Assessment of Adipocyte Differentiation and Maturation-related Gene Expression in the Epididymal Fat of Estrogen Receptor α Knockout (ERαKO) Mouse during Postnatal Development Period

Abstract

The absence of functional estrogen receptor α (Esr1) results in an overgrowth of the epididymal fat, as observed in estrogen receptor α knockout (ERαKO) mouse. The present research was aimed to evaluate expression of various molecules associated with adipocyte differentiation and maturation in the epididymal fat of ERαKO mouse at several postnatal ages by using quantitative real-time polymerase chain reaction. The highest transcript levels of all molecules were detected at 12 months of postnatal age, except leptin which the mRNA level was increased at 5 months of age and was unchanged until 12 months of age. The expression levels of CCAAT enhancer binding protein (Cebp) alpha, androgen receptor, and lipoprotein lipase were decreased at 5 months of age but increased at about 8 months of age. The mRNA levels of Cebp gamma and sterol regulatory element binding transcription factor 1 remained steady until 8 months of age. Continuous increases of transcript levels during postnatal period were found in Cebp beta, estrogen receptor (ER) beta, fatty acid binding protein 4, and delta like non-canonical Notch ligand 1. The increases of peroxisome proliferator-activated receptor gamma and adiponectin mRNA levels were detected as early as 8 months of age. The levels of fatty acid synthase and resistin transcript at 5 and 8 months of age were lower than that at 2 months of age. These findings show the aberrant expression patterns of genes related to adipocyte differentiation and maturation in the postnatal epididymal fat pad by the disruption of ER alpha function.

Keywords: Differentiation; Epididymal fat; Estrogen receptor α knockout (ERαKO) mouse; Maturation; Postnatal development; Real-time polymerase chain reaction (PCR).

Fig. 1.. Expression patterns of Cebpa, Cebpb, and Cebpg in the epididymal fat tissue of ERαKO mouse at different postnatal ages.

The transcript levels of Cebpa (A), Cebpb (B), and Cebpg (C) genes were normalized against its of Rn18s, and relative expression ratios were calculated by 2^−ΔΔCt method (Livak & Schmittgen, 2001 ). Each value represents the mean±SEM of three to five mice. Lower cases on bars indicate statistical significances among experimental groups at p<0.05. M, months of postnatal age; Cebpa, CCAAT enhancer binding protein, alpha; Cebpb, CCAAT enhancer binding protein, beta; Cebpg, CCAAT enhancer binding protein, gamma; 18s ribosomal RNA. ERαKO, estrogen receptor α knockout.

Fig. 2.. Expression patterns of Srebp1, Ar, and Esr2 in the epididymal fat tissue of ERαKO mouse at different postnatal ages.

The transcript levels of Srebp1 (A), Ar (B), and Esr2 (C) genes were normalized against its of Rn18s, and relative expression ratios were calculated by 2^−ΔΔCt method (Livak & Schmittgen, 2001). Each value represents the mean±SEM of three to five mice. Lower cases on bars indicate statistical significances among experimental groups at p<0.05. M, months of postnatal age; Srebf1, sterol regulatory element binding transcription factor 1; Ar, androgen receptor; Esr2, estrogen receptor, beta; 18s ribosomal RNA. ERαKO, estrogen receptor α knockout.

Fig. 3.. Expression patterns of Pparg, Fabp4, and Dlk1 in the epididymal fat tissue of ERαKO mouse at different postnatal ages.

The transcript levels of Pparg (A), Fabp4 (B), and Dkl1 (C) genes were normalized against its of Rn18s, and relative expression ratios were calculated by 2^−ΔΔCt method (Livak & Schmittgen, 2001). Each value represents the mean±SEM of three to five mice. Lower cases on bars indicate statistical significances among experimental groups at p<0.05. M, months of postnatal age; Pparg, peroxisome proliferator-activated receptor, gamma; Fabp4, fatty acid binding protein 4; Dlk1, delta like non-canonical Notch ligand 1; 18s ribosomal RNA. ERαKO, estrogen receptor α knockout.

Fig. 4.. Expression patterns of Fasn and Lpl in the epididymal fat tissue of ERαKO mouse at different postnatal ages.

The transcript levels of Fasn (A) and Lpl (B) genes were normalized against its of Rn18s, and relative expression ratios were calculated by 2^−ΔΔCt method (Livak & Schmittgen, 2001). Each value represents the mean±SEM of three to five mice. Lower cases on bars indicate statistical significances among experimental groups at p<0.05. M, months of postnatal age; Fasn, fatty acid synthase; Lpl, lipoprotein lipase; 18s ribosomal RNA. ERαKO, estrogen receptor α knockout.

Fig. 5.. Expression patterns of Lep, Adipoq, and Retn in the epididymal fat tissue of ERαKO mouse at different postnatal ages.

The transcript levels of Lep (A), Adipoq (B), and Retn (C) genes were normalized against its of Rn18s, and relative expression ratios were calculated by 2^−ΔΔCt method (Livak & Schmittgen, 2001). Each value represents the mean±SEM of three to five mice. Lower cases on bars indicate statistical significances among experimental groups at p<0.05. M, months of postnatal age; Lep, leptin; Adipoq, adiponectin; Retn, resistin; 18s ribosomal RNA. ERαKO, estrogen receptor α knockout.