西伯利亚鲟AdipoR克隆、组织分布及其对禁食的响应

为了探究西伯利亚鲟脂联素受体的表达特征及其对能量状态变化的响应, 研究采用RACE方法克隆获得AdipoR1和AdipoR2的cDNA全长。西伯利亚鲟AdipoR1 cDNA全长为2013 bp, 开放阅读框为1146 bp; AdipoR2 cDNA全长为1590 bp, 开放阅读框为1086 bp。多重序列比对结果显示, 西伯利亚鲟AdipoR1与哺乳类、鸟类、两栖类和鱼类的氨基酸一致性较高, AdipoR2与鱼类氨基酸一致性更高。系统进化树分析显示, 西伯利亚鲟AdipoR1与其他硬骨鱼类聚为一支, AdipoR2与斑点雀鳝聚为一支。实时荧光定量结果显示, AdipoR1和AdipoR2 mRNA在西伯利亚鲟组织中均有表达, AdipoR1在中脑、延脑、前脑、小脑和下丘脑中的表达高于外周组织, AdipoR2在中脑、鳃、瓣肠、性腺和延脑的表达相对较高。此外, 与其他硬骨鱼类似, 西伯利亚鲟AdipoR1和AdipoR2在肌肉中表达较低。当禁食10d时, 西伯利亚鲟幼鱼肌肉中AdipoR1和AdipoR2 mRNA表达量均显著上调, 复投喂后AdipoR1表达显著降低, 而AdipoR2表达无显著变化。由上可知, 西伯利亚鲟AdipoR1和AdipoR2可能参与能量稳态的维持。文章为进一步研究AdipoR1和AdipoR2在肌肉中的作用提供了理论基础。     

The evidence of apelin has the bidirectional effects on feeding regulation in Siberian sturgeon (Acipenser baerii)

Apelin is a peptide, mainly produced in the brain, which participates in several physiologic effects. However, knowledge about the mechanism of appetite regulation in teleosts, including the role of apelin is not well understood. The aim of this study is to explore the effect of feeding status on the expression of apelin mRNA in the whole brain and the effects of injection of apelin on food intake in Siberian sturgeon (Acipenser baerii). In this study, we first cloned the apelin cDNA sequence of the Siberian sturgeon. We obtained a 1046-bp cDNA fragment, including a 237-bp open reading frame (ORF) that encoded 78 amino acids. Apelin was widely distributed in 11 tissues related to feeding regulation, with the highest expression in thewhole brain, followed by the spleen and trunk kidney. In addition, we measured the effects of periprandial (preprandial and postprandial) change, fasting and re-feeding on apelin mRNA expression in whole brain. The level of apelin mRNA was significantly decreased 1 h after feeding. The results of the fasting experiment showed that the expression of apelin mRNA in the brain was significantly reduced after 1 day of fasting but consistently increased throughout the 15-day food deprivation period. When the 15-day fasted fish were re-fed, apelin mRNA expression in the brain was significantly increased as compared to that of the control. These results suggest that apelin may play a bidirectional role in the regulation of food intake in the Siberian sturgeon. In order to further examine the effect of apelin on feeding regulation in Siberian sturgeons, acute and chronic intraperitoneal (i.p.) injection experiments were performed and food intakes were recorded. Results showed that acute i.p. injection of apelin-13 reduced food intake, however, chronic i.p. injection apelin-13 increased the food intake for 7 days in Siberian sturgeons. In conclusion, our results show that apelin has a bidirectional effect on feeding regulation in Siberian sturgeons by acting as a satiety factor in short-term feeding regulation and a starvation factor in long-term feeding regulation.     

