Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium

Journal o f Reproductive Immunology, 16 (1989) 269--284

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Elsevier Scientific Publishers Ireland Ltd.

JRI 00626

Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium

F r a n c e s c o G a l d i e r o a, M a r i a A n t o n i e t t a T u f a n o % L u i s a De M a r t i n o a, C i r o C a p a s s o a, R a f f a e l e Porta% G i a n p i e t r o R a v a g n a n ~, G i a n f r a n c o P e l u s o ~ a n d Salvatore Metafora ~ ~lnstitute o f Microbiolog)', 1st .~ledical School, University o f Naples, rDepartment o f Biochemistry and Biophysics, University o f Naples, CNR Institute o f E.~perintental Medicine, I'iale Carlo Marx 15, Rome and ~CNR Institute o f Protein Biochemistry and En~vmolog.v, |.Ta Toiano 6, 80072 Arco Felice, .Vaples [Italy) (Accepted for publication 31 August 1989)

Summary The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed. Key words: macrophages; phagocytosis," seminal vesicles," protein S V-IV,"

transglutaminase.

Correspondence to: Dr. S. Metafora. 0165-0378/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Introduction

In the seminal plasma of humans and rodents there are several proteins which seem to play an important role in the survival of spermatozoa in both male and female genital tracts (Alexander and Anderson, 1987; James and Hargreave, 1984; Mann and Lutwak-Mann, 1981; Metafora et al., 1987c). SV-IV, an androgen-dependent protein with a MW of 9758, is synthesized and secreted in large amounts by the rat seminal vesicle (SV) epithelium (Mansson et al., 1981; McDonald et al., 1983; Ostrowski et al., 1979). This protein is named SV-IV (seminal vesicle protein no. 4) according to its electrophoretic mobility in SDS-PAGE (Ostrowski et al., 1979). The sequence of its amino acids is known (Pan Yu and Li, 1982) and the molecular biology of the gene(s) coding for it has been extensively investigated (Abrescia et al., 1982; Harris et al., 1983; Kandala et al., 1983; Mansson et al., 1979; McDonald et al., 1983). Proteins immunologically related to SV-IV have been detected in many rat and human organs and body fluids both in males and females (Abrescia et al., 1985; Metafora et al., 1987a; Metafora et al., 1987b). One of these is uteroglobin (UG) (Metafora et al., 1987a), a small mol. wt. (15,000) progesterone-induced and progesterone-binding protein, possessing biological properties similar to those of the protein SV-IV (Levin et al., 1986; Metafora et al., 1989; Metafora et al., 1987c; Miele eta[., 1987; Mukherjee et al., 1983; Paonessa et al., 1984; Porta et al., 1988; Porta et al., 1987; Torkeli et al., 1978). SV-IV and UG are effective substrates (Manjunath et al., 1984; Metafora et al., 1987c; Porta et al., 1988) for transglutaminase (TGase) (Folk, 1980; Lorand and Conrad, 1984), an enzyme present in human seminal plasma (Porta et al., 1986) and in the secretions of the rodent anterior prostate (Wiiliams-Ashman, 1984). In the presence of this enzyme, both proteins are transformed into high mol. wt. (> 106) polymers that, by binding to the surface of rat epididymal spermatozoa, produce a marked suppression of the sperm cell immunogenicity (Manjunath et al., 1984; Metafora et al., 1987c; Mukherjee et al., 1983; Paonessa et ai., 1984; Porta et al., 1987). SV-I[V and UG possess powerful immunosuppressive activities (Metafora et al., 1989; Metafora et al., 1987c; Miele et ai., 1987; Porta et al., 1988) and a remarkable anti-inflammatory property. The latter characteristic is probably related to their ability to inhibit in vitro phospholipase A_, (PLA_,) enzymatic activity (Metafora et al., 1989; Porta et al., 1988). It has been reported that UG has a potent inhibitory effect on both monocyte and neutrophil chemotaxis and phagocytosis in vitro (Miele et al., 1987). On the basis of the structural homology between SV-IV and UG, and by taking into consideration their similar immunosuppressive and antiinflammatory properties, we investigated whether SV-IV has any suppressive effect on macrophage phagocytosing activity in vitro.

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Materials and methods

Every experiment reported in this paper was carried out in triplicate.

