Enhanced estrogen-induced proliferation in obese rat endometrium

Abstract

Objective: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals.

Study design: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol.

Results: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats.

Conclusion: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

Figure 1. Oral glucose tolerance test and plasma glucose and insulin levels in zucker obese and lean rats

Animals were fasted overnight before glucose challenge (2g/kg body weight). Plasma glucose (A) and insulin (B) level were determined before and up to 120min after glucose challenging. * p<0.05, n=3

Figure 2. Comparison of endometrial cell proliferation in zucker obese and lean rats

Immunohistochemical staining of BrdU labeled endometrial cell. BrdU (100mg/kg) were IP injected to rats 90 minutes before they were sacrificed. Uteri tissue sections were stained with anti-BrdU antibody. 10 views were counted for BrdU labeled nuclei in each slide. * p<0.05, n=6

Figure 3. Expression of pro-proliferative genes in the Rat Endometrium

The transcript levels of Cyclin A (A), PCNA (B), and c-Myc (C) in the endometrium from ovariectomized zucker fa/fa rats (Ob) and their lean littermates (Ln) receiving vehicle (Vh) or 17β-estradiol (E2) for 3 days were measured by real-time quantitative RT-PCR. Each sample was assayed in triplicate. The numbers of molecules of cylin A, PCNA and c-Myc in 20 ng total RNA were normalized to and expressed as the percentage of 18S. n = 5∼6. **, p < 0.05 compared with Ln/Vh; ••, p < 0.05 compared with Ob/Vh; ##, p < 0.05 compared with Ln/E2.

Figure 4. Expression of p27Kip1 in the rat endometrium

(A) Immunoblots of rat endometrium lysates against p27Kip1 antibody. 25 ug of protein from the uteri was subjected to immunoblotting with anti- p27Kip1 antibodies. The blots were then stripped and reprobed with anti- actin antibodies. 1. Lean control; 2. Lean E2; 3. Obese control; 4. Obese E2 (B) real-time quantitative RT-PCR analysis of p27Kip1 mRNA level in rat endometrium. 20 ng of endometrial RNA was assayed in triplicate for p27Kip1 transcripts. Absolute values were normalized to and expressed as a percentage of 18S. n = 5∼6. **, p < 0.05 compared with Ln/Vh; ••, p < 0.05 compared with Ob/Vh; ##, p < 0.05 compared with Ln/E2.

Figure 5. Expression estrogen regulated anti-proliferative gene expression in rat endometrium

Transcript level of progesterone receptor (A), sFRP4 (B), and RALDH2 (C) in the endometrium of Zucker fa/fa obese and lean rats. 20 ng of endometrial RNA were assayed in triplicate for RALDH2 transcripts by real-time quantitative RT-PCR. Absolute values were normalized to and expressed as a percentage of 18S. n =6. **, p < 0.05 compared of lean estradiol-treated group/Lean Control; ##, p<0.05, obese estradiol-treated group vs. lean estradiol-treated group

Figure 6. Akt and Erk1/2 Phosphorylation in rat uteri

Western blot analysis of Akt (A) and Erk1/2 (B) activiation. 25 μg of uterine protein from each rat was subjected to immunoblot analysis with antibodies specifically against Ser⁴⁷³ phosphorylated Akt (A) and phosphorylated Erk1/2 MAPK (B) antibody. PVDF membranes were subsequently stripped and reprobed with Akt (A) and Erk (B) antibody. n=6.