Serge Pampfer

Decreased Inner Cell Mass Proportion in Blastocysts From Diabetic Rats

Late morulae and blastocysts were recovered from streptozocin-induced diabetic pregnant rats and individually examined for numbers of inner cell mass (ICM) cells and trophectoderm (TE) cells. Compared with embryos collected from control rats, exposure to maternal diabetes significantly decreased mean ICM cell number of blastocysts recovered on day 5 of gestation, but the TE population of these embryos remained unaffected. The mean ICM proportion was therefore significantly lower than that of control embryos. These differences were not observed between the two groups of morulae collected on day 5, suggesting that the distinctive susceptibility of the ICM was expressed after blastocyst formation. On day 6, a significant inhibitory effect of diabetes was observed on the growth of both ICM and TE cells, but because the reduction was more severe in the ICM than in the TE, the mean ICM proportion of these blastocysts was again significantly lower than in control embryos. A linear quadratic relationship was obtained between the numbers of ICM cells of individual blastocysts and their respective numbers of TE cells in each of the two experimental groups. However, the slope of the curve was slower in the diabetic group than the control group. The disturbed ICM cell growth in the blastocysts from diabetic rats was found to be associated with a significantly increased incidence of cell death predominantly located in the ICM. Because it is known that excessive reduction of the ICM is incompatible with normal embryogenesis after implantation, our results suggest that the differential sensitivity of ICM and TE cells in preimplantation blastocysts may contribute to the pattern of postimplantation defects described in diabetic pregnancies.

Apoptosis at the time of embryo implantation in mouse and rat.

The aim of this review is to summarize the information currently available regarding the occurrence of apoptosis in the developing embryo and in the receptive uterus during the peri-implantation period of gestation. Cell death is detected in the inner cell mass of late pre-implantation embryos as the result of an eliminative process that helps trim the embryonic cell lineages of surplus or dysfunctional stem cells. Cell death is also detected in the epiblastic core of early post-implantation embryos, where the process is implicated in the formation of the pro-amniotic cavity. On the maternal side, uterine epithelial cells situated around the attachment site undergo cell death during the initial phase of implantation in order to facilitate embryo anchorage and access to maternal blood supply. Uterine stromal cells closest to the implantation chamber first transform into decidual cells and then commit suicide to make room for the rapidly growing embryo. Although apoptosis is well recognized as a crucial determinant of successful peri-implantation development, our understanding of the cellular and molecular mechanisms regulating this process clearly lags behind the comprehension of cell death control in other systems.

Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro.

The effect of glucose and insulin on the in vitro development of the rat preimplantation embryo was studied by incubating rat blastocysts recovered on days 5 or 6 of pregnancy in the absence or presence of increasing levels of glucose and/or insulin for 24 or 48 h. A differential cell-staining method allowed the separate counting of inner cell mass (ICM) and trophectoderm (TE) cells at the end of the incubation period. In a high-glucose medium (17 mM), ICM and, to a lesser extent, TE developments were significantly and irreversibly inhibited. Low insulin concentrations (3 pM) stimulated ICM and TE development in the presence of 1.1 or 6 mM glucose. Higher insulin levels (30–600 pM) in a 6-mM glucose medium, resulted in a dose-dependent inhibition of ICM and, to a lesser extent, TE development after both 24 and 48 h. This insulin-induced inhibition was reversible if insulin was removed from the medium after 24 h. In the absence of glucose in the medium, insulin was neither stimulatory nor inhibitory on ICM growth. Dead-cell occurrence in ICM after a 48-h incubation increased with increasing glucose concentration in the medium. Insulin alone did not increase dead-cell number but enhanced the effect of glucose. These results show that, in the presence of glucose, insulin might be stimulatory (at low concentrations) or inhibitory (at higher concentrations) on ICM development. A high glucose level was also inhibitory and increased dead-cell occurrence. The data suggest that insulin and glucose might interact and modulate blastocyst development as a function of their respective concentrations.