Preparation o f rat and mouse peritoneal macrophages Macrophages were obtained by injecting 3 ml 30/0 thioglycolate broth into the peritoneal cavity of rats (Wistar, groups of ten animals). The cells of the peritoneal exudate were washed three times in RPMI 1640 medium. After centrifugation, they were resuspended in the same medium, plated in Falcon flasks and incubated for 3 h at 37°C in 5°-/0 CO 2 in air. At the end of the incubation the adherent ceils were harvested in RPMI 1640, washed three times in the same medium, centrifuged and finally suspended in RPMI 1640 supplemented with 2°7,0 fetal calf serum at a final concentration of 2 × 106 cells/ml. Cell viability was evaluated by Trypan Blue dye exclusion and LDH release into the medium. Similar methods were used to prepare mouse (strain CD-I) peritoneal macrophages.

Microorganisms All microorganisms used (Saccharomyces cerevisiae, Candida aibicans and Staphylococcus epidermidis) were obtained from pure stock cultures grown in our laboratory by conventional procedures.

Purification o f S V-IV, TGase and UG The protein SV-IV was purified to homogeneity from adult rat (FischerWistar strain) seminal vesicle secretion according to Ostrowski et al. (1979). The purity of the protein was verified by 15°/0 PAGE in denaturing and nondenaturing conditions (Metafora et al., 1987a), by amino acid composition analysis and by fingerprint technique (Abrescia et al., 1986). Pure guinea pig liver TGase was obtained from Sigma Chemical Co, Ltd., England. UG was purified from rabbit uterus as previously described (Nieto et al., 1977).

['2-~I]SV-IV binding to rat peritoneal macrophages and yeast cells The binding assay mixtures of ['-'sl]SV-IV, prepared by the chloramine T method (Hunter, 1978), contained 10~ cells (S. cerevisiae, C. albicans or rat peritoneal macrophages), 140 mM NaCi, 30 mM Tris--HCl (pH 7.5), 1.4/ag ['z-q]SV-IV (5 × 106 cpm) and, where indicated, 5 mM dithiothreitol, 2.5 mM CaCI_~ and 1/ag purified guinea pig liver TGase (final vol. 0.3 ml). After incubation, either at 37 °C for 2 h or at 4 °C for different lengths of time (1-30 min) the assay mixtures were centrifuged (12,000 × g at 4°C for 30 s) and the sedimented cells washed with RPMI 1640 until no radioactivity was detectable in the supernatant. The washed cells were transferred before

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lysis in another polypropylene test tube to avoid contamination of final cell lysate with radioactivity not bound to the cells, but absorbed to the assay test tube walls. Cell lysis was carried out by suspending each cell pellet in 60/al of double distilled water. After addition of 30/al of a solution containing 15070 SDS, 15%/~-mercaptoethanol, 190 mM Tris--HCl buffer (pH 6.8) and glycerol, the samples were boiled for 2 min and then analyzed by both TCAprecipitable radioactivity counting and SDS-15% PAGE (Metafora et al., 1987a). Fluorography of dried gels was performed according to the technique of Bonner and Laskey (1974).

Visualization o f phagocytosis by fluorescence and electron microscopy Monolayers of 10 6 peritoneal macrophages in Leighton tubes were covered with 1 ml of RPMI 1640 containing 50/ag of SV-IV and then incubated at 37°C for 2 h in 5% CO, in air. At the end of incubation the monolayers were extensively washed with RPMI, covered with 1 ml of the same medium containing 5 x 10 6 S. cerevisiae cells and incubated with the yeast cells at 37 °C for different lengths of time. The modifications induced by the SV-IV treatment on the phagosomelysosome fusion process were substantiated by electron microscopy analysis performed with a Zeiss electron microscope EM 109. For these ultrastructural studies the macrophages were previously labelled by pre-incubating them for 3 h at 37°C in the presence of ferritin (10 mg/ml). Fixation of the macrophages and their labelling with ferritin were performed according to the technique of D'Arcy Hart and Young (1975). The effect of UG on phagocytosis was evaluated by treatment (37°C for 30 min) of mouse peritoneal macrophages in RPMI 1640 containing 10 mM glutamine and various concentrations of UG. The cells were then incubated at 37°C with latex beads (Sigma) for 15 min and finally washed three times with RPMI 1640. The cells were fixed with 2.5% gluteraldehyde in cacodylate buffer (pH 7.3) and kept at 4°C until processed for electron microscopy. Protein concentration measurement Protein content was measured either by the method of Lowry et al. (1951) or by absorbance at 276 nm. Results

S V-IV decreases rat peritoneal macrophage ability to phagocytose S. cerevisiae, C. albicans and S. epidermidis It is well known that at peritoneal macrophages phagocytose with high efficiency yeast cells, either saprophytic, such as S. cerevisiae, or pathogenic, such as C. albicans.

