Cloning Of CDNAs Encoding The Proopiomelanocortin, Thyrotropin β Subunit And Somatolactin, And Spatio-Temporal Expression Of Pituitary Hormones In Silurus Meridionalis

We cloned and sequenced the full-length cDNAs encoding Silurus meridionalis proopiomelancortin (POMC),thyrotropin beta subunit(TSHβ)and somatolactin(SL)by RT-PCR and RACE,and studied on tissue expression and ontogeny of pituitary hormone by RT-PCR,also on the structure and organogenesis of Silurus meridionalis pituitary by in situ hybridization using homologous antisense RNA probe encoding pituitary hormone.Spatio-temporal expression model of pituitary pormone and pattern formation of adenohypophysis in Silurus meridionalis were made clear,thereby providing a basis for future researches on their physiological functions and the regulation mechanism of pituitary development in telost.Full length of Silurus meridionalis POMC(1003bp)cDNA has an open-reading frame(ORF)of 639bp that encodes a 28-amino-acid signal peptide and a 184-amino-acid mature peptide,and 135bp 5'-untranslated Region(5'UTR),229bp 3'-untranslated Region(3'UTR).The POMC shares 84% amino acid sequence identity with Ictalurus punctatus,41.2～66.7%and 29.2～29.7%wtih other telost and elasmobranch respectively,31.2～41.9%with amphibians,reptiles,aves and mammals, and 37.1%with human.However,the presumed biologically active peptides are highly conserved within all species considered:ACTH shares 52.1～67.6%with elasmobranch and 69.8～94.2%with other vertibtates.The domain ofα-MSH is the same with other species except Bos taurus,Squalus acanthias,Dasyatis akajei,Heterodontus portusjacksoni and Chimaera phantasma to which there have a mutant residue.β-endorphin has 58～83.9%identity to other vertebrates.Full-length of Silurus meridionalis TSHβsubunit cDNA(640bp)includes 53bp 5'UTR, 152bp 3'UTR and 435bp ORF encoding a 19-amino-acid signal peptide and a 135-amino-acid mature peptide.The Silurus meridionalis TSHβsubunit has 55.5～65.3%amino acid sequence identity with the fishes of Cypriniformes,Perciformes and Salmoniformes,and 35.0%,39.1～44.4%with Neoceratodus forsteri and other vertebrates,respectively.Silurus meridionalis TSHβsubunit has the 12 conserved cysteine and N-glycosylation site of the glycoprotein hormone family,and shares 16.4%,29.9%and 42.4%indentity with Silurus meridionalis GTHα,LHβand FSHβ, respectively.Different tissues of Silurus meridionalis expressed variant SL mRNA for alternative splicing. There are three cDNA variants for SL in Silurus meridionalis,SL1 and SL2 were expressed in pituitary,and SL3 in pancreatic gland,spleen,kidney,head kidney,gill,bulbus arteriosus, telencephalon,diencephalon and olfactory bulb.SL1 cDNA variant of 863bp includes 24bp 5'UTR, 140bp 3'UTR and 699bp ORF which encodes 27-amino-acid signal peptide and a 205-amino-acid mature peptide.SL1 is identical to other SL,with 84%amino acid sequence identity to Ictalurus punctatus,and 37.6～53.9%to other fishes.It is worth noting that Silurus meridionalis SL1 and Ictalurus punctatus SL are in low identity with the groups of SLβand SLαwhich were characteristed by Zhu(2004).Phylogenetic analysis of Silurus meridionalis SL1 and various fishes SL indicated that SLs of Siluriformes associate with SLαgroup,which were situated in the base of the phylogenetic tree,so it is need further researches on the phyletic evolution of SL in Siluriformes. The full length of SL2 cDNA variant is 766bp,contains 24bp 5'UTR,250bp 3'UTR and 492bp ORF encoding a 27-amino-acid signal peptide and a 136-amino-acid mature peptide.978bp SL3 cDNA variant containes 24bp 5'UTR,399bp 3'UTR and 555bp ORF encoding a 27-amino-acid signal peptide and a 157-amino-acid mature peptide.Comparative study of these cDNA variants showed that SL2 cDNA lost 97bp from G⁴²⁹to G⁵²⁵in SL1 cDNA,SL3 cDNA inserted 115bp in SL1 cDNA from G⁵²⁵to A⁵²⁶.Comparative study of the deduced amino acid sequence for these cDNA variants indicated that SL2 and SL3 both lose C-terminal three conservative ammothiopropionic acid residues(cys)in SL1,and totally lose the fourth conservative structural domain of PRL/GH/SL family(SLD),and SL2 totally lose their third totally lose(SLC).The discovery will provide new clues for study on SL physiologic function.RT-PCR analysis indicated that pituitary hormones mRNA were not only in pituitary but also in other tissues.POMC mRNA highly expressed in pituitary and diencephalon,low expressed in esophagus,cardia,pancreas,head kiedny,gill,mesencephalon and olfactory bulb.Besides expressed in pituitary,GH mRNA also expressed in esophagus,cardia,pylorus,foregut,midgut,hindgut,liver, pancreas,spleen,gill,ovary,testis,cardiac atrium,cardiac ventricle,bulbus arteriosus,cerebellum and capsula olfactoria.TSHβmRNA expressed in pituitary,cardia,pylorus,foregut,pancreas,spleen, head kidney,gill,cardiac atrium,cardiac ventricle,skin,telencephalon,mesencephalon and olfactory bulb.SL1 expressed in pituitary,midgut,hindgut and liver.While SL3 expressed in esophagus,body of stomach,pylorus,foregnt,pancreas,spleen,kidney,head kidney,gill,testis, cardiac atrium,cardiac ventricle and olfactory bulb.Both SL1 and SL3 expressed in cardia,ovary, muscle,telencephalon,diencephalons,mesencephalon,cerebellum,medulla oblongata and capsula olfactoria.Extrapituitary expression of pituitary hormones implied that these hormones produced by the corresponding tissues can play roles through autocrine or paracrine.We studied the structure of pituitary of Silurus meridionalis by in situ hybridization using homologous antisense RNA probe encoding several pituitary hormone.The results suggest that the distribution pattern of eight kinds of hormone producing cells in Silurus meridionalis