Inhibition of the NF-κB signaling pathway affects gonadal differentiation and leads to male bias in Paramisgurnus dabryanus.

In recent years, sex-controlled breeding has emerged as an effective strategy to enhance the yields of economic animals with different growth characteristics, while increasing the economic benefits of aquaculture. It is known that the NF-κB pathway participates in gonadal differentiation and reproduction. Therefore, we used the large-scale loach as a research model for the present study and selected an effective inhibitor of the NF-κB signaling pathway (QNZ). This, to investigates the impacts of the NF-κB signaling pathway on gonadal differentiation during a critical period of gonad development and after maturation. Simultaneously, the sex ratio bias and the reproductive capacities of adult fish were analyzed. Our results indicated that the inhibition of the NF-κB signaling pathway influenced the expression of genes related to gonad development, regulated the gene expression related to the brain-gonad-liver axis of juvenile loaches, and finally impacted the gonadal differentiation of the large-scale loach and promoted a male-biased sex ratio. Meanwhile, high QNZ concentrations affected the reproductive abilities of adult loaches and inhibited the growth performance of offspring. Thus, our results deepened the exploration of sex control in fish and provided a certain research basis for the sustainable development of the aquaculture industry.

Effects of short-time exposure to atrazine on miRNA expression profiles in the gonad of common carp (Cyprinus carpio).

BACKGROUND: Atrazine is widely used in agriculture and is a known endocrine disrupting chemical. Atrazine can seep into the water body through surface, posing a potential threat to the aquatic ecological environment and human drinking water source. In vertebrate, studies have shown that it can affect reproduction and development seriously, but its molecular mechanism for aquatic animals is unknown. Aquaculture is very common in China, especially common carp, whose females grow faster than males. However, the effects of atrazine on the reproduction of carp, especially miRNA, have not been investigated. RESULTS: In this study, common carp (Cyprinus carpio) at two key developmental stages were exposed to atrazine in vitro. Sex ratio was observed to analyze the effect of atrazine on the sex. MiRNA expression profiles were analysed to identify miRNAs related to gonad development and to reveal the atrazine mechanisms interfering with gonad differentiation. The results showed that the sex ratio was biased towards females. Atrazine exposure caused significant alteration of multiple miRNAs. Predicted targets of differently-expressed miRNAs were involved in many reproductive biology signalling pathways. CONCLUSIONS: Our results indicate that atrazine promoted the expression of female-biased genes by decreasing miRNAs in primordial gonad. In addition, our results indicate that atrazine can up-regulate aromatase expression through miRNAs, which supports the hypothesis that atrazine has endocrine-disrupting activity by altering the gene expression profile of the Hypothalamus-Pituitary-Gonad axis through its corresponding miRNAs.

Selection of suitable candidate genes for miRNA expression normalization in Yellow River Carp (Cyprinus carpio. var).

Yellow River carp is widely cultivated in the world due to its economic value in aquaculture, and the faster growth of females compared to males. It is believed that microRNAs (miRNA) are involved in gonadal differentiation and development. qPCR is the most preferred method for miRNA functional analysis. Reliable reference genes for normalization in qRT-PCR are the key to ensuring the accuracy of this method. The aim of present research was to evaluate as well as identify the efficacy of reference genes for miRNA expression using qRT-PCR in Yellow River carp. Nine ncRNAs (miR-101, miR-23a, let7a, miR-26a, miR-146a, miR-451, U6, 5S, and 18S) were chosen and tested in four sample sets: (1) different tissues in adult carp, (2) different tissues in juvenile carp, (3) different early developmental stages of carp, and (4) different developmental stages of carp gonads. The stability and suitability values were calculated using NormFinder, geNorm, and BestKeeper software. The results showed that 5S was a suitable reference gene in different tissues of adult and juvenile carp. The genes 5S, 18S, and U6 were the most stable reference genes in the early developmental stages of carp. Let-7a and miR-23a were considered as the suitable reference genes in the development of gonads. All these reference genes were subsequently validated using miR-430. The results showed that genes 5S and 18S were the most suitable reference genes to normalize miRNA expression under normal growth conditions in early different developmental stages. The genes Let-7a, and miR-23a were the most suitable in different developmental stages. The present study is the first comprehensive study of the stability of miRNA reference genes in Yellow River carp, providing valuable as well as basic data for investigating more accurate miRNA expression during gonadal differentiation and development of carp.

