membrane boundaries (Fig. 5), thus presenting a pattern that was distinct from the well-described appearance of nuclear fragmentation that is detected in the embryos treated with high glucose alone. The highest nuclear fragmentation index was found in blastocysts that were exposed to a combination of 28 mM glucose and DEVD-CHO, which corresponded to the addition of the values found in blastocysts that had been exposed to high glucose alone ("classical" nuclear fragmentation) and in blastocysts that had been exposed to the DEVD-CHO alone (nuclear bodies). These data suggest indirectly that the inhibitor did not block the induction of nuclear fragmentation by high glucose.
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CAD Expression in Blastocyst
The expression of CAD mRNA in blastocysts was investigated by RT-PCR (Fig. 6). Because the sequence of rat CAD cDNA is not available, a first pair of primers (CAD1/CAD2) was designed based in the existence of conserved regions in mouse and human CAD sequences. Amplification of cDNA preparations from mouse thymus by CAD1 and CAD2 generated an amplicon of 463 bp but produced several amplicons when rat ovary and rat spleen cDNA were tested. The sequence of one of these rat amplicons was found to be highly similar to mouse and human CAD (95%) and thus represented a partial sequence of rat CAD. Based on this information, a second pair of primers, specific for rat CAD (CAD3/CAD4), was designed. Amplification of cDNA preparations by CAD3 and CAD4 generated an amplicon of 205 bp from rat thymus cDNA but no amplicon from mouse thymus cDNA. Detection of this amplicon after amplification of rat blastocyst cDNA demonstrated the transcription of CAD at that developmental stage. RT-PCR analysis was also performed on blastocysts cultured for 24 h, and showed that the expression of CAD mRNA was maintained in vitro.
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The presence of CAD protein in blastocysts was investigated by immunocytochemistry (Fig. 7). Positive immunostaining was detected in all the cells of the blastocysts. CAD immuno-signal was excluded from the nuclei. Negative experiments without primary antibody or with nonimmune rabbit IgG demonstrated that both control of fluorescence and nonspecific background signals were very low.
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Inhibition of CAD Activity
To study the role of CAD in the induction of cell death by high glucose, blastocysts were exposed to aurin, an inhibitor of CAD activity.
Preliminary experiments were conducted to evaluate the possible embryotoxicity of aurin. Blastocysts were incubated for 24 h in 6 mM glucose in the presence of increasing concentrations of the compound (1 µM to 50 µM) and then analyzed for their morphology and their average number of cells per embryo according to the method of Tarkowski. Doses higher than 1 µM were found to significantly decrease the proportion of developing embryos (data not shown). Degenerated blastocysts showed no evidence of cell proliferation when compared with embryos analyzed at the start of the culture period.
Experiments were then performed to test the effect of aurin on the incidence of high glucose-induced nuclear fragmentation and chromatin degradation. Blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of 1 µM aurin. Addition of aurin to 28 mM of glucose resulted in a significant decrease in the chromatin degradation index, but not in the nuclear fragmentation index when compared with blastocysts exposed to high glucose alone (Fig. 8). The chromatin degradation index value in blastocysts exposed to a combination of high glucose and aurin was not statistically different compared with embryos cultured in control culture medium. In contrast, the induction of nuclear fragmentation was not reduced by the addition of the compound in a culture medium containing 28 mM glucose.
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DISCUSSION
It has been recognized that preconceptional maternal diabetes is associated with an increase in the rate of congenital malformations in offspring [13, 14]. Besides the deleterious effect of the metabolic disorder on early fetal organogenesis, several studies have shown that embryo growth is already impaired before its implantation in the uterine wall. Blastocysts exposed to maternal diabetes in utero or to a high concentration of glucose in vitro were characterized by a decrease of the cell number and by an increase in the expression of various markers of apoptosis [7, 8, 12]. In the present study we confirm that blastocysts exposed for 24 h to 28 mM glucose contain fewer cells than control embryos. Higher frequencies of nuclei presenting a pattern of either chromatin degradation or nuclear fragmentation were detected in glucose-exposed blastocysts. Previous investigations [12] substituting nonmetabolizable L-glucose for D-glucose have demonstrated the specificity of the active form of the nutrient. As an additional control measure, the osmolarity of the different culture media was adjusted with NaCl to 285–300 mOs.
Limited observations published recently suggest that embryonic cells that enter into apoptosis within rodent preimplantation embryos use many of the intracellular effectors that are used by other cell types to execute cell suicide [4, 15].