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Figure 1 shows that l-h treatment of 106 macrophages at 37°C with different concentrations of SV-IV produced a marked dose-dependent inhibition of their ability to phagocytose either Saccharomj'ces or Candida. The maximum inhibition (,,-,60o7'0) was observed when 50/ag/ml of SV-IV were used in the assay. Pre-incubation of 106 macrophages for 30 min with SV-IV (50/~g/ mi, final concentration (f.c.)), followed by either washing (Fig. 2, e _ _ e ) or not washing (Fig. 2, A - - & ) of the ceils, was effective in inhibiting their ability to phagocytose S. cerevisiae. Pre-incubation of S. cerevisiae with SVIV-induced inhibition of the macrophage phagocytosing ability either by washing or not washing the target ceils (Fig. 2, © - - O , A__A). Figure 3 shows that SV-IV was also able to inhibit the phagocytosis of S. epidermidis by macrophages with an efficiency similar to that of the phagocytic activities described above in which eukaryotic cells were used as target cells. All the reported data were obtained by using rat peritoneal macrophages. Comparable results were achieved with mouse peritoneal macrophages. By using the Trypan Blue dye exclusion test and other "live/dead" staining techniques, together with phase contrast microscopy, SV-IV was shown to be non-cytotoxic when added at final concentrations between 20

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Fig. 1. Effect of rat peritoneal macrophage treatment ~ith different concentrations of SV-IV on macrophage abilit.~ to phagocytose S. cerevisiae ( 0 - - 0 ) and C. albtcans ( e - - e ) . Rat peritoneal macrophages (10 ") ~ere incubated in Leighton tubes at 37 °C for I h in 0.5 ml of RPMI 1640 medium containing 2% FCS and different amounts of SV-IV. At the end of incubation the cells ~ere washed three times with RPMI 1640 and their phagocytosing ability ~as e~aluated as prex iousl} described (Van Zwet et al., 1975) by using either S. cerevisiae 15 × 10° cells., ml, f.c) or C. albicans (5 x l0 t cells, ml, f.c.).

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Fig. 2. Effect of treatment with SV-IV on either rat peritoneal macrophages or S. cerevisiae for different lengths of time on macrophage ability to phagocytose S. cerevisiae. Rat peritoneal macrophages (8 x l&) and Saccharomyces (8 x lff3 were separately incubated (at 37°C for different times) in 4 ml of RPM! 1640 containing 2% FCS and 200 ~g of SV-IV. At the end of the treatment, aliquots corresponding to 10" cells of both macrophages and Saccharom.vces were '~ashed with RPMI 1640. SV-IV-treated macrophages (l& washed or not ~ashed) were separately mixed ~ith untreated Saccharomyces (5 x 10~' cells/m[, f.c.) and the macrophage phagocytosing ability was e~aluated (Van Zwet et al., 1975). Similarly, SV-IV treated Saccharomyces (washed or not x~ashed; 5 × Iff' c e l l s ml, f.c.) were separately mixed with 106 untreated macrophages and the phagocytosing ability of the latter was evaluated (Van Zwet et al., 1975). O - - e , SV-IV treated and washed macrophages + untreated Saccharomyces ( S a c c ) ; & - - & , SV-IV treated and not v,ashed macrophages + untreated Sacc.; O - - O , untreated macrophages + SVIV-treated and washed Sacc.; A - - A , untreated macrophages + SV-IV-treated and not washed Sacc.

and 2000/ag/ml to the culture medium of different cell types (lymphocytes, macrophages, polymorphonuclear leukocytes, fibroblasts, thyroid or SV epithelial cells, yeast ceils, gram-positive or gram-negative bacteria). The inhibitory effect of SV-IV on the intracellular digestion of S. cerevisiae by rat peritoneal macrophages can be visualized by electron microscopy The data obtained by electron microscopy demonstrated that SV-IV was effective in suppressing the process of fusion between phagosomes and lysosomes. Thin sections of control macrophages, when observed by electron microscopy 45 rain after the beginning of the phagocytosis assay, showed the pres-

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Fig. 3. Effect of treatment with SV-IV of either rat peritoneal macrophages or S. epidermidis for different lengths of time on macrophage ability to phagocytose S. epidermidis cells. The experiments ~ere performed as described in the legend to Fig. 2 by using S. epidermidis to e~aluate the phagocytic abilit.~ of maerophages. • - - • , SV-IV treated and washed macrophages + untreated Staphylococcus (Staph.); &--A, SV-IV treated and not washed macrophages + untreated Staph.: O - - O , untreated macrophages + SV-IV-treated and washed Staph.; A - - A , untreated macrophages + SV-IV.treated and not washed Staph.

