Scales of non-avian reptiles and their derivatives contain corneous beta proteins coded from genes localized in the Epidermal Differentiation Complex.

The evolution of modern reptiles from basic reptilian ancestors gave rise to scaled vertebrates. Scales are of different types, and their corneous layer can shed frequently during the year in lepidosaurians (lizards, snakes), 1-2 times per year in the tuatara and in some freshwater turtle, irregularly in different parts of the body in crocodilians, or simply wore superficially in marine and terrestrial turtles. Lepidosaurians possess tuberculate, non-overlapped or variably overlapped scales with inter-scale (hinge) regions. The latter are hidden underneath the outer scale surface or may be more exposed in specific body areas. Hinge regions allow stretching during growth and movement so that the skin remains mechanically functional. Crocodilian and turtles feature flat and shield scales (scutes) with narrow inter-scale regions for stretching and growth. The epidermis of non-avian reptilian hinge regions is much thinner than the exposed outer surface of scales and is less cornified. Despite the thickness of the epidermis, scales are mainly composed of variably amount of Corneous Beta Proteins (CBPs) that are coded in a gene cluster known as EDC (Epidermal Differentiation Complex). These are small proteins, 100-200 amino acid long of 8-25 kDa, rich in glycine and cysteine but also in serine, proline and valine that participate to the formation of beta-sheets in the internal part of the protein, the beta-region. This region determines the further polymerization of CBPs in filamentous proteins that, together a network of Intermediate Filament Keratins (IFKs) and other minor epidermal proteins from the EDC make the variable pliable or inflexible corneous material of reptilian scales, claws and of turtle beak. The acquisition of scales and skin derivatives with different mechanical and material properties, mainly due to the evolution of reptile CBPs, is essential for the life and different adaptations of these vertebrates.

Effect of the oil from the fatty tissues of Crocodylus siamensis on gut microbiome diversity and metabolism in mice.

Dietary fat can alter host metabolism and gut microbial composition. Crocodile oil (CO) was extracted from the fatty tissues of Crocodylus siamensis. CO, rich in monounsaturated- and polyunsaturated fatty acids, has been reported to reduce inflammation, counter toxification, and improve energy metabolism. The aim of this study was to investigate the effect of CO on gut microbiota (GM) in laboratory mice as well as the accompanying metabolic changes in the animals. Forty-five C57BL/6 male mice were randomly divided into five groups and orally administrated either sterile water (control [C]); 1 or 3% (v/w) CO (CO-low [CO-L] and CO-high [CO-H], respectively); or 1 or 3% (v/w) palm oil (PO-low and PO-high, respectively) for 11 weeks. Body weight gain, food intake, energy intake, blood glucose levels, and blood lipid profiles were determined. Samples from colon tissue were collected and the 16S rRNA genes were pyrosequenced to clarify GM analyses. The results showed that there were no differences in body weight and blood glucose levels. Food intake by the mice in the CO-L and CO-H groups was statistically significantly less when compared to that by the animals in the C group. However, neither CO treatment had a statistically significant effect on calorie intake when compared to the controls. The CO-H exhibited a significant increase in serum total cholesterol and low-density lipoprotein but showed a downward trend in triglyceride levels compared to the control. The GM analyses revealed that both CO treatments have no significant influence on bacterial diversity and relative abundance at the phylum level, whereas increases of Choa1 and abundance-based coverage estimator indexes, distinct ß-diversity, and Proteobacteria abundance were observed in the PO-high group compared with the C group. Furthermore, the abundance of Azospirillum thiophilum and Romboutsia ilealis was significantly higher in the CO-L and CO-H groups which could be associated with energy metabolic activity. Thus, CO may be an alternative fat source for preserving host metabolism and gut flora.

