Maturation antigen of the mouse sperm flagellumII. Origin from holocrine cells of the distal caput epididymisкод для вставкиСкачать
THE ANATOMICAL RECORD 217:146-152 (1987) Maturation Antigen of the Mouse Sperm Flagellum: II. Origin From Holocrine Cells of the Distal Caput Epididymis F.A. FEUCHTER, M.F. GREEN, AND A.J. TABET Department of Anatomy, School of Medicine, University of New Mexico, Albuquerque, NM 87131 ABSTRACT During epididymal transit, the mouse sperm flagellum acquires a surface glycoprotein (SMA4)from epididymal fluid that functions as a sperm antiagglutinin. To determine the origin of this molecule, testes and epididymides of male mice were sectioned for light microscopy and stained with wheat germ agglutinin (WGA)-peroxidase, a probe that has been used previously to examine the biology of SMA4. WGA reactivity was localized to the cytoplasm in a small population of cells in the distal caput epididymis. Testis cells and principle cells of the caput were nonreactive with WGA, while stereocilia were stained on principle cells in the corpus and cauda. The WGA-positive cells in the distal caput were identified as holocrine cells on the basis of morphology, distribution, and PAS+ reaction. At high magnification, intense WGA reactivity was due to the presence of numerous apical granules in the cytoplasm. The location of the cells in distal caput coincided exactly with the region of tubule in which sperm first acquired SMA4 on their flagellae. These data suggest that holocrine cells near the junction of caput and corpus epididymis are the source of the sperm antiagglutinin SMA4. The mammalian epididymis produces a complex secretory product that bathes the sperm during passage through the epididymis. In this environment sperm undergo the final stages of development leading to fertility (Bedford, 1975; Orgebin-Crist et al., 1975). As shown by autoradiography, principle cells of the epithelium are the source of proteins that interact with spermatozoa during this maturational period (Kopecny, 1971; Kanka and Kopecny, 1977; Kopecny et al., 19841, although the functions and identities of the molecules are largely unknown. Additionally, the method of release of secretory products from the principle cells does not appear to be similar to that of typical secretory cells, since they do not possess observable secretory granules (Flickinger, 1979,1981,1985). Histochemical and electronmicroscopic evidence indicates that the epididymal epithelium is not homogeneous but varies in appearance throughout several regions of the tube, and cells other than principle cells are present (Martan, 1969; Hoffer et al., 1972; Hamilton, 1975). Among these subpopulations of cells, it has been proposed that “holocrine” cells may also have a secretory function (Martan and Risley, 1963; Martan and Allen, 1964; Martan et al., 19641, but no specific epididymal products have been traced to these cells. These cells are present in specific regions of the epididymis in mouse (Martan and Allen, 1964),rat (Martan and Risley, 1963; Hoffer et al., 1972; Hamilton, 1975; Brown and Montesano, 1980; Sun and Flickinger, 1980), and human (Martan et al., 1964). Morphological evidence suggests that they undergo a cyclic secretion process similar to that of holocrine cells elsewhere (Martan and Risley, 1963). 0 1987 ALAN R. LISS, INC. In previous studies using monoclonal antibodies, we have shown that a 54-kd epididymal secretory product is acquired by mouse sperm flagellae as they transit the epididymis (Feuchter et al., 1981, 1986; Vernon et al., 1982). This molecule, termed SMA4, is a specific receptor for wheat germ agglutinin (WGA), and its biology can be studied by using WGA as a probe. In a separate report we examine the functional significance of SMA4 as a sperm antiagglutinin (Feuchter et al., 1986). The present study provides evidence that this sperm-binding molecule, which covers the entire sperm flagellum, originates within holocrine cells of the distal caput epididymis in the mouse. MATERIALS AND METHODS Animals ICR retired breeder male mice (Harlan Labs) used for these studies were housed in a controlled environment on a 12 L:12 D (12 h r light, 12 h r dark) cycle and provided with Purina Lab