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Embryo |
Physiology of Human Reproduction Research Unit,3 Université Catholique de Louvain, 1200 Brussels, Belgium
Diabetes and Nutrition Unit,4 Université Catholique de Louvain, 1200 Brussels, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, developmental biology, early development, embryo, environment
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Under certain pathological conditions, an increase of cell death has been observed. Previous studies have shown a decrease in the number of cells and an abnormal high incidence of nuclear fragmentation and chromatin degradation, two nuclear apoptotic markers, in ICM cells of blastocysts recovered from insulin-dependent diabetic rats or mice, or in blastocysts from normal rats or mice incubated in vitro in high concentrations of D-glucose [4, 5]. It is hypothesized that insufficient cell number in the ICM may contribute to an increase risk of subsequent developmental deficiencies, thus providing an explanation for the high occurrence of fetal complications in preconceptional diabetic pregnancy [6, 7].
Several members of two major families of apoptotic genes, the Bcl-2 family and the caspase family, are expressed at the blastocyst stage [8]. However, the understanding of the cellular mechanisms involved in the regulation and execution of apoptosis in blastocysts exposed to high glucose remains far from complete. Recent observations showed that the expression of BAX mRNA and protein, a proapoptotic member of the Bcl-2 family, was increased significantly in blastocysts from diabetic mice or in embryos incubated in vitro in high concentrations of glucose [9, 10]. Other studies using in vitro culture of rat blastocysts in high concentrations of D-glucose reported that specific inhibition of two apoptotic effectors (caspase-3 and caspase-activated deoxyribonuclease [CAD]) protected blastocysts from the chromatin degradation event but not from the nuclear fragmentation [11]. These observations suggest that high glucose concentrations induce apoptosis in blastocysts by an intracellular cascade involving several members of the Bcl-2 and caspase families and that the chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately in rat blastocysts. The chromatin degradation is mediated by the activation of caspase-3 and CAD [11]; the effectors implicated in the induction of nuclear fragmentation remain to be determined. A possible effector for nuclear fragmentation is caspase-6, a member of the family of cysteine proteases that share the ability to cleave their substrates on the carboxyl side of aspartate residues. Each caspase is synthesized as a zymogen that contains an N-terminal prodomain, a large subunit, and a small subunit. The maturation involves caspase-mediated cleavage at aspartate residues. Each mature caspase is a tetramer comprising two identical large and small subunits [12]. Caspase-6 is an effector caspase as caspase-3 and caspase-7. The main function of effector caspases is to mediate the highly specific cleavage of various cellular proteins whose degradation contributes to the biochemical and morphological features associated with apoptosis. The first substrates identified for caspase-6 were the nuclear lamins, and its target recognition sequence was identified as the tetrapeptide motif: Val-Glu-Ile-Asp (VEID) [13]. Different studies selectively inhibiting caspase-6 activity suggested that lamina cleavage might be required for nuclear breakdown during apoptosis [14, 15].
For these reasons, in the present work we determined whether caspase-6 is expressed in rat blastocyst and its role in the induction by high glucose of the two nuclear markers of apoptosis, nuclear fragmentation, and chromatin degradation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 center were mated overnight with males 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 or 28 mM, and the osmolarity was adjusted with NaCl to 285–300 mOs/L. In some experiments, the incubation medium was supplemented with an inhibitor for caspase-6. VEID-CHO (218767, Calbiochem, La Jolla, CA) was used to inhibit the activity of caspase-6-like proteases. VEID-CHO inhibitor was prepared in dimethyl sulfoxide (DMSO) at 1 mM. In each experiment, the final concentration of the inhibitor was adjusted to 100 nM for VEID-CHO by diluting with the culture medium.
Reverse Transcription and Polymerase Chain Reaction
Total RNA was isolated from blastocysts immediately after collection or after a 24 h culture. Reverse transcription was performed using oligo (dT)15 primers (814270, Roche Molecular Biochemicals, Mannheim, Germany) and Expand reverse transcriptase (1785826, Roche).
PCR rat caspase-6-specific primers were designed based on the available rat cDNA sequence (GenBank accession AF025670; Table 1) and verified on Basic Local Alignment Search Tool Programs (BLAST) for cross-reaction. Total cDNA was then amplified in 1.5 mM MgCl2, 0.2 mM of each dNTP, PCR buffer, 0.1 µM for each primers, and 2.5 U Taq polymerase for 32 cycles, with the following profile: 96°C 1 min for one cycle; 96°C for 1 min, 62°C (annealing temperature) for 1 min, and 72°C for 2 min for 30 cycles; and 72°C for 5 min for one cycle with primers.