Intraperitoneal injection urocortin-3 reduces the food intake of Siberian sturgeon (Acipenser baerii)

Urocortin-3 (UCN3), one of the corticotropin releasing factor (CRF) family peptides, which was discovered in 2001, has a variety of biological functions. However, the researches of UCN3 in fish were scarce. In order to understand whether UCN3 play a role in regulating food intake in fish, we first cloned the ucn3 cDNAs sequence of Siberian sturgeon (Acipenser baerii Brandt), and investigated the ucn3 mRNA levels in 11 tissues. The Siberian sturgeon ucn3 cDNA sequence was 1044 bp, including an open reading frame (ORF) of 447 bp that encoded 148 amino acids with a mature peptide of 40 amino acids, a 5ʹ-terminal untranslated region (5ʹ-UTR) of 162 bp and a 3ʹ-terminal untranslated region (3ʹ-UTR) of 435 bp. The result of tissue distribution showed that ucn3 widely distributed in 11 tissues with highest expression in brain. We also assessed the effects of periprandial (pre- and post-feeding), fasting and re-feeding on ucn3 mRNAs abundance in brain. The results showed the expression of ucn3 mRNA in brain was significantly elevated after feeding, decreased after fasting 17 days and increased after re-feeding. To further investigate the food intake role of UCN3 in Siberian sturgeon, we performed intraperitoneal (i.p.) injection of Siberian sturgeon UCN3 (SsUCN3) with three doses (60, 120 or 240 ng/g) and recorded the food intake. Acute and chronic i.p. injection SsUCN3 reduced the food intake in a dose-dependent pattern. In conclusion, this study indicates that SsUCN3 acts as a satiety factor to inhibit the food intake of Siberian sturgeon.     

Glucose Tolerance and Skeletal Muscle Gene Expression in Response to Alternate Day Fasting

Objective: Alternate day fasting may extend lifespan in rodents and is feasible for short periods in nonobese humans. The aim of this study was to examine the effects of 3 weeks of alternate day fasting on glucose tolerance and skeletal muscle expression of genes involved in fatty acid transport/oxidation, mitochondrial biogenesis, and stress response. Research Methods and Procedures: Glucose and insulin responses to a standard meal were tested in nonobese subjects (eight men and eight women; BMI, 20 to 30 kg/m²) at baseline and after 22 days of alternate day fasting (36 hour fast). Muscle biopsies were obtained from a subset of subjects (n = 11) at baseline and on day 21 (12‐hour fast). Results: Glucose response to a meal was slightly impaired in women after 3 weeks of treatment (p < 0.01), but insulin response was unchanged. However, men had no change in glucose response and a significant reduction in insulin response (p < 0.03). There were no significant changes in the expression of genes involved in mitochondrial biogenesis or fatty acid transport/oxidation, although a trend toward increased CPT1 expression was observed (p < 0.08). SIRT1 mRNA expression was increased after alternate day fasting (p = 0.01). Discussion: Alternate day fasting may adversely affect glucose tolerance in nonobese women but not in nonobese men. The gene expression results indicate that fatty acid oxidation and mitochondrial biogenesis are unaffected by alternate day fasting. However, the increased expression in SIRT1 suggests that alternate day fasting may improve stress resistance, a commonly observed feature of calorie‐restricted rodents.     

Dispermic androgenesis in sturgeons with the use of cryopreserved sperm: Production of androgenetic siberian sturgeon and androgenetic hybrids between Siberian and Russian sturgeons

Dispermic androgenesis was used to produce, for the first time, an androgenetic progeny of the Siberian sturgeon (Acipenser baerii) and the androgenetic nucleo-cytoplasmic hybrids (Siberian sturgeon, A. baerii × Russian sturgeon, A. gueldenstaedtii) using cryopreserved sperm. Microsatellite DNA analysis confirmed exclusively paternal inheritance in the androgenetic progeny of Siberian sturgeon. Heterozygotes for certain microsatellite loci were detected among the androgenetic hybrids, thereby confirming a dispermic nature of androgenesis. According to the data of comparative morphological analysis, the obtained androgenetic hybrid, by the age of 15 months old, was completely identical to the paternal species. Both a female and a male were detected in the androgenetic sturgeon progenies, which is of interest for producing bisexual progenies via androgenesis. The data of this study confirm the feasibility of dispermic androgenesis using cryopreserved sperm to preserve and recover the gene pools of endangered sturgeon species.     