Chow and water ad libitum. Tissue Preparation Animals were killed by cervical dislocation, and entire reproductive tracts (testis, caput, corpus, and cauda epididymides, as well as ductus deferens) were excised. After removal of excess fat, tissue was rinsed in modified Tyrode’s solution (MTS) (2.7 mM KCI, 117 mM NaC1,0.5 mM MgClz x 6H20, 0.36 mM NaH2P04 x lHzO, 1.7 mM CaC12 x 2Hz0, 12 mM NaHC03, 0.25 mM Na Pyruvate, 5.5 mM glucose, and 3.3 mM HEPES, pH 7.4, Received August 5,1986; accepted September 24, 1986. SPERM-BINDING GLYCOPROTEIN OF HOLOCRINE CELLS room temperature) and fixed in 3% paraformaldehyde for 24 h r (4°C). Samples were dehydrated in a series of ethanols ranging from 70 to loo%, cleared in toluene, and embedded in paraffin. Eight-micron-thick microtome sections of either whole epididymis or various portions of the reproductive tract were mounted with albumin on acid-alcohol-cleaned glass slides. Frozen sections were prepared from udixed tissue that had been immersed in liquid nitrogen-cooled isopentane and mounted in Cryostat embedding medium. After airdrying, frozen sections were blocked and stained as described below. Lectin-peroxidase Staining of Tissues Staining was carried out a t room temperature by removing paraffin from slides with three changes of xylene and rehydrating in a n ethanol series of 100 to 70%, followed by three rinses in phosphate-buffered saline (PBS) (.E M NaC1, 2.5 mM NaH2P04 x 1H20, and 7.5 mM Na2HP04 x 7H20, pH 7.4). Nonspecific protein binding sites were blocked by incubation in 1% bovine serum albumin (BSA) (Calbiochem) for 30 min. Slides were then rinsed in PBS twice, followed by incubation in 5 pg/ml biotinylated WGA (Vector) for 60 min to 24 hr. After two rinses in PBS, slides were incubated in a mixture of avidin and biotinylated horseradish peroxidase (ABC Kit, V & P Scientific) for 1 hr. After three rinses in PBS of 10 min each, slides were immersed in substrate (.3 mg/ml diaminobenzidene [Sigma] and .005% H202) for 10 min and rinsed in double-distilled water for 10 min. WGA binding sites were localized by a brown reaction product. In order to visualize the tissue that was nonreactive with WGA, slides were immersed in toluidine blue stain (30%ethanol and .05% toluidine blue) for 5 min, followed by dehydration with 50-100% ethanol and two changes of xylene. Slides were air-dried, and coverslips were mounted with Permount for observation by bright-field microscopy. Specificity of WGA binding was confirmed by incubation of slides in .1 M N-acetyl-neuraminic acid, either simultaneously or subsequent to staining with WGA-biotin. Both procedures resulted in no staining of the tissues by WGA. RESULTS The distribution of SMA4 within the male reproductive tract was examined by WGA-peroxidase staining of Fig. 1. Paraffin section of mouse testis, stained with WGA-peroxidase and counterstained with toluidine blue. Sperm were nonreactive with WGA, and no WGA-positive cells were seen within the seminiferous tubules. Therefore, tissue in this micrograph shows only toluidine blue staining (dark circles at periphery of tubule are nuclei of spermatogonial cells). x200. Fig. 2. Paraffin section of mouse caput epididymis, middle region, stained with WGA-peroxidase and toluidine blue. Loosely packed sperm in lumen (SP) were nonreactive with WGA and showed only light blue staining. The columnar epithelium, consisting of principle cells, also showed no WGA staining in either the surface stereocilia (arrows), cytoplasm, or nuclei, which exhibited blue stain of varying density. x225. Fig. 3. Paraffin section of mouse corpus epididymis, stained with WGA-peroxidase and toluidine blue. Densely packed sperm in the lumen (SP) exhibited strong WGA reactivity on their flagellae, and stereocilia (arrows) were heavily stained with brown reaction product as well. Cytoplasm of epithelia1 cells appears dark blue in this micrograph owing to the thickness of section used to illustrate details of the stereocilia. Nuclei were stained with toluidine blue. ~ 2 2 5 . 