Molecular Cloning Of Gonadotropin-releasing Hormones And Their Regulatory Functions On Reproduction In Scatophagus Argus

Gonadotropin-releasing hormone(GnRH)is a neuropeptide that secretes from hypothalamus.Combined with GnRH receptor,GnRH can active the synthesis and release of gonadotropins,which is very important for gonad maturation and reproductive behavior.In the present study,the full length cDNA sequences of three GnRH subtypes(sbGnRH,cGnRH-? ? s GnRH)c DNA were cloned in Scatophagus argus.The expression pattern of GnRH in various tissues was next characterized using RT-PCR.The expression pattern of GnRH during Embryogenesis and ontogenesis was next characterized using quantitative real-time PCR(qPCR).Effects of GnRH on FSH and LH mRNA expressions in female hypothalamus were examined using qPCR in vivo and vitro.Effects of estradiol(E2)on GnRHs mRNA expressions in hypothalamus were examined using qPCR.The main results were as following:1.Molecular cloning and characterization of GnRHs in S.argus.Three subtypes of GnRHs were cloned in S.argus.The full-length cDNA of sbGnRH was 334 bp with 288 bp ORF which encoded 95 amino acids.The complete cGnRH-? cDNA was 284 bp with 258 bp ORF which encoded 85 amino acids,and that of sGnRH was 288 bp with 273 bp ORF which encoded 90 amino acids.Homology comparison revealed that S.argus cGnRH-? was the most conventional kind;the sGnRH was less conventional than cGnRH-?;sbGnRH was the least conventional kind.The phylogenetic analysis revealed GnRH of S.argus closely related to respective GnRH of closely related species,but distally related to the mammalian species.2.Temporal and spatial expression of GnRHs in S.argus.In the detected 12 tissues(brain,stomach,pituitary,testis,ovary,liver,kidney,spleen,intestine,heart,gill and muscle)in S.argus,the sbGnRH was present in all tissues in both male and female;The s GnRH and cGnRH-? expressed only in gonad and brain in both male and female.Results suggested that sbGnRH might involve in ovary mature;During embryogenesis,three Gn RHs expressed highly at the gastrula stage of embryogenesis in S.argus and weakly at the rest of embryogenesis;During ontogenesis,sbGnRH was simultaneously up-regulated with ovary mature in female hypothalamus,while cGnRH-? and sGnRH were not.Results suggested that sbGnRH might involve in ovary mature.3.Effects of GnRHs on the expression of FSH and LH in S.argusIn vivo,0.001,0.01 and 0.1?g/g body weight sbGnRH significantly promoted the expression of FSH in a dose-dependent manner after 3h and 6h treatmeat.0.001 and 0.01?g/g body weight sb GnRH significantly promoted the expression of LH after 3h and 6h treatmeat,while 0.01?g/g body weight sbGnRH don't affect the expression of LH;0.001 and 0.01?g/g body weight sGnRH don't affected the the expression of FSH and LH.0.1ug/g body weight sGn RH significantly promoted the expression of FSH after 3h and 6h treatmeat.0.1?g/g body weight sGnRH significantly promoted the expression of LH in 3h treatment not in 6h treatment;0.1?g/g body weight cGnRH-? significantly promoted the expression of FSH and LH in 6h treatmeat not in 3h treatment,while 0.001 and 0.01?g/g body weight cGnRH-? don't affected the the expression of FSH and LH.In vitro,10?M sbGnRH significantly promoted the expression of FSH and LH after 3h and 6h treatment,while 0.1 and 1?M sbGnRH don't affect the expression of FSH and LH.1 and 10?M sGnRH significantly promoted the expression of FSH after 3h treatment not in 6h,sGnRH didn't affect the the expression of LH.4.Effects of estradiol(E2)on Gonadotropin-releasing hormones(GnRHs)in S.argus in vivo and vitroIn vivo,the expression of sbGnRH m RNA was inhibited significantly(P<0.05)at 6 hours incubated with E2(dose 4?g/g body weight)in vivo in both male and female,while cGnRH-? and sGnRH were not affected.In vitro,the expression of sbGnRH mRNA was inhibited significantly(P<0.05)in a dose-dependent manner at 3,6 and 12 hours incubated with E2(dose from 0.1 to 10?M),while cGnRH-? and sGnRH were not affected.Both FULSTRANRT and MPP can promote the expression of sbGnRH mRNA,while PHTPP can not.Those results indicated that E2 of high concentrations can significantly inhibit the expression of sbGnRH m RNA gene,but FULSTRANRT and MPP can release this inhibition of E2.Results indicated that sbGnRH play a most important role in the reproduction in S.argus.Estrogens regulate the expression of sbGnRH mRNA mainly via estrogen receptor elpha in S.argus.