Growth differentiation factor 9 of Megalobrama amblycephala: molecular characterization and expression analysis during the development of early embryos and growing ovaries.

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factorß superfamily and plays an essential role during follicle maturation in mammals. In the present study, the full-length complementary DNA (cDNA) of gdf9 was obtained from Megalobrama amblycephala. The cDNA sequence is 2,061 bp in length with an open reading frame of 1,287 bp encoding 428 amino acid residues. The deduced amino acid sequence shared identities of about 42-86 % with the homologues of other vertebrates. During the early development of embryos, the gdf9 mRNA was detected in zygote with significantly high level and declined sharply by 47 and 87 % at 4 hours post-fertilization (hpf) and 6 hpf and even to an undetectable level through advancing stages. Expression analysis based on quantitative real-time PCR revealed that gdf9 mRNA was mainly expressed in ovary, but much lower levels were also found in some nonovarian tissues. Within the follicle, gdf9 mRNA was localized both in the oocytes and the follicle layer cells by in situ hybridization. During the ovarian cycle, gdf9 mRNA significantly decreased after the previtellogenic stage and became to increase again after the fully grown stage. The results imply that Gdf9 may play critical physiological functions in M. amblycephala early embryonic development and reproduction.

Megalobrama amblycephala cardiac troponin T variants: molecular cloning, expression and response to nitrite.

Cardiac troponin T (TNNT2), as a member of troponin superfamily, plays important roles during early cardiogenesis, and contraction and relaxation of myocardial cells. In this study, two alternatively spliced variants of Megalobrama amblycephala TNNT2 were identified showing a difference of 19 amino acids in the N-terminal hypervariable region. The longer cDNA (TNNT2-1) was 1,118 bp, encoding 284 amino acid residues, contained conserved central tropomyosin-binding region, cardiac specific signal and C-terminal segments except the N-terminal hypervariable region. The TNNT2 transcripts first appeared at 16 hours post-fertilization (hpf) peaking at 28 hpf (onset of heartbeat). In addition, strong expression of TNNT2 was found in the cardiac muscle. After nitrite exposure, the increased TNNT2 expression levels in the heart indicated that nitrite might induce cardiac injury. Results of semi-quantitative RT-PCR indicated that the two alternatively spliced variants existed in early development stages since their first appearance at 16 hpf and heart, spleen, headkiney of M. amblycephala. The shorter transcript (TNNT2-2) was proved to be dominant in the embryos and heart of M. amblycephala, furthermore, the increase of TNNT2 expression level in the heart after nitrite exposure was mainly caused by TNNT2-2.

Sequence analysis and expression differentiation of chemokine receptor CXCR4b among three populations of Megalobrama amblycephala.

Chemokine (C-X-C motif), receptor 4 (CXCR4), a member of the family of seven transmembrane G-protein-coupled receptors, plays important roles in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration. We characterized the sequences and expression profiles of CXCR4b in Megalobrama amblycephala. The full-length cDNA was 1638bp, encoding 353 amino acid residues. Multiple alignment and phylogenetic analysis indicated that M. amblycephala CXCR4b contained the similar conservative sequences and motifs with other organisms. The CXCR4b expression in different development stages of M. amblycephala showed the mRNA levels before hatching and at 62h post fertilization (hpf) were significantly higher than at other post hatching stages (P<0.05). Besides, CXCR4b was constitutively expressed in a wide range of tissues, at higher levels in headkidney, liver, intestine spleen, blood and gill, where a larger number of immune cells including lymphocytes and macrophages reside, suggesting its specific roles in inflammatory responses. The CXCR4b expression after high nitrite concentration (ρNO(2-)-N: 20.29mg/L) exposure supported a potential pro-inflammatory function for CXCR4b. In order to identify the better population with immune property for breeding, we compared the tissue expression of CXCR4b among Liangzi Lake population (L), Yuni Lake population (Y) and Poyang Lake population (P), it was indicated that the expression levels in the population Y were obviously higher than that of the other two populations.

Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits, HIF-1α/2α/3α of the hypoxia-sensitive freshwater species, Chinese sucker.

Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in oxygen tension in the cellular environment. In this study, we identified full-length cDNAs of HIF-1α, HIF-2α and HIF-3α in an endangered hypoxia-sensitive fish species, Chinese sucker. The HIF-1α/2α/3α cDNAs are 3890, 3230 and 3374 bp in length, encoding 780, 782 and 632 amino acid residues, respectively. The real-time PCR results suggested that HIF-1α and HIF-3α mRNAs were highly expressed in liver and gonads, followed by spleen and muscle. Moreover, HIF-1α and HIF-3α transcription factors revealed similar developmental expression patterns, with the lowest expression at 48h post-fertilization, and reaching the highest expression level at 360 h post-fertilization. Short-term hypoxia exposure (2.2, 2.8 and 3.2mg/L dissolved oxygen for 24h) increased mRNA levels of HIF-1α and HIF-3α. HIF-2α mRNA showed similar expression patterns with that of HIF-1α and HIF-3α, however, its expression was extremely low in the spatio-temporal expression patterns and hypoxia treatment. This is the first report describing the potential to identify hypoxia-sensitive/tolerant fishes according to the number of the serine residues of fish oxygen-dependent degradation (ODD) domain. It was suggested that Cyprinomorpha fishes, with less than 40 serine residues in fish ODD domain were hypoxia-sensitive fishes and more than 40 serine residues in this domain were hypoxia-tolerant fishes.