147 TESTIS D.EFFERENTES PROXIMAL CAPUT 1 DISTAL CAPUT ORPUS CAUDA &VAS DEFERENS Fig. 4. Sketch of mouse testis and epididymis, illustrating region of distal caput (shaded area) where epithelial cells were localized, which stained strongly with WGA. Fig. 5. Paraffin section of distal caput region indicated in Figure 4, stained with WGA-peroxidase and toluidine blue. A subpopulation of epithelial cells in this region exhibited heavy cytoplasmic staining with WGA (arrows). There were about three to eight positive cells per tubular profile, while the principle cells in this region were negative and appear blue in the micrograph. Sperm throughout most of the caput were nonreactive with WGA, but sperm in the distal parts showed WGA reactivity. x 120. Fig. 6. Higher magnification of cells shown in Figure 5. The WGApositive cells are similar to those described by others as holocrine or apical cells, having a narrow or constricted base, a n expanded cell apex toward the lumen, and an apically placed nucleus (large arrow, upper right). In contrast, principle cells had a rectangular shape and basally placed nuclei (small arrowheads). ~ 2 4 0 . Fig. 7. Enlargement of the area inside rectangle in Figure 6. In this micrograph, two darkly stained holocrine cells are flanked by columnar principle cells (with lightly stained nuclei). The apical nucleus (NU) and constricted base of these cells were readily apparent, as well as the granular nature of WGA-positive material within the cells (SG). Granules averaged about 1 pm in diameter. ~ 6 0 0 . SPERM-BINDING GLYCOPROTEIN OF HOLOCRINE CELLS paraffin sections. To rule out the possibility that fixation and paraffin embedding produced staining artifacts, paraffin sections were compared to unfxed frozen sections stained in a similar manner. No differences in staining patterns were observed between the two methods. For ease of handling, paraffin sections were used in most of the studies. Regions of tissue with affinity for WGA were characterized by a dark brown reaction product, while areas that were nonreactive with WGA stained blue, as indicated in the figure legends. In sections of testis, no WGA-reactive cells were found within the seminiferous tubules (Fig. 1).Sperm flagellae were not reactive with WGA, nor were any cells in the region of the rete testis (not shown). Sperm flagellae acquire SMA4 during epididymal transit, and this could be demonstrated by comparing sections of caput, corpus, and cauda, or by using sagittal sections of whole epididymis. Figure 2 illustrates a representative section through caput epididymis stained with WGA-peroxidase and toluidine blue. Sperm in the lumen (SP) were loosely packed and were not reactive with WGA. Principle cells were likewise unreactive in both their cytoplasm and stereocilia (arrows). Throughout the epididymis, nuclei stained with toluidine blue; in thicker sections they appeared dark (Figs. 2,3), while in thinner sections used for clarity at high magnification they appeared lighter in color (Figs. 6, 7). In contrast, sections through corpus epididymis were highly reactive with WGA (Fig. 3). Sperm from this region (SP) were densely packed and exhibited heavily stained flagellae. Stereocilia of the principle cells were also WGA positive (arrows). Cytoplasmic staining of the principle cells was not detected. The zone between unstained and stained sperm was sharply demarcated near the caputlcorpus junction; a zone of transition or zone of intermediate staining was not seen between caput and corpus. In the region where sperm first showed WGA staining, there were many more sperm per luminal profile, and sperm were more densely packed than in proximal parts of the caput. Although the cytoplasm of most epithelial cells in the epididymis was unreactive with WGA, a small population of WGA-positive cells was found in the distal segment of caput epididymis (Fig. 4). The subpopulation of WGA-positive epithelial cells was confined solely to this region and was not found elsewhere in the epididymal ducts. The cells were sparsely distributed within the epithelium, showing about three to eight cells per lumina1 profile (arrows, Fig. 5). The cells were found in the same region where sperm first exhibited staining of the flagellae, and where sperm first became noticeably concentrated within the lumen. At higher magnification (Fig. 6)the WGA-positive cells exhibited a morphology that was similar to