ence of phagosomes containing ingested cells, the phagosome ferritinlabelled membranes juxtaposed to the yeast cell walls ("tight" phago-lysosomal membranes) indicated the occurrence of phago-lysosomal structures. Labelled lysosomes, with their membranes beginning to fuse with the phagosome membranes, were visible in some areas of the cytosol (Fig. 4, panel A). Macrophages (106), pre-treated with SV-IV, were fixed 45 min after the beginning of the phagocytosis assay and their thin sections studied by electron microscopy. In the cytosol of these SV-IV-treated phagocytes no ingested cells were detectable, the Saccharomyces being visible only in the extracellular environment. Ninety minutes after the beginning of the phagocytosis few ingested cells were detectable in the SV-IV inhibited macrophages. The phago-lysosome structures, observable in the controls, were completely undetectable and the ferritin-labelled lysosomes were evident only outside the phagosome membrane (Fig. 4, panel B). Furthermore, the absence of lysosome-phagosome fusions were also suggested by the occur-

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'ig. 4. Electron microscopy (ferritin labelling melhod) following ingeslion of S. cerevisiae by rat periloneal macrophages, unlreated (Panel A) or trealed Panel B) with SV-IV. Experimental details are described in the text. Panel A: Y = yeast cell; DY = digested yeast cells; L = lysosome; FL = lysosome fus~g with phagosome; PLM = phago-lysosomal membrane ("light"). Panel B: Y = yeast cell: t = lysosome; PM = phagosome membrane ("loose").

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rence in the phagosomal structures of "loose" phagosome membranes, not juxtaposed to the membrane of the ingested cells. The treatment of mouse peritoneal macrophages with UG, a structural homologue of SV-IV, was also able to suppress completely phagocytosis at micromolar concentrations (Fig. 5). Other investigators have demonstrated, in addition, that UG inhibits human monocyte and rabbit polymorphonuclear leukocyte chemotaxis in response to formyl peptide attractants in a dose-dependent manner (Vasanthakumar et al., 1988).

Native and TGase-modified forms o f S V-IV bind to the surface o f rat peritoneal macrophages, S. cerevisiae and C. albicans It was thought that SV-IV may bind to the surface of macrophages and/or yeast cells in order to inhibit phagocytosis. Preliminary binding experiments, in which iodinated native SV-IV was incubated with macrophages in RPMI 1640 for different lengths of time (1--30 min) at 4°C, were performed in order to evaluate the number of specific SV-IV sites on the surface of these ceils. The temperature used was chosen to avoid both the internalization of the protein and the interference by TGase released from the macrophages during the binding assay. Scatchard analysis of the radioactivity bound to the phagocyte cell surface either in the absence or in the presence of native unlabelled SV-IV showed the specificity of this binding. The binding sites contained only saturable components completely displaceable by unlabelled SV-IV. The number of specific sites was calculated to be approximately 100,000/cell with a K d of 10-8 M. By using the same binding assay it was found that labelled SV-IV had a similar ability to bind to the surface of the target cells (S. cerevisiae and C. albicans). Since SV-IV was previously shown to be able to bind as a TGase modified form to the surface of rat spermatozoa and human peripheral blood lymphocytes (Paonessa et al., 1984; Metafora et al., 1987c), we investigated whether the modified forms of the protein bound to macrophages a n d / o r yeast cells in the same experimental conditions used in the phagocytosis assay (37 °C, I--2 h incubation time). The experimental results (data not shown) demonstrated a poor binding of native labelled SV-IV to the surface of macrophages and yeast ceils. The increase in the amount of radioactivity bound to the macrophages in the presence of TGase-polymerized SV-IV was most probably due to the binding of the labelled polymers, rather than monomers, to the same number of SVIV receptors. The non-involvement of the macrophage-released TGase in the SV-IV binding to the cell surface was demonstrated by the occurrence of SVIV binding even in the presence of EGTA (an experimental condition in which the TGse activity is inhibited).

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[3 Fig. 5. Inhibitory effect of different concentrations of LrG on mouse peritoneal macrophage ability to phagocytose latex beads. Macrophages were treated for 30 rain at 37°C ~ith 10 ~M (Panel B), 20 ~lkl (Panel C), 50 ~M (Panel D) UG. Panel A sho,~s untreated macrophages with c.vtosol filled with ingested latex beads.

