Isolation and Characterization of a Macromolecular Complex Associated with the Outer Acrosomal Membrane of Bovine Spermatozoa1

Abstract

The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.

Actin, α‐actinin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa

“…The present ultrastructural results confirm earlier findings showing the presence of extraction-resistant acrosomal lamina associated with the luminal aspect of outer acrosomal membrane (Russell et al, 1979;Olson and Winfrey, 1985a;Olson et al, 1985Olson et al, , 1987. From our results it appears that extraction of sperma- Hyamine, not only dissolves the majority of the acrosoma1 contents, but also modifies the continuous structure of the acrosomal lamina into a folded pattern.…”

An Antigenically Related Polypeptide Family is a Major Structural Constituent of a Stable Acrosomal Matrix Assembly in Bovine Spermatozoa1

“…Specific components of the matrix likely regulate the observed differential release of hydrolases during the acrosome reaction [13,27,[33][34][35][36]. We have previously isolated and characterized a localized, stable acrosomal matrix assembly from the bovine acrosome termed the outer acrosomal membrane-associated matrix complex (OMC) [31,37]. This stable matrix assembly exhibits specific binding activity for proacrosin/acrosin and is restricted to the apical and principal segments of the acrosome where it is associated with the luminal surface of the outer acrosomal membrane [31,37].…”

Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa

“…Immunofluorescence techniques have demon¬ strated that calmodulin is present in the head region of mammalian spermatozoa, preferentially around the acrosome (Jones et al, 1980), and closely localized to the region containing phospholi¬ pase A2 (Weinman et al, 1986), an enzyme which has been reported to be modulated by calmodulin (Moskowitz et al, 1983). Moreover, several calmodulin-binding proteins have been identified in the acrosomal region of hamster (Moore & Dedman, 1984) and cattle (Olson et al, 1985) spermatozoa.…”