holocrine cells described in the literature. They had a constricted base, a n expanded apex toward the lumen, and apically placed nuclei (large arrow, Fig. 6). In contrast, the principle cells in the rest of the epithelium were nonreactive with WGA and had basally placed nuclei that were lightly stained with toluidine blue (small arrowheads, Fig. 6). Under oil immersion optics (Fig. 7) morphology of the WGA-reactive material could be observed. Many small, granular deposits were present throughout these cells, with the majority of WGA-positive granules at the cell apex. Granules varied in size, with a n average of about 1pm diameter. 149 DISCUSSION The epididymis produces many kinds of secretory products that interact with spermatozoa during their final maturation processes (Bedford, 1975; Hamilton, 1975). Secretory glycoproteins of the epididymis have been examined in several mammalian species, and some have been shown by indirect immunofluorescence to originate from principle cells of the epididymal epithelium. For instance, bovine forward motility protein (Acott and Hoskins, 19811, acrosome-stabilizing factor in the rabbit (Thomas et al., 1984), and SSEA-1 antigen of mouse sperm (Fox et al., 1982) appear to be produced by principle cells. A protease inhibitor on mouse sperm (Aarons et al., 1984; Poirier and Nicholson, 1984) and three antigens on human sperm (Yan et al., 1984; Tezon et al., 1985a,b)have also been traced to origins in the principle cells. The rat has been most extensively studied, and several glycoproteins that are added to sperm during epididymal transit have been discovered. These include ol-lactalbumin-like proteins of low molecular weight (Jones and Brown, 1982; Byers et al., 1984; Klinefelter and Hamilton, 1985), a 37.5-kd protein (Faye et al., 1980), specific epididymal proteins, or “SEP,” (Garberi et al., 1979; Kohane et al., 1980), a 32-kd acrosomal protein (Jones et al., 1981; Wong and Tsang, 1982; Zeheb and Orr, 1984), “HIS’ proteins of 100 and 66 kd (Rifiin and Olson, 19851, a 50-kd molecule (Dravland and Joshi, 19811, acidic epididymal glycoprotein, or “AEG,” (Lea et al., 1976; Pholpramool et al., 19831, sulfated “DAG’ protein (Sylvester et al., 1984), and several others (Brooks and Tiver, 1984). It is probable that some of these apparently different proteins being investigated in the rat are similar, if not identical; all have been localized to principle cells in various portions of the epididymis by immunocytochemical techniques. The functional significance of most of these molecules is unclear at the present time. There are other cell types in the epithelium of epididymis that present a n appearance suggestive of secretory activity and that may be involved in sperm maturation. The holocrine or apical cells have been considered secretory for over 20 years based on morphological evidence (Martan and Risley, 1963; Martan and Allen, 1964; Martan et al., 1964),yet none of their secretory products have been identified. They have been described in mouse (Martan and Allen, 19641, rat (Martan and Risley, 1963; Sun and Flickinger, 1980), hamster (Flickinger et al., 19781, and human (Martan et al., 1964). They are characterized by swollen cell apices with nuclei placed above the basal row of principle cell nuclei. There generally are few holocrine cells per luminal profile (about three to eight). The cell types known collectively as “holocrine,” “apical,” “basal,” and “clear” cells described by various authors may represent different stages in the life cycle of holocrine secretory cells, and it has been suggested that they may be a variation of principle cells based on electronmicroscopic evidence (Reid and Cleland, 1957; Sun and Flickinger, 1980). The cells can be selectively stained using PAS or alcian blue techniques, indicating a high glycoprotein content (Martan and Risley, 1963; Martan and Allen, 1964).They are rich in mitochondria (Brown and Montesano, 1980) as well as carbonic anhydrase (Cohen et al., 1976; AbouHaila and Fain-Maurel, 1985). Histochemical studies show dehydrogenase, acid phosphatase, and Ca2+ 150 F.A. FEUCHTER, M.F. GREEN, AND A.J. TABET ATPase activities higher than surrounding principle cells (Abou-Haila and Fain-Maurel, 