280

Discussion

The results of the experiments reported in this paper clearly indicate that the protein SV-IV possesses the ability to inhibit the phagocytic activity of rat and mouse peritoneal macrophages in vitro. The cells used as targets of phagocytosis for the macrophages were either eukaryotic (Saceharomyces cerevisiae and Candida albicans) or prokaryotic (Staphylococcus epidermidis). The SV-IV-induced inhibition was detectable when either macrophages or the target cells were pre-treated with the protein. The removal of SV-IV from the reaction medium after cell pre-treatment did not eliminate the inhibitory effect. On the other hand, the presence of SV-IV during both the cell pre-incubation and the phagocytosis assay did not seem to increase the degree of inhibition. In numerous species of mammals, including rat, some seminal plasma proteins can enter the uterus where they appear to play an important role in inhibiting the local immune response (Alexander and Anderson, 1987; Hogarth, 1982; James and Hargreave, 1984; Harper, 1988; Mann, 1964; Nicholson et al., 1983; Overstreet, 1983). The formation of the vaginal plug protein network-induced in rats and mice by the prostatic TGase does not seem to involve SV-IV, the major TGase-clottable protein being the protein SV-IV (Wagner and Kistler, 1987). In fact, prostatic TGase can cross-link the smaller proteins of the rat seminal vesicle secretion, but with the following rate of cross-linking in vitro: SV-II ,> SV-V, SV-VI ~> SVIV (Wagner and Kistler, 1987). Accordingly, the SV-IV cross-linked component of the clot represents only a small fraction of the SV-IV total amount present in seminal plasma. Moreover, the occurrence of polyamines in semen, by producing a TGase-mediated polyamination of SV-IV, is effective in decreasing the ability of TGase to polymerize SV-IV, thus reducing the amount of SV-IV covalently bound to the vaginal plug protein network (Folk et al., 1980; Paonessa et al., 1984). We have demonstrated that native and TGase-modified forms of SV-IV bind to the surface of both macrophages and their target cells, the TGase activity not being involved in the binding process. In addition, we also have unpublished data showing that the SV-IV inhibitory effect on the macrophage phagocytic activity did not change when active TGase was added to the incubation medium of the phagocytosing assay. We can therefore conclude that TGase activity is not involved in the process of macrophage phagocytic inhibition by SV-IV. The binding of SV-IV to its specific phagocytic surface receptors may cause a "perturbation" of the plasma membrane that could not only affect cellular functions such as phagocytosis but might also be the basis of the alterations occurring in those cytosol structures of SV-IV treated macrophages which are involved in the lysosome-phagosome membrane interactions.

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The biological significance and possible clinical implications of SV-IV (as well as UG (Miele et al., 1987)) activities (immunosuppressive, anti-PLA 2, inhibiting phagocytosis) deserve consideration. The significance of the present findings can be assessed by reference to the reproductive process. SV-IV is an immunosuppressive protein secreted in large quantity by the rat SV (Mansson et al., 1981; McDonald et al., 1983; Metafora et al., 1989; Ostrowski et al., 1979) and antigens related to SV-IV have also been detected in human seminal plasma (Abrescia et al., 1985; Metafora et aI., 1987a; Metafora et al., 1987b). It has been demonstrated that the seminal plasma itself can severely impair, either directly or indirectly, the function of most cells of the immune system, including T and B cells. NK cells and macrophages (Alexander and Anderson, 1987, James and Hargreave, 1984). It can also reduce the activity of antibody and complement molecules (Alexander and Anderson, 1987; James and Hargreave, 1984). A number of immunosuppressive substances of high and low MW (UG, TGase, nucleases, proteases, various " s e m i n a l " proteins and peptides, prostaglandins, polyamines, etc.) have been found in the seminal plasma of many mammalian species (Alexander and Anderson, 1987; Hogarth, 1982; James and Hargreave, 1984; Mann and Lutwak-Mann, 1981; Metafora et al., 1987c). Some mechanisms are known by which these factors suppress, in male and female reproductive tracts, the immune response of the local immunocompetent cells to spermatozoa (Alexander and Anderson, 1987; James and Hargreave, 1984; Metafora et al., 1989; Metafora et al., 1987c; Miele et al., 1987). Among others, a direct inhibitory action on T lymphocyte, NK cell and macrophage functions, such as mitogen-induced T lymphocyte blast transformation, cell killing, and activated macrophage reactions (phagocytosis, chemotaxis, spreading, etc.), has been demonstrated (Alexander and Anderson, 1897; James and Hargreave, 1984). The macrophages, present in the male reproductive tract (EI-Demiry and James, 1988), can participate in the complex cell-cell interactions known to occur in either cellular or humoral immune responses by acting both as phagocytic and antigen-presenting cells. The SV-IV inhibition of phagocytosis may provide a local protection against the risk of immunological sensitization to escaped sperm antigens following alterations in the integrity of the bloodtestis barrier. All these effects are believed to play a fundamental role in mammalian reproduction, mainly because the)' prevent the destruction of developing spermatozoa within the male reproductive tract and minimize the risk of rejection of the immunogenic male gametes in the immunologicaIiy competent female environment (Alexander and Anderson, 1987; James and Hargreave, 1984; Metafora et al., 1989; Metafora et al., 1987c; Miele et al., 1987). Moreover, in the case of severe inflammatory responses to invading viruses, bacteria or malignant cells, the anti-inflammatory properties shared by some seminal plasma immunosuppressive factors could be of particlar