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The PCR products were sequenced and compared with the rat caspase-6 sequence (GenBank accession AF025670) to prove that we had well amplified caspase-6 cDNA.
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 a rabbit anti-human-caspase-6 at 0.5 µg/ml (AAP-106; Stressgen Biotechnologies Corp., Victoria, BC, Canada) in PBS with 1% Tween-50 (PBS-T) and 3% BSA. This antibody recognizes only the zymogen form and the active form of caspase-6 in many species (human, monkey, mouse, rat, etc.). Negative control reactions consisted of replacing the primary antibody with a normal rabbit immunoglobulin G (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 60-min incubation at 37°C. The secondary antibody was a goat anti-rabbit IgG-fluorescein isothiocyanate (FITC, F-1262, Sigma Chemicals, St. Louis, MO) at 5.5 µg/ml. 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 Laboratories, Inc., Burlingame, CA) 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-6 (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 [16] 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: vol/vol). 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) [4].
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) and then exposed to a sheep antifluorescein 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 Analyses
Results were presented as means ± SEM. One-way ANOVA coupled to Scheffé F-test was used to identify statistically significant differences between the different culture groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of caspase-6 transcript in blastocysts was investigated by reverse transcription-polymerase chain reaction (RT-PCR; Fig. 1). Amplification of cDNA preparations from freshly collected blastocysts and from blastocysts cultured for 24 h using rat caspase-6-specific primers generated an amplicon with the predicted size and sequence of 452 base pairs (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-6 was detected on Day 5 and was maintained for at least 24 h in vitro.
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The presence of the zymogen form, the active form of caspase-6, or both was investigated by immunocytochemistry after incubation for 24 h in control culture medium (Fig. 2). Positive immunostaining was detected in all cells of the embryos, indicating that expression of caspase-6 is evenly distributed in the two cell lineages. Nuclear counterstaining showed that the caspase-6 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 control of both fluorescence and nonspecific background signals were negligible.
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Apoptotic Effect of High Glucose on Blastocysts
Rat blastocysts were incubated for 24 h in either 6 or 28 mM D-glucose and stained to detect cells showing signs of nuclear fragmentation (bisbenzimide staining) or chromatin degradation (TUNEL staining). The combination of chromatin degradation and nuclear fragmentation within the same cell nucleus was rare regardless of the concentration of glucose added to the medium.
After 24 h of incubation, the blastocysts cultured in the presence of 28 mM D-glucose contained fewer cells (7% cell deficiency) than blastocysts in control medium (P 
0.05; Fig. 3A). The chromatin degradation index was significantly increased in blastocysts exposed to high D-glucose compared with control embryos (P < 0.0001; Fig. 3B). No significant difference was detected in the percentage of cells showing signs of nuclear fragmentation (Fig. 3C).
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Inhibition of Caspase-6 Activity
To study the importance of caspase-6 activity in the apoptotic effect induced by high glucose in blastocysts, VEID-CHO, a cell-permeable peptide inhibitor, was used.
Preliminary experiments were conducted to investigate the possible embryotoxicity of VEID-CHO. Rat blastocysts were incubated for 24 h in 6 mM glucose in the presence of increasing concentrations (10–100 nM) of the compound or in 6 mM glucose in the presence of DMSO (the vehicle for VEID-CHO) and then analyzed for their morphology and average cell number per embryo according to the method of Tarkowski (data not shown) [16]. The concentration of 100 nM was selected for further experiments based on the absence of embryotoxicity and because higher concentrations may diminish its specificity against caspase-6.
An experiment was then performed to test the consequence of VEID-CHO-mediated inhibition of caspase-6 activity on the effect of high glucose in blastocyst development. Blastocysts were incubated for 24 h in either 6 or 28 mM D-glucose in the presence or absence of VEID-CHO (100 nM) and then examined for the number of cells per embryo and the frequencies of nuclear fragmentation and chromatin degradation (Fig. 3). The deleterious impact of high glucose on the number of cells per embryo was not prevented when the blastocysts were cotreated with high glucose and VEID-CHO (Fig. 3A). The induction of chromatin degradation by 28 mM D-glucose in blastocysts was not reduced by the addition of VEID-CHO in the culture medium containing 28 mM D-glucose (Fig. 3B). In contrast, the nuclear fragmentation index value in blastocysts exposed to a combination of high glucose and VEID-CHO was statistically reduced (P 0.05) compared with embryos cultured in the medium containing 28 mM D-glucose alone (Fig. 3C), but no difference was found with the control blastocysts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously shown in separate reports that chromatin degradation and nuclear fragmentation were two nuclear damages induced separately in rat blastocysts by high glucose and that chromatin degradation was apparently mediated by the activation of caspase-3 and caspase-activated deoxyribonuclease (CAD) [11, 19].