Experimental diabetes impairs rat embryo development during the preimplantation period.

SummaryCongenital malformations and early fetal losses are still the main complications of diabetic pregnancy. Whether the diabetic state affects the early embryo development during the preimplantation period is not known. To understand better the early steps of embryo growth, we collected the embryonic structures from the uterine horns of pregnant diabetic rats on day 5 of pregnancy. Diabetes was induced by streptozotocin (50 mg/kg) injection, 7, 14 or 21 days before mating. The morphological analysis revealed a lower rate of blastocysts (72% of all structures) and an increased rate of morulae (19.5%) in diabetic rats, compared to control animals (86.7 and 7.9% respectively). Hence, diabetic rats had fewer blastocysts (5.5±2.9 per rat) and more morulae (1.5±1.7) than control animals (7.2±2.7 and 0.66±1.2 respectively). Moreover, blastocysts from diabetic rats had fewer nuclei (26.9±7.3 per blastocyst) than blastocysts from control animals (31±6.1). In another set of experiments, subdiabetogenic doses of streptozotocin were administered. In rats injected with 25 mg/kg, neither the glycaemia, nor the morphological aspects of the embryos, nor the number of blastocyst nuclei differed from the control animals. In the animals receiving 35 mg/kg, the glycaemia was increased to approximately twice the control group value. However, the embryonic morphology and the nuclei counting of the blastocysts were similar to those of the fully diabetic group injected with 50 mg of streptozotocin. These results show that experimentally induced diabetes, even of a rather mild degree, affects the embryo development during the preimplantation period. The recovered embryos appear less mature and less developed. This observation raises the possibility that diabetes induced early fetal loss and teratogenesis might, to some extent, be anticipated by environmental factors deleterious to the preimplanted embryo.

Increased cell death in mouse blastocysts exposed to high D-glucose in vitro: implications of an oxidative stress and alterations in glucose metabolism

Aims/hypothesis. Signs of apoptosis have been observed in rodent blastocysts exposed to high d-glucose concentrations in vitro. The mechanism underlying the detrimental influence of glucose remains to be identified. It has been postulated that high d-glucose concentrations induced oxidative stress in rat post-implantation embryos in vitro. A decreased glucose uptake has also been implicated in the embryotoxicity of glucose in pre-implantation mouse embryos. We examined whether the high incidence of cell death in high d-glucose-treated embryos was associated with a disrupted redox status and with alterations in glucose transport and metabolism. Methods. After blastocysts were incubated in different concentrations of d-glucose for 24 h, they were examined for the proportion of nuclei showing signs of chromatin degradation using the TUNEL technique, for the generation of reactive oxygen species and for the mitochondrial membrane potential using specific fluoroprobes and the confocal microscopy. Glucose transport and metabolism were assessed using radiolabelled 3-O-methylglucose and glucose, respectively. Results. Compared to the control blastocysts, high d-glucose-treated embryos showed a higher incidence of TUNEL-positive nuclei and reactive oxygen species generation principally in the inner cell mass cells. Decreased glucose transport and glycolytic activity but unmodified pentose phosphate pathway activity were detected in these embryos. Conclusion/interpretation. Incubation in high d-glucose concentrations in vitro increased cell death, induced oxidative stress and decreased glucose transport and metabolism in mouse blastocysts. As only glycolysis was affected, however, we suggest that metabolic inhibition occurred downstream glucose transport and glucose-6-phosphate formation. [Diabetologia (2002) 45: ▪–▪]

In vitro study of the carry-over effect associated with early diabetic embryopathy in the rat.

