红鳍东方鲀精原干细胞鉴定、分离及纯化
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摘要
为获得高比例精原干细胞,开展了不同月龄红鳍东方鲀精原干细胞分离纯化研究。采用组合酶消化法制备不同月龄的精巢单细胞悬液,通过形态学观察和生殖细胞特异基因vasa免疫荧光鉴别精原干细胞,Percoll不连续梯度(10%、30%和50%)纯化精原干细胞。结果显示:14月龄雄鱼精巢生殖细胞主要为A型精原细胞,精原干细胞占比极显著性地高于22月龄(P<0.05)。纯化后精原干细胞主要分布在10%—30%Percoll梯度带中, 14月龄雄鱼生殖细胞主要分布在此层,且纯化后精原干细胞占比远高于纯化前以及22月龄纯化前后。结果表明, 14月龄更适用组合酶消化分离,并通过Percoll梯度离心法获得高比例的红鳍东方鲀精原干细胞。
This study investigated the isolation and purification of spermatogonial stem cells(SSCs) by single cell suspensions after enzyme digestion using testes of various months old tiger puffer. SSCs were enriched by Percoll discontinuous gradient. The SSCs were identified by optical observation and immunolocalization of Vasa protein. The majority of SSCs at 14-month-old fish was type A spermatagonial cells that were significantly higher than those of the 22-month-old males(P<0.05). SSCs were mainly enriched in 10%—30% Percoll gradient belt, where the germ cells were mainly distributed at 14-month-old fish. These results indicate that 14-month-old male fish is a great source for SSCs using the current isolation method.
This study investigated the isolation and purification of spermatogonial stem cells(SSCs) by single cell suspensions after enzyme digestion using testes of various months old tiger puffer. SSCs were enriched by Percoll discontinuous gradient. The SSCs were identified by optical observation and immunolocalization of Vasa protein. The majority of SSCs at 14-month-old fish was type A spermatagonial cells that were significantly higher than those of the 22-month-old males(P<0.05). SSCs were mainly enriched in 10%—30% Percoll gradient belt, where the germ cells were mainly distributed at 14-month-old fish. These results indicate that 14-month-old male fish is a great source for SSCs using the current isolation method.
引文
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[20]Leal M C,Cardoso E R,Nóbrega,Rafael H,et al.Histological and stereological evaluation of zebrafish(Danio rerio)spermatogenesis with an emphasis on spermatogonial generations[J].Biology of Reproduction,2009,81(1):177-187
[21]Loir M.Spermatogonia of rainbow trout:Ⅱ.In vitro study of the influence of pituitary hormones,growth factors and steroids on mitotic activity[J].Molecular Reproduction and Development,1999,53(4):434-442
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[23]Zhongkai W,Jinning G,Huayu S,et al.Sexually Dimorphic Expression of vasa Isoforms in the Tongue Sole (Cynoglossus semilaevis)[J].PLoS One,2014,9(3):e93380
[24]Chen Q Y,An L L.Advances in fish spermatogonial stem cells[J].Guangdong Agricultural Science,2015,6:105-110[陈秋宇,安立龙.鱼类精原干细胞的研究进展.广东农业科学,2015,6:105-110]
[25]Nakagawa T,Sharma M,Nabeshima Y,et al.Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment[J].Science,2010,328(5974):62-67
[26]Yoon C,Kawakami K,Hopkins N.Zebrafish vasa homologue RNA is localized to the cleavage planes of 2-and 4-cell-stage embryos and is expressed in the primordial germ cells[J].Development,1997,124:3157-3165
[27]Xu H,Gui J,Hong Y.Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp(Carassius auratus gibelio),a bisexually and gynogenetically reproducing vertebrate[J].Development Dynamics An Official Publication of the American Association of Anatomists,2005,233:872-882
[28]Muren H,Davey R J,Hill J R.Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation[J].Cell and Tissue Research,2007,330(2):321-329
[29]Shen F,Zhang C,Zheng H,et al.Long-term culture and transplantion of spermatogonial stem cells from BALB/cmice[J].Cells Tissues Organs,2010,191(5):372-381
[30]Lacerda S M S N,Batlouni S R,Silva S B G,et al.Germ cells transplantation in fish:the Nile-tilapia model[J].Animal Reproduction,2006,3(2):146-159
[2]Matsuura S,Naito T,Shincho M,et al.Gonadal differentiation in tiger puffer,Takifugu rubripes[J].Aquaculture Science,1994,42(4):619-625
[3]Suzuki N,Okada K,Kamiya N.Gonadal development,sexual differentiation and sex ratio of tiger puffer Takifugu rubripes[J].Bulletin of the Nasei National Fisheries Research Institute(in Japanese),1996,29(2):39-48
[4]Suzuki N.Histological study on intersexual gonads observed in wild tiger puffer,Takifugu rubripes[J].Bulletin of the Nansei National Fisheries Reserch Institute,1997,30:101-113
[5]Lee K H,Yamaguchi A,Rashid H,et al.Estradiol-17βtreatment induces intersexual gonadal development in the pufferfish,Takifugu rubripes[J].Zoologicalence,2009,26(9):639-645
