一氧化氮合酶在绵羊卵母细胞体外成熟及胚胎发育中的表达

摘要

To understand the expression of three isoforms of nitric oxide synthase (NOS) in early embryos and investigate w hether NO plays a role in sheep preimplantation development ,the mature oocytes and pre-implantation embryos(2-cell embryo ;4-cell embryo ;8-cell embryo ;morula ,blastula) were obtained though in vitro culture ;and the relative expression levels of neuronal nitric oxide synthase (nNOS) ,endothelial ni-tric oxide synthase (eNOS) ,inducible nitric oxide synthase (iNOS) ,mRNA were detected with real-time PCR .The results indicated that :①nNOS ,eNOS ,iNOS mRNA were expressed in sheep oocytes and preim-plantation embryos ;②The expressions of nNOS were higher (P<0 .05) ,compared with eNOS and iNOS in oocyte maturation ,and the expressions were remarkable higher in mature oocytes than that in immature oo-cytes(P<0 .05);③During the preimplantation embryo development ,the expressions of nNOS ,iNOS were higher and similar(P<0 .05) .Compared with 2-cell stage ,the expressions in 4-cell stage were decreased ;and the expression siginificantly increased in 8-cell stage(P<0 .05);moreover ,the expression in the morula reached the highest level ;and the expression in blastula significantly decreased (P<0 .05) .The results sug-gested that NO plays essential physiological roles in sheep oocyte maturation and preimplantation embryo development ,especially have the parenchymatous significance in the differential stages of 8-cell to morula . Meanwhile the production of NO may be mediated by nNOS in oocyte maturation ,and may be mainly medi-ated by nNOS ,iNOS in preimplantation embryo development .%为了解一氧化氮合酶（NOS）的3个亚型在绵羊早期胚胎发育过程中的表达情况，明确一氧化氮（NO）是否参与调控绵羊早期胚胎发育。运用体外培养体系获得成熟卵母细胞及不同阶段早期胚胎（2-细胞胚胎、4-细胞胚胎、8-细胞胚胎、桑葚胚、囊胚），应用实时荧光定量PCR技术检测目标基因神经元型一氧化氮合酶（nNOS）、内皮型一氧化氮合酶（eNOS）、诱导性一氧化氮合酶（iNOS）相对表达情况。结果显示，①在绵羊未成熟卵母细胞、成熟卵母细胞和各发育阶段的早期胚胎，nNOS、eNOS、iNOSmRNA均有表达；②绵羊卵母细胞成熟过程中，nNOSmRNA相对表达量在未成熟卵母细胞期及成熟卵母细胞期均显著高于eNOS、iNOS的表达量（P＜0．05），并且在成熟卵母细胞中的表达显著高于未成熟卵母细胞（P＜0．05）；③早期胚胎发育过程中，nNOS、iNOSmRNA相对表达水平显著高于eNOS的表达（P＜0．05），并且表达趋势相似，具体呈现出4-细胞期较2-细胞有降低趋势；8-细胞期的表达量显著升高（P＜0．05）并在桑葚胚期达到最高；到囊胚期出现显著降低（P＜0．05）的表达趋势。研究结果显示，NO可能参与调控绵羊卵母细胞成熟和早期胚胎发育过程，尤其在8-细胞期到桑葚胚期这一特定阶段可能具有实质性的生理意义；并且在绵羊卵母细胞成熟过程中的NO产生可能主要由nNOS介导，而早期胚胎发育过程中的NO可能主要由nNOS和iNOS共同介导调控。