Features of inheritance of several morphological characters in the hybrids between Siberian sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae) and in gynogenetic individuals of Siberian sturgeon

Comparative analysis of several morphological characters is conducted in experimentally obtained hybrids between Siberian sturgeon Acipenser baerii and beluga A. huso, in parental species, and in gynogenetic progeny of Siberian sturgeon. The gynogenetic individuals are similar to Siberian sturgeon by all characters used for differentiation between Siberian sturgeon and beluga that supports gynogenetic origin of these individuals. Based on the data obtained on the hybrid specimens, the features of inheritance of the studied characters in sturgeons and their variation in postlarval ontogeny are discussed.     

Comparison of the Effects of Gonadotropic Preparations of the Carp and Stellate Sturgeon Pituitaries onin vivoand in vitroOocyte Maturation in the Siberian Sturgeon Acipenser baeriBrandt

Injections of 2.5 mg/kg of stellate sturgeon (Acipenser stellatusPall.) pituitary extract and 5 mg/kg of carp (Cyprinus carpioL.) pituitary extract in Siberian sturgeon (Acipenser baeriBrandt) females did not reveal significant differences in the effects of these preparations. There were no differences in the percentage of females that responded by ovulation, duration of the period from injection to ovulation, rate of ovulation, or quality of mature eggs as estimated by the percentage of fertilization or percentage of normal embryos at the small yolk plug stage. Thus, an insufficient efficiency in the artificial reproduction of the Siberian sturgeon grown in captivity is not related to the use of the carp pituitary preparation as a stimulus. Estimation of the ratio of specific activities of the pituitary extracts and purified gonadotropins of the stellate sturgeon and carp by in vitrooocyte maturation has shown that it varies within wide limits as a function of the medium composition and physiological state of follicles. Hence, the ratio of activities of the gonadotropins of different species as determined by in vitromaturation of sturgeon oocytes may markedly differ from that upon injection of these preparations in breeders.     

The metabolic effects of prolonged starvation and refeeding in sturgeon and rainbow trout

The present study examines the particular metabolic strategies of the sturgeon Acipenser naccarii in facing a period of prolonged starvation (72 days) and subsequent refeeding (60 days) compared to the trout Oncorhynchus mykiss response under similar conditions. Plasma metabolites, endogenous reserves, and the activity of intermediate enzymes in liver and white muscle were evaluated. This study shows the mobilization of tissue reserves during a starvation period in both species with an associated enzymatic response. The sturgeon displayed an early increase in hepatic glycolysis during starvation. The trout preferentially used lactate for gluconeogenesis in liver and white muscle. The sturgeon had higher lipid-degradation capacity and greater synthesis of hepatic ketone bodies than the trout, although this latter species also showed strong synthesis of ketone bodies during starvation. During refeeding, the metabolic activity present before starvation was recovered in both fish, with a reestablishment of tissue reserves, plasmatic parameters (glucemia and cholesterol), and enzymatic activities in the liver and muscle. A compensatory effect in enzymes regarding lipids, ketone bodies, and oxidative metabolism was displayed in the liver of both species. There are metabolic differences between sturgeon and trout that support the contention that the sturgeon has common characteristics with elasmobranchs and teleosts.     

Induced maturation and spawning in an incidentally caught adult pair of critically endangered European sturgeon, Acipenser sturio L.

Described is how a pair of wild spawners of the critically endangered common Atlantic sturgeon, Acipenser sturio L., were induced to provide progeny despite their unsuitable reproductive states. When captured, the female revealed signs of over‐maturation. The male, caught in brackish water, yielded no sperm; after 5 days in fresh water and with pituitary injection, motile spermatozoa were produced. A batch of 30% fertilized eggs was produced with a two‐injection sequence. As opposed to the Siberian sturgeon, A. baeri, the A. sturio L. hatchlings showed no positive phototaxis.     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms

