Morphological aspects of cytoplasmic droplets of some plethodontid salamander spermatozoa.

Ultrastructural studies of cytoplasmic droplets of the spermatozoa of bulls, rams, rabbits, rats (Bloom & Nicander, 1961) and bats (Fawcett & Ito, 1965) revealed only numerous membranous vesicles and tubules in the matrix of the droplets. These authors agreed that the membranous elements were probably of endoplasmic reticulum and/or Golgi complex origin. The simplicity of the ultrastructure and lack of other organelles and inclusions in the droplet matrix led Fawcett & Ito (1965) to doubt suggestions that cytoplasmic droplets contain endogenous substrates for epididymal sperm survival. Recent biochemical and cytochemical analyses of cytoplasmic droplets in spermatozoa of different domestic animals (Dott & Dingle, 1968; Harrison & White, 1972; Moniem & Glover, 1972) showed a rather high activity of hydrolytic and glycolytic enzymes. Harrison & White (1972) suggested that at least some of the enzymes found in the seminal plasma may have their origin in disrupted cytoplasmic droplets. In our study (Martan, Brandon & Wortham, 1973) of interspecific variation in sperm morphology within the salamander family, Plethodontidae, we have observed that the spermatozoa have a characteristic cytoplasmic droplet. The seven genera studied were Desmognathus, Eurycea, Typhlotriton, Aneides, Ensatina, Plethodon and Bolitoglossa. Cytoplasmic droplets of sixteen species were examined with the light microscope, and those of Plethodon glutinosus were also examined with the electron microscope. For light microscopy, seminal fluid was removed from the ductus deferens of freshly killed or anaesthetized males, spot-smeared on slides lightly coated with albumin, and fixed for 30 min in 95% ethanol. Slides for cytological details were stained in Heidenhein iron haematoxylin, while slides for local¬ ization of RNA were stained by the azure technique (Swift, 1955). Control slides were exposed for 2 hr to RNAse before staining (Barka & Anderson, 1963). Living spermatozoa were supravitally stained with Janus green. For electron microscopy, spermatozoa were obtained by the same procedure and immediately fixed at 4° C in paraformaldehyde (Lynn, Martin & Race, 1966) and post-fixed in 1 % osmium tetroxide buffered with s-collidine buffer (Bennett & Luft, 1959) at pH 7-4. The fixed spermatozoa were resuspended in 2% agar after centrifugation and cut into 0-5-mm blocks and stained with 1 %