Neonatal diethylstilbestrol exposure alters the metabolic profile of uterine epithelial cells

Abstract

Developmental exposure to diethylstilbestrol (DES) causes reproductive tract malformations, affects fertility and increases the risk of clear cell carcinoma of the vagina and cervix in humans. Previous studies on a well-established mouse DES model demonstrated that it recapitulates many features of the human syndrome, yet the underlying molecular mechanism is far from clear. Using the neonatal DES mouse model, the present study uses global transcript profiling to systematically explore early gene expression changes in individual epithelial and mesenchymal compartments of the neonatal uterus. Over 900 genes show differential expression upon DES treatment in either one or both tissue layers. Interestingly, multiple components of peroxisome proliferator-activated receptor-γ (PPARγ)-mediated adipogenesis and lipid metabolism, including PPARγ itself, are targets of DES in the neonatal uterus. Transmission electron microscopy and Oil-Red O staining further demonstrate a dramatic increase in lipid deposition in uterine epithelial cells upon DES exposure. Neonatal DES exposure also perturbs glucose homeostasis in the uterine epithelium. Some of these neonatal DES-induced metabolic changes appear to last into adulthood, suggesting a permanent effect of DES on energy metabolism in uterine epithelial cells. This study extends the list of biological processes that can be regulated by estrogen or DES, and provides a novel perspective for endocrine disruptor-induced reproductive abnormalities.

Fig. 1.

DES alters uterine gene expression primarily in the epithelium. (A) Hierarchical clustering heatmap of differentially regulated genes by DES in the UE and UM. Green to red, color range gradient of mean abundance (−0.7 to 0.7). Each column represents a pool of more than three animals. Red box, genes whose expression was altered by DES primarily in the UE; green box, genes regulated by DES similarly in the UE and UM; black box, genes altered by DES primarily in the UM. (B) Star glyph distribution of the fold-change of differentially regulated gene in the uterus; log2 values of the mean fold-change. Red, UM; blue, UE. (C) Venn diagram of UE and UM DRGs. (D) Pie chart showing classification of DRGs based on Gene Ontology Consortium biological processes. Note that some genes might have multiple functions and could be represented in more than one category.

Fig. 2.

DES affects lipid metabolism and transport. (A) Hierarchical clustering heatmap of genes involved in lipid trafficking and metabolism. Green to red, color range gradient of mean abundance (−1.0 to 1.0). Red box, genes whose expression was altered by DES primarily in the UE. (B) Real-time RT-PCR survey of genes involved in adipocyte differentiation in the uterus. Expression of each gene was normalized to that of Rpl7; normalized expression by oil UE was considered to be 1.0. (C) Western blot on whole uterine lysates showed markedly increased PPARγ protein in DES-treated uteri. GAPDH served as a loading control. (D,E) Immunohistochemistry of PPARγ showed increased staining in the DES-treated UE. Inset shows that PPARγ was predominantly detected in the UE nuclei (arrow). (F,G) Increased KLF4 protein was detected in the UE nuclei by immunofluorescence. Red, KLF4; blue, nuclei. Insets show magnified immunofluorescence signal of the boxed region. (H) Real-time RT-PCR validated genes involved in fatty acid transport and metabolism in oil- or DES-treated UE and UM. Data are presented as mean + s.d. of three samples analyzed in each treatment group from corresponding tissues. *P<0.01, **P<0.05.

Fig. 3.

DES-induced lipogenesis in UE is mediated through PPARγ. (A–D) TEM of UE at P5 revealed that DES treatment alone caused accumulation of electron-dense droplets in the UE close to the basement membrane (B, arrows, compare with A). Co-injection of PPARγ inhibitor BADGE (C) or GW9662 (D) dampened this effect, resulting in a lower number of droplets. (E–H) ORO staining identified droplets as neutral lipid droplets (F) and confirmed that co-treatment of DES with PPARγ inhibitors severely attenuated this effect (G,H). (I) Quantification of lipid droplets from TEM images confirmed DES-induced accumulation of lipid droplets in the UE. Co-treatment of DES with PPARγ inhibitors resulted in reduced number and size of the droplets. *P<1×10⁻⁴. (J) PPARγ agonist pioglitazone (TZD) induced lipid droplet formation in the UE without specific subcellular localization (arrowheads). (K) Co-injection of TZD and DES resulted in intense ORO staining on the basal side of the UE (arrows).

Fig. 4.

DES alters glucose transport and metabolism in the UE. (A,B) Immunofluorescence against GLUT1 showed a dramatic increase and translocation to the basal-lateral membrane of the protein upon DES treatment. (C–F) Immunofluorescence of HK2 (C,D) and G6PD (E,F) revealed that both were expressed at low levels in the UE at P5, but were markedly upregulated by neonatal DES treatment. Insets show magnified immunofluorescence signal of the boxed area in each panel. (G) Glucose uptake assay showed a 1.4-fold increase in isolated DES-treated UE compared with controls. **P<0.025. (H) Western blot of HK2 and G6PD confirmed their upregulation by DES in P5 whole uterine lysates.

Fig. 5.

Neonatal DES treatment alters adult UE metabolic homeostasis. (A–F) ORO staining of uteri of ovariectomized (OVX) adult mice. No staining was detected in the OVX UE of control animals (A), whereas a number of lipid droplets were observed in the OVX UE neonatally exposed to DES (D, arrows). Dashed lines outline the UE. DES induced ORO staining in the luminal UE of both groups (B,E, arrows), without affecting the glandular epithelium (B, arrowhead). E2 treatment induced ORO staining in the UE of animals previously exposed to DES (F, arrow), but had little effect on neonatally oil-treated UE (C). (G–L) Expression of G6PD assessed by immunofluorescence. G6PD was higher in the UE with neonatal DES exposure (J) and remained high when treated with DES or E2 (K,L). Neonatally oil-treated UE had lower G6PD levels regardless of hormone treatment in adulthood (G–I).