Bouma Gerrit J, Cloud Joseph G, Nagler James J
The Jackson Laboratory, Bar Harbor, Maine 04609, USA.
J Exp Zool A Comp Exp Biol. 2005 Aug 1;303(8):698-703. doi: 10.1002/jez.a.197.
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 investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) 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.
鱼类性腺片段的体外培养仍然是回答基础科学问题和应用科学问题的重要实验方法。本研究的目的是测试培养一周或更长时间的幼年虹鳟（Oncorhynchus mykiss）睾丸在生理上是否具有功能。将鳟鱼睾丸片段（约1平方毫米）放在平底培养板插入滤膜上，置于一滴液体培养基（改良的Leibovitz's L - 15）中，并漂浮在多孔培养板的培养基表面。培养板盖上盖子，在15摄氏度的空气中孵育。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，将体外培养不同时间段直至8天的睾丸片段与完整睾丸的组织学外观进行比较，结果表明没有明显差异。其次，在添加浓度为2.5%和25%牛犊血清（BCS）的培养基中孵育的睾丸片段，通过核5 - 溴 - 2'-脱氧尿苷掺入法测量，间质细胞和精囊肿细胞的增殖明显更大。最后，培养5天并移植回供体鱼体内的睾丸片段，在接受鲑鱼垂体提取物注射方案后出现早熟性成熟，并产生了可育精子，这通过它们成功使卵子受精的能力得以确定。这些实验证明了该方法用于幼年虹鳟睾丸片段体外培养的实用性。