Identification of differentially-expressed genes in early developmental ovary of Yellow River carp (Cyprinus carpio var) using Suppression Subtractive Hybridization.

Ovary development appears to be under polygenic control, and is influenced by multiple genetic factors that may vary from organism to organism. To gain a better insight into the molecular mechanisms of carp ovary development, Suppression Subtractive Hybridization (SSH) DNA libraries in two species of Yellow River carp were analyzed. Primordial gonads and stage II ovaries were used as testers, and adult ovaries as drivers. One hundred and fifty differentially-expressed candidate genes were examined by Southern blot microarray hybridization. We identified 41 differentially-expressed genes in the PG (Primordial gonad) library and 37 in the stage II ovary library. Gene Ontology Biological Pathway analysis showed the genes were involved in signal transduction, proteolysis process, cell differentiation, TGF-ß signal and other biological responses. Twenty-two candidate genes were selected and further characterized using qRT-PCR. Pvalb, epd, and MYH were found specifically expressed in PG, while bmp2b, desmin and fp1 were specifically expressed in stage II ovary. Our results indicate that these genes could be used as biomarkers of the early development of carp ovary. This finding will provide a basis for further understanding of the complex gonad developmental molecular mechanisms in Yellow River carp.

Oxidative stress, genotoxicity and cytotoxicity of 1-methyl-3-octylimidazolium chloride on Paramisgurnus dabryanus.

This study evaluated the toxicity of 1-methyl-3-octylimidazolium chloride ([C8mim]Cl) on Paramisgurnus dabryanus by enzyme analysis, comet assay, and apoptosis analysis. The study showed that [C8mim]Cl had an obvious toxic effect inducing oxidative stress, genotoxicity, and cytotoxicity to fish liver cells. [C8mim]Cl also induced changes in the activities of superoxide dismutase and catalase, and the glutathione content and malondialdehyde level in fish exposed at 20-80mgL-1. With increased exposure concentration and time, the four antioxidant enzyme activities, three different comet parameters and apoptosis rates of tested cells were significantly increased, with significant differences (P<0.05 or P<0.01) observed between control group and each treatment group. This study shows that [C8mim]Cl could be a threat to aquatic organism health when accidentally released into aquatic ecosystems.

Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization.

The limited number of gonad-specific and gonad-related genes that have been identified in fish represents a major obstacle in the study of fish gonad development and sex differentiation. In common carp Cyprinus carpio from China's Yellow River, the ovary and testis differ in volume and weight in adult fish of the same age. Comparing sperm, egg, and somatic cell transcripts in this carp may provide insight into the mechanisms of its gonad development and sex differentiation. In the present work, gene expression patterns in the carp ovary and testis were compared using suppression subtractive hybridization. Two bidirectional subtracted complementary DNA (cDNA) libraries were analyzed in parallel using testis or ovary as testers. Eighteen nonredundant clones were identified in the male library, including 15 known cDNAs. The expression patterns of selected genes in testis and ovary were analyzed using reverse transcriptase polymerase chain reaction. Tektin-1, GAPDS, FGFIBP, IGFBP-5, and an unknown gene from the Ccmg4 clone were observed to be expressed only in testis. GSDF, BMI1b, Wt1a, and an unknown gene from the Ccme2 clone were expressed at higher levels in testis than in ovary at sexual maturity. Thirty functional expressed sequence tags (ESTs) were identified in 43 sequenced clones in the female library, including 28 known cDNAs, one uncharacterized cDNA (EST clone), and one novel sequence. Eight identified ESTs showed significant differences in expression between the testis and the ovary. ZP3C and Psmb2 were expressed exclusively in ovary, whereas the expression levels of IFIPGL-1, Setd6, ATP-6, CDC45, AIF-1, and an unknown gene from the Ccfh2 clone were more strongly expressed in ovary than in testis. In addition, the expression of ZP3C, Wt1a, and Setd6 was analyzed in male and female gonads, heart, liver, kidney, and brain. ZP3C was expressed only in ovary. Setd6 expression was significantly stronger in female tissues than that in the male, except in the liver, and Wt1a expression showed sexual dimorphism in the kidney and liver. Results suggest that these genes could play key roles during carp growth, both in the gonad and other tissues. The results provide a resource for further investigation of molecular mechanisms responsible for gonad development and sex differentiation in Yellow River common carp.

