Harris P D, Cable J, Tinsley R C
Department of Adult Education, University of Nottingham, UK.
Parasitol Res. 1997;83(6):533-8. doi: 10.1007/s004360050294.
A rapid fluorescent staining method demonstrating spermatozoa within gyrodactylid monogeneans is described. Gyrodactylids fixed and stained in 2% acetic acid containing 1 microgram ml-1 bisBenzimide (Hoechst 33258) were viewed using epifluorescence microscopy. In addition to staining sperm in the testis, seminal vesicle and seminal receptacle, the technique highlighted sperm in the gut and cytoplasmic lining of the uterus, locations from which they had not previously been recorded. The technique was more rapid than transmission electron microscopy (TEM), which was used to confirm the observations. Fluorescence microscopy provided an overview of sperm orientation and distribution that could otherwise be obtained only from serial ultrathin sections. Using the fluorescence technique and/or TEM we have located migrating sperm in the tissues of Gyrodactylus turnbulli, G. bullatraudis and G. gasterostei, although these sperm do not appear to enter the tissues of embryos in utero. The technique can be used to study insemination patterns in gyrodactylid populations and in experimental studies of gyrodactylid reproduction.
本文描述了一种用于显示三代虫单殖吸虫体内精子的快速荧光染色方法。将三代虫固定于含1微克/毫升双苯甲酰亚胺（Hoechst 33258）的2%醋酸中进行染色，然后用落射荧光显微镜观察。该技术除了能对睾丸、精囊和受精囊中的精子进行染色外，还突出显示了肠道和子宫细胞质内衬中的精子，这些部位此前未有精子存在的记录。该技术比用于确认观察结果的透射电子显微镜（TEM）更快。荧光显微镜提供了精子取向和分布的总体情况，而这原本只能从连续超薄切片中获得。通过荧光技术和/或TEM，我们在特恩布尔三代虫、布拉特罗氏三代虫和腹棘鱼三代虫的组织中定位到了迁移的精子，不过这些精子似乎并未进入子宫内胚胎的组织。该技术可用于研究三代虫种群的受精模式以及三代虫繁殖的实验研究。
