Molecular cloning and expression analysis of adiponectin and its receptors (AdipoR1 and AdipoR2) in the hypothalamus of the Huoyan goose during different stages of the egg-laying cycle

Abstract

Background: Adiponectin and its receptors (AdipoR1 and AdipoR2) are novel endocrine systems that act at various levels to regulate metabolic homeostasis and reproductive processes. We cloned and characterized the cDNA of adiponectin and its receptors from the hypothalamus of the Huoyan goose to reveal the influence of these factors on the process of goose egg-laying. We also determined the mRNA and protein expression profiles during different stages of the egg-laying cycle.

Methods: Hypothalamus tissues were obtained from 36 Huoyan geese in the pre-laying, early-laying, peak-laying, and ceased periods. The cDNA sequences of goose adiponectin and its receptors (AdipoR1 and AdipoR2) were cloned and characterized using the 5'-RACE and 3'-RACE methods. Multiple alignments and phylogenetic analyses of the deduced amino acid sequence were conducted using bioinformatics tools. The expression profiles of mRNA and protein in the hypothalamus during the pre-laying, early-laying, peak-laying and ceased periods were examined using real-time PCR (qRT-PCR) and Western blotting techniques.

Results: The cDNA of adiponectin, AdipoR1 and AdipoR2 consisted of 738, 1131 and 1161 bp open reading frame encoding 245, 376 and 386 amino acids, respectively. The deduced amino acid sequence of goose adiponectin, as well as AdipoR1 and AdipoR2 showed a closer genetic relationship to the avian species than to other mammal species. The expression level of adiponectin mRNA and protein increased from the pre-laying period to the peak-laying period, reached its peak in the peak-laying period, and then decreased during the ceased period. Conversely, the expression levels of AdipoR1 and AdipoR2 mRNA and protein decreased in the early-laying period, peak-laying period, and ceased period compared with the pre-laying period.

Conclusions: This study is the first to obtain full-length cDNA sequences of goose adiponectin and the genes of its receptors from the hypothalamus, and demonstrate that the egg-laying cycle affects the expression of the goose adiponectin system. Our results suggest the potential role of adiponectin as a key neuromodulator of reproductive functions.

Fig. 1

Relative expression of adiponectin, AdipoR1 and AdipoR2 mRNA in the hypothalamus of Huoyan geese during different stages of the egg-laying cycle. The expression levels of adiponectin, AdipoR1 and AdipoR2 were normalized to 18S rRNA. The expression levels, calculated by the 2^−ΔΔCt method, are presented as arbitrary units (AU). The presented values are the means ± SEM. The data were analysed by ANOVA followed by Tamhane’s T2 test post hoc test. Bars with different superscripts are significantly different (P < 0.05).

Fig. 2

Relative expressions of adiponectin, AdipoR1 and AdipoR2 protein in the hypothalamus of Huoyan geese during different stages of the egg-laying cycle. A comparison of adiponectin, AdipoR1 and AdipoR2 protein content were determined by western blotting analysis. Protein band density was analysed with GelQuant software. Beta-Actin was used as the internal control. Upper panels: representative immunoblots. Lower panels: densitometric analysis of adiponectin, AdipoR1 and AdipoR2 proteins relative to actin protein. Values are expressed as the mean ± SEM of arbitrary optical density units. Data were analysed by ANOVA followed by Tamhane’s T2 test post hoc test. Bars with different superscripts are significantly different (P < 0.05).