Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

MOLECULAR REPRODUCTION AND DEVELOPMENT 2 6 1 2 2 3 (1990)

Protein Synthesis and Secretion in the Human Epididymis and Immunoreactivity With Sperm Antibodies PETER ROSS,*p4 FREDERICK W.K. KAN? PIERRE ANTAKI? NORMAND VIGNEAULT? ALCIDE CHAPDELAINE,3.4 AND KENNETH D. ROBERTS'*4 Departments of 'Biochemistry, 'Anatomy, and "Medicine, University of Montreal, and 4Maisonneuue-Rosemont Hospital Research Center, Montrea€, Quebec, Canada

ABSTRACT The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Sig- nificant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. lmmunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bonds of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.

Key Words: Epididymal proteins, Spermatozoa, Sperm antigens, Sperm maturation

INTRODUCTION The mammalian epididymis and its role in sperm

maturation have been studied extensively in many species (for recent reviews, see Cooper, 1986; Setchell and Brooks, 1988; Robaire and Hermo, 1988). During their transit through the epididymal duct, spermatozoa un- dergo specific physical, biochemical, and morphological changes. These changes coincide with variations in the

content of the epididymal lumen due to the synthesis, metabolism, and secretion of products such as steroids, carnitine, and proteins (for review, see Cooper, 1986).

Epididymal proteins have been the subject of many studies in view of their interaction with spermatozoa and their possible key role in the maturation of the male gamete. Experimental evidence such as the incorporation of radiolabeled precursors by epididymal tissue has shown that the pattern of secreted proteins differs in each segment of the epididymis of the rat (Cameo and Blaquier, 1976; Jones et al., 1980; Brooks, 1981; Sylvester et al., 1984; Klinefelter and Hamilton, 19851, mouse (Murphy and Carroll, 1987; Rankin et al., 1987; Holland and Orgebin-Crist, 19881, rabbit (Jones et al., 1981; Orgebin-Crist et al., 1987), and hamster (Gonzalez Echeverria et al., 1982).

Since access to human tissues is hampered by ethical problems, the involvement of the human epididymis in sperm maturation remains to be clarified. Although limited in number, some results have been obtained using these tissues. The binding capacity of human spermatozoa to zona-free hamster oocytes increases along the length of the epididymis (Hinrichsen and Blaquier, 1980; Moore et al., 1983; Sutherland et al., 1985). Moreover, Tezon et al. (1985a,b) have recently identified five androgen-dependent proteins synthesized by the human epididymis in organ culture. Three of these, with molecular weights of 38,29, and 21 kDa, were also found on the acrosomal cap of spermatozoa obtained from the cauda but not the caput epididymis. Furthermore, the authors have demonstrated the secretion of two major proteins (70 and 37 kDa), which may be secreted in the caput and corpus regions of the human epididymis; these proteins bind to the plasma membrane of the spermatozoa during their transit

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Received August 9, 1989; accepted November 24, 1989. Address reprint requests to Kenneth D. Roberts, Maisonneuve-Rose- mont Research Center, 5415, Boul. l'hsomption, Montreal (Que), Canada H1T 2M4.

0 1990 WILEY-LISS, INC.

HUMAN EPIDIDYMAL PROTEINS 13

(Tezon et al., 1987). An antiserum recognizing the proteins of 38, 29, and 21 kDa has demonstrated a quantitative reduction as well as an abnormal distribution of these antigens on spermatozoa collected from a population of infertile patients (Blaquier et al., 1987).

Although previous studies tend to implicate the epididymis in sperm maturation, there are reports of human pregnancy resulting from fertilization with sperm that have not passed through the corpus or cauda regions of the epididymis (Schmidt et al., 1976; Schoys- man and Bedford, 1986; Silber, 1987). Furthermore, Silber (1988) has reported three pregnancies occurring with sperm that had not been in contact with any epididymal region, although it should be noted that this has not been confirmed by other groups.

Thus, it is of great importance to resolve this contro- versy and elucidate the exact role of the human epididymal tract in sperm maturation. The present report is a preliminary study of the incorporation of radiolabeled methionine into newly synthesized proteins by various regions of human epididymides obtained from patients who had undergone castration for prostatic carcinoma and from cadavers via an organ donor pro- gram. Subsequently, the partial characterization of secreted epididymal proteins with regard to molecular weight and epididymal region of origin was achieved using antibodies raised against human spermatozoa.