1985). In the present study, we demonstrate the localization of a sperm-binding epididymal secretion (SMA4) in holocrine cells of the distal caput epididymis. SMA4 is a glycoprotein that coats the sperm flagellae during epididymal transit (Feuchter et al., 1981; Vernon et al., 1982) and that is a specific receptor for wheat germ agglutinin (F'euchter e t al., 1986). WGA has been useful in examining the biology of this molecule, since it appears to be the only receptor for WGA on sperm surfaces. On SDS-PAGE blots it stains a single, well defined 54kd band of glycoprotein, and the staining of blots, sperm flagellae, and holocrine cells can be prevented by incubation with 0.1 M n-acetylneuraminic acid (Feuchter et al., 1986). WGA-reactive holocrine cells were found in the distal caput, in a region initially identified using SMA4 antibodies (Vernon et al., 1982). The region is similar to regions IV and V of the mouse epididymis described by Abou-Haila (Abou-Haila and Fain-Maurel, 1984) and region B of Pavlok (Pavlok, 1974). Several secretory proteins have been traced to caput epididymis (Lea et al., 1976; Sylvester et al., 19841, but none have been traced to this particular region. The location of these cells coincides exactly with the region of the tubule where sperm first exhibit staining of the flagellum, a n observation that supports the hypothesis that these cells secrete the tail-coating glycoprotein, SMA4. In addition, sperm first become condensed and tightly packed within the lumen immediately distal to the region of WGA-positive cells, a n observation that may be of functional significance. The packing of sperm in the lumen of corpus and cauda results from absorption of much of the fluid by principle cells of the latter half of the caput (Flickinger et al., 1978). In a separate study we have shown that one of the functions of SMAC is to prevent tail-to-tail agglutination of sperm (Feuchter et al., 1986). Caput sperm, when diluted into saline, show rapid tail-to-tail agglutination, whereas corpus and cauda sperm, which possess SMA4 on their flagellae, do not. When caput sperm are incubated with purified SMA4, they become coated with it and do not agglutinate. The addition of this molecule to sperm flagellae during epididymal transit prevents their agglutination when packed together in the corpus and cauda. Addition to the surface must necessarily precede condensation of sperm into a tight mass, in order that they may untangle and become free-swimming at ejaculation. Therefore, the location of these cells in the area immediately proximal to where sperm first stain for SMA4 and where they first become tightly packed is appropriate for their function. The WGA-reactive material in the cell cytoplasm appears as large granules, whose size (about 1 pm) and distribution near the cell apex are reminiscent of typical secretory granules. Electron microscopic studies have determined that cells with the characteristics of holocrine cells contain numerous large vesicles with lightly flocculent material near the cell apex (Flickinger et al., 1978; Sun and Flickinger, 1980). Since their size and location correspond to the WGA-positive granules pointed out in this study, it is likely that these vesicles contain the WGA-reactive material. Future electron microscopic studies should confirm this. Principle cells of the epithelium did not stain with WGA. Although principle cells have been confirmed to be secretory by autoradiography (Flickinger, 1979,1981), the mode of secretion from these cells is unknown, since they have no recognizable secretory granules. It is possible that secretion of certain proteins occurs from numerous small coated vesicles or associated Golgi vesicles of principle cells (Flickinger, 1985). Secretion products that have been localized in principle cells of mouse and other species are glycoprotein in nature, although they do not appear to be as heavily glycosylated as SMA4 (Lea et al., 1976; Garberi et al., 1979; Jones et al., 1981; Wong and Tsang, 1982; Zeheb and Orr, 1984). Thus, their mechanisms of secretion may be different. The principle cells distal to the region where SMA4 is secreted by holocrine cells were stained on their stereocilia. Stereocilia are