Pacchiarini T, Olague E, Sarasquete C, Cabrita E
ICMAN-Institute of Marine Science of Andalusia, Spanish National Research Council, Puerto Real, Cádiz, Spain.
Histol Histopathol. 2014 May;29(5):619-28. doi: 10.14670/HH-29.10.619. Epub 2013 Dec 27.
Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20÷-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg⁻¹ busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.
白消安是一种用于治疗慢性粒细胞白血病的细胞毒性烷化剂，对哺乳动物生殖细胞有影响。在鱼类中，这种化合物在种内和种间生殖细胞移植方面具有特殊意义。为了确定白消安对鱼类的影响，之前设计了一项范围查找实验。测定了孵化后150天（150DAH）的塞内加尔鳎幼鱼的存活率和生长率。在第二项实验中，分析了亚致死剂量的白消安对鱼类生殖细胞耗竭和体细胞组织的影响。在体细胞和性腺组织中测定了几种白消安处理（注射后10天的B10、注射后20天的B20、注射后20天双倍注射的B20÷）的亚致死效应。通过组织病理学技术、TUNEL（细胞凋亡）进行变化记录，并使用瓦萨mRNA（Ssvasa1 - 2和Ssvasa3 - 4 mRNA）作为塞内加尔鳎幼鱼生殖细胞的分子标记在分子水平（Q - PCR分析）进行量化。观察到40 mg kg⁻¹白消安（一种非致死剂量）有几种亚致死效应，例如：肝脏细胞核固缩、脾脏中黑色素巨噬细胞中心增加和血液停滞以及性腺发育中断。在生殖细胞破坏方面，雌性比雄性受白消安处理影响更大，因为在处理过的雌性而非雄性的性腺中发现Ssvasa1 - 2和Ssvasa3 - 4标记的表达显著降低。处理后10天（B10），通过Q - PCR证实雌性生殖细胞增殖已经减少。Ssvasa表达被证明是直接评估白消安对塞内加尔鳎性腺发育影响的可靠工具，证明白消安可以是一种合适的处理方法，用于在生殖细胞移植中使受体性腺产生短暂不育。
