Fig 1.
Establishment of a diabetic pregnancy model with transient reduction of blood glucose levels.
(A) A schematic diagram showing the experimental design in establishing the model. (B) Blood glucose (BG) levels in pregnant mice before treatment and after treatment with phlorizin (PHZ) or vehicle (CON) for 12 hrs. Animals with blood glucose levels ≥16.7 mmol/L (dotted line) were defined as diabetic. Data are expressed as mean ± SEM of 5–6 mice in each group. Statistical analysis was conducted using paired t-test. GD, gestational day; STZ: streptozotocin.
Table 1.
Summary of the primer sequence for real-time quantitative RT-PCR.
Fig 2.
Reduction of maternal blood glucose level by PHZ treatment up-regulates Cyp26a1 expression but not Cyp26b1 and Cyp26c1 expressions.
(A-H) Representative embryos showing the expression of Cyp26a1, detected by whole-mount in situ hybridization, in GD 9 mouse embryos after treatment of PHZ or an equivalent volume of vehicle as control (CON). (A-D) In the anterior region of embryos, Cyp26a1 was expressed in the facial and cervical mesenchyme (circled) and along the maxillary-mandibular cleft (arrowhead). (E-H) In the posterior region of embryos, Cyp26a1 was highly expressed in the tailbud region. About 15–20 embryos from 3 litters were examined in each group. Scale bar representing 0.5 mm in A-D and 0.1 mm in E-H. (I-J) The mRNA expression levels of Cyp26a1, Cyp26b1 and Cyp26c1 relative to β-actin in tailbud region of embryos (I) and embryonic trunk (J). The tailbud region was defined as the region from the caudal end to the presomitic region at a level one-somite length caudal to the last somite. Data are expressed as mean ± SEM of 5 litters in each group (embryonic tissues from the same litter were pooled as 1 sample). Statistical analysis was conducted using two-way ANOVA followed by Bonferroni test.
Fig 3.
Normalization of RA degrading efficiency in different embryonic tissues upon reduction of maternal blood glucose levels.
(A) A schematic diagram showing the experimental design of measurement of in vitro RA degrading efficiency. (B-C) RA degrading efficiency in the tailbud region of embryos (B) and embryonic trunk (C). Tissues were incubated in 50 nM RA with 1.6 mg/mL NADPH and 0.3 mg/mL DTT for 2 hrs. Data are expressed as mean ± SEM of 5–6 reactions for the tailbud region and 18 reactions for the embryonic trunk. Statistical analysis was conducted using two-way ANOVA followed by Bonferroni test.
Fig 4.
Reduction of maternal blood glucose reduces RA levels in tissues and abolishes the increased embryonic susceptibility to various RA-induced malformations in diabetic pregnancy.
(A) A schematic diagram showing the experimental design. (B-C) Amount of RA in the tailbud region of embryos (B) and embryonic trunk (C) at 3 hrs after injection of 25 mg/kg dose of RA. Data are expressed as mean ± SEM of 11–12 individual tailbud/embryonic trunk examined for each group. (D) The severity of caudal regression is represented by TL/CRL ratio. (E-F) Percentage of near-term fetuses per litter with cleft palate (E) and renal malformations (F). Data are expressed as mean ± SEM of 7–8 litters for each group. Statistical analysis was conducted using two-way ANOVA followed by Bonferroni test.
Fig 5.
Whole embryo culture of GD 9 rat embryos in varying concentrations of D-glucose for 48 hrs demonstrates a dose-dependent suppression of Cyp26a1 and caudal regression.
(A) Representative embryos showing the expression of Cyp26a1 in the anterior and posterior regions of embryos detected by whole-mount in situ hybridization. Dose-dependent down-regulation of Cyp26a1 in rat embryos cultured in serum supplemented with different concentrations (2, 3 and 4 mg/mL) of D-glucose (D-Glu) or DMEM (Control) for 48 hrs from GD 9 (equivalent to GD 7.5 of mouse embryo). Around 20–25 embryos from 3–4 litters in each group were examined. Cranial and cervical mesenchyme were circled. Scale bar representing 0.5 mm for whole embryo and 0.25 mm for posterior region. (B-C) The mRNA expression levels of Cyp26a1, Cyp26b1 and Cyp26c1 relative to β-actin in tailbud region of embryos (B) and embryonic trunk (C). Data are expressed as mean ± SEM with 7 samples for each group. Embryonic tissues from 3 embryos were pooled as one sample. Statistical analysis was conducted using Pearson’s correlation test.