Physiological Functions Of Pituitary Gonadotropin In Turbot(Scophthalmus Maximus.L)

The histomorphology,cell types and the distribution of gonadotropins(FSHβ and LHβ)in the pituitary of female turbot and mouse were compared by tissue section and in situ hybridization.The full-length cDNA sequences of gonadotropin common glycoprotein α subunit(CGα)and LHβ of turbot were obtained and their bioinformatics characteristics were analyzed by rapid amplification of cDNA ends(race)and real-time fluorescence quantitative PCR.Moreover,the expression of FSHβ,LSHβ,CGα,Gn RH,Kiss and Kissr during larval development of turbot was investigated.The optimal internal reference genes were selected from the hypothalamus,pituitary,ovary and liver during the ovarian development of turbot by quantitative real-time PCR.Meanwhile,the recombinant proteins of FSHβ and LHβ of turbot were successfully obtained by prokaryotic expression in E.coli.These results strengthen the content of reproductive endocrine regulation mechanism of turbot,and provide effective technical support for standardized production of turbot breeding.The results are as follows:1.Anatomical comparison of the pituitary and cell lines in turbot and mouseHistological and histochemical analyses of the pituitary in the adult turbot and mouse.Results showed that turbot pituitary is typically chicken heart-shaped,dorsoventral,located on the ventral surface of the diencephalon,and closely connected with the hypothalamus via the pituitary stalk.By contrast,the mouse pituitary is located in the pituitary fossa of the sella turcica at the skull base.It is oval with a gray-white center and gray-red on the sides.Two well-distinguished areas in the turbot and mouse were identified,namely,the adenohypophysis(AH)and the neurohypophysis(NH).Turbot AH comprised the rostral pars distalis(RPD),the proximal pars distalis(PPD),and the pars intermedia(PI).NH was not pronounced and with finger-like protrusions into PPD.However,mouse AH only comprised the pars distalis(PD)and PI.NH distribution was semicircular.Three main types of cells(acidophilic,basophilic,andchromophobic cells)were distributed in the mouse PD region,whereas PPD,RPD,and PI were observed in the turbot pituitary.In addition,the percentage of mouse chromophobic and basophilic cells was higher and lower than that of turbot,respectively.The diameter of the three above mentioned cells in the mouse pituitary was significantly higher than that of the turbot.The ovary development of turbot can be divided into five stages: previtellogenesis,early vitellogenesis,late vitellogenesis,migratory-nucleus,atresia.fshβ-and lhβ-immunoreactive signals were identified in turbot-distinct pituitary cells that primarily occupied the peripheral and central regions of AH.However,mouse fsh and lh-immunoreactive cells were expressed in the same cells and were present in the PD.The pituitary morphology,cell lines,and gonadotropin(fshβ and lhβ)expression in turbot and mouse significantly differed.2.Optimal selection of internal reference genes during ovarian development of turbotIn this study,the stability of six genes,namely,18 S ribosomal RNA(18s),beta-actin(actb),elongation factor 1-alpha(ef1α),glyceraldehyde-3-phosphate-dehydrogenase(gapdh),cathepsin D(ctsd),and beta-2-microglobulin(b2m)were evaluated as potential references for q RT-PCR analysis.The genes were examined in the hypothalamus-pituitary-ovary-liver(HPOL)axis throughout turbot ovarian development via using the ge Norm,Norm Finder and Best Keeper algorithms.Results showed that the most stable reference genes were ef1α,actb,ctsd and actb in the hypothalamus,pituitary,liver and ovary,respectively.The best-suited gene combinations for normalization were 18 s,ef1α,and ctsd in the hypothalamus;actb,ctsd,and 18 s in the pituitary;actb,and ctsd in the ovary;gapdh and ctsd in the liver.Moreover,the expression profile of estrogen receptor α(erα)manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development.However,no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development.In summary,these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of q RTPCR data in HPOL axis-related gene expression analysis during turbot ovarian development.3.cDNA cloning of gonadotropin subunits CGα and LHβ and expression of HPG axis key genes during turbot larval developmentIn this study,full-length sequences coding for common glycoprotein α subunit(CGα)and luteinizing hormone β(LHβ)were isolated from female turbot(Scophthalmus maximus)pituitary by homology cloning.Results showed that the two cDNAs consisted of 669 and 660 nucleotides encoding 129 and 139 amino acids,respectively.CGα and LHβ manifested typical characteristics of glycoprotein hormones,high homologies with the corresponding sequences of available teleosts,and high homology with that of Hippoglossus hippoglossus.CGα,FSHβ,and LHβ m RNAs were abundant in the pituitary,but less expressed in extra-pituitary tissues.The cgα,fshβ,and lhβ were detected at 1-day post-hatching(dph)and peaked simultaneously at early-metamorphosis(22 dph).cgα and fshβ m RNA levels were significantly increased at pre-metamorphosis,peaked in early metamorphosis,and then gradually decreased until metamorphosis was completed.Conversely,lhβ m RNA levels gradually decreased at pre-metamorphosis,dramatically peaked at early metamorphosis,and then decreased during metamorphosis.In addition,the m RNA levels of cgα were significantly higher than those of fshβ and lhβ during turbot larval metamorphic development,whereas no significant difference was found between fshβ and lhβ.However,c Gn RHⅡ、s Gn RH、sb Gn RH 、 Kiss1 、 Kiss2 and Kissr,which are related to gonadotropin in turbot,all increased rapidly and reached the maximum value within 5 days after hatching,and began to decline to 14 days after 5 days,and the expression level between 14 days and30 days was low and fluctuated up and down.4.Prokaryotic expression of gonadotropin FSHβ and LHβ genes in turbotIn this study,we designed specific primers for FSHβ and LHβ,and amplified them by PCR using cDNA template of pituitary gland,connect the amplified fragment to the p Cold I vector and select the positive clone for sequencing,the recombinant expression plasmids p Cold I-FSHβ and p Cold I-LHβ were constructed.The recombinant plasmid was transferred into BL21 cells and induced by IPTG.SDS-PAGE showed that the size of FSHβ recombinant protein was about 20.1 KDa.In addition,the expression of p Cold I was weak between 29.0 and 116 KDa.The expression of LHβ was similar to that of FSHβ.The recombinant protein was about 23.0 KDa in size.This laid a foundation for the preparation of FSHβ and LHβ specific antibodies.