Characterization and expression analyses of somatolactin-α and -ß genes in rare minnows (Gobiocypris rarus) following waterborne cadmium exposure.

Using reverse transcription-polymerase chain reaction (RT-PCR) and RACE (rapid amplification of cDNA ends), somatolactin-α (rmSLα) and -ß (rmSLß) were identified from the pituitary gland of rare minnows (Gobiocypris rarus). The full-length cDNAs of these two genes were 1288 and 801 bp, encoding prepeptides of 250 and 228 amino acids residues, respectively. rmSLß can be detected in the brain (including the pituitary), ovary, testis, and gill, while rmSLα was mainly expressed in the brain. On the other hand, rmSLα was expressed in all the fetal developmental stages; however, rmSLß can just be detected in the stages since from 14 h post-fertilization (hpf). After exposure to acute waterborne cadmium (Cd), rmSLα was distinctly upregulated in juvenile rare minnows at all detected time points, from 24 to 96 h and 10 days, while rmSLß was significantly altered only in 96 h or 10-day treatment groups. As for adults, acute Cd exposure caused alterations of both rmSLα and rmSLß in the brain (containing the pituitary) at the 24 h; subchronic waterborne Cd treatment led to upregulation of rmSLα, while decrease of mSLß in the brain. Alteration of rmSL transcripts following waterborne Cd exposure further confirmed the endocrine disruption of this heavy metal. Besides, exposure to as low as 5 µg/L Cd caused alteration of rmSLα, which suggested that rmSLα might be a potential biomarker for risk assessment of aquatic Cd.

The secretion, synthesis, and metabolism of cortisol and its downstream genes in the H-P-I axis of rare minnows (Gobiocypris rarus) are disrupted by acute waterborne cadmium exposure.

The H (hypothalamic)-P (pituitary)-I (interrenal) axis plays a critical role in the fish stress response and is regulated by several factors. Cadmium (Cd) is one of the most toxic heavy metals in the world, but its effects on the H-P-I axis of teleosts are largely unknown. Using rare minnow (Gobiocypris rarus) as an experimental animal, we found that Cd only disrupted the secretion and synthesis of cortisol. Neither hormones at the H or P level nor the expressions of their receptor genes (corticotropin-releasing hormone receptor (CRHR) and melanocortin receptor 2 (MC2R)) were affected. Steroidogenic acute regulator (StAR), CYP11A1 and CYP11B1, which encode the key enzymes in the cortisol synthesis pathway, were significantly up-regulated in the kidney (including the head kidney). The level of 11ß-HSD2, which is required for the conversion of cortisol to cortisone, was increased in the kidney, intestine, brain, and hepatopancreas, whereas the expression of 11ß-HSD1, which encodes the reverse conversion enzyme, was increased in the gill, kidney and almost unchanged in other tissues. The enzyme activity concentration of 11ß-HSD2 was increased in the kidney as well. The level of glucocorticoid receptor (GR) decreased in the intestine, gill and muscle, and the key GR regulator FK506 binding protein5 (FKBP5) was up-regulated in the GR-decreased tissues, whereas the level of nuclear receptor co-repressor 1 (NCoR1), another GR regulator remained almost unchanged. Thus, GR, FKBP5 and 11ß-HSD2 may be involved in Cd-induced cortisol disruption.

Effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae.

The objective of this study was to investigate the effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae. After ammonia-N exposure at a constant concentration of 7.21 ± 0.10 mg L(-1) for 96 h, B. reevesae was challenged with Aeromonas hydrophila. Quantitative PCR analysis showed predominant and significant expression of HSP70 in liver, gill, skin, spleen and kidney (P < 0.05), with significantly upregulated expression of the mRNA transcript in these tissues after sublethal ammonia-N exposure and A. hydrophila challenge. Furthermore, following A. hydrophila challenge after ammonia-N exposure, HSP70 mRNA expression was significantly upregulated in kidney and gill tissues, although its expression levels were significantly lower than those detected following A. hydrophila challenge or ammonia-N exposure individually. These results indicate that B. reevesae HSP70 is involved in resistance to pathogenic bacteria. It is hypothesized that ammonia-N results in the downregulation of HSP70 mRNA in immune organs after an A. hydrophila challenge, thus lowering their resistance to pathogenic stress.

cDNA cloning, pituitary location, and extra-pituitary expression of pro-opiomelanocortin gene in rare minnow (Gobiocypris rarus).

A cDNA encoding pro-opiomelanocortin (POMC) gene was cloned from the pituitary gland of the rare minnow (Gobiocypris rarus), a small freshwater fish endemic to China. This was achieved by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Data showed that the predicted rare minnow POMC (rmPOMC) cDNA consisted of 846bps coding for the following sequences, flanked by proteolytic cleavage sites: signal peptide (SP, Met(1)-Ala(28)), N-terminal peptide (Gln(29)-His(105)), ACTH (Ser(108)-Met(146)), α-MSH (Ser(108)-Gal(121)), CLIP (Pro(126)-Met(146)), ß-LPH (Glu(149)-His(221)), γ-LPH (Glu1(49)-Ser(186)), ß-MSH (Asp(170)-Ser(186)), and ß-endorphin (ß-EP, Tyr(189)-Gln(221)). Sequence analysis showed no region was homologous to γ-MSH (a tetrapod POMC feature). The amino acid sequence is highly similar to POMC-I and POMC-II of the common carp (92.4%), according to homologous alignment. It was POMCα through the phylogenetic analysis. Pituitary and extra-pituitary expression were studied using RT-PCR and in situ hybridization. The rmPOMC-positive cells were mainly located in the rostral pars distalis (RPD) and pars intermedia (PI). Some rmPOMC-positive cells were detected in the proximal pars distalis (PPD) as well, according to in situ hybridization. In the extra-pituitary tissues, positive signals were observed in the brain, intestines, gonads, hepatopancreas, spleen, and gills by RT-PCR analysis.