Alarelin免疫绵羊对垂体GnRHR、FSHR和LHR基因表达与生殖激素分泌的作用

Alarelin active immunization affects expressions of GnRHR,FSHR and LHR mRNAs in the pituitary gland and reproductive hormone secretion of ewes

目的探讨GnRHa主动免疫对绵羊腺垂体GnRHR、FSHR和LHR表达及生殖激素的作用。方法 42只5～6月龄母绵羊均分为6组。EG-Ⅰ、EG-Ⅱ和EG-Ⅲ分别皮下注射阿拉瑞林抗原200、300和400μg,0和14 d各1次;EG-Ⅳ和EG-Ⅴ皮下注射阿拉瑞林抗原200、300μg,0、7、14和21 d各1次;对照组皮下注射药物的溶媒。于70 d无菌切取腺垂体、子宫及卵巢。实时荧光定量PCR测定GnRHR、FSHR和LHR mRNA的表达。ELISA测定血清GnRH、FSH、LH和E2浓度。结果实验组GnRHR、FSHR和LHR mRNAs的2-(ΔΔCt)值均低于对照组,EG-Ⅳ和EG-Ⅴ的GnRHR、FSHR和LHR mRNA的2-(ΔΔCt)值低于EG-Ⅰ和EG-Ⅱ(P<0.05)。EG-Ⅰ和EG-Ⅱ血清GnRH在21和28 d达到谷值(P<0.05),EG-Ⅲ、EG-Ⅳ和EG-Ⅴ则在45d达到谷值(P<0.01)。实验组血清FSH始终高于对照组(P<0.05),EG-IV和EG-V在45和60 d达到峰值(P<0.01)。EG-Ⅰ、EG-Ⅱ和EG-Ⅲ血清LH分别在21、21和28 d抵至谷值(P<0.05),EG-Ⅳ和EG-Ⅴ在35 d抵至谷值(P<0.01);35 d时,EG-Ⅳ和EG-Ⅴ低于EG-Ⅰ、EG-Ⅱ和EG-Ⅲ。结论阿拉瑞林主动免疫可抑制垂体GnRHR、FSHR和LHR mRNAs表达,促进FSH的合成与释放,降低GnRH和LH的合成与释放,增加抗原的注射剂量和次数,作用更明显。

Objective To explore the expressions of GnRHR,FSHR and LHR mRNAs in the pituitary,as well as the secretion of reproductive hormones in ewes actively immunized with different concentrations of alarelin antigen.Methods Forty-two ewes were divided into six groups.Eves in EG-Ⅰ,EG-Ⅱ and EG-Ⅲ were injected twice with 200,300 and 400 μg Alarelin antigen respectively.Animals in EG-Ⅳ and EG-Ⅴwere injected（at day 0,7,14 and 21） with 200 and 300 μg Alarelin antigen.The control group was injected 2.0 mL solvent twice（at day 0 and 7）.PCR amplification,reverse transcription and fluorescent quantitative RT-PCR were implemented.Serum concentrations of GnRH,FSH,LH and estradiol were detected using ELISA.Results The 2-（ΔΔCt） values of GnRHR,FSHR and LHR mRNAs in EG-Ⅰ,EG-Ⅱ and EG-Ⅲ decreased.The 2-（ΔΔCt） values in EG-Ⅳ and EG-Ⅴ were lower than that in EG-Ⅰand EG-Ⅱ.Serum GnRH concentrations in EG-Ⅰ and EG-Ⅱ reached the bottom values at day 21 and 28（P0.05）.GnRH concentrations in EG-Ⅲ,EG-Ⅳ and EG-Ⅴ also reached the bottom at 45 d（P0.01）.FSH concentrations in EGs were higher than that in CG.EG-Ⅳ and EG-Ⅴ reached the peak values at 45 d and 60 d（P0.01）.Serum LH concentrations in EGs declined.LH concentrations in EG-Ⅳ and EG-Ⅴwere lower than those in EG-Ⅰ,EG-Ⅱ and EG-Ⅲ at day 35.Conclusions Alarelin active immunization can suppress the expression of GnRHR,FSHR and LHR mRNAs in the pituitary gland of ewes,promote the secretion and release of FSH and decrease the serum level GnRH and LH.The effects are strengthened with increasing injection dose and times of alarelin.