Possible mechanisms of action of diabetes on the early embryo

Several mechanisms were shown to play a role in diabetes-induced early embryopathy in animals. Elucidation of these mechanisms of action may help to understand the human situation. We will, therefore, discuss these mechanisms mainly based on experimental animal models.

Apoptosis

Hyperglycemia induced excessive cell death in the ICM of rat blastocysts, which was characterized mainly by nuclear fragmentation. It was shown that these cells contain a large amount of the clusterin transcripts, a gene associated with apoptosis. The over-expression of clusterin in blastocysts of STZ diabetic rats indicates that these embryos may be affected by subtle disruptions in the expression pattern of critical developmental genes.16 Similar to the previous study, blasto-cysts from diabetic rats and mice showed increased nuclear chromatin degradation in the ICM cells.17,18 These and other studies demonstrate that in pre-implantation embryogenesis, hyperglycemia triggers increased apoptosis, especially at the blastocyst stage. Maternal hyperglycemia caused a decrease in the expression of facilitative glucose transporter genes such as GLUT1, GLUT2, and GLUT3, which was associated with the reduction in glucose transport to the embryo and a decrease in intra-embryonic free glucose.18 This mechanism acts as a signal for cell death which triggers p53 activity. The hyper-glycemia-induced cell death signal increases the expression of the pro-apoptotic protein BAX, which is a member of the Bcl-2 family.18 In addition, mRNA levels and protein expression of BAX were higher in blastocysts cultured in hyperglycemic culture medium or in blastocysts obtained from STZ-induced diabetic mice.18 In rat blastocysts in culture, transcription of the Bcl-2 gene and the activity of the protein were also markedly increased in the presence of high glucose concentrations in the culture medium.19 These events induced by hyperglycemia, lead to activation of caspases, to DNA fragmentation, and morphologic changes consistent with apopto-sis. This cascade (Figure 22.1), connecting the expression of Bcl-2 and/or BAX with cell death signals involving p53, suggests that the hyperglycemia induces embryonic hypoglycemia due to the decrease in the glucose transport expression and induces the p53 apoptotic cascade.18,19

Of the different glucose transporters existing in the early embryo, GLUT8 was recently found to be one of the most important.20 This transporter is regulated by insulin. During early differentiation of the mouse blastocyst there is a significant increase in glucose demand, and insulin causes GLUT8 to

Figure 22.1 Hyperglycemia-induced apoptosis. (Modified from Keim et al.21)

move to the plasma membrane, thus increasing the uptake of glucose, which is then converted to lactic acid. It is presumed that, similar to other glucose transporters, hyperglycemia decreases GLUT8 hence reducing the uptake of glucose by the ICM, inducing cell death.

Mouse blastocysts, genetically BAX deficient (BAX-/-) obtained from diabetic dams, showed lower chromatin degradation and apoptosis than BAX-positive (BAX+/+) embryos. Furthermore, the embryos from the BAX deficient diabetic mice had lower rates of malformations and resorption on day 14 of pregnancy.18 These data propose that increased apopto-sis in the blastocysts might indicate future increased early embryonic death or malformations, as observed in diabetic pregnancies.

The second apoptotic compound, Bcl-2, belongs to a family of proteins that operate in the effectors phase of apoptosis and may either promote or inhibit apoptosis. An increase in the expression of the anti-apoptotic Bcl-2 mRNA was observed in rat blastocysts which were cultured in 28 mM glucose for 24 h, compared to blastocysts incubated in 6 mM glucose.19 When the Bcl-2 expression was inhibited, using antisense oligodeoxynucleotide, there was an increase of chromatin degradation in blastocysts incubated in high glucose concentrations. The addition of specific inhibitors to caspase-3 and caspase-activated-deoxyribonuclease (CAD) prevented the degradation of rat blastocysts.19

A third apoptotic compound, clusterin, a disulfide-linked heterodimeric protein associated with the clearance of

Possible mechanisms of action of diabetes on the early embryo 169

cellular debris and apoptosis, was twice higher in embryos of diabetic rats than in control embryos.17 When rat and mouse blastocysts were incubated with high glucose concentrations, there was an increase in BAX, in clusterin expression and nuclear chromatin degradation.18,19

It can be concluded that the significant loss of progenitor cells from the ICM makes the embryos more sensitive to later developmental deficiencies. Furthermore, it was reported that normal embryogenesis can occur only if sufficient number of functional ICM cells are available.21 Increased apoptosis at this early stage of development may lead to spontaneous miscarriage or congenital malformations. Figure 22.1 summarizes the steps leading to diabetes-induced increased apoptosis.