Population genetics and molecular identification of Crocodylus acutus and C. moreletii (Crocodilia: Crocodylidae) in captive and wildlife populations / Genética de poblaciones e identificación molecular de Crocodylus acutus y C. moreletii (Crocodilia: Crocodylidae) en poblaciones de cautiverio y de vida libre

Abstract Introduction: There is low evidence of genetic diversity and hybridization processes within Crocodylus acutus and C. moreletii populations. Objetive: To evaluate genetic diversity and some phylogenetic relationships in wild and captive populations of C. acutus and C. moreletii using the Barcode of Life Data System (COX1, cytochrome C oxidase subunit 1 gene). Methods: 28 individuals phenotypically like C. acutus located in the state of Guerrero, Oaxaca and Quintana Roo were sampled, as well as animals belonging to C. moreletii located in the states of Tabasco, Campeche, and Quintana Roo. 641 base pairs of nucleotide sequence from COX1 were used to obtain the haplotype and nucleotide diversity per population, and a phylogenetic and network analysis was performed. Results: Evidence of hybridization was found by observing C. moreletti haplotypes in animals phenotypically determined as C. acutus, as well as C. acutus haplotypes in animals classified as C. moreletti. Low haplotypic diversity was observed for C. acutus (0.455 ± 0.123) and for C. moreletii (0.505 ± 0.158). A phylogenetic tree was obtained in which the sequences of C. acutus and C. moreletii were grouped into two well-defined clades. Organisms identified phenotypically as C. acutus but with C. moreletii genes were separated into a different clade within the clade of C. moreletii. Conclusions: There are reproductive individuals with haplotypes different from those of the species. This study provides a small but significant advance in the genetic knowledge of both crocodile species and the use of mitochondrial markers, which in this case, the COX1 gene allowed the detection of hybrid organisms in wild and captive populations. Conservation efforts for both species of crocodiles should prevent the crossing of both threatened species and should require the genetic identification of pure populations, to design effective conservation strategies considering the possibility of natural hybridization in areas of sympatry.

Resumen Introducción: Existe poca evidencia de la diversidad genética y los procesos de hibridación dentro de las poblaciones de Crocodylus acutus y C. moreletii. Objetivo: Evaluar la diversidad genética y algunas relaciones filogenéticas en poblaciones silvestres y cautivas de C. acutus y C. moreletii utilizando el Sistema de Código de Barras de la vida (COX1, subunidad I del gen del citocromo C oxidasa). Métodos: Se muestrearon 28 individuos fenotípicamente similares a C. acutus ubicados en los estados de Guerrero, Oaxaca y Quintana Roo, así como animales pertenecientes a C. moreletii ubicados en los estados de Tabasco, Campeche y Quintana Roo. Se utilizaron 641 pares de bases de la secuencia de nucleótidos de la subunidad I del gen del citocromo C oxidasa para obtener el haplotipo y la diversidad de nucleótidos por población, y se realizó un análisis filogenético y de redes. Resultados: Se encontró evidencia de hibridación al observar haplotipos de C. moreletti en animales determinados fenotípicamente como C. acutus, así como haplotipos de C. acutus en animales clasificados como C. moreletti. Se observó una baja diversidad haplotípica para C. acutus (0.455 ± 0.123) y para C. moreletii (0.505 ± 0.158). Se obtuvo un árbol filogenético en el que las secuencias propias de C. acutus y C. moreletii se agruparon en dos grandes y bien definidos clados. Los organismos identificados fenotípicamente como C. acutus pero con genes de C. moreletii se separaron en un clado diferente dentro del clado de C. moreletii. Conclusiones: Existen individuos reproductores con haplotipos diferentes a los de la especie. Este estudio aporta un pequeño pero significativo avance en el conocimiento genético tanto de las especies de cocodrilos como del uso de marcadores mitocondriales, que, en este caso, el gen COX1 permitió la detección de organismos híbridos en poblaciones silvestres y cautivas. Los esfuerzos de conservación para ambas especies de cocodrilos deben evitar el cruce de ambas especies amenazadas y deben requerir la identificación genética de poblaciones puras, para diseñar estrategias de conservación efectivas considerando la posibilidad de hibridación natural en áreas de simpatría.

Community and shotgun metagenomic analysis of Alligator mississippiensis oral cavity and GI tracts reveal complex ecosystems and potential reservoirs of antibiotic resistance.