MATERIALS AND METHODS Tissues were obtained from eight subjects; four sam-

ples came from patients who had undergone bilateral castration for prostatic carcinoma (ages 68,70, 72, and 78 years), and the remaining four were surgically removed from cadavers (ages 29, 52, 56, and 65 years). All samples were obtained within a 4 hr period. None of the eight subjects had received prior hormonal treatment or radiotherapy. The epididymal tissue was dis- sected free of the testis and connective tissue and separated into the proximal and distal segments of the caput, corpus, and cauda regions of the epididymis (Fig. 1).

The criteria established for the use of these epididy- ma1 tissues were as follows: no prior hormonal treatment, normal aspect of the tissues, and the presence of viable (motile) spermatozoa in the cauda epididymis (or vas deferens). Tissue samples from each region were found to have a conserved epithelium, with no evidence of necrosis as judged by histological examination under light microscopy.

Incorporation of Labeled Methionine The incubations were performed according to the

technique of Brooks (1981 ), with slight modifications. Essentially, segments from each region were cut into small pieces (1-2 mm3) and washed free of spermatozoa and luminal fluid by stirring them in a petri dish with Dulbecco's phosphate-buffered saline (PBS-D) for 20- 30 min. Twenty-five milligrams of tissue were incu-

co

Pig. 1. Schematic drawing of the human epididymis (Baumgarten et al., 1971). The tissue was divided into the proximal (PI and distal ID) regions of the caput, corpus, and cauda, as indicated.

bated in a silicone-coated scintillation vial (20 ml) containing 1 ml of incubation medium. The latter was composed of PBS-D (Gibco), 1 g/liter of glucose, 0.01 g/liter of phenol red, and 0.1 mM of amino acids except methionine. [35S]methionine was added in quantities ranging from 20 to 40 pWml of medium immediately preceding the incubations that were performed at 33°C in a shaker bath. After the incubation, the medium was removed and stored at -20°C. The tissues were homogenized in 2 ml of 10 mM Tris HC1,250 mM sucrose, 1.5 mM MgCl,, pH 7.4, with a mixture of protease inhibitors (10 mM benzamidine HC1,0.5 mM phenylmethyl- sulfonylfluoride, 3 pM aprotinin, 3 p,M pepstatin, and 11 pM leupeptin). Following ultracentrifugation at 100,OOOg for 30 min at 4"C, the supernatant was recov- ered and stored at -20°C.

To measure the levels of incorporation of radiolabeled precursors into newly synthesized proteins, 50 111 aliquots (in triplicate) were collected from the media and cytosolic fractions and incubated for 120 min at 25°C in 200 ~1 of nonlabeled precursor (0.1% methionine). The latter step was performed to remove the non- specific labeling of the proteins. Subsequent precipitation of acid-insoluble products was achieved within 30 min using 5 ml of cold 10% trichloroacetic acid (TCA). The samples were then filtered through Whatman GFI C filters and successively washed with 6 ml of 10% TCA containing 0.1% of unlabeled precursor, 3 ml of 70%~ ethanol, and 3 ml of ether. The filters were placed

14 P. ROSS ET AL.

in scintillation vials, dissolved in 0.5 ml of 90% NCS tissue solubilizer (Amersham) for 30 min at 50°C, neu- tralized with acetic acid, and counted in 10 ml of tolu- ene scintillation solution.

Electrophoresis and Fluorography Electrophoresis was carried out on 10-15%. linear

gradient acrylamide slab gels (13 x 14 x 1.5 cm) according to Laemmli (1970). The proteins used as molecular weight standards were lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhy- drase (31.0 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa), phosphorylase B (92.5 kDa), p- galactosidase (116.2 kDa), and myosin (200 kDa). Sil- ver staining of proteins was achieved using the method of Morrissey (1981).

To visualize the radiolabeled proteins after electrophoresis, the gels were incubated 30 min in Amplify (Amersham), dried on filter paper, and exposed at -80°C to preflashed RX Fuji films. To increase the detection levels of proteins in the media, the latter were desalted by centrifugation in CF-25 Amicon filter cones and subsequently concentrated by freeze drying the samples.

Western Blotting The transfer of secreted proteins from the electro-

phoresis gels to nitrocellulose paper was performed according to Towbin et al. (1979). Molecular weight standards were visualized using a solution of methanol: acetic acid:water (45:10:45) containing 0.1% amido black (Bio Rad) and subsequently washed in methanol: acetic acid:water (90:2:8).

