Chapter 29 Fixation of Mammalian Spermatozoa for Electron Microscopy

Abstract

This chapter discusses methods for preparing mammalian spermatozoa in situ (for example, in the epididymis) or in suspension (for example, in semen) for the transmission electron microscope (TEM). The best fixation of spermatozoa is obtained when the glutaraldehyde is buffered with collidine. The period of fixation is not critical. Both the epididymis and testis are difficult tissues to fix by immersion. In the case of the testis, seminiferous tubules tend to move apart when the tissue is dissected. Because of this, after the tissue is embedded and cut, thin sections may contain more empty plastic than tissue. The epididymis can be equally problematic because when the tubules are cut up during fixation, spermatozoa may wash out. Not only does this reduce the number of spermatozoa in the tissue, but the displacement of spermatozoa caused by cutting may disturb the orderly arrangement of spermatozoa within the tubules. Tissue damage can be minimized by cutting the tissue carefully. Alternatively, the intact tissue can be fixed by perfusing the blood vessels with fixative and dissecting the tissue afterward. When spermatozoa are fixed in suspension, it is important to create a pellet where the cells are tightly packed without compromising fixation or damaging or decapitating the sperm with too high a centrifugal force. It is necessary to pack the sperm tightly, because searching for perfect cross sections of a sperm tail or exact sagittal sections of a sperm head takes less time if the sperm are closer together. It is easier to find a field with several sperm cut in the desired orientation. In addition, relatively high centrifugal force tends to pack spermatozoa in register, which may facilitate the location of cross sections of several spermatozoa in the same field. The chapter provides hints on helpful practices for conducting the tests.