pituitary is similar to other species investigated.Corticotrophs were arranged into cords and islets in the rostralis pars distalis(RPD),forming a border between neurohypophysis and the zone of lactotrophs,and corticotrophs were also in proximal pars distalis(PPD).Lactotrophs were main kind of RPD and also arranged in PPD and pars intermadia(PI).Somatotrophs,the follicle-stimulating hormone-producing cells(FSH cells)and the luteotrophic hormone-producing cells(LH cells)were mingled with each other which spreaded all over the PPD,and also distributed in the dorsal-ventral boundery of RPD and posterior border of PI.Melanotrophs were in major in PI which were devided into some island-shape mass by the developed nerve branches and arranged in the PPD as well. Somatolactotropes were around the melanotrophs groups and the nerve branches in PI,and in RPD, PPD as well.Thyrotrophs distributed maily in PI nearby PPD,dispersed in everywhere of PPD and there also had few thyrotrophs in dorsal and ventral edge of RPD.The comparative studies on the structure of the FSH cells and the LH cells in adult and immature Silurus meridionalis showed that they both had these two cell types,and in immature fish,there had less FSH cells than LH cells. This implys that FSH and LH both play important roles at the early stage of the development of gonad,with stimulateing spermatogenesis and vitellogenesis,and LH palys principal role,which is in contrast to majority including salmonids in which FSH cells are more numerous than LH cells during the early stages of spermatogenesis and vitellogenesis,and FSH mainly stimulates spermatogenesis and vitellogenesis,while LH mainly plays important roles in stimulating maturity of oocyte and ovulation.Furthermore,We firstly report POMC-positive big froth cells among the neurocrine fibers in PI and spindly thyrotrophs in dorsal and ventral verge of RPD and among the neurocrine fibers in PI.POMC-positive big froth cells had some vacuolated appearance in adult fishes implys that they are relative to development of gonad.The secretory cell among the neurocrine fibers havn't been reported before.RT-PCR analysis on ontogeny of pituitary hormones indicated that PRL mRNA started to expresse at 8.5 hours after fertilization(hpf)when embryogenesis reachs to early gastrula.17.5hpf when at late gastrula,POMC mRNA started to express.TSHβmRNA began to express at the time of 28.5hpf.GTHαsubunit mRNA,GH mRNA and SL mRNA expressed at 6h,12h and 20h after hatching,respectively.The larvals expressed not only SL1 but also SL3 from 80h after hatchingIn situ hybridization of pituitary formation indicated that the anterior part of diencephalon abdomen first occurrenced line arranging lactotrophs at 38.5hpf.Then lactotrophs gradually moved to core at 40.5hpf,locating anterior part of diencephalon abdomen and displayed bilateral symmetry; and then they moved back when stomodeum appeared at 42.5hpf.At 44.5hpf,lactotrophs gathered to be a mass below anterior part of diencephalon abdomen,section in situ hybridization observation indicated that most of the subregion distributed positive cells and little were negative cells.At 47.5 hpf,lactotrophs of initial hatched larvals were at anterior part of adenohypophysis anlage.POMC-positive cells began to occur at 44.5hpf,located in anterior region of diencephalon abdomen and displayed two distinct domains in both sides.POMC-positive cells gathered in the middle of the diencephalons abdomen at 32h after hatching,section in situ hybridization observation discovered that POMC-positive cells located at the front of infundibulum,the anterior part of adenohypophysis anlage distributed few POMC cells.Few POMC-positive cells spreaded all over the adenohypophysis at 32h after hatching.The number of POMC-positive cells increased significantly at 44h after hatching.Somatotrophs differentiated and appeared in the middle of adenohypophysis anlage at 12h after hatching,and then spreaded to forward and backward along the back and abdomen edge.Few Gonadotropin-α-producing cells appeared in posterior abdomen of adenohypophysis anlage at 12h after hatching,and then located in the middle and posterior adenohypophysis at 56h after hatching.The initial differentiated somatolactotropes located in posterior adenohypophysis at 32 hours after hatching,and then extended to the middle of adenohypophysis.LH cells began to differentiate at 128h after hatching at the posterior adenohypophysis,and always were there till 30d.FSH cells were not observed till 30d after hatching. All the researches make it clear that the adenohypophysis anlage of Silurus meridionalis originates from basal plate ectoderm at neural ridge,expresses in a bilateral fashion.At 38.5hpf,a list of adenohypophysis anlage cells firstly differentiate into lactotrophs from left to right,and then lactotrophs move to core with whole adenohypophysis anlage,and inhabite most part of adenohypophysis anlage which will form RPD.The posterior region with few PRL-negative cells will then proliferate and form PPD and PI.Corticotrophs and melanotrophs differentiate in anterior and posterior part of adenohypophysis anlage,respectively.Somatotrophs firstly differentiate in the middle of adenohypophysis anlage,and then extend to PPD.FSH cells,LH cells,Thyrotrophs and somatolactotropes all differentiate initially in posterior part of adenohypophysis anlage.The spatial distribution of pituitary hormone producing cell form gradually.Compared to the pattern formation of zebrafish adenohypophysis,we found that there were obvious differences in adenohypophysis earlier period spatial distribution.So it is need further research on pattern formation process of fish Pituitary.