Caspase-3在黄鳝性腺性逆转过程中的表达及定位分析
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摘要
为探究黄鳝(Monopterus albus)Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝caspase-3的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝caspase-3基因cDNA序列,推导的氨基酸序列对比发现,黄鳝caspase-3与大黄鱼(Larimichthys crocea)和鳜(Siniperca chuatsi)亲缘关系最近。实时定量PCR(qRT-PCR)结果显示,caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。
Monopterus albus, an important aquaculture species in China, is a protogynous hermaphrodite fish that changes sex from female through intersex to male phase during its life. Caspases-3, as a member of the cysteine-aspartic acid protease(caspase) family, is a prominent mediator of apoptosis. The part sequences of caspase-3 cDNA were amplified to improve the knowledge of ricefield eel Caspases-3 functions during sex reversal. The mRNA expression, relative content and immunolocalization of Caspase-3 in different developmental stages of gonad were also investigated. The 432 bp cDNA sequence of ricefield eel caspase-3 was obtained by PCR. Ricefield eel Caspase-3 showed high homologies with those of other teleost, especially with Larimichthys crocea and Siniperca chuatsi. The results of real-time quantitative PCR(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA) showed that the mRNA expression of caspase-3 was detected in all gonadal development stages and decreased along with sex reversal. However, the relative content of Caspase-3 increased along with sex reversal and peaked at the late intersexual stage. There were strong immunoreactive signals in primary growth stage oocytes, which indicated a large portion of cells undergo apoptosis. Furthermore, accompanying the sex reversal of gonad, the positive signals were detected in the cytoplasm of vitellogenic stage oocytes and nucleus of cortical alveoli stage oocytes. Taken together, the expression of Caspase-3 was closely related to the natural sex reversal, which might be involved in the process of oocyte apoptosis during sexual reversal.
Monopterus albus, an important aquaculture species in China, is a protogynous hermaphrodite fish that changes sex from female through intersex to male phase during its life. Caspases-3, as a member of the cysteine-aspartic acid protease(caspase) family, is a prominent mediator of apoptosis. The part sequences of caspase-3 cDNA were amplified to improve the knowledge of ricefield eel Caspases-3 functions during sex reversal. The mRNA expression, relative content and immunolocalization of Caspase-3 in different developmental stages of gonad were also investigated. The 432 bp cDNA sequence of ricefield eel caspase-3 was obtained by PCR. Ricefield eel Caspase-3 showed high homologies with those of other teleost, especially with Larimichthys crocea and Siniperca chuatsi. The results of real-time quantitative PCR(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA) showed that the mRNA expression of caspase-3 was detected in all gonadal development stages and decreased along with sex reversal. However, the relative content of Caspase-3 increased along with sex reversal and peaked at the late intersexual stage. There were strong immunoreactive signals in primary growth stage oocytes, which indicated a large portion of cells undergo apoptosis. Furthermore, accompanying the sex reversal of gonad, the positive signals were detected in the cytoplasm of vitellogenic stage oocytes and nucleus of cortical alveoli stage oocytes. Taken together, the expression of Caspase-3 was closely related to the natural sex reversal, which might be involved in the process of oocyte apoptosis during sexual reversal.
引文
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[15]艾文娜,吴润,刁小龙.PRNP基因SYBR Green I双标准曲线法荧光定量PCR检测方法的建立及初步应用[J].中国兽医科学,2013,43(12):1240-1245.
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[17]徐富翠,郭勇,梅欣明,等.卵泡发育与其细胞凋亡[J].四川解剖学杂志,2002,10(2):99-104.
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[20]Okuthe G.Investigation of apoptosis as a biomarker for earlier detection of impending sex inversion in zebrafish(Danio rerio)[J].T S Afri Philos Soc,2016,71(2):129-135.
[21]ThoméR G,Domingos F F T,Santos H B,et al.Apoptosis,cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish[J].Tissue Cell,2012,44(1):54-62.
[22]赵瑞杰,李引乾,王会,等.Caspase家族与细胞凋亡的关系[J].中国畜牧杂志,2010,46(17):73-78.
[23]王永霞,马娜,钟兴明,等.Caspase-3、TNF-α、bcl-2、bax在人胎儿卵巢中的表达[J].中国医药导报,2015,12(31):21-24.
[24]Danilovi■ L J,Kora■ A,Milo■evi■ I,et al.Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism:Light and electron microscopy approach[J].Micron,2016,90:33-42.
[25]刘修业,崔同昌,王良臣,等.黄鳝性逆转时生殖腺的组织学与超微结构的变化[J].水生生物学报,1990,14(2):166-169.
