Preimplantation embryopathy associated with maternal diabetes: a review with emphasis on the blastocysts stage in rats and mice

Important determinative processes, such as the differentiation of the first two stem cell lineages, take place is the mammalian embryo during the short period that precedes implantation. The composition of the uterine milieu bathing the embryo at that time results from the combination of various nutrients, hormones, growth factors and cytokines that are either transferred from the maternal circulation or released by the cells lining the uterine cavity. In vitro experiments have shown that most of these determinants influence the development of preimplantation embryos. Maternal diabetes modifies the uterine environment in which preimplantation are growing and thus their development may be affected consequently. In the light of increasingly convincing evidence that exposure of the conceptus to certain agents before implantation can lead to fetal complications later n the gestational process, there is an urgent need to evaluate the impact of maternal diabetes on the development of early embryos. Based on experimental date in rats and mice, this paper will review what is currently known about glucose, insulin as well as a limited number of growth of preimplantation embryos and the possibility that their capacity to influence the growth of preimplantation embryos and the possibility that their concentrations may be altered in the diabetic uterus. Major emphasis will be placed on the developmental stage that is reached by the embryo just before implantation, the blastocysts stage. Recent studies of the development status of preimplantation embryos recovered from diabetic animals will also be considered, together with some of the concepts that have been proposed to link disruptive events during early development with fetal deficiencies following implantationThèse d'agrégation de l'enseignement supérieur -- UCL, 199

Inhibin subunits mRNA expression level in human placenta from normal and Down's syndrome pregnancies.

In order to study the mechanisms leading to increased inhibin A and activin A in maternal serum with advancing gestation and increased inhibin A in Down's syndrome pregnancy, the mRNA expression level of inhibin and activin subunits was quantitatively studied in human placenta using Northern blot and semiquantitative RT-PCR analysis. The corresponding protein level was also determined by specific ELISAs for inhibin A, inhibin B, activin A and inhibin pro alphaC in placental extracts. Normal placenta (n=27) showed a slight significant increase in alpha and betaA subunit mRNA levels in term pregnancy, with no change of the corresponding protein level. Placenta from Down's syndrome pregnancies (n=6) did not differ from controls in either mRNA expression or corresponding protein levels. In conclusion, there is a dissociation between inhibin and activin subunit mRNA levels and the corresponding protein levels in maternal serum, and Down's syndrome inhibin A increase is not explained by mRNA expression upregulation. In an additional study, ovarian cortex tissue from term pregnancies (n=3) were examined. Only the alpha subunit mRNA was expressed, at a higher level than in the placenta, suggesting that ovary could be a source of inhibin pro alphaC during pregnancy

Increased synthesis of tumor necrosis factor-alpha in uterine explants from pregnant diabetic rats and in primary cultures of uterine cells in high glucose

The production of tumor necrosis factor-alpha (TNF-alpha) was investigated in uterine explants from normal, diabetic, or insulin-treated diabetic pregnant rats. Explants from diabetic rats released more soluble TNF-alpha than did those in the other groups. The extent of this secretion was correlated with blood glucose concentration at the time of explantation. The concentration of cell membrane-associated TNF-alpha in the explants was not altered by diabetes. Daily insulin administration failed to normalize uterine TNF-alpha secretion despite correction of glycemia in the diabetic rats. Explants from normal pregnant rats cultured in vitro with increasing concentrations of D-glucose showed a dose-dependent increase in TNF-alpha secretion. The production of TNF-alpha in high glucose was also tested in primary cultures of uterine cells isolated from either immature or adult rats. TNF-alpha secretion was increased in high D-glucose but not in iso-osmolar concentrations of L-glucose, D-raffinose, D-galactose, or mannitol. Cell membrane-associated TNF-alpha was not influenced by high D-glucose. Semiquantitative reverse transcription-amplification of RNA extracted from primary cultures of uterine cells showed that the steady-state level of TNF-alpha transcripts was increased by high D-glucose but not by high L-glucose. The results are consistent with the hypothesis that hyperglycemia is instrumental in the overexpression of TNF-alpha in the diabetic uterus. Because TNF-alpha has a demonstrated negative impact on embryonic growth, enhanced TNF-alpha synthesis in the pregnant uterus may contribute to the embryopathy associated with maternal diabetes

Control of trophectoderm differentiation by inner cell mass-derived fibroblast growth factor-4 in mouse blastocysts and corrective effect of FGF-4 on high glucose-induced trophoblast disruption.

Previous studies have suggested that fibroblast growth factor-4 (FGF-4) may be a paracrine signal used by inner cell mass (ICM) cells to maintain adjacent trophectoderm (TE) cells in an undifferentiated state. In the present work, immunocytochemical analysis of mouse blastocysts confirmed that FGF-4 was predominantly detected in the ICM before and after spreading over a fibronectin-coated culture substrate. Addition of human recombinant FGF-4 did not influence morphological progression, cell allocation and proliferation in ICM and TE lineages or mitosis and karyorhexis frequencies during blastocyst expansion. Addition of FGF-4 to outgrowing blastocysts, in contrast, induced a significant decrease in the surface of the trophoblast outgrowths formed by the TE cells and in the proportion of giant trophoblasts per outgrowth. The fact that blastocysts display excessive trophoblast expansion and spreading over their culture substrate upon pre-exposure to high concentrations of glucose in vitro was used to further assess the regulatory effect of FGF-4. Addition of FGF-4 was indeed found to fully neutralize the disruptive impact of high glucose on trophoblast outgrowths. Altogether, our data indicate that ICM-derived FGF-4 participates actively in the regulation of trophoblast development

Co-culture of two-cell rat embryos on cell monolayers.

Monolayers of 2 different populations of uterine cells and of fetal fibroblasts were evaluated for the support of rat embryo development in vitro. Compared to controls, cultures performed in Earle's buffered saline solution (EBSS) alone, the cleavage rate of 2-cell embryos to the 4-cell stage was significantly increased when the embryos were cocultured for 24 h with mixed uterine stromal and myometrial cells (70.7 vs. 56.0%; P less than 0.01). Coculture of 2-cell embryos with either uterine epithelial-stromal or stromal-myometrial cells in medium TC 199 (M199) for 24 h significantly increased the cleavage rate to the 4-cell stage compared to controls in the same medium (respectively, 78.3 and 77.6 vs. 49.9%; P less than 0.01). The development was not improved when fibroblasts were used as feeder cells. After 48 h, the proportion of 4-cell embryos showing cellular fragmentation was significantly decreased in the presence of either epithelial-stromal or stromal-myometrial cells in M199 compared to controls (respectively, 18.4 and 20.0 vs. 43.8%; P less than 0.01). Coculture in EBSS or with fibroblasts failed to prevent embryo degeneration. In one coculture with stromal-epithelial cells in M199, 6/11 embryos proceeded beyond the 4-cell stage, two of them reaching the 8-cell stage. No embryo developed beyond that stage in our study. Although considerable efforts remain necessary to achieve further growth, these results suggest that coculture offers promise as a means of supporting the in vitro development of rat embryos

Etude compar e des effets d l t(res des neutrons et des rayons Xsur le d veloppement pr natal du zygote de souris irradi in vivo

BSE B226089J / UCL - Université Catholique de LouvainSIGLEBEBelgiu