Junquera Concepción, Colás Carmen, Martínez-Ciriano Carmen, Serrano Pedro, Castiella Tomás, Cebrian-Perez José A, Muiño-Blanco Teresa
Departamento de Anatomía e Histología Humanas, Facultad de Medicina, Universidad de Zaragoza, and Hospital Clínico Universitario Lozano-Blesa, Zaragoza, Spain.
Microsc Res Tech. 2007 Aug;70(8):657-62. doi: 10.1002/jemt.20460.
The study of the ultrastructure of spematozoa by means of transmission electron microscopy (TEM) often presents with problems of interpretation according to the method employed, depending on whether samples are either centrifuged previously to the fixation or immersed in viscous gels. The major problems of interpretation are changes in the location of vesicles originated during the maturation process and modifications in the adsorption of seminal plasma proteins to the sperm membrane surface. The aim of our study is to communicate an original new method for the treatment of spermatozoa for ultrastructural study. Our method is based on the use of animal tissues as biological containers, inside which the spermatic suspensions are included. We developed this method using fresh sperm samples taken from mature Rasa Aragonesa rams. As biological container, we used 2.5-cm long segments of the intestine of 1-week-old chickens (Gallus gallus) (diameter around 4 mm). In order to avoid any influence of digestive enzymes of the mucosa on the sperm surface, we put each intestine fragment inside out by means of microdissection forceps under a bifocal optical microscope and cold light. One of the edges was tied with thin suture silk. The sperm suspension was injected in the optimal experimental condition and amount. Finally, the still-open edge of the intestine segment was tied with silk in the same way as the other segment edge. By using this technique, we can perform a suitable morphological study at an ultrastructural level. In addition, the functional relationship of the ultrastructural components of the target cells is correctly preserved.
通过透射电子显微镜（TEM）对精子超微结构进行研究时，根据所采用的方法，往往会出现解释方面的问题，这取决于样本在固定前是否经过离心处理，或者是否浸泡在粘性凝胶中。主要的解释问题包括成熟过程中产生的囊泡位置变化以及精浆蛋白吸附到精子膜表面的改变。我们研究的目的是传达一种用于精子超微结构研究的全新原始处理方法。我们的方法基于使用动物组织作为生物容器，将精子悬浮液包含在其中。我们使用从成年阿拉贡拉萨公羊采集的新鲜精子样本开发了这种方法。作为生物容器，我们使用了1周龄雏鸡（家鸡）长2.5厘米的肠段（直径约4毫米）。为了避免黏膜消化酶对精子表面产生任何影响，我们在双焦点光学显微镜和冷光下，用显微解剖镊子将每个肠段翻过来。其中一个边缘用细缝合丝线系住。在最佳实验条件下，以最佳量注入精子悬浮液。最后，用丝线以与另一段边缘相同的方式系住肠段仍开口的边缘。通过使用这种技术，我们可以在超微结构水平上进行合适的形态学研究。此外，目标细胞超微结构成分的功能关系得到了正确保留。