The genetic polymorphism of ten sturgeon species that inhabit the territory of the Russian Federation (Russian sturgeon, Siberian sturgeon, Amur sturgeon, Sakhalin sturgeon, Persian sturgeon, ship sturgeon, sterlet, stellate sturgeon, beluga, and kaluga) was examined at five microsatellite loci (Afug41, Afug51, An20, AoxD161, AoxD165) (in total, 3821 individuals). The examined loci were successfully amplified with the same primer set in all species tested and demonstrated a high level of variation. Alleles specific to different species have been identified, which allows them to be used to identify species of sturgeon and their food products. In addition, the possibility of identifying hybrid forms was demonstrated. The assignment test performed in the STRUCTURE software program showed a high probability of correctly assigning each individual to its species based on genotyping with five microsatellite loci examined (96–98%, on average). However, for Russian and Persian sturgeon, the rate of proper species assignment was considerably lower (75 and 84%, respectively).

     

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10 min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5 °C and 30 min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.     

Comparative coelomic fluid composition of sterlet sturgeon Acipenser ruthenus Linnaeus, 1758, Siberian sturgeon Acipenser baerii Brandt, 1869, and Russian sturgeon Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833

The objectives of this study were to compare the physicochemical properties of coelomic fluid (CF) in three sturgeon species, sterlet sturgeon Acipenser ruthenus (age 5–8 years), Siberian sturgeon Acipenser baerii (age 15–20 years), and Russian sturgeon Acipenser gueldenstaedtii (age 13–18 years). For the study, CF was collected by plastic pipette from the eggs of mature female sterlet sturgeon (N = 10), Siberian sturgeon (N = 7) and Russian sturgeon (N = 4); osmolality, pH, ionic composition (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?), glucose, cholesterol and total protein of the CF were then analyzed. Mean concentration of K⁺ (4.4–6.11 mmol/L), Cl^? (89.8–98.0 mmol/L), Ca²⁺ (0.87–0.96 mmol/L), Mg²⁺ (0.57–0.67 mmol/L), cholesterol (0.13–0.16 mmol/L), total protein (2.41–3.57 g/L), pH (7.92–7.98), and osmolality (190–213 mOsm/kg) of CF were not significantly different among these three species. However, the mean concentration of Na⁺ was significantly lower in sterlet sturgeon (104.6 ± 7.7 mmol/L; p < .05) than in the Siberian (126.4 ± 6.2) and Russian (123.0 ± 5.9) sturgeon. For these three species, Na⁺, K⁺, Cl^?, and Mg²⁺ were the dominating ions; several significant correlations were observed among these ions and other physiochemical properties of CF. This is the first report on the ionic and biochemical composition of the CF of sterlet, Siberian and Russian sturgeon, which can be used as a reference point for further development of artificial media for the short‐term storage of unfertilized sturgeon eggs as well as for the standardization of the fertilization protocol for these species in controlled reproduction.     

Gene characterization and expression pattern of Mx and Viperin genes in Dabry’s sturgeon Acipenser dabryanus

Mx and Viperin are important interferon‐stimulated genes that mediate the antiviral immune response. In this study, we cloned the Mx and viperin genes from Dabry's sturgeon (Acipenser dabryanus). The Mx cDNA sequence contained an open reading frame (ORF) of 1,449 nucleotides, encoding a putative protein of 392 aa, which is significantly shorter than other animal Mx proteins. Although the similarity and identity were low between sturgeon Mx and other animal Mx proteins, sturgeon Mx contains the conserved tripartite GTP binding motif and a dynamin family signature. The sturgeon Mx gene contains eight exons split by seven introns. The sturgeon viperin cDNA sequence contained an ORF of 1,047 bp encoding a putative protein of 349 aa, which is relatively well conserved among species. Sturgeon viperin proteins show 82% similarity with those of Xiphophorus maculatus platyfish and Poecilia formosa Amazon molly. The sturgeon viperin gene has a six exon/five intron structure with the same size of second, third, fourth, and fifth exons between different species. The expression of Mx and viperin was detectable in all tissues examined, with the highest expression in skin for Mx and in peripheral blood for viperin. After mock infection using polyinosine‐polycytidylic acid, Mx and viperin showed significantly upregulated expression in primary spleen leukocytes from 3 hr to 72 hr. Lipopolysaccharide could also induce their expression. These results suggested Mx and Viperin could play a vital antiviral role in the innate immune system of Dabry's sturgeon.     

