Effect of Maternal Gradient Nutritional Restriction during Pregnancy on Mammary Gland Development in Offspring

Abstract

We aimed to investigate the effect of different levels of nutritional restriction on mammary gland development during the embryonic period by gradient nutritional restriction in pregnant female mice. We started the nutritional restriction of 60 female CD-1(ICR) mice from day 9 of gestation based on 100%, 90%, 80%, 70% and 60% of ad libitum intake. After delivery, the weight and body fat of the offspring and the mother were recorded (n = 12). Offspring mammary development and gene expression were explored by whole mount and qPCR. Mammary development patterns of in offspring were constructed using Sholl analysis, principal component analysis (PCA) and regression analysis. We found that: (1) Mild maternal nutritional restriction (90-70% of ad libitum intake) did not affect offspring weight, while body fat percentage was more sensitive to nutritional restriction (lower at 80% ad libitum feeding). (2) A precipitous drop in mammary development and altered developmental patterns occurred when nutritional restriction ranged from 80% to 70% of ad libitum intake. (3) Mild maternal nutritional restriction (90% of ad libitum intake) promoted mammary-development-related gene expression. In conclusion, our results suggest that mild maternal nutritional restriction during gestation contributes to increased embryonic mammary gland development. When maternal nutritional restriction reaches 70% of ad libitum intake, the mammary glands of the offspring show noticeable maldevelopment. Our results help provide a theoretical basis for the effect of maternal nutritional restriction during gestation on offspring mammary development and a reference for the amount of maternal nutritional restriction.

Keywords: mammary development; nutritional restrictions; offspring; pregnancy.

Figure 1

Body weight and body fat of gestational nutritional restriction female mice. (A) Body weight of female mice during gestation. (B) Postpartum weight of female mice. (C) Body fat percentage and X-ray images of female mice after parturition. Identical letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05), determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Different colors represent different groups. Asterisk stands for p < 0.05 and no asterisk means no significant difference.

Figure 2

The number, body weight, body fat and X-ray images of maternally nutritionally restricted offspring. (A) Litter size in different nutrient-restricted groups. (B) Birth weight of offspring mice in different nutritionally restricted groups. (C) Body fat percentage of offspring mice in different nutrient restriction groups. (D) X-ray images of offspring mice in different nutritionally restricted groups. Identical letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05), determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Different colors represent different groups.

Figure 3

Whole-mount and Sholl analysis of #4 inguinal mammary glands from maternally restricted offspring. (A) Whole-mount image of mammary glands in different nutrient-restricted groups. (B) Bubble plot of mammary gland Sholl analysis. (C) Linear Sholl plots of mammary glands. (D) Principal component analysis (PCA) biplot of mammary gland Sholl results.

Figure 4

Regression analysis of mammary whole-mount results from maternally restricted offspring. Regression analysis between (A) mammary enclosing radius and mammary epithelial area (MEA), (B) mammary enclosing radius and sum inters and (C) mammary enclosing radius and Sholl regression coefficient (k). The dots represent individual measured values, the black lines represent linear regressions, and the gray areas represent their 95% confidence intervals.

Figure 5

Mammary gland proliferation- and hormone-related gene expression in offspring. Identical letters or no letters are not significant difference (p > 0.05), while different letters indicate significant difference (p < 0.05) determined by one-way ANOVA followed by a Sidak multiple-comparison test. Bar charts represent mean, error bars represent SD. Sox10: SRY (sex-determining region Y)-box 10. Axin2: Axis inhibition protein 2. Elf5: E74-like factor 5. Aldh1a1: aldehyde dehydrogenase family 1. Wnt5a: wingless-type MMTV integration site family, member 5A. Lgr5: leucine-rich repeat-containing G-protein-coupled receptor 5. Procr: protein C receptor. ERα: estrogen receptor 1 (alpha). ERβ: estrogen receptor 2 (beta). Pr: progesterone receptor. K18: keratin 18. K5: keratin 5.