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The critical role of the G protein-coupled receptor 1 (GPER1), a member of the seven-transmembrane G protein-coupled receptor family, in the functional regulation of oocytes accumulated abundant theories in the early research on model animals. However, the full-length cDNA encoding GPER1 and its role in the folliculogenesis has not been illustrated in crocodilians. 0.5, 3, and 12 months old Alligator sinensis cDNA samples were used to clone the full-length cDNA encoding GPER1. Immunolocalization and quantitative analysis were performed using Immunofluorescence technique, RT-PCR and Western blot. Simultaneously, studies on GPER1's promoter deletion and cis-acting transcriptional regulation mechanism were conducted. Immunolocalization staining for the germline marker DDX4 and GPER1 demonstrated that DDX4-positive oocytes were clustered tightly together within the nests, whereas scarcely any detectable GPER1 was present in the oocytes nest in Stage I. After that, occasionally GPER1-positive immunosignal was observed in oocytes and somatic cells additional with the primordial follicles, and it was mainly located at the granulosa cells or thecal cells within the early PFs in the Stage III. The single mutation of the putative SP1 motif, double mutating of Ets/SP1 and SP1/CRE binding sites all depressed promoter activities. This result will help to investigate the role of GPER1 in the early folliculogenesis of A. sinensis., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “G-protein couple receptor (GPER1) plays an important role during ovarian folliculogenesis and early development of the Chinese Alligator”., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Although oviductal sperm storage are essential steps in reproduction for female animals with internal fertilisation, no systematic study on the identification of genes involving sperm storage has been performed in crocodilian species. In the present research, the relationship between morphological variation related to sperm storage in the oviduct and gene expression patterns derived from RNA sequencing analyses between active period (AP), breeding period (BP), and hibernation period (HP) were investigated. The corresponding results indicated that sperm were observed not only in the ciliated cells within infundibulum and mucosal layer of uterus during BP, but also been detected in the spermatosperm storage tube (SST) in the anterior uterus at HP stage. The further transmission electron microscopy analysis indicated that the differences in the number and activity of the secretory cells likely to attributed to the seasonal variation of microenvironment related to the sperm storage. Based on the RNA-sequecing, 13147 DEGs related to the Peroxisome proliferator-activated receptors (PPARs) and FOXO signalling were identified, including these, the down-regulated ATG12 and BCL2L11 in the HP group may thus constitute an important point of convergence between autophagy and apoptosis involving the FOXO1 pathway. The genes involved in the PPARs pathway might modulate the immune response and thereby contribute to prolong the life span of stored spermatozoa in Alligator sinensis . The outcomes of this study provide fundamental insights into the mechanism of sperm storage in A. sinensis .

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., (Copyright © 2021. Published by Elsevier B.V.)

Hibernation is a physiological state for Chinese alligators to cope with cold weather. In mammals, gene expression changes during hibernation and their regulatory mechanisms have been extensively studied, however, these studies in reptiles are still rare. Here, integrated analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) reveals the molecular mechanisms of the hypothalamus, liver, and skeletal muscle in hibernating and active individuals. During hibernation, the number of genes increased in the hypothalamus, liver, and skeletal muscle was 585, 282, and 297, while the number of genes decreased was 215, 561, and 627, respectively, as compared with active individuals. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, the differential expressed genes were mainly enriched in DNA damage repair, biological rhythm, energy metabolism, myoprotein degradation, and other related items and pathways. Besides, 4740 miRNAs were identified in three tissues. Through the comprehensive analysis of miRNA and mRNA abundance profiles, 12,291, 6997, and 8232 miRNA-mRNA pairs all showed a negative correlation in the hypothalamus, liver, and skeletal muscle, respectively. Some miRNA target genes were related tobiological rhythm and energy metabolism, suggesting that miRNA may play an important role in the physiological metabolism of the hibernating adaptability of Chinese alligators. Moreover, 402, 230, and 130 differentially expressed lncRNAs were identified in the hypothalamus, liver, and skeletal muscle, respectively. The targeting relationship of four lncRNA-mRNA pairs were predicted, with the main function of target genes involved in the amino acid transportation. These results are helpful to further understand the molecular regulatory basis of the hibernation adaptation in Chinese alligators., (© 2020 Wiley Periodicals LLC.)

