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. 2015 Feb 15;8(2):1669-76.
eCollection 2015.

Expression of the P2Y2 receptor in the terminal rectum of fetal rats with anorectal malformation

Yuan-Mei Liu  1 , Meng Kong  1 , Zhu Jin  1 , Ming-Mei Gao  1 , Yan Qu  1 , Ze-Bing Zheng  1
Affiliations

Expression of the P2Y2 receptor in the terminal rectum of fetal rats with anorectal malformation

Yuan-Mei Liu et al. Int J Clin Exp Med. .

Abstract

Objective: The expression and distribution of a subtype of purine receptors (P2Y2) in the terminal rectum of fetal rats with anorectal malformations (ARM) were examined to investigate their possible impact on the development of the enteric nervous system (ENS).

Methods: Pregnant Sprague-Dawley rats were randomly divided into a control group (5 rats) and an experimental group (20 rats). The experimental group was treated with ethylene thiourea (ETU). On gestational day 20, the intrauterine fetal rats were collected from both groups of pregnant rats. Sagittal sections of the pelvic perinea were stained with HE. P2Y2 protein and mRNA expression in the terminal recta of the fetal rats in the control group, the ARM group, and the ETU-treated group that exhibited no malformations (the ETU group) were detected by immunohistochemistry, western blot, and qRT-PCR.

Results: The fetal rats in the control group showed normal position of the anal opening, with no malformation. The incidence of ARM was 89.2% for the fetal rats in the experimental group. The immunohistochemistry results showed that P2Y2 was expressed in the cytoplasm of the cells in the terminal rectum submucosa and myenteric plexus of the fetus rats in the control group, the ETU group, and the ARM group. The average integrated optical density (IOD) value for the ARM group was significantly lower than the IOD value for the control and ETU groups (186.48 ± 23.03 vs. 493.18 ± 19.70; 186.48 ± 23.03 vs. 479.48 ± 41.71, P<0.01), while the IOD value for the ETU group was comparable to the control group IOD (493.18 ± 19.70 vs. 479.48 ± 41.71, P = 0.360). The western blot and qRT-PCR results showed that the P2Y2 protein and mRNA expressions were significantly lower in the terminal rectum of the fetal rats in the ARM group than in the control and ETU groups (0.28 ± 0.08 vs. 0.51 ± 0.10, 0.28 ± 0.08 vs. 0.48 ± 0.12; 48.91 ± 12.17 vs. 98.03 ± 15.68, 48.91 ± 12.17 vs. 92.53 ± 10.43; P<0.01), while the P2Y2 protein and mRNA levels in the control group were comparable to the ETU group (0.51 ± 0.10 vs. 0.48 ± 0.12, P = 0.494; 98.03 ± 15.68 vs. 92.53 ± 10.43, P = 0.058).

Conclusion: P2Y2 may participate in and affect the development of ENS in the terminal rectum of fetal rats with ARM.

Keywords: Anorectal malformation; P2Y2; enteric nervous system; fetal rats.

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Figures

Figure 1
Figure 1
A. Sagittal plane from the control group: normal anal opening, with a clear outline; B. Sagittal plane of the ETU group: normal anal position and opening, with observable meconium in the rectum; C. Sagittal plane of the ARM group: the anal opening was a closed blind end.
Figure 2
Figure 2
A. Cross-section from the normal group: the terminal rectum submucosa and myenteric plexus exhibited a large number of darkly staining spherical or ellipsoid neurons with a large nuclear volume, abundant cytoplasm and clear axons; B. Cross-section from the ETU group: a large number of darkly staining neurons with a large nuclear volume; C. Cross-section from the ARM group: a smaller number of neurons, with smaller nuclear size, shrinkage, light staining, and irregular shapes.
Figure 3
Figure 3
Comparison of the number of submucosal and myenteric neurons from the fetal rats in the normal group (A), the ETU group (B), and the ARM group (C).
Figure 4
Figure 4
A. High expression of P2Y2 in the control group; B. High expression of P2Y2 in the ETU group; C. Low expression of P2Y2 in the ARM group.
Figure 5
Figure 5
Comparison of the IOD values for P2Y2 in the terminal rectum of fetal rats in the control group (A), the ETU group (B), and the ARM group (C).
Figure 6
Figure 6
P2Y2 and β-actin expression levels in the three groups. 1 and 2: the ARM group; 3 and 4: the ETU group; 5 and 6: the control group.
Figure 7
Figure 7
Comparison of the P2Y2 protein expression in the terminal rectum of fetal rats from the control group (A), the ETU group (B), and the ARM group (C).
Figure 8
Figure 8
Comparison of the P2Y2 mRNA expression levels in the terminal rectum of fetal rats in the control group, the ETU group, and the ARM group.
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