Oxidative stress and antioxidants

Many studies implied that the causes of diabetic embryopathy may be secondary cellular damage from overproduction of reactive oxygen species (ROS) or/and decreased antioxidant defense mechanism in the embryonic cells.15,22-24 The source of ROS is complex and non-specific. The main question is whether deranged oxidant antioxidant status can occur at this early stage of pre-implantation embryonic development. We found that serum from diabetic women can induce oxidative stress in the mouse blastocysts,15 apparently in a way similar to that induced in post-implantation embryos.22 This was evidenced by reduced concentrations of low molecular weight antioxidants (LMWA) such as glutathione and vitamins C and E. The pre-implantation mouse embryos cultured in serum from diabetic pregnant women had lower concentration of LMWA compared to embryos cultured in serum from nondiabetic women. It seems, therefore, that diabetic metabolic factors may induce embryotoxicity in pre-implantation embryos through derangement of the antioxidant defense mechanism. Leunda-Casi et al.24 found that hyperglycemia may increase ROS generation, and this might be one of the reasons for increased cell apoptosis as evidenced in this study by the TUNEL technique. Similar findings were reported by others in mouse zygote and blastocysts.25 One of the key mediators that was suggested as essence for apoptosis is hydrogen perox-ide.26 Violation of this balance by high glucose concentrations can cause massive cell damage, increase in apoptotic events and defective embryonic development. When there are high levels of glucose they need to be degraded, and the result is a high production of ketone bodies and an increase in the production of ROS.

Fertilization and embryonic development take place in an environment of low oxygen tension. Oxygen tension is gradually increasing with advanced gestation, once placentation is well established and maternal uterine arterioles are not obliterated by trophoblastic cells.27,28 Oxidative stress also seems to play an important role in the early phases of embryonic development and hence antioxidants may play a significant role in preventing damage to the embryos.13,22,26-28

ROS mediate their action through many of the proinflammatory cytokines. They can influence the oocyte, sperm and embryos. During pregnancy, there are increased numbers of polymorphonuclear leucocytes (PMNL) which can cause superoxide ions increase. This oxidative stress may regulate the expression of cytokine receptors in the placenta, cytotrophoblastic cells, vascular endothelial cells, and smooth muscle cells. In addition, several studies have also demonstrated the significant role of free radicals in placental function. Oxidative damage to the trophoblastic cells early in pregnancy or to the placenta during the establishment of its maternal circulation, may also cause early pregnancy loss.26-28

Growth factors and cytokines

Cytokines and growth factors play an active role in the normal implantation process. They also have important roles in the pathogenesis of diabetes-induced organ damage, and those that interrupt the reproductive tract are able to cause pre-implantation embryopathy.29-31 There are only few reports that refer to cytokines expression in the diabetic uterus. Insulin-like growth factor 2 (IGF-II) synthesis is down-regulated and, to the contrary, tumor necrosis factor-alfa (TNF-a) synthesis is up-regulated around the implantation sites in diabetic rat females.32,33 Wuu et al.34,35 showed a correlation between reduction in the concentrations of mRNA encoding IGF-II and embryonic growth retardation 2 days after initiation of implantation in C57B1/6J pregnant mice. However, later observations demonstrated that maternal diabetes did not affect the uterine IGF-I expression. Detection of TNF-a revealed over-expression in the mRNA as well as in the protein concentrations in the pre-implantation uterus of STZ-induced diabetic Wistar rats.32 The majority of TNF-a protein synthesis was located in the epithelium lining the uterine lumen. Despite normalization of glycemia by addition of insulin to the diabetic animals, it did not prevent the overproduction of TNF-a in the uterus.33 Incubation of rat and mouse uterine cells in different glucose concentrations induced stimulation of TNF-a secretion in uterine epithelial cells, apparently mediating the release of other cytokines, i.e. interleukin ip from the subepithelial population of macrophages after their direct activation by hyperglycemia. There is evidence that high levels of TNF-a in utero can be harmful to the embryonic development at the implantation phase. Embryos exposed to high levels of TNF-a and surviving to term were significantly smaller than control embryos.34-36 Mouse embryonic stem cells (ES) indicate that TNF-a inhibit cell proliferation in the ICM lineage and decreased their differentiation potential.35 Further evidence for the hypothesis that TNF-a contributes to the harmful influence of maternal diabetes on pre-implantation development was found in blastocysts from Wistar rats. Culture medium was produced from normal and diabetic uterine cells; blastocysts of Wistar rats incubated in these media showed diminished growth in the diabetic medium, but improved significantly (not completely) by pre-treating the blastocysts with anti-sense oligonucleotide which blocked the embryonic TNF-a receptors.36

To conclude, there is increased secretion of TNF-a in diabetic rats and mice at the pre-implantation period which may harm the embryos, interfering with normal embryonic growth.

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