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. 2006 Oct;147(10):4772-80.
doi: 10.1210/en.2006-0437. Epub 2006 Jul 20.

Peroxisome proliferator-activated receptor alpha activation during pregnancy severely impairs mammary lobuloalveolar development in mice

Qian Yang  1 , Reiko Kurotani, Atsushi Yamada, Shioko Kimura, Frank J Gonzalez
Affiliations

Peroxisome proliferator-activated receptor alpha activation during pregnancy severely impairs mammary lobuloalveolar development in mice

Qian Yang et al. Endocrinology. 2006 Oct.

Abstract

To identify the potential functions of peroxisome proliferator-activated receptor alpha (PPARalpha) in skin development, transgenic mice were generated to target constitutively activated PPARalpha (VP16PPARalpha) to the stratified epithelia by use of the keratin K5 promoter. In addition to marked alterations in epidermal development, the transgenic mice had a severe defect in lactation during pregnancy resulting in 100% pup mortality. In this study, the alteration of mammary gland development in these transgenic mice was investigated. The results showed that expression of the VP16PPARalpha transgene during pregnancy resulted in impaired development of lobuloalveoli, which is associated with reduced proliferation and increased apoptosis of mammary epithelia. Mammary epithelia from transgenic mice also showed a significant reduction in the expression of beta-catenin and a down-regulation of one of its target genes, cyclin D1, which is thought to be required for lobuloalveolar development. Furthermore, upon PPARalpha ligand treatment, similar effects on lobuloalveolar development were observed in wild-type mice, but not in PPARalpha-null mice. These findings suggest that PPARalpha activation has a marked influence in mammary lobuloalveolar development.

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Figures

Figure 1
Figure 1
Generation of transgenic mice and analysis of transgene expression. (A) Schematic representation of the 7-kb construct used for generating tetracycline response element-VP16PPARα (TREVP16PPARα) transgenic mice. PminhCMV, minimal human cytomegalovirus promoter. (B) VP16 immunostaining in mammary gland. Sections of Wt glands and Tg glands in the absence of dox were stained with anti-VP16 antibody. (C) PPARα immunostaining in mammary gland. Sections of Wt glands and Tg glands in the absence or in the presence of dox were stained with anti-PPARα antibody. In both B and C, positive cells were visualized with DAB (brown, shown by red arrows). Nuclei were counter-stained with hematoxylin (blue). Bars: 125 μm. (D) Northern blot analysis of RNA from day 1 of parturition. The expression of the VP16PPARα transgene was observed only in Tg animals in the absence of dox.
Figure 2
Figure 2
Impaired lobuloalveolar development in the transgenic mammary glands during pregnancy. (A) Morphological observation by whole mount. Wt and Tg mammary glands in the absence of dox at different time during pregnancy from day 12.5 to day 1 of parturition (L1) were compared. Note although continued side branching, alveolar development in Tg glands was severely hypoplastic as compared with Wt controls. (B) Histological analyses by H&E staining. Wt and Tg glands in the absence of dox at L1 were compared. Top panels are low magnification of glands. Note there is a scarce distribution of alveoli in Tg glands. Middle panels are medium magnification of glands. Note some alveolar structure was disrupted in Tg glands (representative shown by red arrow). Bottom panels are high magnification of glands. Note there are accumulated lipid droplets in Tg glands (representative shown by red arrow). Bars: (A) 1.25 mm; (B) top: 1.25 mm; middle: 250 μm; bottom: 125 μm. (C) Relative expression of milk protein genes. Expression of β-casein and WAP mRNAs were analyzed using total RNA extracts from Wt and Tg glands in the absence of dox at L1 by quantitative real-time PCR.
Figure 3
Figure 3
Impaired epithelial proliferation in transgenic mammary glands. (A) BrdU incorporation and detection. BrdU was administrated to Wt and Tg mice in the absence or in the presence of dox at L1 and the mammary glands were removed 2 hr later. BrdU incorporated into mammary glands was detected by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). (B) BrdU labeling indices. 300 - 400 epithelial cell nuclei were examined per sections of Wt and Tg glands. The values represent the average fraction of BrdU-positive epithelial cells per total number of epithelial cells of three different mice, p < 0.001. Note Tg glands without dox had decreased numbers of BrdU positive cells compared with Wt controls or Tg in the presence of dox. (C) PCNA detection. PCNA was detected in mammary glands of Wt and Tg glands in the absence or in the presence of dox at L1. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox had decreased numbers of PCNA positive cells compared with Wt or Tg with dox glands. (D) TUNEL assay. Paraffin sections of mammary glands at L1 from Wt and Tg glands in the absence or in the presence of dox were subjected to TUNEL analysis. Positive cells were visualized with ABC (brown, representative shown by red arrow). Nuclei were counter-stained with a hematoxylin (blue). (E) Percentage of apoptotic cells in mammary epithelial cells. 300 - 400 epithelial cell nuclei were examined per sections. The values represent the average fraction of TUNEL positive epithelial cells per total number of epithelial cells of three different mice, p < 0.001. Note Tg glands without dox displayed increased numbers of apoptotic cells compared with Wt or Tg with dox glands. Bars: 125μm.
Figure 4
Figure 4
Defect in the β-catenin-cyclin D1 pathway in transgenic mouse mammary glands. (A) immunohistochemistry of cyclin D1. Cyclin D1 was detected in Wt and Tg mammary glands in the absence or in the presence of dox at L1 by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox displayed reduced levels of cyclin D1 expression compared with Wt glands or Tg with dox. (B) Northern blot analysis of cyclin D1 expression in Wt and Tg whole mammary glands in the absence of dox. Average relative expression levels are indicated below with arbitrary value. (C) immunohistochemistry of β-catenin. β-catenin was detected in Wt and Tg mammary glands in the absence or in the presence of dox at L1 by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox showed reduced levels of β-catenin compared with high levels of cytoplasmic β-catenin expression in Wt or Tg with dox glands. Bars: (A) 125μm; (C) 250μm.
Figure 5
Figure 5
Impaired lobuloalveolar development in wild-type mouse mammary glands upon Wy-14,643 treatment during pregnancy. Pregnant Wt and PPARα-null female mice were treated with Wy-14,643 from indicated time, and the mammary glands were removed at L1. (A) Morphological observation by whole mount. Note lobuloalveolar development was inhibited by Wy-14,643 in wild-type mice in a time dependent manner, but not in PPARα-null animals. (B) Histological analyses by H&E staining. Note the small alveoli observed in Wt mice upon Wy-14,643 treatment from day 7.5, but not in PPARα-null mice. PPARα-null mice have accumulated lipid droplets in the lumen. Bars: 125μm.
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