abundant on cells throughout the epididymis and probably function in the absorption of material from the epididymal lumen (Hamilton, 1975; Turner, 1979,1984). The observation that sterocilia were stained only distal to the point where sperm first acquire WGA reactivity suggests that they may be coated with SMA4 to prevent adherence or entanglement of sperm during passage. They also may be involved in the recovery of excess SMA4 from epididymal fluid. In our earlier studies, SMA4 was also localized to large apical vesicles in a few of the principle cells (Vernon et al., 1982).This observation can be reconciled with the present data by postulating that the principle cells are involved in absorption of excess SMA4. Secretion from principle cells does not involve typical large secretory granules but rather involves a small vesicle system of coated vesicles and associated Golgi elements (Flickinger, 1985). Furthermore, there is evidence that secretory antigens from the proximal epididymis are absorbed by principle cells distal to the secretion site (Faye et al., 1980; Lea et al., 1976). Biochemical studies on SMA4 confirm that it is a heavily glycosylated molecule (Feuchter et al., 1986). Prime receptors for WGA on glycoproteins are carbohydrate side chains with terminal or internally situated N-acetyl-D-glucosamine residues; more recently it has been shown that WGA also reacts specifically with Nacetyl-neuraminic acid (sialic acid) and has a lower aFinity for N-acetyl-D-galactosamine (Goldstein, 1980). We have reported that the enzyme N-acetyl-D-glucosaminidase has no effect on the staining of sperm flagellae by WGA, whereas N-acetyl-neuraminidase abolishes all WGA reactivity (Feuchter et al., 1986). Therefore, terminal carbohydrate residues of SMA4 appear to be rich in sialic acid. Several species of spermatozoa have been reported to increase in sialic acid content during epididymal transit (Bedford, 1963; Bedford et al., 1973; Holt, 1980), although the functional consequences of sialic acid addition to the sperm are unknown. Addition of the antiagglutinin SMA4 to mouse sperm is probably the mechanism by which they increase in sialic acid content. Since the addition of sialic acid during epididymal maturation appears to be a common phenomenon among species, it may be a general mechanism by which sperm agglutination is prevented. On other cell types in which agglutination is undesirable, such as erythrocytes, high surface sialic acid content is thought to play a role in preventing agglutination. SPERM-BINDINGGLYCOPROTEIN OF HOLOCRINE CELLS Holocrine cells of the epididymis exhibit similarities in morphology, distribution, and secretory product to cells in other epithelia, such as goblet cells of the intestine. Both have a distinctive shape, a sparse arrangement within the epithelium, large secretory vesicles, and a product rich in carbohydrate. The lubricating and protective function of intestinal mucus can be compared to the antiagglutination and surfactant functions of SMA4. These cells may represent a general subset present in many epithelia that perform similar functions. The discovery of a sperm-binding epididymal secretion that originates from holocrine cells in the distal caput raises questions about the functions of holocrine cells elsewhere in the epididymis and about their relationship to sperm maturation. The observation that an epididymal secretion functions as an antiagglutinin suggests that epididymal dysfunction may be one cause of unexplained sperm agglutination, particularly in humans in which no antisperm antibodies are present. LITERATURE CITED Aarons, D., J.L. Speake, and G.R. Poirier (1984) Evidence for a protein* ase inhibitor binding component associated with murine spermatozoa. Biol. Reprod., 31:811-817. Abou-Haila, A., and M.A. Fain-Maurel (1984) Regional differences in the proximal part of mouse epididymis: Morphological and histochemical characterization. Anat. Rec., 209:197-208. Abou-Haila, A., and M.A. Fain-Maurel(1985)Postnatal differentiation of the enzymatic activity of the mouse epididymis. Int. J. Androl., 8:441-458. Acott, T.S., and D.D. Hoskins (1981) Bovine sperm forward motility protein: Binding to epididymal spermatozoa. Biol. Reprod., 