Ghrelin and ghrelin receptor (GHSR) in Chinese alligator, alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 '-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 '-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5'-UTR of 375-bp, an ORF of 1059-bp and a 3' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core "GSSF" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.

Importance of the postcranial skeleton in eusuchian phylogeny: Reassessing the systematics of allodaposuchid crocodylians.

Our current knowledge on the crocodyliform evolution is strongly biased towards the skull morphology, and the postcranial skeleton is usually neglected in many taxonomic descriptions. However, it is logical to expect that it can contribute with its own phylogenetic signal. In this paper, the changes in the tree topology caused by the addition of the postcranial information are analysed for the family Allodaposuchidae, the most representative eusuchians in the latest Cretaceous of Europe. At present, different phylogenetic hypotheses have been proposed for this group without reaching a consensus. The results of this paper evidence a shift in the phylogenetic position when the postcranium is included in the dataset, pointing to a relevant phylogenetic signal in the postcranial elements. Finally, the phylogenetic relationships of allodaposuchids within Eusuchia are reassessed; and the internal relationships within Allodaposuchidae are also reconsidered after an exhaustive revision of the morphological data. New and improved diagnoses for each species are here provided.

Molecular cloning, characterization, and gene expression behavior of glucocorticoid and mineralocorticoid receptors from the Chinese alligator (Alligator sinensis).

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.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic-pituitary-ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle.

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.

Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines.

Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3-120.2% target recovery at 0.1-10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian.

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

Alligator aromatase cDNA sequence and its expression in embryos at male and female incubation temperatures.

In all species of crocodilians, sex is determined not by genetic mechanisms, but by the temperature at which the egg is incubated. In the American alligator (Alligator mississippiensis) the thermosensitive period (TSP) for sex determination is a 7- to 10-day window within stages 21-24 of development, around the middle third of the incubation period. Treating embryos with estrogen during the TSP produces female offspring, even at male incubation temperatures. Conversely, blocking embryonic estrogen synthesis at female-inducing temperature prevents development of the female phenotype. Therefore, it has been suggested that estrogen plays a role in determination of sex in the alligator. Estrogen is produced from an androgen substrate by cytochrome P450 aromatase (CYP19). If estrogen plays a critical role in sex determination, there should be differences in aromatase expression between embryos at male- and female-producing temperatures during the TSP. Therefore, to address this question, we cloned and characterized the alligator CYP19 cDNA. Based on the sequence information, a quantitative kinetic reverse transcriptase-polymerase chain reaction (TaqMan) assay was designed to measure expression of the alligator aromatase gene in RNA extracted from the gonadal and brain regions of alligator embryos incubated at male- or female-producing temperatures from prior to the TSP through hatching. Aromatase expression was detected in the brain region from the earliest stage tested (stage 20) through hatching. The hypothalamus had significantly higher expression than the forebrain or hindbrain in both male and female embryos. Expression was not significantly different in the gonadal region between embryos at male and female temperatures until after the TSP, when there was a dramatic increase in expression at female temperature. These data indicate that aromatase expression and, thus, estrogen production, are not the initial trigger for sex determination but play an essential role in ovarian differentiation in the alligator. J. Exp. Zool. 290:439-448, 2001.