Genotoxic effects of 8-hydroxylquinoline in loach (Misgurnus anguillicaudatus) assessed by the micronucleus test, comet assay and RAPD analysis.

This study was a preliminary step in evaluating the genotoxic effects of 8-hydroxylquinoline (8-HOQ) in loach (Misgurnus anguillicaudatus) using the micronucleus, comet and RAPD assays. In the micronucleus test and comet assay, the micronuclei rate (%) and three comet parameters (trailing rate, tail length and tail moment) in treated fish increased with increasing 8-HOQ concentration and treatment time. These results showed that exposure to 8-HOQ significantly induced genetic toxicity in loach blood cells. A subsequent RAPD assay also showed that 8-HOQ induced a genotoxic effect in loach. Among the 23 tested RAPD primers, 11 primers produced unique polymorphic band patterns and generated RAPD profile variations that displayed the band intensity, disappearance of bands and appearance of new bands of amplified DNA in the 8-HOQ-treated genomic DNA samples. In addition, the variation in RAPD profiles was time- and concentration-dependent. These results suggested that 8-HOQ is potentially harmful to fish and may be a toxic contaminant in the aquatic environment.

[Identification of a novel male-specific DNA marker in loach (Misgurnus anguillicaudatus)].

The loach is widely distributed in China and cultivated enormously with the price increased in recent years. Its sex determination type and sex chromosome has not been recognized. In the present study, we report a novel sex-specific DNA marker in loach Misgurnus anguillicaudatus. The animals used in our experiments were collected from a wild population in wetland on the ancient Yellow river, Yanjin Country, Henan Province. 220 random primers were used for random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with the genomic DNA from two sex individuals as template. When using the S2111 primer, a novel 1040 bp sex-specific PCR product was amplified from known male fish. This fragment of DNA was cloned and sequenced. Two primers (MAmf1-P1 and -P2) were designed from the cloned sex-specific sequence to amplify the male-specific fragment using PCR for sexing, with the house-keeping gene beta-actin as positive control. Sex-specific bands in the gel were represented in the males but none were found in the females. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned male-specific DNA fragment. Clear hybridization signals were found only in the male loach. The same result was obtained from dot blotting hybridization. The fragment is a repetitive DNA, and presents its closely related species Paramisgurnus dabryanus but not linked to sex. The cloning of the male-specific DNA laid a good foundation for the isolation of gender-specific chromosome segments with chromosomal walking or gene library, and improved the mechanism exploration of sex chromosome evolution in fish.

Cloning and study of adult-tissue-specific expression of Sox9 in Cyprinus carpio.

The Sox9 gene is one of the important transcription factors in the development of many tissues and organs, particularly in sex determination and chondrogenesis. We amplified the genomic DNA of Cyprinus carpio using degenerate primers, and found that there were two versions of Sox9 in this species: Sox9a and Sox9b, that differ in having an intron of different length (704 bp and 616 bp, respectively) in the conserved HMG box region that codes for identical amino acid sequences. We used a two-phase rapid amplification of cDNA ends (RACE) for the isolation of full-length cDNA of Sox9b. Sequence analyses revealed a 2447-bp cDNA containing 233-bp 5' untranslated region, a 927-bp 3' untranslated region, including poly(A), and a 1287 bp open reading frame (ORF) encoding a protein of 428 amino acids. The HMG box of 79 amino acid motif was confirmed from positions 96-174. Sequence alignment showed that the identity of amino acids of Sox9 among ten animal species, including C. carpio, is 75%, indicating that the Sox9 gene is evolutionarily quite conserved. The expression level of Sox9b gene varied among several organs of adult C. carpio, with the level of expression being highest in the brain and testis.

