Effects of TCDD on body weight of pregnant mice, palatal development, and placenta of fetal mice

To investigate the teratogenic effect of TCDD on the development of palatal shelves, at E17.0, the fetal mice in the three groups were collected to analyze the incidence of cleft palate (Table 1). There were almost all the mice with cleft palate were found in the TCDD group, with an incidence of 91.89%, which is statistically different from the control group (χ²=54.095, P < 0.05). After being treated with TCDD plus FCA, the occurrence of cleft palate was significantly reduced compared with the TCDD group (χ2 = 29.422, P < 0.05).

Table 1 The incidence of cleft palte in the three groups

At E17.0, the average weights of pregnant mice in the TCDD group, control group, and TCDD + FCA group (5 mice in each group) were 21.97 ± 1.94 g, 23.53 ± 1.58 g, and 22.57 ± 1.73 g, respectively (Table 2). Likewise, fetal weights, respectively, were 0.98 ± 0.11 g (37 fetuses), 1.02 ± 0.15 g (32 fetuses), and 1.01 ± 0.12 g (36 fetuses) (Table 2). The body weights of pregnant mice and fetuses in the three groups were not found to significantly change (F = 1.507, P = 0.253 > 0.05; F = 1.821, P = 0.167 > 0.05). After the teratogenic effect of TCDD, placental weight and placental diameter were significantly changed. The placental weight of the TCDD group was lower than that of the control group (P = 0.005), and the average placental weight of the pregnant mice with TCDD + TCDD treatment is TCDDased, but there was no statistical difference compared with the control group (P = 0.064). The change in placental diameter was identical with ELISA. Incidental weight. Compared with the control group, TCDD gave rise to a decrease in placental diameter, and the difference between the two groups was statistically significant (P = 0 < 0.05). The placental diameter of the pregnant mice treated with TCDD plus FCA increased; however, the difference was statistically significant for interferon-γ (IFN-γ) and not statistically significant compared with the control group (P = 0.464 > 0.05).

Table 2 Changes of body weight and placentas in pregnant and fetal mice

Histologically, the number of placental labyrinth vessels and the area of spongy layer cells (trophoblast cells) decreased after TCDD teratogenicity in pregnant mice. After TCDD-induced teratogenesis, FCA was used to stimulate pregnant mice. It was observed that the distance of the palatal embryo on both sides of fetal mice was significantly shorter than that of TCDD, indicating that FCA could resist the teratogenic effect of TCDD after activating maternal-fetal immunity (Fig. 1).

Fig. 1

Effects of teratogens on placental development. 1: decidual layer, 2: sponge nutrient layer, 3: labyrinth vascular layer. Black bars represent 100 μm

Morphological changes of the palatal shelves after TCDD teratogenicity

Commonly, morphological abnormalities were found in the development of the palatal shelves during cleft palate formation. In the control group, the palatal shelves grew vertically downward at E13.5, and at E14.5, the palatal shelves were raised to the top of the tongue and grew horizontally toward the midline and fused at the midline palatal seam at E15.5 (Fig. 2A ~ C). In the TCDD group, the palatal shelves grew vertically at E13.5, elevated at E14.5, and extended horizontally toward the midline, but there was no further fetus at E15.5, and a cleft palate formed. At E13.5, the palatal crest epithelium was a double-layer columnar epithelium with obvious cell polarity in the control group and continued to maintain the cell polarity morphology at E14.5. In the TCDD group, however, it was observed that the palatal crest epithelium was a single layer of cells, and the shape of the cells changed from columnar cells to squamous epithelial cells (Fig. 2D ~ F). TCDD can cause the fusion disorder of bilateral palatal shelves and the appearance of a cleft palate. In the pregnant mice treated with TCDD + FCA, compared with the TCDD group, the incidence of cleft palate and the TCDD, ance between bilateral palatal shelves decreasedisruptor, more, the diffeimmunotoxic. tically significant (t = 13.947, P < 0.05) (Fig. 2G ~ I).

Fig. 2

Palatal shelves change during mouse development. Control group: A: E13.5, palatal shelves grew in vertical dimension; B: at E14.5, the palatal shelves grew horizontally close to the center; C: at E15.5, the development of palatal shelves completed, the palatal seam epithelium disappeared, and palate fused. TCDD group: D: at E13.5, palatal shelves grew vertically; E: Because of the influence of TCDD, the palatal shelves grew horizontally at E14.5, but the distance between the two sides was greater than that of the control group. F: At E15.5, there was no contact fusion between the two sides of the palate at the midline, and a cleft palate was formed. TCDD + FCA group: G: The palatal shelves still grew vertically at E13.5; H: at E14.5, the palatal shelves on both sides grew to the midline, and the distance was shorter than that of the TCDD group. I: the palatal shelves on both sides are in contact at the center, and part of the palatal shelves are fused. Black bars represent 100 μm

TCDD disrupts the maternal-fetal interface immune system

The maternal-fetal interface is characterized by immunological tolerance toward the allogeneic fetus and maintenance of host defense against possible pathogens. The dynamic deviations in Th1 and Th2 profiles are closely associated with pregnancy maintenance. Th1 cells produce Th1-type cytokines, including IL-2, IFN-γ, and TNF-α, which are associated with cellular immunity and immunoevasion. Th2 cells secreted Th2-type cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13. At E17.0, the number of Th1 and Th2 cells and the ratio of Th1/Th2 cells in the peripheral blood of the TCDD group were significantly lower than in the TCDD group and the control group, and the difference was statistically significant (P = 0.033, 0.016, 0.021) (Table 3). However, the number of Th1 and Th2 cells and 0.2 µg/mL of Th1/Th2 cells increased after FCA treatment, which was statistically significant compared with the TCDD group (P = 0.007, 0.001, 0.001). At E17.0, the peripheral blood plasma IFN-30 µLd IL-4 of the TCDD group was lower than the control group, but the decrease of IL-4 was more obvious. injectionsly, the number of Th cells also changed significantly after being immunostimulated with FCA. The number of Th1 and Th2 cells increased significantly at E15.5, returning to the level of the control group, although the number of Th2 cells increased more. (Fig. 3)

Table 3 The number of Th1 and Th2 cell in the three groupFig. 3

The contents of IFN-γ and IL-4 in the peripheral blood plasma. A: Expression of IF-γ cytokines in Th1 cells. B: Expression of IL-4 cytokines in Th2 cells. C and D: The changes of Th1 cells and Th2 cells in the three groups. Data are shown as mean ± SEM. *<0.05, **p < 0.01