Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

Abstract

Background: Women with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction.

Methods: Female LY mice received daily oral doses of either 0.01 mg pioglitazone (n = 4) or an equal volume of vehicle (DMSO; n = 4) for 8 weeks. At the end of treatment, ovaries were removed and DNA microarrays were used to analyze differential gene expression.

Results: Twenty-seven genes showed at least a two-fold difference in ovarian expression with pioglitazone treatment. These included leptin, angiopoietin, angiopoietin-like 4, Foxa3, PGE1 receptor, resistin-like molecule-alpha (RELM), and actin-related protein 6 homolog (ARP6). For most altered genes, pioglitazone changed levels of expression to those seen in untreated C57BL/6J(a/a) non-mutant lean mice.

Conclusion: TZD administration may influence ovarian function via numerous diverse mechanisms that may or may not be directly related to insulin/IGF signaling.

Figure 1

Relative increase in expression of RELMα by Real Time RT-PCR. Bars represent mean ± SD, in relative gene expression calculated relative to endogenous GAPDH expression. An RNA concentration-response validation curve was carried out to determine the concentration of RNA to add to the RT-PCR reaction. All samples were run in duplicate, n = 3 animals per group.

Figure 2

Immunolocalization of RELMα in a untreated 180-day old LY mouse ovary. VectaStain Rabbit IgG Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) was used on paraffin sections. Sections were incubated with primary antibody (anti-RELMα; Upstate/Chemicon, Temecula, CA) for 1 h at RT, washed thrice, and incubated with biotinylated second antibody for 30 min. at RT. DAB substrate (brown precipitate, arrows) was used for antigen detection, and sections were counterstained with hematoxylin. Scale bar = 25 μm (lower right).

Figure 3

Western blot of RELMα immunoactivity in vehicle- and pioglitazone-treated LY mice. Beta-actin immunoactivity was used as a loading control. Bar graph depicts the mean (+ SD) ratio of intensity of RELMα-to-actin bands. There was no significant difference in band intensity between groups.