We report here the community structure and functional analysis of the microbiome of the Alligator mississippiensis GI tract from the oral cavity through the entirety of the digestive tract. Although many vertebrate microbiomes have been studied in recent years, the archosaur microbiome has only been given cursory attention. In the oral cavity we used amplicon-based community analysis to examine the structure of the oral microbiome during alligator development. We found a community that diversified over time and showed many of the hallmarks we would expect of a stable oral community. This is a bit surprising given the rapid turnover of alligator teeth but suggests that the stable gumline microbes are able to rapidly colonize the emerging teeth. As we move down the digestive tract, we were able to use both long and short read sequencing approaches to evaluate the community using a shotgun metagenomics approach. Long read sequencing was applied to samples from the stomach/duodenum, and the colorectal region, revealing a fairly uniform and low complexity community made up primarily of proteobacteria at the top of the gut and much more diversity in the colon. We used deep short read sequencing to further interrogate this colorectal community. The two sequencing approaches were concordant with respect to community structure but substantially more detail was available in the short read data, in spite of high levels of host DNA contamination. Using both approaches we were able to show that the colorectal community is a potential reservoir for antibiotic resistance, human pathogens such as Clostridiodes difficile and a possible source of novel antimicrobials or other useful secondary metabolites.

Xenobiotic estradiol-17ß alters gut microbiota of hatchling American alligators (Alligator mississippiensis).

Environmental oestrogens pose serious concerns for ecosystems through their effects on organismal survival and physiology. The gut microbiome is highly vulnerable to environmental influence, yet the effects of oestrogens on gut homeostasis are unknown because they are poorly studied in wildlife populations. To determine the influence of environmental oestrogens (i.e., xenoestrogens) on the diversity and abundance of gut microbiota, we randomly assigned 23 hatchling American alligators (Alligator mississippiensis) to three ecologically relevant treatments (control, low, and high oestrogen concentrations) for 10 weeks. We predicted that xenoestrogen exposure would decrease microbial diversity and abundance within the digestive tract and that this effect would be dose-dependent. Microbial samples were collected following diet treatments and microbial diversity was determined using 16S rRNA gene-sequencing. Individuals in oestrogen-treatment groups had decreased microbial diversity, but a greater relative abundance of operational taxonomic units than those in the control group. In addition, this effect was dose-dependent; as individuals were exposed to more oestrogen, their microbiome became less diverse, less rich and less even. Findings from this study suggest that oestrogen contamination can influence wildlife populations at the internal microbial-level, which may lead to future deleterious health effects.

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.

Molecular cloning, immunological characterization, and expression analysis of gonadotropin-releasing hormone (GnRH) in the brain of the Chinese alligator during different stages of reproductive cycle.

The neurohormone gonadotropin-releasing hormone (GnRH) plays an essential role in the control of reproductive functions in vertebrates. However, the full-length complementary DNA (cDNA) encoding the GnRHs precursor and it role in the reproductive cycles regulating has not been illustrated in crocodilian species. In the present study, full-length cDNAs encoding GnRH1 forms, its predominant localization within brain and peripheral tissues, and GnRH1 peptide concentrations in the hypothalamus and pituitary in relation to seasonal gonadal development of Chinese alligator were investigated. The cDNA of GnRH1 is consisted of 282 bp open reading frame encoding 93 amino acids. The deduced amino acid sequence of alligator GnRH1 contains several conserved regions and shows a closer genetic relationship to the avian species than to other reptile species. The GnRH1 immunopositive cells were not only detected widely in cerebrum, diencephalon, medulla oblongata but also observed in peripheral tissues, these widespread distribution characteristics indicated that GnRH1 possibly possess the multi-functionality in Chinese Alligator. GnRH1 peptide concentration within hypothalamus were observed be the highest in RP group (P < 0.05), in association with an peak value in GSI and emerging of late vitellogenic follicles in the ovary. Taken together, our results suggested that GnRH1 was predominantly involved in the vitellogenesis process of seasonal gonadal development of Chinese Alligator.

Reptilian ß-defensins: Expanding the repertoire of known crocodylian peptides.

