Monoclonal antibody labelling of human spermatozoa: an electron microscope study

Abstract

The antigenic determinants recognized by six anti-human sperm monoclonal antibodies were localized at the subcellular level using an indirect peroxidase immunoelectron microscopic method. Labelling was performed using fresh spermatozoa, and after cell permeabilization (by osmotic shock or freeze-thawing) or detergent demembranation. Two antibodies bound to distinct regions of the plasma membrane, one over the acrosome and the other on the tail, but both also bound to intracellular sites on damaged cells. The internal organelles labelled by the other four antibodies were identified as the acrosomal membrane of the equatorial segment, structures in the connective piece, mitochondrial membranes and axonemal microtubules, respectively. These results are compared with those of a previous immunofluorescence study (Villarroya & Scholler, 1986) and the advantages of joint light and electron microscopy for sperm immunocytochemistry are discussed.

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

“…The non-ionic detergent Triton X-100 has been used to demembranate the spermatozoa while leaving the intracellular structures intact. The model of demembranated sperm has allowed a number of experimental manipulations on the flagellar motility to be performed (Gibbons, 1975;Lindemann, 1978;Mohri & Yanagimachi, 1980;White & Voglmayr, 1986) and to directly detect the antigens on the internal organelles of spermatozoa (Jones et al, 1983;Huneau et al, 1987). In the present study this model was successfully used to reveal the intracellular localization of antigenic determinants recognized by the Mab TUS 10 in the rat epididymal spermatozoa.…”

“…The model of demembranated sperm has allowed a number of experimental manipulations on the flagellar motility to be performed (Gibbons, 1975;Lindemann, 1978;Mohri & Yanagimachi, 1980;White & Voglmayr, 1986) and to directly detect the antigens on the internal organelles of spermatozoa (Jones et al, 1983;Huneau et al, 1987). The model of demembranated sperm has allowed a number of experimental manipulations on the flagellar motility to be performed (Gibbons, 1975;Lindemann, 1978;Mohri & Yanagimachi, 1980;White & Voglmayr, 1986) and to directly detect the antigens on the internal organelles of spermatozoa (Jones et al, 1983;Huneau et al, 1987).…”

“…These results are not specific to rat epididymal spermatozoa. Since Triton X-100 removes sperm surface membrane, but does not greatly damage the internal structure of the sperm tail (Huneau et al, 1987), we reasoned that antigenic determinants reacted with Mab TuSlO must be also on the surface of the intracellular organelle, most likely on the fibrous sheath. At the light microscopic level in the present study, the intact sperm from the caput epididymidis showed strong affinity to the Mab TUS 10, but the demembranation significantly decreased the affinity.…”

“…O n the other hand, the cauda sperm both intact and demembranated showed similar affinity to the Mab TuS10, which suggests that the antigenic determinanats are localized both on the plasma membrane and on the internal organelles. Since Triton X-100 removes sperm surface membrane, but does not greatly damage the internal structure of the sperm tail (Huneau et al, 1987), we reasoned that antigenic determinants reacted with Mab TuSlO must be also on the surface of the intracellular organelle, most likely on the fibrous sheath. Our immunoelectron microscopy has substantiated this hypothesis, namely the reaction products were localized on the plasma membrane overlying the principal piece of the spermatozoa collected both from the caput and cauda epididymidis, and on the external surface of the fibrous sheath of the cauda spermatozoa, but not on that of caput spermatozoa.…”