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advantage to prevent in the male and female reproductive tracts the production of large amounts of inflammatory mediators, detrimental to the survival of spermatozoa and embryo. On the other hand, an abnormal quantitative modulation of these immunosuppressive substances in the male genital tract may cause a particular vulnerability of this region to chronic bacterial or viral infections, to malignancies or to autoimmune disorders which may eventually lead to infertility. Acknowledgements We thank Professor A.B. Mukherjee (N.I.H., USA) for performing the experiments showing the anti-phagocytic properties of uteroglobin. We are also very grateful to him and to Professor H.G. Williams-Ashman for the critical reading of the manuscript. This work x~.as supported by a CNR grant from Progetto Finalizzato "Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie", a CNR Grant from Progetto Finalizzato "Oncologia" and a grant from the Italian Ministry of Education (40%). References Abrescia. R., Corbo, L. and Metafora, S. (19861 Maturation in different translational s.~'stems of the protein RSV-IV from the rat seminal xesicle epithehum. Bull. Mol. Biol. Med. [1, 19--33. Abrescia, P.. Guardiola, J., Felsani, A. and Metafora, S. ~1982) Expression in male and genomic organization of the gene(s) coding for a major protein ~ecreted by the rat seminal ~esicle epithelium. Nucleic Acids Res. 10, 1159--1174. Abrescia, P., Lombardi, G., De Rosa, M., Quagliozzi, L.. Guardio[a. J. and Metafora. S. 11985) Identification and preliminar.~ characterization of a sperm-binding protein in normal h u m a n semen. J. Reprod. Ferti[. ~3, 7 1 - - T 7. Alexander, N.J. and Anderson, D.J. 11987) Immunolog~ of semen. Fertil. Steril. 47, 192--205. Bonnet, ~3, .M. and Laskex, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in pob, acDlamide eels. Eur. d. Biochem. 46, 83--88. D'Arcx Hart, P. and Young, M.R. 11975) Interference v, ith normal phagosome-I.~sosome fusion in macrophages, using ingested yeast cells and s u r a m m . Nature 256, 4"--49. EI-Demir.,., M. and James, K. (19881 L.xmphoc.x te sub~et,~ and macrophages in the male genital tract in health and disease. Eur. Lirol. 14, 2261235. Folk, J.E. c 1980) Transglutaminases. Ann. Re,,. Biochem. 49.51-~--531. Folk. J.E., Park, M.H.. Chung. S.I.. Schrode, J., Lester, E.P. and Cooper, H.L. 119801 P o b a m i n e s as physiological substrates for tran~glutaminase. J. Biol. Chem. 255. 3695--3700. Harper, M.J.K. (19881 Gamete and z.~gote transport. In: The Phy~iolog} of Reproduction IKnobil, E., Neill, J.D., Ewing, L.L., Gree,,~ald, G.S., Markert, C.D. and Pfaff, D.W., eds.~, pp. 103--134. Raven Press, New York. Harris, S.E., Mansson, P.E.. l-ully, D.B. and Burkhar~, B. 11983) Seminal ',e:icle ¢,ecretion IX' gene: allelic difference due to a series of 20 base-pair direct tandem repeats ,,~ithin an intron. Proc. Natl. Acad. Sci. USA 80, 6460--6464. Hogarth. P.J. (1982) Immunological Aspects of Mammalian Reproduction. Blackie, Glasgov, and London.

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