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., (© 2020 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The purpose of this study was to study the reproductive characteristics of the Nile crocodile and Siamese crocodiles after introduction into China since the time this occurred near the end of the last century. The data for the eggs and young crocodiles (recently hatched crocodiles) of two introduced species were collected at a Sanya crocodile breeding farm in Hainan. The characteristic variables of crocodile eggs were statistically analyzed, and the results indicated that: egg mass of the Nile and Siamese crocodile was significantly correlated with the egg length and width. Regression analyses were used to develop the linear equation between the egg length, egg width and egg mass. There was a strong positive correlation between the egg mass and initial weight of young crocodiles. The linear equation for assessing egg mass and initial weight of young crocodile was developed for regression analyses. There was no significant linear relationship between clutch size and egg characteristics. Mating time of the Nile crocodile in Hainan (November-April) and the spawning season (March-May) are significantly earlier than in the Zimbabwe region of origin. The average of clutch size and the mean size of eggs for Nile crocodiles in their native habitat is greater than the introduced region as indicated by analyzing data using a two-sample t-test. The Siamese crocodile spawning time was similar in the Hainan and Zimbabwe regions, but the size of clutches and the mean size of eggs in the introduced region were greater than in their native region as indicated by results using a two-sample t-test., (Copyright © 2018. Published by Elsevier B.V.)

The Chinese alligator Alligator sinensis is a critically endangered species endemic to China. Knowledge about reproductive strategies of a species contributes to their conservation. Little is, however, known about the reproductive strategies and its impact on the population. In the present study, an easy and non-invasive genetic method was used to improve the understanding of mating system of Chinese alligators and its effect on the population genetic diversity by nine polymorphic microsatellite loci. There was a high incidence of multiple paternity among 50 clutches, with a total 60% of the clutches having multiple paternity and up to three males contributing to single clutches. In addition, polyandry females choose to mate with males that are more distant in relatedness compared with monogamy females. Multiple paternity can decrease the inbreeding coefficient, while there is no significant difference between single and multiple paternity (P>0.05). Furthermore, there was an increased allelic diversity (though not heterozygosity) in multiple paternity sired offspring compared with the single paternity sired offspring in F2 generations (P<0.05), as predicted by the genetic diversity hypothesis. Multiple paternity may function as an important inbreeding avoidance compensation mechanism leading to the potential of the species to avoid extinction. These findings will not only enhance the understanding of the mating system and the biological traits of the Chinese alligator, but also improve the captive breeding program management and conservation strategies of the endangered species., (Copyright © 2017. Published by Elsevier B.V.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, the regional distribution and histological localization of somatostatin (SS) immunoreactive (IR) perikarya and fibers was investigated for the first time in the brain of adult Chinese alligator by immunohistochemical method. The results showed SS-IR perikarya and fibers were widely distributed in various parts of the brain except for olfactory bulbs. In the telencephalon, SS-IR perikarya were predominantly located in the cellular layer and deep plexiform layer of dorsomedial and medial cortex, less in the dorsal and lateral cortex, while SS-IR fibers were found in all layers of the cerebral cortex. SS-IR perikarya and fibers were also detected in the dorsal ventricular ridge, hippocampus cortex, accessory olfactory bulb nuclearus, lenticular nucleus, and caudate nucleus. In the diencephalon, SS-IR perikarya and fibers were mainly present in supraoptic nucleus, paraventricular nucleus of hypothalamus, recessus infundibular nucleus, median eminence, the pineal gland and pituitary gland, in which the IR-fibers were abundant, appearing dot-shaped and varicosity-like. In the mesencephalon, they were present in tectum cortex, ependyma of cerebral aqueduct and the periaqueductal grey matter. Additionally, they were also detected in Purkinje's cellular layer of cerebellum, in the reticularis nucleus and raphe nucleus of medulla oblongata. The distribution pattern of SS-IR perikarya and fibers in the brain of Chinese alligator is generally similar to that reported in other reptiles, but also has some specific features. The wide distribution indicated that SS might be a neurotransmitter or neuromodulator which acts on many kinds of target cells with a wide range of physiological functions. Anat Rec, 300:507-519, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The Chinese alligator Alligator sinensis is an endangered species endemic to China, up to date, little is known about the regulation of its growth and development. Insulin-like growth factor I (IGF-I) plays a vital role in regulating vertebrate growth and development. In this study, the full-length cDNA of IGF-I in Chinese alligator (caIGF-I) was obtained for the first time, it contains 890-bp nucleotides encoding a 153-amino acid precursor, the mature caIGF-I consists of 70 amino acids by cleaving the signal peptide and C-terminal extension (E domain). The caIGF-I contains all the features of IGF-I peptide with B, C, A, and D domains and the six conservative cysteine residues involved in the stable tertiary structure. Multiple alignment analysis showed that the amino acid sequence of caIGF-I shares high identity with American alligator Alligator mississippiensis (100%) and birds (95-97%). Phylogenetic tree analysis of the IGF-I amino acid sequences indicated that alligators cluster into the bird branch. Real-time quantitative PCR technique showed that caIGF-I is widely expressed in all the examined tissues with the highest expression level in liver, higher in pancreas and oviduct while lower in heart, spleen, lung, kidney, stomach, intestines, ovary and muscles. During hibernation, the caIGF-I expression level decreased significantly in liver, pancreas, oviduct and kidney, while did not significantly change in heart, spleen, lung, stomach, small intestine, ovary and muscles. The mRNA expression changes during the two periods implicate that caIGF-I might play an important role in the regulation of feeding and growth in the Chinese alligator., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

