The Pituitary Structure Of Rare Minnow (Gobiocypris Rarus) And Cloning, Analysis Of POMC CDNA

Rare minnow (Gobiocypris rarus), belonging to order of Cypriniformes, family of Cyprinidae, subfamily Danioninae, genus Gobiocypris, is a small freshwater fish endemic to China.The structure of rare minnow pituitary was observed by histology and histochemistry methods. As a kidney, the pituitary of rare minnow was comprised of neurohypophysis and adenohypophysis, attaching to the hypothalamus by a pituitary stalk. Adenohypophysis was divided into three parts, RPD, PPD and PI according to the distinct histochemistrical characteristics. The curved branches of neurohypophysis were inserted in every part of adenohypophysis, and were most plentiful in PI. The blood vessels were rich in all parts of the pituitary.cRNA probes were designed according to the cloned cDNAs of pituitary hormones. Data shows that:PRL positive cells were mainly distributed in RPD, and few were scattered in PPD. POMC positive cells located in most part of RPD and PI, with patterns of dispersed and compact along the PN branches, respectively. Inaddition, there were some POMC positive cells observed in PPD. GH positive cells were mainly grouped in PPD, few found in RPD, in a shape of round, oval or triangle. FSHβpositive cells located in RPD, PPD and few in PI, in a shape of round, oval or irregular. LHP positive cells distributed in PPD alone or in group, and few cells were also found in RPD and PI. There was a dividing line in PPD and PI, formed by LHβpositive cells. TSHPmainly grouped in the dorsal part of PPD, few scattered in RPD and PI. GTHapositive cells with a large distributing area, detected in every part of the pituitary, mostly in PPD, and formed a distinct limit between PPD and PI like the LHβ positive cells. There were few SL positive cells detected, and these cells were grouped or scattered only in posterior-ventral par of PI. The distributing patterns of hormone producing cells were different from each samples.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, Met1-Ala28), N-terminal peptide (Gln29-His105), ACTH (Ser108-Met146),α-MSH (Ser108-Gal121), CLIP (Pro126-Met146),β-LPH (Glu149-His221),γ-LPH (Glul49-Ser186),β-MSH (Asp 170-Ser186), andβ-endorphin (β-EP, Tyr189-Gln221). Sequence analysis showed no region was homologous toγ-MSH (a tetrapod POMC feature). The amino acid sequence is highly similar to POMC-Ⅰand POMC-Ⅱof the common carp (92.4%), which supporting the viewpoint of rare minnow belongs to subfamily Gobioninae according to homologous alignment. It was POMCαthrough the phylogenetic analysis.Pituitary and extra-pituitary expression were studied using RT-PCR and in situ hybridization. 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, showing that the paracrine pathway was existed in these tissues.