Puberty is a critical window for the impact of diet on mammary gland development in the rabbit

Abstract

Background: Nutritional changes can affect future lactation efficiency. In a rabbit model, an obesogenic diet initiated before puberty and pursued throughout pregnancy enhances mammary differentiation, but when started during the neonatal period can cause abnormal mammary development in early pregnancy. The aim of this study was to investigate the impact of an unbalanced diet administered during the pubertal period only.

Results: Consuming an obesogenic diet at puberty did not affect either metabolic parameters or certain maternal reproductive parameters at the onset of adulthood. In contrast, at Day 8 of pregnancy, epithelial tissue showed a lower proliferation rate in obesogenic-diet fed rabbits than in control-diet fed rabbits. Wap and Cx26 genes, mammary epithelial cell differentiation markers, were upregulated although Wap protein level remained unchanged. However, the expression of genes involved in lipid metabolism and in alveolar formation was not modified.

Conclusion: Taken together, our results demonstrate that the consumption for 5 weeks of an obesogenic diet during the pubertal period initiates mammary structure modifications and affects mammary epithelial cell proliferation and differentiation. Our findings highlight the potentially important role played by unbalanced nutrition during critical early-life windows in terms of regulating mammary epithelial cell differentiation and subsequent function in adulthood.

Keywords: diet; mammary epithelial cells; mammary gland; puberty; rabbit.

Figure 1

Average weight (A) and food intake (B) of female receiving either CD (n = 9) or OD (n = 7) during the peri‐pubertal period. Data are expressed as means ± SEM

Figure 2

Histological analyses of mammary gland on Day 8 of pregnancy. Hematoxylin and eosin staining of 5‐μm mammary sections observed at different magnifications from CD (A,C) and OD (B,D) fed rabbits. Representative scans of total (A,B) and low magnified (C,D) sections. The scale bars represent 2 mm (A,B) and 100 μm (C,D). Relative quantification of mammary gland tissues (E). Data are expressed as means ± SEM. Significant differences (P < .05) between the groups are indicated by asterisks (*). Number of animals per group: CD: n = 9; OD: n = 7

Figure 3

Mammary epithelial enrichment by laser capture microdissection. A, Cresyl violet‐stained rabbit mammary gland sections on Day 8 of pregnancy at low magnification (×20). The nuclei of epithelial and myoepithelial cells are in blue. B, High magnification (×120) of epithelial alveoli isolated by LCM

Figure 4

Characterization of mammary cells captured by LCM. Analyses were performed in the two groups of rabbits according to the diet received during the 8 to 13 week period (CD: n = 9; OD: n = 7). RT‐PCR were assessed for specific markers from myoepithelial cells (Krt14) and epithelial cells (Krt8). Data are expressed as means ± SEM

Figure 5

Expression of genes in MEC captured by LCM. Analyses were performed in the two groups of rabbits according to the diet received during the 8 to 13 week period (CD or OD). The expression of transcripts was assessed for κ casein, Wap, Lalba, Scd, FasN, Elf5, Ki67, Itgb1, ZO‐1, and Cx26 and normalized with Tbp as the housekeeping gene. Data are expressed as means ± SEM. Significant differences (at least P < .05) between the groups are indicated by asterisk (*)

Figure 6

Immunolocalization and quantitative expression of Wap protein in rabbit mammary tissue on Day 8 of pregnancy. (A) Wap localization (green) was performed in CD and OD mammary tissue. DAPI was used to stain the nuclei (blue). Scale bar = 20 μm. (B) Western blot analysis of Wap and β‐actin proteins in CD and OD mammary gland and histogram quantification. Quantification was performed with all animals (CD: n = 9; OD: n = 7); one representative blot is shown. Data are expressed as means ± SEM

Figure 7

Proliferative and apoptotic status of mammary epithelial tissus on Day 8 of pregnancy. A, Ki67 immunostaining (green) was performed on mammary sections in CD and OD animals. Histogram summarizes the percentage of Ki67 positive nuclei in all animals (CD: n = 9; OD: n = 7) of both groups. Concerning tData are expressed as means ± SEM. Significant differences (at least P < .05) between the groups are indicated by asterisks (*). (B) TUNEL staining (red) was performed on mammary sections of CD and OD animals. Negative (C−) and positive (C+) controls are shown. DAPI was used to stain the nuclei (blue). Magnification ×400. Scale bar = 20 μm

Figure 8

Immunolocalization of E‐cadherin in rabbit mammary tissue on Day 8 of pregnancy. E‐cadherin localization (green) was performed in CD and OD mammary tissue. DAPI was used to stain the nuclei (blue). Scale bar = 20 μm