| In order to study the cytological and molecular mechanisms of the hyperpigmentation phenomenon on the blind side of tongue sole(Cynoglossus semilaevis Günther), three kinds of chromatophores including melanophore, xanthophore and iridophore were identified. Furthermore, the full-length c DNA sequences of three hyperpigmentation-related genes including proopiomelainocortin(POMC), pro-melanin concentrating hormone(p MCH) and melanin concentrating hormone receptor(MCHR) were isolated. In addition, their structure, phylogenetic role, spatial and temporal expression patterns were determine using quantitative PCR methods, the relationship between gene expression and blind-side hypermelanosis was analyzed. Results from the present study will be helpful for better understanding of the hyperpigmentation on the blind side of half-smooth tongue sole. The main results are listed as follows: 1. The morphological feature of blind-side hypermelanosis in tongue sole- pigment cells and scalesThree kinds of chromatophores, namely melanophore, xanthophore and iridophore, were identified in tongue sole. The melanophore is containing black and brown pigment granules with a relative larger nucleus, and generally it could be divided into two types based on their morphology. In contrast, nucleus of xanthophore and iridophore is relatively small. The distribution density and pattern of these three chromatophores in eyed-side and blind-side skin were examined and compared. Results showed that, no melanophore is found in normal skin of blind side, but the melanophore density in blind-side skin with hypermelanosis is significantly lower than that of eye-side normal skin(P<0.05), and so does the iridophore. In addition, the melanophore distributed in blind-side skin mainly with radial branches, whereas in eye side the melanophore mainly in non-radial condition, similar with those in the scales. In life history, the scales from the blind side changed from ctenoid scale to cycloid scale, but the scales locating at the blind-side hyperpigmentation area unchanged. Moreover, the number of spines on ctenoid scales is different among eye-side skin, blind-side normal skin and blind-side pigmentation skin, which hinted that the morphology and its endocrine mechanism both changed during blind-side hypermelanosis. 2. Molecular cloning and expression patterns of pro-opiomelanocortin(POMC) in half-smooth tongue soleThe full-length c DNAs encoding of POMC-a and POMC-b were firstly isolated from the pituitary of tongue sole using RACE methods. The POMC-a c DNA is 910 bp in length containing 642 bp open reading frame(ORF) encoding 213 amino acids, a 5’ un-translated region(5’-UTR)(113 bp) and a 3’ un-translated region(3’-UTR)(154 bp). POMC-b c DNA is 844 bp in length containing 537 bp ORF which encoded 178 amino acids, a 5’-UTR(87 bp) and a 3’-UTR(210 bp). The mature peptides of both genes consist of Adrenocorticotropin(ACTH), α-melanocyte stimulating hormone(α-MSH), β-melanocyte stimulating hormone(β-MSH), γ-lipotrophic hormone(γ-LPH) and β-endorphin, whereas the γ-melanocyte stimulating hormone(γ-MSH) and most joining region are lost. The predicted molecular weight is 23.7KDa for POMC-a and 19.5KDa for POMC-b, and the isoelectric point are 6.51 and 6.91, respectively. The amino acid sequence identity and phylogenetic relationship between tongue sole POMC and other teleosts were compared and determined.The spatial expression patterns of POMC-a and POMC-b m RNA in tongue sole were determined by real-time PCR method. The results showed that POMC m RNA exhibited highest expression level in pituitary. In addition, secondary higher expression levels of POMC-a m RNA was found in brain, intestine, eye-side skin and blind-side skin with hypermelanosis. As for POMC-b m RNA, it has secondary higher expression in spleen, brain, liver and head-kidney. The level of blind-side hyperpigmentation skin POMC m RNA is significantly higher than that of eye-side and blind-side normal skin. Moreover, the pituitary and skin POMC m RNA levels are closely related with pigmentation degree on blind side, wherein both pituitary POMC-a and POMC-b levels peaked in fish with 50% pigmentation on blind-side skin, then significantly decreased, both skin POMC-a and POMC-b levels peaked in fish with 10% pigmentation on blind-side skin, then significantly decreased. 3. Molecular cloning and expression patterns of melanin concentration hormone(MCH) gene in half-smooth tongue soleThe full-length c DNA of p MCH2 was isolated from the brain of tongue sole, the c DNA sequence consists of 626 bp nucleotides, including a 444 bp ORF, 32 bp 5’-UTR and 150 bp 3’-UTR, encodes 147 amino acids. MCH1 core sequence locates at the 352-402 bp position of p MCH1 and encodes 17 amino acids. MCH2 core sequence locates at the 379-441 bp position of p MCH2 and encodes 21 amino acids. Their predicted molecular weight are 14.8KDa and 16.2KDa and the isoelectric point are 6.22 and 5.80, respectively. The amino acid sequence identity and phylogenetic relationship between tongue sole p MCH and other teleosts were analyzed and determined.The spatial expression pattern of p MCH m RNA were determined by real-time PCR method: p MCH m RNA exhibited highest expression level in pituitary. In addition, p MCH1 showed secondary higher expression levels in intestine, eye-side skin, brain and gonad, whereas p MCH2 m RNA showed secondary higher expression levels in brain, gill, eye-side skin and blind-side normal skin. Moreover, p MCH1 in blind-side pigmentation skin was significantly higher than that in blind-side normal skin, but p MCH2 showed contrary expression pattern. Temporal expression investigation showed that both pituitary and skin p MCH1 m RNA reached maximum levels in fish with 10% of pigmentation on blind side, then followed by decrease in expression levels. In contrary, the pituitary and skin p MCH1 m RNA expression levels increased with the pigmentation degree of the blind side. These results indicated that the pituitary and skin p MCH both are included into the regulation of blind-side hypermelanosis in tongue sole. 4. Molecular cloning and expression patterns of melanin concentration hormone(MCHR) gene in half-smooth tongue soleThe full-length c DNAs encoding MCHR1 and MCHR2 were firstly isolated from the brain of tongue sole. The MCHR1 and MCHR2 sequences consist of 1685 bp and 1626 bp nucleotides, respectively. MCHR1 c DNA encodes an 1080 bp ORF, 262 bp 5’-UTR and 343 bp 3’-UTR. MCHR2 c DNA encodes an 1044 bp ORF, 213 bp 5’-UTR and 369 bp 3’-UTR. MCHR1 and MCHR2 c DNA sequences encode 359 and 347 amino acids respectively, they both have 7 trans-membrane domains and the polyadenylation signalAATAA. Their predicted molecular weight are 23.7KDa and 19.5KDa and the isoelectric point are 6.51 and 6.91, respectively. The amino acid sequence identity and phylogenetic relationship between tongue sole MCHR and other teleosts were compared and determined.Tongue sole MCHR1 m RNA exhibit highest expression level in gill followed by spleen, gonad and eye-side skin. MCHR2 m RNA is highly expressed in eye-side skin followed by gonad. There are significant difference of MCHR expression levels between blind-side hyperpigmentation skin, eye-side skin and blind-side normal skin. Pituitary MCHR1 m RNA significantly increased at the initial stage of blind-side pigmentation and peaked in fish with 50% blind-side pigmentation, skin MCHR1 m RNA maintained high levels during the pigmentation on blind-side. Both pituitary and skin MCHR2 m RNA levels peaked in fish with 10% blind-side pigmentation, then significantly decreased with the pigmentation degree of the blind side. These results showed that the MCHR is directly or indirectly involved into the regulation of blind-side hypermelanosis in tongue sole. |
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