Smithsonian Conservation Biology Institute, Front Royal, VA, 22360, United States of America.
Hawaii Institute of Marine Biology, 46-007 Lilipuna Rd, Kaneohe, HI, 96744, United States of America.
Sci Rep. 2018 Apr 18;8(1):6149. doi: 10.1038/s41598-018-24269-3.
As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.
随着全球生物多样性的减少，生物标本的价值不断增加。冷冻保存的二倍体精原细胞符合两个目标：产生高质量的分子序列数据；并再生新个体，从而有可能防止物种灭绝。目前，自然历史博物馆的收藏中没有能够进行这种缓解措施的冷冻保存精原细胞，因为没有收集它们的标准方案。传统上，脊椎动物标本，尤其是鱼类，都是用福尔马林固定和酒精保存的，这使它们非常适合形态学研究和作为博物馆凭证，但不适合分子序列数据。鱼类的分子研究通常使用保存在乙醇中的组织；然而，用这种方法保存的组织随着时间的推移可能会产生降解的序列。作为组织固定方法的替代方法，我们通过流式细胞术对鱼类睾丸细胞进行门控和计数，以鉴定假定的 A 型精原细胞，从而评估和比较了先前发表的冷冻保存方法。在这里，我们描述了一种从 Asterropteryx semipunctata（一种海洋虾虎鱼）的睾丸中冷冻保存组织的方案，该方案可从睾丸中获得高比例的活精原细胞。使用该方案冷冻保存的材料代表了首次在自然历史博物馆中冷冻和解冻后仍具有活力的鱼类精原细胞，为物种的重新衍生、DNA 保存和分析提供了更好质量的材料。