24:234240. Bedford, J.M. (1963) Changes of electrophoretic properties of rabbit spermatozoa during passage through the epididymis. Nature, 200:1178-1180. Bedford, J.M. (1975) Maturation, transport, and,fate of spermatozoa in the epididymis. Handbook Physiol., 5303-317. Bedford, J.M., H. Calvin, and G.W. Cooper (1973) The maturation of spermatozoa in the human epididymis. J. Reprod. Fertil. [Suppl.], 18:199-213. Brooks, D.E., and K. Tiver (1984) Analysis of surface proteins of rat spermatozoa during epididymal transit and identification of antigens common to spermatozoa, rete testis fluid and cauda epididymal plasma. J . Reprod. Fertil., 71:249-257. Brown, D., and R. Montesano (1980) Membrane specialization in the rat epididymis. I. Rod-shaped intramembrane particles in the apical (mitochondria-rich)cell. J. Cell Sci., 45:187-198. Byers, S.W., P.K. Qasba, H.L. Paulson, and M. Dym (1984)Immunocytochemical localization of alpha-lactalbumin in the male reproductive tract. Biol. Reprod., 30:171-178. Cohen, J.P., A.P. Hoffer, and S.Rosen (1976) Carbonic anhydrase localization in the epididymis and testis of the rat: Histochemical and biochemical analysis. Biol. Reprod., 14:339-346. Dravland, E., and M.S. Joshi (1981) Sperm coating antigens secreted by the epididymis and seminal vesicle of the rat. Biol. Reprod., 25:649-658. Faye, J.C., L. Duget, M. Mazzuca, and F. Bayard (1980) Purification, radioimmunoassay, and immunohistochemical localization of a glycoprotein produced by the rat epididymis. Biol. Reprod., 23:423432. Feuchter, F.A., R.B. Vernon, and E.M. Eddy (1981) Analysis of the sperm surface with monoclonal antibodies: Topographically restricted antigens appearing in the epididymis. Biol. Reprod., 24:1099-1110. Feuchter, F.A., A.J. Tabet, and M.F. Green (1986) Maturation antigen of the mouse sperm flagellum, I. Analysis of its secretion, association with sperm, and function. Submitted to Am. J. Anat. Flickinger, C.J. (1979) Synthesis, transport and secretion of protein in the initial segment of the mouse epididymis as studied by electron microscope autoradiography. Biol. Reprod., 20: 1015-1030. Flickinger, C.J. (1981) Regional differences in synthesis, intracellular transport, and secretion of protein in the mouse epididymis. Biol. Reprod., 25:871-883. Flickinger, C.J. (1985)Radioautographic analysis of the secretory path- 151 way for glycoproteins in principle cells of the mouse epididymis exposed to [3H]fucose.Biol. Reprod., 32:377-389. Flickinger, C.J., S.S. Howards, and H.F. English (1978)Ultrastructural differences in efferent ducts and several regions of the epididymis of the hamster. Am. J. Anat., 152557-586. Fox, N., I. Damjanov, B.B. Knowles, and D. Salter (1982) Teratocarcinoma antigen is secreted by epididymal cells and coupled to maturing sperm. Exp. Cell Res., 137:485-488. Garberi, J.C., A.C. Kohane, M.S. Cameo, and J.A. Blaquier (1979) Isolation and characterization of specific rat epididymal proteins. Mol. Cell. Endocrinol., 13:73-82. Goldstein, I.J. (1980) N-acetyl-D-glucosamine-binding lectins. Qualityline (Miles Labs), Winter, 1980,4:1-7. Hamilton, D.W. (1975) Structure and function of the epithelium lining the ductuli efferentes, ductus epididymidis, and ductus deferens in the rat. Handbook Physiol., 5259-301. Hoffer, A.P., D.W. Hamilton, and D.W. Fawcett (1972)The ultrastructure of the principle cells and intraepithelial leucocytes in the initial segment of the rat epididymis. Anat. Rec., 175:169-202. Halt, W.V. (1980) Surface-bound sialic acid on ram and bull spermatozoa: Deposition during epididymal transit and stability during washing. Biol. Reprod., 23:847-857. Jones, R., and C.R. Brown (1982) Association of epididymal secretory proteins showing a-lactalbumin-like activity with the plasmamembrane of rat spermatozoa. Biochem. J., 206;161-164. Jones, R., C. Pholpramool, B.P. Setchell, and C.R. Brown (1981) Labelling of membrane glycoproteins on rat spermatozoa collected from different regions of the epididymis. Biochem. J., 200:457-460. Kanka, J., and V. Kopecny (1977) An autoradiographic study of macromolecular synthesis in the epithelium of the ductus epididymis in the mouse. I. DNA, RNA and protein. Biol. Reprod., 16421-427. Klinefelter, G.R., and