Purification and Partial Characterization of Plasma Membranes from Bovine Spermatozoa

Abstract

Bovine epididymal spermatozoa were subjected to nitrogen cavitation (600 psi for 10 min) to remove plasma membrane. Examination of the cavitated cells by electron microscopy revealed that the plasma membrane was preferentially removed from the periacrosomal and flagellar regions. Nuclear, mitochondrial and acrosomal membranes remained intact and attached to the spermatozoa, but the cytoplasmic droplets were frequently disrupted and their internal membrane-bound vesicles were released. Lower pressures (less than 200 psi) were relatively ineffective in removing the periacrosomal plasma membrane, while an intermediate pressure (400 psi) removed this membrane from about 70% of the spermatozoa. No apparent selectivity for removal of the periacrosomal and flagellar plasma membrane was observed as a function of cavitation pressure. The cavitated cells were separated from the plasma membranes by differential followed by linear sucrose density gradient centrifugation. Two distinct membrane populations were resolved on sucrose gradients and were designated Band I and Band II. Band I contained only spherical vesicles which arose from the plasma membrane. Surface labeling of intact cells confirmed the plasma membrane as the origin of Band I. The membranes of higher density comprising Band II were heterogeneous consisting of both spherical and flattened vesicles. When purified cytoplasmic droplets were cavitated and centrifuged on the sucrose gradient only Band II was obtained. These studies indicate that nitrogen cavitation of bovine epididymal spermatozoa can result in significant contamination of plasma membrane fractions by cytoplasmic droplet membranes unless appropriate differential centrifugation is used to separate the membrane fractions.

Activation of mouse sperm T-type Ca 2+ channels by adhesion to the egg zona pellucida

“…[18][19][20]. While the Ca 2ϩ buffering and efflux mechanisms of sperm are not well understood (44,45), it is unlikely that such processes could permit T channels to produce sustained Ca i 2ϩ responses. Alternatively, a transient Ca 2ϩ influx through zona pellucida-activated T channels may activate Ca 2ϩ -dependent Ca i 2ϩ elevation.…”

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

“…To avoid contamination by the acrosomal membranes, a nitrogen pressure of 600-650 PSI has usually been used in the preparation of plasma membranes from mammalian spermatozoa (Noland et al 1983). We have not examined the ultrastructural changes induced in trout sperm by this pressure, but protein yields and fraction compositions indicate that detachment of membrane may not be as complete and the purity not as high at 600 compared to 900 PSI.…”

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

“…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;…”

“…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).…”

“…Further microscopic analyses of our final plasma membrane preparation failed to reveal double-walled vesicles, these vesicles being 125 distinctively indicative of cytoplasmic droplet contamination (Noland et al, 1983;Parks et al, 1987). Table 2 showed specific enzyme activities for plasma or other membranes of spermatozoa in the final plasma membrane preparation.…”