Exotic sturgeons in the Vistula Lagoon in 2011, their occurrence,diet and parasites,with notes on the fishery background

The Siberian sturgeon Acipenser baerii and the sterlet Acipenser ruthenus were recorded for the first time in the Vistula Lagoon in 2011. Among 66 sturgeons collected between April and December 2011, the Siberian sturgeon was the most numerous species (77%); however, a significant seasonal variability was observed, with sterlet dominating in the catches in late autumn of the same year. The stomach contents of the two species differed widely: Siberian sturgeon (14.9–42.2 cm standard length, SL) fed on crustaceans (Cercopagis pengoi, Oithona sp., Neomysis integer) (10.7% IRI), larvae and pupas of insects (Chaoborus sp., Chironomus sp., Polypedilum sp., Procladius sp., Culex sp.) (88.9% IRI) and fishes (Neogobius melanostomus, Osmerus eperlanus) (0.5% IRI), whereas sterlet (24.0–34.4 cm SL) consumed crustaceans (N. integer) (64.3% IRI), larvae of insects (Chironomus sp., Polypedilum sp.) (20.3% IRI) and fish (N. melanostomus) (15.4% IRI). Single Siberian sturgeon (4.3%) were found to harbour the parasitic nematode (Raphidascaris acus).     

Overexpression of a novel imprinted gene,<Emphasis Type="Italic">PEG10</Emphasis>, in human hepatocellular carcinoma and in regenerating mouse livers

Many of the promising applications of the microarray technology are pertinent to identifying abnormalities in gene expression that contribute to malignant progression. We developed a bioinformatics tool to identify differentially expressed genes in human hepatocellular carcinoma (HCC). This involved the construction of a liver EST database (http://lestdb.nhri.org.tw) and in silico verification of differentially expressed genes with a human hepatoma microarray database. The stringency of the search was reinforced with a statistical analysis. A novel imprinted gene,Paternally Expressed 10 (PEG10) was identified as having an elevated level of expression in the majority of the HCC samples and was also induced to express during G₂/M phase of regenerating mouse liver. Ectopic expression ofPEG10 in 293T cells affects cell cycle progression. PEG10 is distributed in the cytosol and associates with the nuclear membrane. This is the first time that an imprinted gene has been found to reexpress in both human HCC and in the regenerating mouse liver. This result indicates that the induction of the paternally imprinted gene may play an important role during liver regeneration or carcinogenesis of the human hepatocyte. Understanding the molecular basis of the abnormal imprinting ofPEG10 will shed new light on the process that leads to liver disease.     

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) α. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARα and are less responsive to treatment with PPARα agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARα activation in these tissues (P<.05). Fasted pigs had a higher activity of γ-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARα activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Ig heavy chain of the sturgeon Acipenser baeri : cDNA sequence and diversity

 To investigate the gene organization of the IGH locus, and the VH diversity of the Siberian sturgeon, a cDNA library was constructed and screened with VH-specific probes from two holostean fish. Isolated clones were analyzed and domain-specific probes used in rescreening of the library, Southern blot analysis, and northern blots. It was concluded that the Siberian sturgeon has one IGH locus with a translocon type of organization. Two allelic variants of the mu gene were found, with identities ranging from 80 to 100% for the different domains (highest for CH4 and lowest for CH2). Sturgeon CH sequences are most closely related to those of holostean fish. There are three distinct VH families, VHI grouping with mammalian clan III, VHII grouping with the teleost clan, and VHIII grouping with the archaic clan. The variability of the CDR 3 region is substantial, and we identified a number of conserved motifs in the D segment. Further, we deduced that there are at least nine different JH segments in the locus, contributing to the antibody repertoire of the sturgeon. The variable segments of the three families can be associated with any of the D or JH segments in the rearrangement. Sturgeon, in addition to the random rearrangement of VH, D, and JH segments, have exonuclease activity, and an introduction of N and probably P nucleotides at the site of rearrangement. Received: 2 March 1998 / Revised: 20 May 1998