Improved In Vitro Culture Conditions to Enhance the Survival, Mitotic Activity, and Transplantability of Rainbow Trout Type A Spermatogonia
Improved In Vitro Culture Conditions to Enhance the Survival, Mitotic Activity, and Transplantability of Rainbow Trout Type A Spermatogonia
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DOI：
10.1095/biolreprod.109.082123
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发表时间：
2010-08-01
影响因子：
3.6
通讯作者：
Yoshizaki, Goro
中科院分区：
文献类型：
--
作者：
Shikina, Shinya;Yoshizaki, Goro
精子发生起源于一小群精原干细胞，它们具有自我更新和产生分化生殖细胞的能力。我们以前建立了一个替代亲鱼技术，使用异种移植的精原细胞在鲑鱼。这项技术有望成为生产对市场或濒危物种有价值的目标种子的有效工具。我们一直在尝试建立一种技术，生产种子通过移植精原细胞增殖在培养皿中。然而，我们以前的培养精原细胞的方法有几个缺陷。首先，残留的睾丸体细胞浸润过度增殖的文化，并最终胜过精原细胞。第二，精原细胞总数在培养过程中逐渐减少，即使有丝分裂得到证实。第三，与完整精原细胞的能力相比，移植后培养的精原细胞融入受体性腺的能力较差。为了克服这些缺陷，本研究对精原细胞培养条件进行了改进。将培养液中的胎牛血清浓度调整为1%，可以抑制睾丸体细胞的过度生长。在培养液中加入可溶性因子，如牛血清白蛋白、腺苷和鲑鱼血清，可以提高精原细胞的存活率、有丝分裂活性和可移植性。在新开发的条件下，我们延长了培养期。此外，移植试验表明，精原细胞在改良培养基中培养42天后仍具有移植性。本研究代表了有价值的步骤，建立一种培养方法，使精原细胞在体外扩大用于种子生产与替代亲鱼技术。
Spermatogenesis originates from a small population of spermatogonial stem cells, which have the ability to both self-renew and produce differentiated germ cells. We previously established a surrogate broodstock technique using xenotransplantation of spermatogonia in salmonids. This technique promised to be an efficient tool for producing target seeds that are valuable to markets or endangered species. We have been attempting to establish a technique to produce seeds by transplanting spermatogonia proliferated in culture dishes. However, our previous methods for culturing spermatogonia had several defects. First, residual testicular somatic cells infiltrated excessively proliferating cultures and eventually outcompeted spermatogonia. Second, the total number of spermatogonia gradually decreased during culture periods even though mitosis was confirmed. Third, the cultured spermatogonia were less able to be incorporated into the recipient gonads following transplantation as compared to the ability of intact spermatogonia. To overcome these defects, in the present study we improved upon spermatogonia culture conditions. The overgrowth of testicular somatic cells could be suppressed by adjusting fetal bovine serum concentration in the medium to 1%. The addition of soluble factors, such as bovine serum albumin, adenosine, and salmonid serum, to the medium would enhance spermatogonial survival, mitotic activity, and transplantability. Under newly developed conditions, we extended the culture periods. Furthermore, a transplantation assay showed that spermatogonia cultured in the modified medium for 42 days still possessed their transplantability. The present study represents valuable steps toward establishing a culture method enabling spermatogonia to expand in vitro for use in seed production with surrogate broodstock technology.