Cloning,Prokaryotic Expression And Functional Verification Of Gonadotropin Hormones From The Scatophagus Argus

In the process of animal life,reproduction is an important part to maintain the indispensable life activities of the continuation of the race.In vertebrates,gametogenesis and its maturation is mainly affected by the formation of the brain-pituitary-gonadal axis(BPG)and hypothalamus-pituitary-gonadal axis(HPG).Hypothalamus of animals secrete gonadotropin-releasing hormone(GnRH),which promote purity secrete gonadotropin hormone(GtH),including follicle-stimulating hormone(FSH)and luteinizing hormone(LH),so as to accelerate gametogenesis and the maturation of gonad.In teleost,function of FSH and LH dependent on the combination with their receptor,for activating the downstream signal transduction pathway,and activating related steroidogenic proteins to promote the process of sex hormones synthesis,which in order to accelerate gametogenesis and the maturation of gonad.The present study with Scatophagus argus as experimental objects,applying follwing techniques: RACE for cloning,prokaryotic expression system for obtaining recombinant proteins,histological observation,Real-time quantitative fluorescence PCR,enzymoimmunoassay(ELISA),proceed studies including cloning and sequence analysis of GtH,constructing recombinant vector,obtaining and using of recombinant proteins,express level of related genes which effect on HPG axis in objects that treated with recombinant Gt H,as well as measurement and analysis of the concentration of sex steroid hormones in serum.Results of the study are summarized as follows:(1)Gene cloning: designing primers and applying SMART-RACE to clone GtH of Scatophagus argus,which including FSH and LH.The full-length of FSH cDNA was542 bp,containing a 363-bp ORF,and encoded a precursor protein of 120 amino acids(aa).Besides,the full-length of LH cDNA was 608 bp,containing a 438-bp ORF,and encoded a precursor protein of 145 amino acids(aa).Gt H of spotted scat showed high sequence identity with those of perciform fishes.Phylogenetic analyses suggest that theGtH of spotted scat closely are related to those of other teleost species and evolutionary status is conservative.(2)Recombinant proteins: constructing recombinant plasmid with the GtH gene segment and pET-28 a vector,so as to obtain recombinant fusion proteins containing r-FSH and r-LH by means of inducible express under the condition after optimized.Size of r-FSH and r-LH is about 12.95 kDa and 16.30 kDa,respectively.Purified r-FSH and r-LH are obtained after Ni2+-NTA affinity chromatography column purified,then the spotted scat and medaka(Oryzias latipe)following treatment with purified recombinant proteins.(3)Histological observation: after the treatment with purified recombinant proteins,gonad tissues of the spotted scat and medaka which treated were observed with Scanning Electron Microscope(SEM)and Hematoxylin-eosin staining(H&E).After treated,gonadal maturation of spotted scat and medaka was advanced than these control group and blank group.In females,ovary development was more advanced than that of the other groups;yolk granules were mature.In males,testes development was more rapid than that of the other groups;the sperm of the treatment group possessed tails.In the control and blank groups,sperm matured relatively slowly.(4)Expression level of related genes: measuring the level of GtHR,Amh,Star,17β-hsd.The qRT-PCR results revealed that FSHR and LHR level was significantly increased(p<0.05,p<0.01).Following treatment with r-SaGt H,Amh expression was significantly decreased(p<0.05),Star expression was significantly increased(p<0.05),and 17β-hsd expression was significantly increased(p<0.05).(5)Serum concentrations of sex steroid hormones: concentrations of E2 and 11-kT in serum were measured with ELISA,respectively.Results as following: In female spotted scats,the plasma E2 levels of the treatment group were 312.77 pg/ml at 0 dpt,337.90 pg/ml at 7 dpt,and 474.57 pg/ml at 14 dpt(p<0.05).In males,the plasma 11-kT levels of the treatment group were 70.88 pg/ml at 0 dpt,65.67 pg/ml at 7 dpt,and135.65 pg/ml at 14 dpt,revealing a significant increase over time(p <0.01).The present study characterized the mechanisms through which GtH affect the HPG axis in the spotted scat.We found that gonadotropins LH and FSH activate sexual steroid signaling and up-regulate expression of GtHR,Amh,Star,and 17β-hsd,which are involved in steroid hormone(E2 and 11-kT)biosynthesis,as well as promoting gonadal development and gametogenesis.In addition,inhibition of Amh expression stimulated vitellogenesis and spermatogenesis by inducing production of E2 and 11-kT.These findings provide insight into hormonal control of reproduction in the spotted scat and other teleost.In addition,we obtained recombinant proteins that can be used as feedadditives to accelerate sexual maturity in fish,as well as in future studies of the functions of GtH.