Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

Abstract

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.

Plasma membrane of trout spermatozoa: I. Isolation and partial characterization

“…In bands 1 and 2, there is a 6.3 to 6.9-fold enrichment of cholesterol and a 4.2 to 5.4-fold enrichment of phospholipid. These values and that for 5'-nucleotidase either approach those for mammals, or, are lower (Gillis et al 1978;Mack et al 1986). Lower values may result from the smaller size of trout compared to mammalian spermatozoa, which implies a higher membrane protein/total cell protein ratio in fish than in mammals (there is, for instance, 5 to 10 times more protein in human (Mack etal.…”

“…This method overcomes the problems associated with the use of detergent, mechanical homogenization, hypotonic shock or sonication (McNamee 1989). The" yield of membrane protein following release of the plasma membrane by nitrogen cavitation was always equal or greater than 1% of the total cell protein (Gillis et al 1978:1%;Mack et al 1986: 1.6%), i.e., according to Gillis et al (1978) and Parks and Hammerstedt (1985) 20 to 30% of the membrane protein was obtained by this procedure.…”

“…Several methods have been used to detach the plasma membrane of sperm cells (McNamee 1989), including sonication, mechanical homogenization, hypotonic shock, and nitrogen cavitation. According to Gillis et al (1978), the latter technique is the most effective. While our study was in progress, Lou et al (1990) opted to prepare plasma membranes from salmon sperm by sonication.…”

Biochemical composition of the spermatozoal plasma membrane in normal and heat-stressed boars

“…When examining initial purification techniques, a good percentage of this past work has been performed in the boar (Lunstra et al, 1974;Gillis et al, 1978;Peterson et al, 1980;Soucek and Vary, 1984;Nikolopoulou et al, 1985;Canvin and Buhr, 1989;Parks and Lynch, 1992) with less done in the ram and goat (Hathaway and Hartree, 1963;Srivastava et al, 1970;Srivatava et al,, 1970;Brown and Hartree, 1974;Ivanov and Propfirov, 1981;Holt and North, 1985;Parks and Hammestedt, 1985;Hinkovska et al, 1989;Rana and Majumder, 1989), bull (Hathaway and Hartree, 1963;Multamaki and Niemi, 1972;Berstein and Teichman, 1973;Multamaki et al, 1975;Noland et al, 1983;Casaii et al, 1985;Parks et al, 1987;Parks and Lynch, 1992), stallion (Parks and Lynch, 1992) and rat (Jones, 1986;Agrawal et al, 1988 (i.e., exposure to pure, pressurized for 10 minutes followed by rapid decompression to atmospheric conditions which allows Nj to form gas vesicles which disrupt the membrane when Nj escapes into the surrounding atmosphere).…”

“…Once capacitation has been completed, the acrosome reaction can proceed, involving progressive fusion, vesiculation and/or exocytosis of the acrosomal membrane to the overlying plasma membrane; this vesiculation/exocytosis allows for the concomitant release of the acrosomal enzymes which are subsequently involved in the sperm's penetration of the mucoproteinaeous investments surrounding the oocyte. Both capacitation and the acrosome reaction have been linked to cellular modifications in membrane structure of the lipid bilayer to produce protein-free zones via protein alteration and/or migration which may also include polar/neutral lipid efflux (Ahuji et al, 1975;Yanagimachi, 1981;Austin, 1985;Hammerstedt and Parks, 1987;Phelps et al, 1990;Ravinik et al, 1990 (Hathaway and Hartree, 1963;Bernstein and Teichman, 1973;Schill, 1973), extraction with detergents (Srivastava et al, 1970;Multamaki et al, 1975), physical removal using glass beads (Hathaway and Hartree, 1963;Morton and Lardy, 1967;Multamaki and Niemi, 1972), freeze-thawing (Pedersen, 1972;Brown and Hartree, 1974), sonication (Lunstra et al, 1974;Esbenshade and Clegg, 1976;Clegg et al, 1975;Hinkovska et al, 1986), homogenization (Moore and Hibbit, 1976;Soucek and Vary, 1984;Holt and North, 1985;Casali et al, 1985;Holt and North, 1986), hypotonic shock (Ivanov and Profirov, 1981;Rana and Majumder, 1989) and nitrogen cavitation (Gillis et al, 1978;Peterson et al, 1980;Noland et al, 1983;Nikolopoulou et al, 1985;Parks and Hammerstedt, 1985;…”