Chemical Dissection of Mammalian Spermatozoa

Abstract

Spermatozoa from several mammalian species have been dissected by chemical methods to yield free heads, tails with attached midpieces, and tails from which the mitochondrial components of the midpiece were removed . Mouse and rat spermatozoa were cleaved by brief treatment with trypsin to yield free heads and tails, while human, guinea pig, and rabbit spermatozoa were cleaved by trypsin only after incubation with 2-mercaptoethanol or dithiothreitol . Spermatozoa were also cleaved at the junction of the head and the tail by treatment with acid and base . Mitochondria were removed from intact spermatozoa or isolated tails by mechanical shear after treatment with 2-mercaptoethanol or dithiothreitol . The dissected components of spermatozoa were fractionated with good yield and high purity by density gradient centrifugation . Ultrastructural analysis indicates that proteolytic cleavage to yield separated heads and tails occurs at a specific location in the neck of the spermatozoon, leaving the basal plate attached to the head of the cell . In contrast, after acid cleavage the basal plate remains with the midpiece . Proteolytic treatment has no apparent effect on any other spermatozoan structures, whereas acid or base treatment results in damage to the plasma membrane, the acrosome, and other structures . The specificity of the proteolytic cleavage suggests that a particular protein or group of proteins may be responsible for the linkage between the sperm head and tail .

Targeted Disruption of the Transition Protein 2 Gene Affects Sperm Chromatin Structure and Reduces Fertility in Mice

“…This procedure breaks the tails off from the heads but leaves the sperm tails intact and has been reported not to cause any apparent damage to sperm head structures (45).…”

A Simplified Method for the Isolation of Sperm Heads from the Caput Epididymidis of Rodents and from Ejaculated Rabbit Seminal Plasma

“…As reported previously [6,9], treatment of mouse and rat spermatozoa with trypsin resulted in rapid quantitative cleavage of the sperm heads and tails without apparent damage to the heads and tails as oberved by phase contrast microscopy. Lability of the basal plate which links the head and the midpiece of sperm tails to trypsin suggests that the structural protein in this region is richly composed of basic amino acids such as i.-arginine and i.-lysine.…”

“…Previously, there have been several biochemical studies of isolated spermatozoa from vas deferens of several animal species [9] and from the epididymides of mice [8] and rat [2,3,5,7], However, the method ologies of sperm isolation differed widely. The reports also failed to provide substantial proof of purity of the isolated sperm heads.…”

“…Consequently, the isolation of highly purified sperm heads be comes the first essential step for such investigations. Although there have been numerous biochemical studies of isolated sperms from mouse [8,9], rat [3,7,9] and rabbit [2,9], the morphological evidence for purity and homogeneity of isolated sperm heads has not yet adequately been provided.…”

Cryopreservation of Rat Sperm

“…Mitochondria, the site of ATP generation due to oxidative phosphorylation, are localized solely in the midpiece of sperm (Millette et al, 1973). The oxidative production of ATP through the Krebs cycle is an essential function of the midpiece mitochondria for motility (Suarez et al, 2007).…”