Previous studies have shown that caspase-3 is expressed at the mRNA level in mouse blastocysts and that this protein plays a critical role during development [16, 17]. In caspase-3-deficient mice, there is a decrease of apoptosis during brain development, resulting in a variety of hyperplasias.
The expression of caspase-3 in rat blastocysts was evidenced here by RT-PCR and immunocytochemistry. Caspase-3 mRNA was detected in freshly recovered blastocysts and appeared to be maintained in vitro at least for 24 h. Caspase-3 protein was visualized by immunocytochemistry in the cytoplasm of cells and no difference was observed in the distribution of the immuno signal between the two differentiating cell lineages. An unpublished observation has shown that caspase-3 activity was only detected in a very limited percentage of cells in these embryos, indicating that most of the caspase-3 protein detected by immunocytochemistry was still in its zymogen form.
Experiments were performed to determine whether caspase-3 plays a role in the induction of cell death by high glucose. When caspase-3-like activity was inhibited by DEVD-CHO, no increase was found in the number of cells showing signs of chromatin degradation upon exposure to high glucose. The inhibitory effect is probably explained by the fact that caspase-3 is responsible for the cleavage of ICAD and the release of CAD activity in cells [18, 19]. The effect of DEVD-CHO on high glucose-induced nuclear fragmentation was more difficult to analyze because the compound was responsible for the production of numerous condensed nuclei and scattered micronuclei in blastocysts. Most of the condensed nuclei were contained into cytoplasmic vesicles and had an aspect that was reminiscent of the ill-defined sporotic bodies that are detected in certain transformed cell lines [20]. At the present time, this unexpected effect of DEVD-CHO on blastocyst cells remains unexplained.
The second intracellular proapoptotic effector that we studied was an endonuclease involved in chromatin degradation and a target of caspase activation, CAD. CAD mRNA was detected in freshly recovered blastocysts. This expression was maintained in blastocysts for at least 24 h. Immunocytochemistry showed the presence of the CAD protein mainly in the cytoplasm of the embryonic cells.
CAD activity was inhibited with aurin (or rosolic acid) in rat blastocysts exposed to high glucose. It has been shown previously that aurintricarboxylic acid (ATA) can specifically inhibit CAD activity [21]. The inhibitory effect of ATA, however, may be due either to its anionic properties, to the chelation of ions, or to the binding of metals necessary for cell death to occur [22, 23]. Therefore, aurin, a structural analogue of ATA that lacks the three carboxylic groups of ATA, was preferred to ATA. When CAD activity was inhibited with 1 µM aurin in blastocysts incubated in high glucose, the rate of chromatin degradation was similar to that of embryos cultured in control medium. High glucose-induced nuclear fragmentation was not modified by aurin. The fact that the CAD inhibitor inhibited chromatin degradation but not nuclear fragmentation supports the idea that CAD directly induces chromatin degradation but not nuclear fragmentation [24]. Thus the two nuclear events of apoptosis may be secondary to different effector pathways toward apoptosis. This absence of correlation between chromatin degradation and nuclear fragmentation has also been observed in rat blastocysts [12] and ES cells treated with TNF- [25]. In these cellular models, TNF- induced chromatin degradation without nuclear fragmentation.
In conclusion, our data show that caspase-3 and CAD are expressed in rat blastocysts and that their activity is important for the induction of chromatin degradation, but not nuclear fragmentation, by high glucose. The identity of one or more apoptotic effectors involved in nuclear fragmentation will be investigated in further experiments.
ACKNOWLEDGMENTS
We are grateful to I. Vanderheyden, S. Cordi, and C. Marchand for their excellent technical assistance and to Professor P. Courtoy for providing access to the confocal laser scanning microscope (grant 9 4531 94, FNRS)
FOOTNOTES
1 Supported by Action de Recherche Concertée de la Direction Générale de la Recherche de la Communauté Française de Belgique (grant 96/01-96) and by the Juvenile Diabetes Foundation International. L.H. is the holder of a research fellowship from FRIA. S.P. is Chercheur Qualifié, FNRS. 
2 Correspondence: Serge Pampfer, OBST 5330 Research Unit, University of Louvain, School of Medicine, 53 Avenue Mounier, B-1200 Brussels, Belgium. FAX: 32 2 764 5396; pampfer{at}obst.ucl.ac.be
Accepted: September 20, 2000.
Received: April 20, 2000.
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