Mammalian spermatozoa must mature in the epididymis before they can fertilize an egg. It is known that modification of the protein composition of the sperm surface is an important part of the maturation process. In this paper, we present data on two related glycoproteins that can be extracted from mature but not immature spermatozoa. Cell surface radioiodination has shown that these proteins are on the sperm surface, and immunofluorescence microscopy, by use of monospecific antibodies to the proteins, has indicated that their localization is restricted to the periacrosomal region of the sperm head. We have also shown that in vitro, these proteins will bind to the identical region of immature sperm. Immunohistochemical localization of the proteins in the epididymis shows that they are produced and secreted by the cauda region. The significance of the addition of these proteins to the sperm surface in both maturation and fertilization is discussed.As they pass from the caput to the cauda of the epididymis, spermatozoa undergo a group of cellular modifications termed maturation. These modifications are dependent on the epididymal environment and must occur before the spermatozoa can fertilize an egg. Several general classes of changes have been described, which include the development of progressive forward motility, changes in the morphology of the acrosome, an increase in the disulfide cross-linking of several sperm organelles, and alterations in the properties of the plasma membrane (for reviews see references 3, 26, and 37).Maturation-dependent changes of the sperm plasma membrane are of particular interest because of this membrane's contact with the epididymal environment and the role it plays in fertilization (for review see reference 25). Examples of membrane changes are an increase in the net negative surface charge (9, 19), changes in lectin binding patterns (21, 24), and differences in the proteins and glycoproteins that are available to various nonpenetrating probes (23,33). Some of these surface changes result from interactions with proteins produced by the epididymis (7,16,20,34) that may either bind to the plasma membrane, or otherwise modify it in some way which may be important in making the spermatozoon capable of fertilizing an egg. There have been reports of several proteins found in epididymal fluid that are thought to have a functional role in sperm maturation These include forward motility protein (1), acrosome stabilizing factor (ASF) (11), and an epididymal glycoprotein thought to contribute to the development of the ability of spermatozoa to bind to the zona pellucida (27).To select specific proteins from the great number present in the epididymal environment, we have chosen to look for extrinsic membrane proteins that appear on the sperm surface during maturation. In this paper, we present data on the identification, characterization, and localization of two related polypeptides that fit these criteria. MATERIALS AND METHODSAnimals: Adult male Sprague-Dawley rats Were kept on a 12...
Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.
In exploring the evolution and adaptive significance of epididymal function, we have studied the male excurrent duct and spermatozoa of a monotreme mammal--the echidna. Sperm maturation in the echidna excurrent duct appears simpler than that in most therians examined. Furthermore, neither the duct nor the spermatozoa of the echidna display specific therian characteristics; they bear a much closer resemblance to those of non-passerine birds. The echidna spermatozoon is filiform, the sperm tail has no distinctive features, and the anterior seventh of the undulating nucleus is covered by a modest acrosome. Immediately behind this a restricted apposition between plasma membrane and nuclear envelope constitutes a post-acrosomal ring. This is evident also in some reptiles and marsupials, whereas in Eutheria such a membrane association appears as the posterior ring at the base of the sperm nucleus. Maturation of spermatozoa in the Wolffian duct of the echidna appears to be expressed only in a changing capacity for motility and in loss of the cytoplasmic droplet. Neither surface, structural nor acrosomal changes that characterize sperm maturation in therian mammals have been detected in maturing echidna spermatozoa. The echidna duct displays little of the regional complexity of the epithelium that typifies this duct in the Theria. Of five regions distinguishable on the basis of epithelial morphology, the first two appear to be counterparts of efferent ducts by virtue of a low columnar, partially ciliated epithelium. The tall pseudo-stratified Golgi-rich epithelium of the major portion of the duct broadly resembles that of the therian epididymis, but it displays only two structurally distinguishable regions, the more distal being the site of a dense luminal secretion. The foamy epithelial cells of the fifth and terminal region, characterized by a mass of supra-nuclear vesicles and rough ER, suggest a secretory function that may in some way contribute significantly to the ejaculate, for accessory glands are poorly developed in monotremes. The possibility is considered that the relative complexity of epididymal function and sperm structure in therian mammals could have been determined by evolutionary change in the milieu of the female tract, and/or in the character of the egg vestments that the fertilizing spermatozoon must penetrate.
Summary. The extent to which specific IgG can reach the lumen of the rabbit cauda epididymidis was investigated by comparison of the concentration in serum and fluid of the cauda epididymidis of a specific IgG raised against dinitrophenylated bovine gamma globulin (DNP\p=n-\BGG).This specific IgG reached the epididymal lumen although in much lower concentration than the levels in serum. The IgG was measured by a specific sensitive radioimmunoassay and in 13 normal males there was a mean molar ratio of 4\m=.\0\m=x\10\m=-\3 (range: 2\m=.\5\p=n-\11\m=.\0\m=x\ 10\ m=-\ 3) between the epididymal lumen and blood; the mean ratio between cerebrospinal fluid and blood was 1\m=.\7\m=x\10\m=-\3 (4 males). Calculations, based on the absolute concentration of anti-DNP IgG in epididymal fluid in relation to total number of spermatozoa and estimated fluid volume in the cauda epididymidis, indicated approximately 40 000 molecules anti-DNP\p=n-\BGG IgG per spermatozoon. This ratio was not affected 6 days after castration or 3\p=n-\4months after vasectomy, but it was about 10 times higher than that of the controls in the cryptic epididymis subjected for 6 days to body temperature.
RIA of specific IgG in serum and epididymal fluids following systemic immunization of male rabbits with DNP‐BGG showed a mean IgG epididymal fluid/serum molar ratio of 4.0 times 10‐3, and in epididymal fluid a mean concentration of 3.8 times 10‐7 moles/1 or the equivalent in the caudal lumen of 40 000 anti‐DNP IgG molecules/spermatozoon there. This concentration did not change after the distension of the duct seen in the first months after vasectomy nor very markedly in the first 6 days after androgen withdrawal brought by castration. But the mean luminal level of specific IgG did rise some ten‐fold in the cauda subjected to body temperature for 6 days. Consideration is given to the prospect that such levels of antibody could compromise fertility if this were directed against some component that is essential for sperm function.
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