Recent studies indicate that proteolytic cleavages of a set of key proteins by activated caspases play a role in the accomplishment of nuclear fragmentation. Disassembly of the nuclear lamina, the supporting structure of the nuclear envelope, is an essential feature of nuclear breakdown in apoptosis. This process depends on caspase-mediated degradation, perhaps by caspase-6, a good lamin protease [20–22].
The expression of caspase-6 in rat blastocysts was evidenced here by RT-PCR and immunocytochemistry. Caspase-6 mRNA was detected in freshly recovered blastocysts and appeared to be maintained in vitro at least for 24 h. Immunocytochemistry showed the presence of the caspase-6 protein, under the zymogen and/or active form, mainly in the cytoplasm of the embryonic cells. No difference was observed in the distribution of the immuno signal between the two differentiating cell lineages, ICM and trophectoderm.
In the present work, we confirm that blastocysts exposed for 24 h to 28 mM glucose contain fewer cells than control embryos. Higher frequency of nuclei presenting a pattern of chromatin degradation was detected in glucose-exposed blastocysts. However, the increase of nuclear fragmentation, usually present when blastocysts are exposed to high concentrations of glucose [4, 11, 19], was observed here only marginally, not reaching statistical significance.
Experiments were performed to determine whether caspase-6 played a role in the induction of cell death by high glucose. When caspase-6 activity was inhibited by VEID-CHO, the deleterious impact of high glucose on the number of cells per embryo was not prevented and the induction of chromatin degradation was not reduced. The fact that the caspase-6 inhibitor did not prevent chromatin degradation supports the idea that caspase-6 is not implicated in high glucose-induced chromatin degradation in rat blastocyst.
The nuclear fragmentation index value, although not significantly increased in the presence of high glucose alone, was significantly reduced in rat blastocysts exposed in vitro to high glucose concentration when caspase-6 activity was inhibited by VEID-CHO. These results suggest that the activity of caspase-6 may be important in the nuclear fragmentation. The difference of the effect of the inhibition of caspase-6 activity on the two nuclear markers of apoptosis confirms the idea that the intracellular cascades leading to nuclear fragmentation and chromatin degradation may be either completely independent or diverge downstream of a common trigger mechanism [11, 19].
This absence of correlation between chromatin degradation and nuclear fragmentation has been already observed in other models. Hardy [23] and Hardy et al. [24] found that the morphological features of apoptosis are not always associated in human blastocysts: fragmented nuclei without a positive TUNEL signal are observed, and thus, conversely, TUNEL-positive nuclei are sometimes observed in the absence of morphological features of apoptosis. In rat blastocysts treated with TNF-
, chromatin degradation without nuclear fragmentation was induced [4].
A lot of studies reported an interdependent pathway between caspase-3 and caspase-6 for their activation of procaspase into caspase [25–27]. In some studies, caspase-6 maturation has been reported to depend on caspase-3 activation. However, in other studies, caspase-6 activation occurred without caspase-3 activation, before caspase-3 activation, or could even activate the caspase-3. The inhibition of caspase-6 activity in rat blastocyst exposed to high glucose did not prevent the caspase-3-dependent-chromatin degradation. These findings support the idea that the activation of caspase-3 is independent of caspase-6 activity in rat blastocysts.
In conclusion, our data show that caspase-6 is expressed in the rat blastocyst and that its activity is not cardinal for the induction of chromatin degradation in rat blastocysts exposed to high glucose. In contrast, caspase-6 seems to play a role in the nuclear fragmentation, indicating that these two nuclear damages are induced separately during apoptosis caused by high glucose concentration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: René De Hertogh, Unité de diabétologie et nutrition, UCL/DIAB 5474, Av. Hippocrate, 54, B-1200 Brussels, Belgium. FAX: 32 2 764 5418; dehertogh{at}obst.ucl.ac.be
Received: 5 August 2002.
First decision: 27 August 2002.
Accepted: 6 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in vitro. Development 1997 124:4827-4836[Abstract]This article has been cited by other articles:
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