[2]Fischer U,Janicke R U,Schulze O K.Many cuts to ruin:a comprehensive update of caspase substrates[J].Cell Death Differ,2003,10(1):76-100.
[3]Zhan Y,Van D W B,Wang Y,et al.The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells[J].Oncogene,1999,18(47):6505-6512.
[4]Boone D L,Tsang B K.Caspase-3 in the rat ovary:localization and possible role in follicular atresia and luteal regression[J].Biol Reprod,1998,58(6):1533-1539.
[5]Papandile A,Tyas D,Omalley D M,et al.Analysis of caspase-3,caspase-8 and caspase-9 enzymatic activities in mouse oocytes and zygotes[J].Zygote,2004,12(1):57-64.
[6]Peluffo M C,Young K A,Stouffer R L.Dynamic expression of caspase-2,-3,-8,and-9 proteins and enzyme activity,but not messenger ribonucleic acid,in the monkey corpus luteum during the menstrual cycle[J].J Clin Eenocr Metab,2005,90(4):2327-2335.
[7]Morais R D,ThoméR G,Lemos F S,et al.Autophagy and apoptosis interplay during follicular atresia in fish ovary:a morphological and immunocytochemical study[J].Cell Tissue Res,2002,347(2):467-478.
[8]Nozu R,Horiguchi R,Murata R,et al.Survival of ovarian somatic cells during sex change in the protogynous wrasse,Halichoeres trimaculatus[J].Fish Physiol Biochem,2013,39(1):47-51.
[9]Ito L S,Takahashi C,Yamashita M,et al.Warm water induces apoptosis,gonadal degeneration,and germ cell loss in subadult pejerrey Odontesthes bonariensis(Pisces,Atheriniformes)[J].Physiol Biochem Zool,2008,81(6):762-774.
[10]丁瑞华.四川鱼类志[M].成都:四川科学技术出版社,1994.
[11]He Y,Shang X,Sun J,et al.Gonadal apoptosis during sex reversal of the rice field eel:implications for an evolutionarily conserved role of the molecular chaperone heat shock protein 10[J].J Exp Zool(Mol Dev Evol),2010,314B:257-266.
[12]Liu C K.Rudimentary hermaphroditism in the symbranchoid eel,Monopterus javanensis[J].Sinensia,1944,15(1):1-8.
[13]肖亚梅.黄鳝繁殖生物学研究-II黄鳝的雌性发育[J].湖南师范大学自然科学学报,1995,18(4):45-51.
[14]肖亚梅,刘筠.黄鳝由间性发育转变为雄性发育的细胞生物学研究[J].水产学报,1995,19(4):297-304.
[15]艾文娜,吴润,刁小龙.PRNP基因SYBR Green I双标准曲线法荧光定量PCR检测方法的建立及初步应用[J].中国兽医科学,2013,43(12):1240-1245.
[16]杨晓菁,姚红军,王玉凤.哺乳动物卵母细胞凋亡的研究进展[J].动物学杂志,2004,39(6):101-106.
[17]徐富翠,郭勇,梅欣明,等.卵泡发育与其细胞凋亡[J].四川解剖学杂志,2002,10(2):99-104.
[18]刘梦鹤.绵羊卵母细胞内CTSB、CTSS和caspase3基因和蛋白表达及活性变化规律[D].保定:河北农业大学,2015.
[19]Izawa M,Nguyen P,Kim H,et al.Expression of the apoptosis-related genes,caspase-1,caspase-3,DNA fragmentation factor,and apoptotic protease activating factor-1,in human granulosa cells[J].Fertil Steril,1998,70(3):549-552.
[20]Okuthe G.Investigation of apoptosis as a biomarker for earlier detection of impending sex inversion in zebrafish(Danio rerio)[J].T S Afri Philos Soc,2016,71(2):129-135.
[21]ThoméR G,Domingos F F T,Santos H B,et al.Apoptosis,cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish[J].Tissue Cell,2012,44(1):54-62.
[22]赵瑞杰,李引乾,王会,等.Caspase家族与细胞凋亡的关系[J].中国畜牧杂志,2010,46(17):73-78.
[23]王永霞,马娜,钟兴明,等.Caspase-3、TNF-α、bcl-2、bax在人胎儿卵巢中的表达[J].中国医药导报,2015,12(31):21-24.
[24]Danilovi■ L J,Kora■ A,Milo■evi■ I,et al.Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism:Light and electron microscopy approach[J].Micron,2016,90:33-42.
[25]刘修业,崔同昌,王良臣,等.黄鳝性逆转时生殖腺的组织学与超微结构的变化[J].水生生物学报,1990,14(2):166-169.