One of the major families of host defense peptides (HDPs) in vertebrates are ß-defensins. They constitute important components of innate immunity and have remained an interesting topic of research for more than two decades. While many ß-defensin sequences in mammals and birds have been identified and their properties and functions characterized, ß-defensin peptides from other groups of vertebrates, particularly reptiles, are still largely unexplored. In this review, we focus on reptilian ß-defensins and summarize different aspects of their biology, such as their genomic organization, evolution, structure, and biological activities. Reptilian ß-defensin genes exhibit similar genomic organization to birds and their number and gene structure are variable among different species. During the evolution of reptiles, several gene duplication and deletion events have occurred and the functional diversification of ß-defensins has been mainly driven by positive selection. These peptides display broad antimicrobial activity in vitro, but a deeper understanding of their mechanisms of action in vivo, including their role as immunomodulators, is still lacking. Reptilian ß-defensins constitute unique polypeptide sequences to expand our current understanding of innate immunity in these animals and elucidate core biological functions of this family of HDPs across amniotes.

Deimination Protein Profiles in Alligator mississippiensis Reveal Plasma and Extracellular Vesicle-Specific Signatures Relating to Immunity, Metabolic Function, and Gene Regulation.

Alligators are crocodilians and among few species that endured the Cretaceous-Paleogene extinction event. With long life spans, low metabolic rates, unusual immunological characteristics, including strong antibacterial and antiviral ability, and cancer resistance, crocodilians may hold information for molecular pathways underlying such physiological traits. Peptidylarginine deiminases (PADs) are a group of calcium-activated enzymes that cause posttranslational protein deimination/citrullination in a range of target proteins contributing to protein moonlighting functions in health and disease. PADs are phylogenetically conserved and are also a key regulator of extracellular vesicle (EV) release, a critical part of cellular communication. As little is known about PAD-mediated mechanisms in reptile immunology, this study was aimed at profiling EVs and protein deimination in Alligator mississippiensis. Alligator plasma EVs were found to be polydispersed in a 50-400-nm size range. Key immune, metabolic, and gene regulatory proteins were identified to be posttranslationally deiminated in plasma and plasma EVs, with some overlapping hits, while some were unique to either plasma or plasma EVs. In whole plasma, 112 target proteins were identified to be deiminated, while 77 proteins were found as deiminated protein hits in plasma EVs, whereof 31 were specific for EVs only, including proteins specific for gene regulatory functions (e.g., histones). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed KEGG pathways specific to deiminated proteins in whole plasma related to adipocytokine signaling, while KEGG pathways of deiminated proteins specific to EVs included ribosome, biosynthesis of amino acids, and glycolysis/gluconeogenesis pathways as well as core histones. This highlights roles for EV-mediated export of deiminated protein cargo with roles in metabolism and gene regulation, also related to cancer. The identification of posttranslational deimination and EV-mediated communication in alligator plasma revealed here contributes to current understanding of protein moonlighting functions and EV-mediated communication in these ancient reptiles, providing novel insight into their unusual immune systems and physiological traits. In addition, our findings may shed light on pathways underlying cancer resistance, antibacterial and antiviral resistance, with translatable value to human pathologies.

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.

Next generation sequencing and RNA-seq characterization of adipose tissue in the Nile crocodile (Crocodylus niloticus) in South Africa: Possible mechanism(s) of pathogenesis and pathophysiology of pansteatitis.

