Freeze‐substitution: a method for the rapid arrest and chemical fixation of spermatozoa

SUMMARY A procedure for the freeze‐substitution fixation of spermatozoa has been developed. Physical fixation of the motile cells is effected very rapidly by immersing the sperm in Freon‐22 at −146°C. Chemical fixation, with glutaraldehyde, is then imposed on the immobilized sperm after substitution of the ice matrix at −50°C. The substitution is achieved with the solvent ethylene glycol in the manner described by Pease (1967). (Subsidiary experiments confirm that substitution of ice does occur at −50°C and that chemical fixation by glutaraldehyde occurs at temperatures at least as low as −20°C.) The spermatozoa are then embedded in Epon and examined as thin sections. Other techniques—substitution in acetone containing osmium tetroxide, the inclusion of formaldehyde with ethylene glycol as a chemical fixative, and the inclusion of agar in the diluent used to suspend the spermatozoa—have been tried but were unsatisfactory. Freezing stops the movement very rapidly and, as little subsequent disturbance occurs, it should be possible to preserve transient changes in the fine structure of the flagellum which might be related to a mechanism for the generation of bends. The observations in this paper are confined to the behaviour of the proximal part of the midpiece of the rat spermatozoon. It is demonstrated that bending of this part of the flagellum is restricted, to a remarkable degree, to one plane, the plane in which the sperm nucleus is flattened. This plane is almost exactly perpendicular to the plane containing the two central tubules of the axoneme. It has been confirmed that the axonemal complex has a straight course through the midpiece during those beats of the flagellum which are not accompanied by rotation of the entire cell. Lastly, it appears that the nine dense fibres do not undergo transient displacements as the flagellum bends.