Crocodiles are susceptible to infection with a wide array of external and internal gastrointestinal helminths, yet little is known on the histopathology following infection or the effects of these parasites. The present study was aimed at evaluating the impact of infection by Ortleppascaris sinensis (Nematoda: Ascaridoidea) on the stomach of captive Alligator sinensis. The histological examination of the stomach revealed presence of superficial ulcer in mucous layer and granulomatous inflammation in submucous layer at entire gastric walls of the Alligator sinensis. Our findings also confirm that development of Ortleppascaris sinensis is in close association with the wall of the stomach., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

The Chinese alligator Alligator sinensis is an endangered species endemic to China, it has a highly specialized reproductive pattern with low fecundity. Up to date, little is known about the regulation of its female reproductive cycle. Follicle-stimulating hormone (FSH), a glycoprotein hormone, plays a key role in stimulating and regulating ovarian follicular development and egg production. In this study, the complete FSHβ cDNA from the ovary of the Chinese alligator was obtained for the first time, it consists of 843-bp nucleotides, including 120-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 327-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHβ with a signal peptide of 18 amino acids followed by a mature protein of 113 amino acids. Its deduced amino acid sequence shares high identities with the American alligator (100%) and birds (89-92%). Phylogenetic tree analysis of the FSHβ amino acid sequence indicated that alligators cluster into the bird branch. Tissue distribution analyses indicated that FSHβ mRNA is expressed in ovary, intestine and liver with the highest level in the ovary, while not in stomach, pancreas, heart, thymus and thyroid. Expression of FSHβ in ovary increases in May (breeding prophase) and peaks in July (breeding period), it is maintained at high levels through September, then decreases significantly in November (post-reproductive period) and remains relatively low from January to March (hibernating period). These temporal changes of FSHβ expression implicated that it might play an important role in promoting ovarian development during the female reproductive cycle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The follicle-stimulating hormone (FSH) plays a central role in vertebrate reproduction, with the actions of FSH mediated by FSH receptors (FSHRs) on the granulosa cells of the ovary. The present study reports the cloning and characterization of FSHR in Chinese alligator, Alligator sinensis (caFSHR), and its tissue distribution and mRNA expression changes during the reproductive cycle. The mature protein of caFSHR displays typical features of the glycoprotein hormone receptor family, but also contains some remarkable differences when compared with other vertebrate FSHRs. The deduced amino acid sequence of the caFSHR shares identity of 85% with Chinese softshell turtle, 84-87% with birds, 77-78% with mammals, 67-73% with amphibians and 51-58% with fishes. Phylogenetic tree analysis of the FSHR amino acid sequence indicated that alligators cluster into the bird branch. Tissue expression analysis showed that caFSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid. Expression of caFSHR in the ovary is high in May (breeding prophase) and peaks in July during the breeding period, where it is maintained at high levels through September (breeding anaphase). Expression decreases significantly in November (hibernating period) and then remains relatively low from January to March (hibernating period). These temporal changes in FSHR expression suggest that it plays an important role in promoting ovarian development during the female reproductive cycle of Chinese alligator., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Decabromodiphenyl ethane (DBDPE), a replacement for decabromodiphenyl ether (deca-BDE), was investigated in captive Chinese alligators from China. DBDPE was detected in adult tissues, neonates and eggs of Chinese alligators with concentrations ranging from 4.74-192, 0.24-1.94, and 0.01-0.51 ng g(-1) lipid weight, respectively. Compared to PBDEs and PCBs, DBDPE contamination was limited in Chinese alligators. Additionally, DBDPE concentrations in adult muscles were one to three orders of magnitude higher than those in neonates and eggs, suggesting the limited maternal transfer potential of DBDPE in Chinese alligators. This is the first study to report the occurrence of DBDPE in Chinese alligators.