D.W. Hamilton (1985)Synthesis and secretion of proteins by perfused caput epididymal tubules, and association of secreted proteins with spermatozoa. Biol. Reprod., 33:1017-1027. Kohane, A.C., M.S. Cameo, L. Pinero, J.C. Garberi, and J.A. Blaquier (1980) Distribution and site of production of specific proteins in the rat epididymis. Biol. Reprod., 23:181-187. Kopecny, V. (1971) Epididymal lumina1 contents labelling after 14C-or 3H-lysine administration in the mouse. Acta Histochem. (Jena), 40:116-122. Kopecny, V., J.E. Flechon, and J. Pivko (1984) Binding of secreted proteins to spermatozoa in the mammalian epididymis: A finestructure autoradiographic study. Anat. Rec., 208:197-206. Lea, O.A., P. Petrusz, and F.S. French (1976) Purification and localization of acidic epididymal glycoprotein (AEG): A sperm coating protein secreted by the rat epididymis. Int. J. Androl., [Supp1.]2:592-607. Martan, J. (1969) Epididymal histochemistry and physiology. Biol. Reprod., 1:134-154. Martan, J., and P.L. Risley (1963) Holocrine secretory cells of the rat epididymis. Anat. Rec.; 146:173-183. Martan, J., and J.M. Allen (1964) Morphological and cytochemical properties of the holocrine cells in the epididymis of the mouse. J. Histochem. Cytochem., 12:628-639. Martan, J., P.L. Risley, and Z. Hruban (1964) Holocrine cells of the human epididymis. Fertil. Steril., 15:180-187. Orgebin-Crist, M.C., B.J. Danzo, and J. Davies (1975) Endocrine control of the development and maintenance of sperm fertilizing ability in the epididymis. Handbook Physiol., 5319-338. Pavlok, A. (1974) Development of the penetration activity of mouse epididymal spermatozoa in uiuo and in uitro. J. Reprod. Fertil., 36:203-205. Pholpramool, C., O.A. Lea, P.V. Burrow, H.M. Dott, and B.P. Setchell (1983)The effects of acidic epididymal glycoprotein (AEG)and some other proteins on the motility of rat epididymal spermatozoa. Int. J. Androl., 6:240-248. Poirier, G.R., and N. Nicholson (1984) Distribution of a proteinase inhibitor of epididymal origin in the tissues and secretions of the male reproductive tract of mice. J. Exp. Zool., 230:465-471. Reid, B.L., and K.W. Cleland (1957) The structure and function of the epididymis. I. The histology of the rat epididymis. Aust. J. Zool., 5:223-246. Rifiin, J.M., and G.E. Olson (1985) Characterization of maturationdependent extrinsic proteins of the rat sperm surface. J. Cell Biol., 100:1582-1591. Sun, E.L., and C.J. Flickinger (1980) Morphological characteristics of cells with apical nuclei in the initial segment of the adult rat epididymis. Anat. Rec., 196:285-293. Sylvester, S.R., M.K. Skinner, and M.D. Griswold (1984) A sulfated glycoprotein synthesized by Sertoli cells and by epididymal cells is a component of the sperm membrane. Biol. Reprod., 31:1087-1101. 152 F.A. FEUCHTER, M.F. GREEN, AND A.J. TABET Tezon, J.G., E. Ramella, M.S. Cameo, M.H. Vazquez, and J.A. Blaquier (1985a) Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa. Biol. Reprod., 32591-597. Tezon, J.G., M.H. Vazquez, L. Pineiro, M.A. De Larminat, and J.A. Blaquier (1985b)Identification of androgen-induced proteins in human epididymis. Biol. Reprod., 32584-590. Thomas, T.S., A.B. Reynolds, and G. Oliphant (1984)Evaluation of the site of synthesis or rabbit sperm acrosome stabilizing factor using immunocytochemical and metabolic labelling techniques. Biol. Reprod., 30:693-705. Turner, T.T. (1979) On the epididymis and its function. Invest. Urol., 16:311-319. Turner, T.T. (1984) Resorption versus secretion in the rat epididymis. J. Reprod. Fertil., 72:509-514. Vernon, R.B., C.H. Muller, J.C. Hem, F.A. Feuchter, and E.M. Eddy (1982) Epididymal secretion of a mouse sperm surface component recognized by a monoclonal antibody. Biol. Reprod., 26:523-535. Wong, P.Y., and A.Y. Tsang (1982) Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa. Biol. Reprod., 27;1239-1246. Yan, Y.C., S.M. Mitsudo, L.F. Wang, and S.S. Koide (1984) Immunolocalization of a sperm membrane protein in human male reproductive organs. Fertil. Steril., 42:614-617. Zeheb, R., and G.A. Orr (1984) Characterization of a maturation-associated glycoprotein on the plasma membrane of rat caudal epididymal sperm. J. Biol. Chem., 2592339-848.