Impact of glucose on rat embryogenesis : apoptosis in blastocysts and differentiation of trophoblast cells

Le diabète maternel est une perturbation métabolique ayant de graves conséquences sur le développement embryonnaire. Le diabète maternel ou une exposition in vitro à de hautes concentrations en glucose durant la phase pré-implantatoire provoque une diminution du nombre de cellules par blastocyste ainsi qu'une incidence accrue de la dégradation de la chromatine et de la fragmentation nucléaire, deux marqueurs nucléaires de l'apoptose. Ces perturbations pourraient engendrer des complications foetales. Durant ce travail, nous avons cherché à mieux comprendre les mécanismes par lesquels le glucose induit l'apoptose. La voie des caspases fut explorée. Nous avons montré que la caspase-3 et CAD (caspase-activated deoxyribonuclease) étaient exprimées dans le blastocyste de rat et activées in vitro par des concentrations élevées de glucose. L'inhibition de ces enzymes réduit l'incidence de l'apoptose, en réduisant la dégradation de la chromatine, mais n'a pas d'effet sur la fragmentation nucléaire. La caspase-6, exprimée également dans le blastocyste de rat, n'exerce pas d'effet sur la dégradation de la chromatine, mais pourrait intervenir sur la fragmentation nucléaire. Ces observations suggèrent l'existence de voies partiellement distinctes conduisant, d'une part, à la dégradation de la chromatine et, d'autre part, à la fragmentation nucléaire. Des travaux antérieurs ont montré que le diabète maternel affecte la phase péri-implantatoire. L'exposition au diabète entraîne des perturbations de la différenciation du trophectoderme. Celui-ci comprend une différenciation excessive des trophoblastes en cellules géantes. Lors de ce travail, nous avons utilisé la lignée cellulaire Rcho-1 exposées à de hautes concentrations en glucose sont caractérisées par un processus d'endoreduplication perturbé (chaque noyau comprend plus d'ADN et leur taille est plus petite). Nous avons également mis en évidence que de hautes concentrations en glucose provoque une diminution de la capacité steroïdogénique des cellules géantes. La sécrétion de progestérone, d'androsténédione et de DHEA est diminuée lorsque les cellules sont exposées à une haute concentration en glucose lors de leur différenciation en cellules géantes. La diminution de l'expression de l'enzyme P450scc explique en partie la diminution de la production des stéroïdes par les cellules exposées à des concentrations élevées de glucose. Des études à l'aide de précurseurs radioactifs suggèrent qu'une activité plus importante de l'enzyme P450c17 contribue à amplifier le déficit de la production de progestérone par les cellules exposées à de hautes concentrations en glucose. Les conséquences de cette réduction de production de progestérone ne sont pas claires. Il est proposé que la production locale de progestérone par le placenta de rongeur agirait comme immunosuppresseur à l'interface maternel ce qui préviendrait le rejet du foetus. Si cete hypothèse est correcte, le faible taux de progestérone produit par les cellules géantes exposées à des concentrations élevées en glucose peut expliquer en partie le taux élevé de pertes pré-natales lors de grossesses diabétiquesMaternal diabetes is a metabolic disorder with severe consequences on the embryo development. The effects induced by an exposure to high concentrations of glucose during the pre-implantation period of pregnancy include a decrease in the number of cells per blastocysts and in a concomitant increase in two nuclear markers of apoptosis : chromatin degradation and nuclear fragmentation. It is hypothesis that insufficient of cells may contribute to increasing the risk of subsequent development deficiencies. The purpose of this work is to better understand the role of caspases and caspase-activated deoxyribonuclease (CAD) in apoptosis induced by high glucose. Our data show that caspase-3 and CAD, in rat blastocysts exposed in vitro to high glucose, indicate that their activity is important in the induction of chromatin degradation but not for the nuclear fragmentation. Furthermore, specific inhibition of caspase-6 activity indicates that its activity is not cardinal for the induction of chromatin degradation, specific inhibition on the two nuclear markers of apoptosis suggest that the intracellular cascades leading to nuclear fragmentation and chromatin degradation are either completely independent or divergent downstream of a common trigger mechanism. Several reports have shown that maternal diabetes affects the peri-implantation period of pregnancy. The exposition to diabetes disturbs the proliferation and expansion of the trophectoderm which is associated with an increased number of trophoblast cells into giant cells. In this study, we have used the Rcho-1 cells line to investigate the direct impact of high giant cells. Our data show that high glucose concentration impairs several features of trophoblast differentiation. The Rcho-1 cells exposed to high glucoses were characterized by impaired endoreduplication process (more DNA per nucleus and smaller nuclear size). The functional consequence of higher DNA content remains unsettled. Indeed, the role of endoreduplication is unclear. We have also observed that a high concentration of glucose disturbed the steroidogenic capacity of giant trophoblast cells. Progesterone, androstenedione and DHEA secretions are decreased when the cells are exposed to high glucose during differentiation. The decrease of P450scc mRNA expression might explain in part the decrease of steroid production in cells exposed to high glucose. As suggested by radioactive precursors conversion studies, the higher activity of P450c17 in these cells might contribute to amplify the deficit in progesterone production in cells exposed to high glucose. The consequences of the reduced placental progesterone production are difficult to assess. Several reports propose that the local production of progesterone by placenta in rodent acts as an immunosuppressant at the maternal interface preventing the rejection of the fetal allograft. If this hypothesis is correct, the low level of progesterone produced by trophoblast cells exposed to high glucose might help to explain the high level of resorption observed in diabetic pregnanciesThèse de doctorat en sciences biomédicales (biologie du développement) -- UCL, 200

Identification of caspase-6 in rat blastocysts and its implication in the induction of apoptosis by high glucose

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 caspase-6 is expressed in rat blastocysts, using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Caspase-6 is detected in all cells of the blastocyst and is excluded from the nucleus. To assess the role of caspase-6 in the glucose-induced apoptosis, rat blastocysts were incubated for 24 h in either 6 or 28 mM glucose in the presence or absence of a specific inhibitor of caspase-6 (VEID-CHO, 100 nM). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation and nuclear fragmentation. Addition of VEID-CHO was found to inhibit nuclear fragmentation, but did not prevent the increase in chromatin degradation 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. Furthermore, nuclear fragmentation in rat blastocysts is apparently mediated by the activation of caspase-6

Activin receptor expression and induction of apoptosis in rat blastocysts in vitro.

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-beta 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 betaA 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 betaA 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

Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.

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 microM] against caspase-3 and aurin [1 microM] 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