Objective: Although intestinal parasites are commonly detected in either wild or farmed crocodiles in other parts of the world, yet few reports have ever been on the captive bred Chinese alligators (Alligator sinensis) since they are only distributed in the southern areas of the Yangtze River. The current work was undertaken to investigate the intestinal parasites and their diversity in the Chinese crocodilian species., Methods: In May and October of 2013, we randomly collected a total of 328 fecal samples of the captive Chinese alligators in a breeding centre in south Anhui province., Results: Three genera of protozoa (Entaoeba, Eimeria and Isospora), five genera of nematodes (Ascaris, Dujardinascaris, Capillaria, Toxocara and Strongylus), two genera of trematodes (Echinostoma and Clonorchis) and two families of trematodes (Schistosomatidae and Cryptogonimidae) were identified, in which Dujardinascaris was the most prevalent and led to the highest infection rate (14.33%), and Entaoeba ranked the second (13.11%). The number of species, richness index, diversity index and evenness index were higher in July and August, and those indexes were relatively most in juvenile and subadult alligators., Conclusions: Our findings suggest that the intestinal parasites infection was prevalent in captive Chinese alligators, and this condition requires our attention, whatever it is for controlling or preventing the intestinal parasitic disease from spreading to humans or conservation of this endangered species., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

The digestive tract is the largest endocrine organ in the body; the distribution pattern of endocrine cells varies with different pathological and physiological states. The aim of the present study was to investigate the distributed density of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS) and vasoactive intestinal peptide (VIP) immunoreactive (IR) cells in the digestive tract of Alligator sinensis during the active and hibernating period by immunohistochemical (IHC) method. The results indicated that 5-HT-IR cells were distributed throughout the entire digestive tract, which were most predominant in duodenum and jejunum. The density increased significantly in stomach and duodenum during hibernation. GAS-IR cells were limited in small stomach and small intestine. The density decreased significantly in small stomach during hibernation, while increased in duodenum. What's more, most of the endocrine cells in duodenum were generally spindle shaped with long cytoplasmic processes ending in the lumen during hibernation. SS-IR cells were limited in stomach and small stomach. The density increased in stomach while decreased in small stomach during hibernation, meanwhile, fewer IR cells occurred in small intestine. VIP-IR cells occurred in stomach and small stomach. The density decreased in small stomach, while increased in stomach during hibernation. These results indicated that the endocrine cells in different parts of digestive tract varied differently during hibernation, their changes were adaptive response to the hibernation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

While a number of studies have reported residual levels of persistent halogenated compounds (PHCs) in crocodilia, there is still a dearth of information on the Chinese alligator, a critically endangered crocodilian species. In the present study, several PHCs, including polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane and its metabolites (DDTs), were detected in the adult tissues, neonates, and eggs of captive Chinese alligators from China. The concentrations of ΣPBDEs, ΣPCBs, and ΣDDTs in Chinese alligators ranged from 0.11 to 16.1, 1.12 to 22.2, and 6.03 to 1020ngg(-1) wet weight, respectively, with higher levels of ΣPCBs and ΣDDTs in the neonates and eggs than in muscle tissues. The ΣDDT residues in the studied Chinese alligators were at the high end of reported ranges from crocodilia around the world, and some results exceeded levels known to cause a female-biased sex ratio in crocodilians., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Expression of nerve growth factor (NGF) in structures of the partial central nervous system of the Yangtze alligator, Alligator sinensis (Reptilia, Crocodylia) was examined during early postnatal growth using immunohistochemistry and Western blot assays. In animals 0-2 years of age NGF-positive cells in the cerebral cortex increased gradually in number and size, and were predominantly distributed in the molecular layer. NGF-positive cells in the midbrain showed similar increases but with predominant distribution in the ependymal layer. NGF-positive cells increased in the cerebellum between 0 and 1 years of age, with increased NGF expression being seen during the first 2 years of life mostly in the ependymal layer. NGF-positive cells were mainly found in the gray matter of the spinal cord with decreasing cell numbers, NGF expression levels being seen from 0 to 2 years and small processes without synaptic connection from 1 to 2 years. These results suggest that NGF is involved in the early postnatal growth of several structures of Yangtze alligator partial central nervous system, suggesting a possible role of NGF in the Yangtze alligator partial central nervous system., (Copyright © 2013 Wiley Periodicals, Inc.)