BACKGROUND: Concerted efforts to identify the pathogenesis and mechanism(s) involved in pansteatitis, (a generalized inflammation of the adipose tissue), that was attributed to the recent crocodile die off in the Olifants River and Loskop Dam in Kruger National Park, Mpumalanga, South Africa have been in the forefront of research in recent time. As part of the efforts, molecular characterization of healthy and pansteatitis adipose tissue was carried out by RNA sequencing (RNA-Seq) using Next Generation Sequencing (NGS) and de novo assembly of the adipose transcriptome, followed by differential gene expression analysis. METHODOLOGY: Healthy adipose tissue consisting of fifty samples was collected from the subcutaneous, visceral, intermuscular adipose tissues and the abdominal fat body of ten 4 years old juvenile crocodiles from a local crocodile farm in Pretoria, South Africa. Ten pansteatitis samples were collected from visceral and intermuscular adipose tissues of five crocodiles that were dying of pansteatitis. RESULTS: Forty-two thousand, two hundred and one (42,201) transcripts were assembled, out of which 37, 835 had previously been characterized. The de novo assembled transcriptome had an N50 (average sequence) of 436 bp, percentage GC content of 43.92, which compared well with previously assembled transcripts in the saltwater crocodile. Seventy genes were differentially expressed and upregulated in pansteatitis. These included genes coding for extracellular matrix (ECM) signaling ligands, inflammatory cytokines and tumour necrosis factor alpha (TNFα) receptors, fatty acid synthase and fatty acid binding proteins, peroxisome proliferator-activated receptor gamma (PPARγ), nuclear factor and apoptosis signaling ligands, and mitogen activated protein kinase enzymes among others. Majority (88.6%) of the upregulated genes were found to be involved in hypoxia inducible pathways for activation of NFkß and inflammation, apoptosis, Toll-like receptor pathway and PPARγ. Bicaudal homologous 2 Drosophila gene (BICD2) associated with spinal and lower extremity muscle atrophy was also upregulated in pansteatitis while Sphingosine -1-phosphate phosphatase 2 (SGPP2) involved in Sphingosine -1- phosphate metabolism was downregulated. Futhermore, Doublesex-mab-related transcription factor 1 (DMRT1) responsible for sex gonad development and germ cell differentiation was also downregulated. CONCLUSION: Thus, from the present study, based on differentially expressed genes in pansteatitis, affected Nile crocodiles might have died partly due to their inability to utilize stored triglycerides as a result of inflammation induced insulin resistance, leading to starvation in the midst of plenty. Affected animals may have also suffered muscular atrophy of the lower extremities and poor fertility.

Assessing impacts of precocious steroid exposure on thyroid physiology and gene expression patterns in the American alligator (Alligator mississippiensis).

The thyroid gland is sensitive to steroid hormone signaling, and many thyroid disrupting contaminants also disrupt steroid hormone homeostasis, presenting the possibility that thyroid disruption may occur through altered steroid hormone signaling. To examine this possibility, we studied short-term and persistent impacts of embryonic sex steroid exposure on thyroid physiology in the American alligator. Alligators from a lake contaminated with endocrine disrupting contaminants (Lake Apopka, FL, USA) have been shown to display characteristics of thyroid and steroid hormone disruption. Previous studies suggest these alterations arise during development and raise the possibility that exposure to maternally deposited contaminants might underlie persistent organizational changes in both thyroidal and reproductive function. Thus, this population provides a system to investigate contaminant-mediated organizational thyroid disruption in an environmentally-relevant context. We assess the developmental expression of genetic pathways involved in thyroid hormone biosynthesis and find that expression of these genes increases prior to hatching. Further, we show that nuclear steroid hormone receptors are also expressed during this period, indicating the developing thyroid is potentially responsive to steroid hormone signaling. We then explore functional roles of steroid signaling during development on subsequent thyroid function in juvenile alligators. We exposed alligator eggs collected from both Lake Apopka and a reference site to 17ß-estradiol and a non-aromatizable androgen during embryonic development, and investigated effects of exposure on hatchling morphometrics and thyroidal gene expression profiles at 5 months of age. Steroid hormone treatment did not impact the timing of hatching or hatchling size. Furthermore, treatment with steroid hormones did not result in detectable impacts on thyroid transcriptional programs, suggesting that precocious or excess estrogen and androgen exposure does not influence immediate or long-term thyroidal physiology.

Embryonic estrogen exposure recapitulates persistent ovarian transcriptional programs in a model of environmental endocrine disruption.

Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL, are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a nonaromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses; however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.

Molecular cloning of androgen receptor and gene expression of sex steroid hormone receptors in the brain of newborn Chinese alligator (Alligator sinensis).

