Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa
نویسندگان
چکیده
Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.
منابع مشابه
Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death.
Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid ana...
متن کاملP-142: Effects of A Synthetic Antioxidant (4-Hydroxy Tempo) Additive to The Semen Extender on the Ejaculated Spermatozoa Characteristics before and after Freezing in Water Buffaloes (Bubalus Bubalis)
Background: Semen cryopreservation is the most important section of artificial insemination programs; it allows preservation of semen fertility for a long time. Materials and Methods: The aim of the present study was to detect the effect of in vitro supplementation of 4-hydroxy Tempo on fresh and frozen spermatozoa quality of buffalo bulls. Five healthy buffalo bulls (5 ejaculates from each bul...
متن کاملThe effect of ouabain on membrane potential and flagellar wave in ejaculated bull spermatozoa.
Ejaculated bull spermatozoa were exposed to various concentrations of ouabain to ascertain the effect on membrane potential, intracellular concentrations of sodium and potassium and motility. Membrane potenital, measured by electrophysiological methods, decreased. Intracellular potassium decreased and intracellular sodium increased. Progressive motility decreased. In addition, the motility chan...
متن کاملO-12: Studies on Sequestration of PDC-109 Protein on Cryodamage and In Vitro Fertility of Crossbred Bull Spermatozoa
Background: Plasma membrane of the spermatozoa interacts with and is altered by abundant seminal PDC-109 present in their immediate milieu in vivo with damaging effect in a time- and concentration- dependent manner. Therefore we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial for cryopreservation of sperm cells. To this aim we evaluated the effect of s...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- The Journal of Cell Biology
دوره 102 شماره
صفحات -
تاریخ انتشار 1986