Our study used MHC class IIb gene exon3 complete sequence as markers to investigate genetic variability, selection and population differentiation in Chinese alligator. In this study, 282 bp MHC IIb exon3 complete sequence was got, none of the sequences contained insertions/deletions or stop codons, suggesting that all sequences might come from functional molecules in the genome. The neighbor-joining (NJ) tree revealed that Xuangzhou and Changxing populations were genetically close related, while Wild population showed the most diverse from the other. Gene flow (Nm) was very higher than one, suggesting that inter-group gene flow may have occurred. Furthermore, the results showed that MHC IIb gene might be a good molecular marker, we think that this technology could be used for Chinese alligator breeding and releasing in future.

Crocodilians are a group of reptiles that are closely related to birds and are thought to possess a strong immune system. Here we report that the IgH locus in the Siamese crocodile and the Chinese alligator contains multiple μ genes, in contrast to other tetrapods. Both the μ2 and μ3 genes are expressed through class-switch recombination involving the switch region and germline transcription. Both IgM1 and IgM2 are present in the serum as polymers, which implies that IgM class switching may have significant roles in humoural immunity. The crocodilian α genes are the first IgA-encoding genes identified in reptiles, and these genes show an inverted transcriptional orientation similar to that of birds. The identification of both α and δ genes in crocodilians suggests that the IgH loci of modern living mammals, reptiles and birds share a common ancestral organization.

The DNA sequences encoding beta-keratin have been obtained from Marsh Mugger (Crocodylus palustris) and Orinoco Crocodiles (Crocodylus intermedius). Through the deduced amino acid sequence, these proteins are rich in glycine, proline and serine. The central region of the proteins are composed of two beta-folded regions and show a high degree of identity with beta-keratins of aves and squamates. This central part is thought to be the site of polymerization to build the framework of beta-keratin filaments. It is believed that the beta-keratins in reptiles and birds share a common ancestry. Near the C-terminal, these beta-keratins contain a peptide rich in glycine-X and glycine-X-X, and the distinctive feature of the region is some 12-amino acid repeats, which are similar to the 13-amino acid repeats in chick scale keratin but absent from avian feather keratin. From our phylogenetic analysis, the beta-keratins in crocodile have a closer relationship with avian keratins than the other keratins in reptiles.

Crocodilians are quite vocal relative to other reptile groups, and the alligators are among the most vocal of the crocodilians. The Chinese alligator, Alligator sinensis, is usually solitary but engages in bellowing choruses in certain waters during the mating season. This paper reports the organization of Chinese alligator's bellowing choruses based upon field observations and playback experiments. Alligators of both genders engaged in the choruses, remaining immobile throughout and inclining toward bellowing synchronously (i.e., starting and finishing at about the same time). The choruses lasted about 10 min with abrupt onset and offset. Moreover, playback experiments revealed that both male and female alligators responded equally to bellowing stimuli from the same and opposite sexes and that none of the tested alligators approached the loudspeaker in spite of playback of male or female stimuli. These suggest that Chinese alligators may not bellow to compete for or attract mates during the choruses. Instead, when their ecological behaviors, namely, dispersed inhabitation, multi-copulation, restricted mating season, etc., are considered, we hypothesize that they may synchronize bellows to enhance group detectability for assembling individuals into certain waters for subsequent copulations.

