An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis
An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis
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DOI：
10.1002/jez.a.197
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发表时间：
2005-08-01
影响因子：
2.8
通讯作者：
Nagler, JJ
中科院分区：
文献类型：
--
作者：
Bouma, GJ;Cloud, JG;Nagler, JJ
鱼类性腺碎片的体外培养仍然是回答基本和应用科学问题的重要实验方法。本研究的目的是测试养殖一周或以上的虹鱼幼鱼精巢是否具有生理能力。将鲑鱼睾丸碎片(类似于1 mm(2))放在平板培养板内置滤器上，滴入改良的Leibovitz‘s L-15液体培养基中，在多孔培养板中漂浮在培养基面上。覆盖培养板并在15℃的空气中孵化，使用三个不同的终点来测试培养的睾丸片段是否保持健康和功能。首先，比较了体外培养不同时期至8天的完整睾丸的睾丸碎片的组织学形态，结果表明没有明显的差异。其次，5-溴-2‘-脱氧尿嘧啶核苷掺入实验表明，在添加2.5%和25%牛小牛血清(BCS)的培养液中孵育的小鼠睾丸间质细胞和精囊细胞的增殖明显增强。最后，培养了5天的睾丸碎片被移植回供体鱼体内，作为对注射鲑鱼脑垂体提取物的反应，它们经历了性早熟，并产生了受精卵成功受精的能力。这些实验证明了该方法在虹鳟鱼幼体睾丸碎片体外培养中的应用。
In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present invesitigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) 2) testes cultured for a week or more were physiologically competent. Trout testis fragments (similar to 1mm(2)) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.