Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

Estrogens play critical roles in reproductive physiology via estrogen receptors (ESRs) in vertebrates, including reptiles. Chinese alligator (Alligator sinensis) is an endemic and endangered reptile species in China. In the present study, we cloned ESR2 gene from the ovary using rapid amplification of cDNA ends (RACE), investigated the spatial expression of ESRs in various tissues and temporal expression of ESRs in the pituitary glands during the reproductive cycle in Chinese alligators by quantitative real-time PCR (qPCR). Bioinformatics and phylogenetic analysis of deduced ESR2 protein were also performed. The full-length cDNA of the ESR2 is 1647 bp in length, with an open-reading frame encoding 548 amino acids. The bioinformatics analysis indicated that the deduced amino acid sequence of alligator ESR2 was highly conserved with that of other vertebrate species. In addition, compared to human ESR2, the 14 amino acids in the alligator ESR2 that are essential for specific recognition of estradiol are entirely conserved. The phylogenetic analysis showed that alligators were more closely related to birds than to other reptiles. The results of qPCR showed that the tissue distribution patterns of both ESR subtype mRNAs appeared to be different. In male tissues, the highest mRNA level of both ESRs is in the liver. While in female tissues, ESR1 and ESR2 showed the highest mRNA level in the hypothalamus and pituitary gland, respectively. During the female reproductive cycle, the expression level of ESR1 mRNA increased from the initial post-hibernation period to the reproductive period, reached its peak in the reproductive period, and then decreased in the autumn active period and hibernation period. Conversely, the highest transcription level of ESR2 was observed in the hibernation period.