The complete sequence of mitochondrial genome of Siamensis Crocodile (Crocodylus siamensis) is determined by using PCR amplification, clone and primer-walking sequencing methods. The genome is 16836 bp in size, containing 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mtDNA genome of Siamensis Crocodile is similar to most vertebrates in gene component, order, orientation, tRNA structure, low percentage of guanine and high percentage of thymine. The nonstandard stop codes (T) in two protein genes (Cox III and Cyt b) are more than those of most vertebrates. Transfer RNA genes range in length from 61 to 76 nucleotides, and their planar structure present characteristic clover leaf, except for tRNA-Ser (AGY) because of lacking the "DHU" arm.

The polymorphism of MHC class II B gene in 14 Chinese alligators was analyzed, which came from three different areas: a wild population from Xuancheng, Anhui, a captive population from Changxing, Zhejiang, and a captive population from Anhui Research Center for Reproduction of Chinese Alligators. The gene fragment was amplified using a pair of specific primers designed from the MHC gene sequence of the spectacled caiman. A total of 34 sequence haplotypes of exon 3 were detected in the sampled Chinese alligators. The numbers of haplotypes of the 3 Chinese alligator populations were 15, 10, and 9, respectively. The overall estimation of the MHC polymorphism in the Chinese alligator population was higher than those in mammals and in cyprinid fish. The rates of nonsynonymous substitutions (d(N)) occurred at a significantly lower frequency than that of synonymous substitutions (d(S)), which were not consistent with the common rule. This result might suggest that the polymorphism of exon 3 seemed not to be maintained by the balancing selection. The neutrality test of Tajima excluded the null hypothesis that the polymorphism of exon 3 was generated by a random drift, and the fact that D = -0.401 indicated an excess of rare mutations in the Chinese alligator. The nucleotide diversity of the sequences and the phylogenetic relations were also analyzed, and the results suggested that there was no significant difference in genetic diversity among the 3 populations of Chinese alligator.

The nucleotide sequence of the complete mitochondrial DNA (mtDNA) molecule of the salt-water crocodile (Crocodylus porosus) was determined in this article. The molecule is 16,917 base pairs (bp) in length, and codes for 22 tRNAs, 13 protein-coding genes, 2 rRNAs, as well as a control region (D-loop), as is characteristic for mitochondrial genomes of other metazoans. The gene order conforms to that of other crocodilians sequenced, but the arrangement of some tRNA genes differs from other vertebrates. It shows that the gene order of crocodilians is remarkably conserved. In this study, the relationships among crocodilians were examined in the phylogenetic analysis based on the control conserved regions of 17 crocodilians. The results suggest that the gharial (Gavialis gangeticus) joins the false gharial (Tomistoma schlegelii) on a common branch, and then constitutes a sister group to traditional Crocodylidae. Thus, the result supports that G. gangeticus belongs to Crocodylidae. The analyses also suggest that the African slender-snouted crocodile (Crocodylus cataphractus) can be treated as an isolated genus, and constitutes a sister group to Crocodylus.

In order to reveal the reasons of the dramatic decline of wild Chinese alligator population, an investigation on its habitat status was made across the National Natural Conservation Regions of Chinese alligator in south Anhui Province from 2002 to 2003. Water samples from 21 sites were collected, and their heavy metals (Cu, Zn, Cd and Pb) content and pH value were measured. The results showed that the pH value was 7.84 +/- 0.62, with no significant difference between the waters with and without Chinese alligator, but with significant difference (P< 0.01) among 3 Chinese alligator habitats. In the waters with and without Chinese alligator, no significant difference was observed in the contents of test metals except Pb. The possible pollution source of Pb in wild Chinese alligator habitat was analyzed, which could provide reference for Chinese alligator conservation.

In June and July of 2003, a field survey on the vegetation of 22 Chinese alligator habitats distributed in Nanling county, Jingxian county, Guangde county, Langxi county and Xuancheng city of Anhui province was conducted, and the plant species of the habitats were recorded and analyzed. The results showed that the vegetation of the whole habitats was secondary, and there were 294 species vascular plants, belonging to 92 families. Pteioblastus amarus shrubbery could be found in all sites, and part differences of the vegetation consisted in different habitats. The relationship between Chinese alligator and habitat vegetation was also analyzed. All of these could offer some basic botanical data for the further protection of the Chinese alligator.