Sex steroid hormones play an important role in mediating physiological responses and developmental processes through their receptors across all vertebrates. Chinese alligator (Alligator sinensis) is a critically endangered reptile species unique to China. In this study, we have cloned one of the sex steroid hormone receptor genes, androgen receptor (AR) from the brain of Chinese alligator for the first time. The full-length AR cDNA is 2717 bp in length with an open reading frame (ORF) encoding 722 amino acids. Amino acid alignment analyses indicated that the ARs exhibit highly conserved functional domains. Especially, the P-box and D-box, which are essential to ensure that receptor binding to the androgen response elements, are completely conserved in selected species. Using the quantitative real-time PCR (qPCR), the spatial expression of four receptor mRNAs in all newborn brain tissues and temporal expression of them in the cerebrum during the embryonic development in Chinese alligators were investigated. The results of qPCR showed ubiquitous expression of the four receptor mRNAs in all newborn brain tissues examined and significant changes in the expression levels of these receptor mRNAs in the embryonic development. These results suggest that sex steroid hormones might play an important role in the regulation of complex neuroendocrine activities in newborn Chinese alligator. Furthermore, these data provide an important foundation for further studies on endocrinology and molecular biology of non-mammalian sex steroid hormone receptors.

[Molecular Cloning,Sequence Analysis,and Expression of Pepsinogens C Gene from Adult Alligator Sinensis Stomach].

Objective To investigate the molecular clone and structural features of pepsinogen C(PGC) gene in the stomach of Alligator sinensis,explore the phylogenetic relationships and tissue distribution,and analyze the variation of PGC expression in the stomachs of adult Alligator sinensis at different life stages. Methods The full-length cDNA of PGC gene of Alligator sinensis was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends and then sequenced.The physical and chemical parameters and advanced structures of the PGC protein were predicted by bioinformatics methods and tools.The PGC amino acid sequences of the Alligator sinensis and other vertebrates were compared by Clustal X software.The neighbor-joining phylogenetic tree was built by MEGA 6 software.Immunohistochemistry was used to locate PGC in the gastric mucosa of Alligator sinensis.The variation of the PGC mRNA levels in the stomach at different life stages was detected by quantitative real-time polymerase chain reaction.Results Reverse transcription polymerase chain reaction and rapid amplification of cDNA ends revealed a 1568 bp cDNA full-length sequence containing 1167 bp open reading frame,which encoded 388 amino acids.The PGC gene of Alligator sinensis had been deposited in the GenBank Data Libraries under the accession number of KY799383.Bioinformatics analysis predicted that the Alligator sinensis PGC had a theoretical relative molecular mass of 41 998 with a theoretical isoelectric point of 4.16.In addition,the three-dimensional structure of the PGC was constructed by homology modeling to predict its active site with two essential aspartyl residues and six essential cysteine residues involved in forming three disulphide bonds.The neighbor-joining phylogenetic tree of vertebrates from the amino acids sequences of PGC showed all crocodiles were clustered as a group,and the PGC of Alligator sinensis was the closest to Alligator mississippiensis.Alligator sinensis PGC was specifically expressed in the gastric mucosa,and its expressions significantly differed during reproduction and hibernation significantly(P<0.05).Conclusions Alligator sinensis PGC gene is highly conserved in evolution.Its protein is a gastric specific digestive proteinase that belongs to a aspartic proteinase family.

Comparative Analysis of Epidermal Differentiation Genes of Crocodilians Suggests New Models for the Evolutionary Origin of Avian Feather Proteins.

The epidermis of amniotes forms a protective barrier against the environment and the differentiation program of keratinocytes, the main cell type in the epidermis, has undergone specific alterations in the course of adaptation of amniotes to a broad variety of environments and lifestyles. The epidermal differentiation complex (EDC) is a cluster of genes expressed at late stages of keratinocyte differentiation in both sauropsids and mammals. In the present study, we identified and analyzed the crocodilian equivalent of the EDC. The gene complement of the EDC of both the American alligator and the saltwater crocodile were determined by comparative genomics, de novo gene prediction and identification of EDC transcripts in published transcriptome data. We found that crocodilians have an organization of the EDC similar to that of their closest living relatives, the birds, with which they form the clade Archosauria. Notable differences include the specific expansion of a subfamily of EDC genes in crocodilians and the loss of distinct ancestral EDC genes in birds. Identification and comparative analysis of crocodilian orthologs of avian feather proteins suggest that the latter evolved by cooption and sequence modification of ancestral EDC genes, and that the amplification of an internal highly cysteine-enriched amino acid sequence motif gave rise to the feather component epidermal differentiation cysteine-rich protein in the avian lineage. Thus, sequence diversification of EDC genes contributed to the evolutionary divergence of the crocodilian and avian integuments.