[The cloning and expression analysis of Sox9b in Cyprinus carpio].

Sox9 gene is one of the important transcription factors involved in developments of many tissues and organs, particularly in sex determination and chondrogenesis. In this article, the genomic DNA of Cyprinus carpio were amplified using degenerate primers. It was found for the first time that there were two versions of Sox9 in C. carpio, Sox9a and Sox9b with the same deduced amino acid sequences in the HMG box. There is one intron in this box with the length 704bp in Sox9a and 616bp in Sox9b respectively. Furthermore, a two-phase rapid amplification of cDNA ends(RACE) was used for the isolation of the full length cDNA of Sox9b. Sequence analysis revealed a 2447bp cDNA encoding a protein of 428 amino acid. HMG box of 79-amino-acid motif was confirmed to be from 96 to 174. Sequence alignment exhibited the identity rate of amino acid of Sox9b among ten kinds of animals including C. carpio, which is above 75%. The result indicated the Sox9 gene is very conservative in the progress of evolution. The tissue-specific expression of Sox9b was studied with semi-quantity RT-PCR. The result showed that this gene expresses widespread in several organs of adult C. carpio, with the level of expression in brain and testis higher than any others.

[Cloning of hemoglobin alpha-chain cDNA sequences from five fishes in cypriniformes].

Oxygen content in water is one of the limit factors to the fish growing. The tolerance of fish to low oxygen content depends on the affinity of hemoglobin to oxygen. The tempt of transgenic fish study is to transfer hemoglobin gene from the fish that tolerates to low oxygen content in water to the fish sensitive to low oxygen condition. Therefore, using hemoglobin gene to breed new varieties may become a main trend in the near future. To explore the underline mechanisms for the variation of hemoglobin property, we have cloned hemoglobin-alpha-chain from five species of Cypriniformes fishes. A pair of consensus degenerate primes were designed basing on N-terminal and C-terminal conservative amino acid of alpha-chain. Using RT-PCR method, alpha-globin gene was amplified and cloned from total RNA which was extracted from blood of five fishes which are widely distribution in China (Cyprinus carpio, Ctenopharyngodon idellus, Carassius auratus, Misgurnus anguillicaudatus, Paramisgurnus dabryanus). Sequence comparison with the existing hemoglobin alpha-chain confirmed that these cDNA fragments were alpha-globin gene of five fishes. The accession numbers in GenBank are AF528156, AF528157, AF528197, AF528198, AF528199 respectively. The cDNA which coding amino acid are all 429 bp. Compared with the amino acid sequences of five fishes, it can be concluded that in five fishes the highest similarity of two fishes which are Paramisgurnus dabryanus and Misgurnus anguillicaudatus is 99.41%, whereas the lowest similarity of two fishes which are Ctenopharyngodon idellus and Paramisgurnus dabryanus is 83.92%. In addition, the sequence similarity and phylogenetic relationship were compared with Within-group method. The result shows that there is much consistent between similarity and phylogenetic relationships from morphological of those near relation species and much connection between similarity and environment of those distant relation species.

[New advances in Sox gene family].

The Sox family of transcription factors are found throughout the animal kingdom. They are characterized by the presence of a HMG domain, involved in the regulation of such diverse developmental processes of early embryogenesis as sex determination,chondrogenesis,haemopoiesis,neural development and lens development. The deletion or mutation of SOX proteins results in developmental defects and congential disease in human. One Sox gene has different effects on the same promoters in the different cells or the different promoters in a cell. Some Sox gene exhibit a remarkable crosstalk and functional redundancy among each other. This paper reviewed the advances related to the Sox gene family in recent years.