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The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptor genes coding from the kidney of Alligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is 2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

SOX9 plays a crucial, extensive and conservative role in the process of somatic tissue development and adult regeneration through the positive self-regulation mediated by SOM across all vertebrates. In this study, we have cloned SOX9 from the kidney of hatchling Alligator sinensis. The full-length of SOX9 cDNA is 3878 bp with an open reading frame encoding 494 amino acids. Amino acid alignment analyses indicated that the SOX9 exhibit highly conserved functional domains. Using the droplet digital PCR, the mRNA abundances of SOX9 during nephrogenesis in A. sinensis showed prominent changes in the embryonic development, suggesting that SOX9 might combines a vital role in the regulation of complex renal development. Interestingly, we detected the nucleocytoplasmic shuttling of SOX9 protein using immunofluorescence, implying that nucleocytoplasmic shuttling is critical to the regulation of SOX9 in the renal embryonic development. Collectively, these data provide an important foundation for further studies on renal developmental biology and molecular biology of non-mammalian SOX9. Furthermore, it provides new insights into the phenomenon of SOX9 nucleocytoplasmic shuttling in Alligator sinensis, which is probably of great significance to the development of kidney metanephros embryo.