Purification, characterization, cloning and structural analysis of Crocodylus siamensis ovotransferrin for insight into functions of iron binding and autocleavage.

Ovotransferrin (OTf), the major protein constituent of egg white, is of great interest due to its pivotal role in biological iron transport and storage processes and its spontaneous autocleavage into peptidic fragments with alternative biological properties, such as antibacterial and antioxidant activities. However, despite being well-investigated in avian, a detailed elucidation of the structure-function relationship of ovotransferrins in the closely related order of Crocodilia has not been reported to date. In this study, electron paramagnetic resonance (EPR) confirmed the presence of two spectroscopically distinct ferric iron binding sites in Crocodylus siamensis OTf (cOTf), but implied a five-fold lower quantity of bound iron than in hen OTf (hOTf). In addition, quantitative estimation of free sulfhydryl groups revealed slight differences to hOTf. To gain a better structural understanding of cOTf, we found a cOTf gene consisting of an open reading frame of 2040bp and encoding a protein of 679 amino acids. In silico prediction of the three-dimensional structure of cOTf and comparison with hOTf revealed four evolutionarily conserved iron-binding sites in both N- and C-lobes, as well as the presence of only 13 of the 15 disulfide bonds in hOTf. This evolutionary loss of disulfide linkages in conjunction with the lack of hydrogen bonding from a dilysine trigger in the C-lobe are presumed to affect the iron binding and autocleavage character of cOTf. As a result, cOTf may be capable of exerting a more diverse array of functions compared to its avian counterparts; for instance, ion buffering, antioxidant and antimicrobial activities.

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 1647bp 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.

Improved genome assembly of American alligator genome reveals conserved architecture of estrogen signaling.

The American alligator, Alligator mississippiensis, like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

Molecular cloning, characterization, tissue distribution and mRNA expression changes during the hibernation and reproductive periods of estrogen receptor alpha (ESR1) in Chinese alligator, Alligator sinensis.

Chinese alligator, Alligator sinensis, is a critically endangered reptile species unique to China. Little is known about the mechanism of growth- and reproduction-related hormones gene expression in Chinese alligator. Estrogens play important roles in regulating multiple reproduction- and non-reproduction-related functions by binding to their corresponding receptors. Here, the full-length cDNA of estrogen receptor alpha (ERα/ESR1) was cloned and sequenced from Chinese alligator for the first time, which comprises 1764bp nucleotides and encodes a predicted protein of 587 amino acids. Phylogenetic analysis of ESR1 showed that crocodilians and turtles were the sister-group of birds. The results of real-time quantitative PCR indicated that the ESR1 mRNA was widely expressed in the brain and peripheral tissues. In the brain and pituitary gland, ESR1 was most highly transcribed in the cerebellum. But in other peripheral tissues, ESR1 mRNA expression level was the highest in the ovary. Compared with hibernation period, ESR1 mRNA expression levels were increased significantly in the reproductive period (P<0.05) in cerebellum, pituitary gland, liver, spleen, lung, kidney and ovary, while no significant change in other examined tissues (P>0.05). The ESR1 mRNA expression levels changes during the two periods of different tissues suggested that ESR1 might play an important role in mediation of estrogenic multiple reproductive effects in Chinese alligator. Furthermore, it was the first time to quantify ESR1 mRNA level in the brain of crocodilians, and the distribution and expression of ESR1 mRNA in the midbrain, cerebellum and medulla oblongata was also reported for the first time in reptiles.