[6]Kawai T,Matsubara H.Hermaphrodism of the Tiger Puffer,Takifugu rubripes(Actinopterygii:Tetraodontiformes:Tetraodontidae),from Hokuto,Hokkaido,Japan[J].Bulletin of Fisheries Sciences,Hokkaido University,2011,61(1):1-4
[7]Okutsu T,Shikina S,Kanno M,et al.Production of trout offspring from triploid salmon parents[J].Science,2007,317(5844):1517-1517
[8]Sato M,Morita T,Katayama N,et al.Production of genetically diverified fish seeds using spermatogonial transplantation[J].Aquculture,2014,S422-423:218-224
[9]Seki S,Kusano K,Lee S,et al.Production of the medaka derived from vitrified whole testes by germ cell transplantation[J].Scientific Reports,2017,7:43185
[10]Yoshizaki G,Fujinuma K,Iwasaki Y,et al.Spermatogonial transplantation in fish:A novel method for the preservation of genetic resources[J].Comparative Biochemistry and Physiology Part D Genomics and Proteomics,2011,6(1):55-61
[11]Wong T T,Saito T,Crodian J,et al.Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae[J].Biology of Reproduction,2011,84(6):1190-1197
[12]Nóbrega R H,Greebe C D,Van d K H,et al.Spermatogonial stem cell Niche and spermatogonial stem cell transplantation in Zebrafish[J].PLoS One,2010,5(9):1-16
[13]Silva M A,Costa G M,Lacerda S M,et al.Successful xeogeneic germ cell transplantaion from Jundia catfish(Rhamdia quelen)into adult Nile tilapia(Oreochromis niloticus)testes[J].General and Comparative Endocrinology,2016,230-231:48-56
[14]Zhao Y X,Zhou Q,Yu Q H,et al.Development and application prospects of fish germ cell transplantation technology[J].Journal of Fishery Sciences of China,2017,24(2):414-423[赵雅贤,周勤,于清海,等.鱼类生殖细胞移植技术的发展及应用前景.中国水产科学,2017,24(2):414-423]
[15]Schulz R W,Frana LR de,Lareyre J J,et al.Spermatogenesis in fish[J].General and Comparative Endocrinology,2009,165(3):390-41
[16]Xu H Y,Li M Y,Gui J F,et al.Fish reproductive cells[J].Scientia Sinica(Vitae),2010,40(2):124-138[徐红艳,李名友,桂建芳,等.鱼类生殖细胞.中国科学:生命科学,2010,40(2):124-138]
[17]Okutsu T,Shikina S,Sakamoto T,et al.Successful production of functional Y eggs derived from spermatogonia transplanted into female recipients and subsequent production of YY supermales in rainbow trout,Oncorhynchus mykiss[J].Aquaculture,2015,446(11):298-302
[18]Takeuchi Y,Higuchi K,Yatabe T,et al.Development of spermatogonial cell transplantation in Nibe Croaker,Nibea mitsukurii(Perciformes,Sciaenidae)[J].Biology of Reproduction,2009,81:1055-1063
[19]Zhang X L,Gao Y M,Zhao S W,et al.Experimental study on the separation and purification of spermatogonia in neonatal mouse[J].Journal of Shandong University(Health Sciences),2005,43(8):674-677[张晓丽,高英茂,赵舒武,等.新生小鼠精原细胞分离和纯化的实验研究.山东大学学报(医学版),2005,43(8):674-677]
[20]Leal M C,Cardoso E R,Nóbrega,Rafael H,et al.Histological and stereological evaluation of zebrafish(Danio rerio)spermatogenesis with an emphasis on spermatogonial generations[J].Biology of Reproduction,2009,81(1):177-187
[21]Loir M.Spermatogonia of rainbow trout:Ⅱ.In vitro study of the influence of pituitary hormones,growth factors and steroids on mitotic activity[J].Molecular Reproduction and Development,1999,53(4):434-442
[22]Chen Y D,Zou Z H,Wang Y L,et al.Progress in studies of vasa gene[J].Chinese Journal of Zoology,2010,45(4):173-180[陈玉东,邹志华,王艺磊,等.Vasa基因研究进展.动物学杂志,2010,45(4):173-180]
[23]Zhongkai W,Jinning G,Huayu S,et al.Sexually Dimorphic Expression of vasa Isoforms in the Tongue Sole (Cynoglossus semilaevis)[J].PLoS One,2014,9(3):e93380
[24]Chen Q Y,An L L.Advances in fish spermatogonial stem cells[J].Guangdong Agricultural Science,2015,6:105-110[陈秋宇,安立龙.鱼类精原干细胞的研究进展.广东农业科学,2015,6:105-110]
[25]Nakagawa T,Sharma M,Nabeshima Y,et al.Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment[J].Science,2010,328(5974):62-67
[26]Yoon C,Kawakami K,Hopkins N.Zebrafish vasa homologue RNA is localized to the cleavage planes of 2-and 4-cell-stage embryos and is expressed in the primordial germ cells[J].Development,1997,124:3157-3165
[27]Xu H,Gui J,Hong Y.Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp(Carassius auratus gibelio),a bisexually and gynogenetically reproducing vertebrate[J].Development Dynamics An Official Publication of the American Association of Anatomists,2005,233:872-882
[28]Muren H,Davey R J,Hill J R.Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation[J].Cell and Tissue Research,2007,330(2):321-329
[29]Shen F,Zhang C,Zheng H,et al.Long-term culture and transplantion of spermatogonial stem cells from BALB/cmice[J].Cells Tissues Organs,2010,191(5):372-381
[30]Lacerda S M S N,Batlouni S R,Silva S B G,et al.Germ cells transplantation in fish:the Nile-tilapia model[J].Animal Reproduction,2006,3(2):146-159