SummaryEmbryos were recovered from diabetic rats on day 5 of pregnancy and incubated in vitro for up to 72 h. Compared to control embryos, blastocysts from diabetic rats showed a marked impairment in growth that resulted at 48 h in a higher rate of degeneration and a lower morphological score in the developing population. After 72 h in vitro, fewer developing blastocysts from diabetic rats formed trophoblastic outgrowths and fewer of those implanted developed an inner cell mass when compared with the control group. When assessed for their cell content, blastocysts from diabetic rats contained fewer cells than control embryos at the start of the culture. This difference persisted, and even worsened, during the ensuing incubation period. The increasing cellular deficiency in blastocysts from diabetic rats was primarily located to their inner cell mass lineage but trophoblast growth was also affected. When trophoblast outgrowths were compared for their surface area and number of nuclei, those collected from diabetic rats were smaller, contained fewer nuclei and had a higher proportion of giant nuclei than control outgrowths. Our data thus demonstrate that despite their removal from the abnormal intra-uterine environment, blastocysts from diabetic rats remain functionally affected by their early exposure and fare less well than control embryos cultured under the same standard conditions.

Maternal insulin treatment improves pre-implantation embryo development in diabetic rats

SummaryPre-implantation embryos were recovered from control, diabetic and insulin-treated diabetic rats on day 5 of pregnancy. Compared to control animals, diabetic rats had a 20 % reduction in the number of embryos per rat and blastocysts recovered from diabetic rats showed a 19 % decrease in total cell number. The cellular decrease observed in blastocysts was mainly at the expense of the inner cell mass. Insulin replacement therapy was started on day 1 of pregnancy and normalized the glycaemia of diabetic rats but failed to raise the number of embryos per rat toward the control value. Insulin treatment, however, fully restored the normal cell number in both the inner cell mass and trophectoderm of blastocysts. The dead cell index, which was significantly elevated in the inner cell mass of blastocysts from diabetic rats, also returned to the control value following insulin treatment. Our data suggest that diabetes-induced impairment of pre-implantation development can be partly prevented by insulin treatment started shortly after conception.

Dysregulation of the cytokine network in the uterus of the diabetic rat.

Insulin‐dependent (type 1) diabetes is an auto‐immune disorder that produces secondary complications in numerous non‐immunological systems. Changes in the synthesis and action pattern of several cytokines have been associated with the development of these alterations. Based on the clinical facts that the pregnant and non‐pregnant functions of the reproductive system are also disrupted by diabetes, our laboratory has decided to concentrate its research activities on the hypothesis that cytokines may be implicated in the uteropathy and embryopathy associated with the metabolic disorder. This review article summarizes our major findings concerning the synthesis of TNF‐α and IL‐1β in the uterus of diabetic rats, and in cultures of rodent uterine cells upon their exposure to high concentrations of glucose. The paper also reviews evidence that both the peri‐implanting embryo and the epithelial cell layer lining the uterine lumen are targets for the deleterious influence of excess TNF‐α. If confirmed in the uterus of diabetic patients, these observations may explain how cytokines contribute to the dysregulation of crucial reproductive events like menstruation and embryo implantation in humans.

Possible role for TNF-alpha in early embryopathy associated with maternal diabetes in the rat.

Tumor necrosis factor (TNF) bioactivity was assessed in culture media conditioned with uterine cells collected from control or diabetic rats on days 5 and 8 of pregnancy. On both days, diabetic uterine cells released significantly more biologically active TNF than did control cells, and this activity was significantly decreased by the addition of anti-TNF-α antibodies but not by the addition of normal IgG when WEHI 164 cells were used as a target. When uterine tissues from day 5 or day 8 pregnant diabetic rats were tested by Northern blot analysis, TNF-α mRNAs were twofold more abundant than in control samples, but the difference was not statistically significant (P = 0.086 and 0.100, respectively). Immunohistochemical analysis of diabetic day 5 uterine sections revealed that most of the TNF-α synthesis occurs in the epithelium lining the uterine lumen. Finally, the growth of day-5 embryos in culture medium conditioned with day-5 diabetic uterine cells was significantly reduced when compared with that of embryos in medium conditioned with control cells. Embryonic development was markedly improved when anti-TNF-α antibodies were added to the diabetic-cell conditioned medium. Our data support the hypothesis that TNF-α may be implicated in the developmental deficiencies observed in preimplantation embryos from pregnant diabetic rats.

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

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.