Preparation of Antisera Antibodies were raised against a preparation of hu-

man spermatozoa. Fresh ejaculated semen obtained from the fertility clinic was processed according to Ahl- uwalia and Holman (1969) t o obtain sperm heads (298% pure). This preparation was washed twice with 5 ml of 0.02% Triton X-100, 10 mM Hepes, 150 mM NaC1, 50 mM benzamidine, at pH 7.4. The treatment with a low concentration of Triton has been shown to release the plasma membrane and outer acrosomal membrane of bull (Wooding, 19731, mouse (Flaherty and Breed, 1983), hamster (Legault, 19791, and human (Langlais, 1985; Antaki et al., 1984) spermatozoa as evidenced by electron microscopy. These studies also showed that the subacrosomal components were not affected by this treatment. After centrifugation (940g, 5 rnin), the supernatants were pooled and separated into aliquots containing the equivalent of 98 x 10" sperm heads and freeze dried. The antigen preparation was resuspended in a 0.05 M Tris HCl buffer (pH 7.4), emulsified with an equal volume of Freund's adjuvant (complete for the first injection and incomplete for the following ones), and injected intradermally along the back of New Zealand white female rabbits every month

for 4 months. The rabbits were killed 2 weeks after the last injection. Immunoglobulins (-350 pg/ml) were purified on a protein A-Sepharose column as described by Sullivan and Bleau (1985).

Indirect Immunofluorescence Aliquots (10 p1) of ejaculated sperm (lo5 cells,

washed twice with PBS) were added to a slide and incubated at 37°C for 30 min. The spermatozoa were fixed in methanol for 30 min and subsequently washed in PBS for 2 min. Fifteen microliters of the specific or preimmune antibody (diluted 112 with PBS) were added and incubated for 60 min. Excess antibody was removed by washing twice with PBS. Sperm were then treated with a tenfold diluted solution of goat antirabbit IgG tagged with fluorescein isothiocyanate containing 0.1% Evans blue. Another incubation of 60 min was carried out followed by two washings with PBS. A drop of 0.1% p-phenylenediamine (in 10% PBS, 90% glycerol, pH 8.0) was added to increase fluorescence. Immunof luorescent staining was observed on an American Optical Microscope 110 equipped with epif- luorescence and photographed on Fuji Ectachrome Film ASA 400.

Label-Fracture Immunocytochemistry Human semen from fertile donors was allowed to liq-

uify at room temperature and processed to separate the spermatozoa from seminal plasma by the "swim-up" technique. Motile spermatozoa were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 hr a t room temperature. The fixed cells were washed three times in 0.1 M cacodylate buffer and processed for label fracture. The label-fracture technique as described by Pinto da Silva and Kan (1984) was employed for surface immunolabeling of the cells. Samples of 0.1 ml of packed cells were used. For surface immunolabeling, 1 mg of lyophilyzed antibody was dissolved in 0.01 M PBS, pH 7.4. Spermatozoa were first resuspended in 1 ml of 0.01 M PBS containing 1% ovalbumin for 5 min and then incubated overnight at 4°C in the polyclonal antibody solution. After labeling, spermatozoa were washed three times by centrifugation and resuspension to remove excess unbound antibody. They were then resuspended in 1 ml of 0.01 M PBS containing 1% ovalbumin followed by incubation for 1 hr with protein A- gold complex. Colloidal gold particles (8 nm diameter) were prepared according to Slot and Geuze (1985). Af- ter labeling with the protein A-gold complex, the cells were washed three times with PBS and processed for freeze fracture.

Freeze Fracture Fixed, labeled cells were impregnated with 30% glyc-

erol/PBS, mounted on double-replica copper discs, fro- zen in Freon 22 cooled by liquid nitrogen, freeze fractured at - 130°C in a Balzers 400T freeze-etch unit, and replicated by Pt/C evaporation. Instead of the conven-


tional cleaning of replicas in acids or bases, replicas were thawed and washed by four successive floatings on distilled water (15 min per wash). Label-fracture preparations revealed a surface distribution of the label coincident with the Pt/C replica of exoplasmic halves of the sperm plasma membrane (see Kan and Pinto da Silva, 1989, for technical details and interpre- tation).

Immunoreaction of Secreted Epididymal Proteins With Antiserum Antibodies

The nitrocellulose membranes containing epididy- ma1 proteins (30,000 cpm/lane, which corresponds to -100 pg of protein) released into the media were soaked in a Tris-buffered saline solution (TBS; 20 mM Tris HCl, 100 mM NaC1,l mM CaCl,, 1 mM MgCl,, pH 7.41, saturated for 1 hr in 3% gelatin in TBS, and incubated for 2 hr in a 1:20 dilution of antisperm immunoglobulins. Antibodies that recognized epididymal proteins were revealed by a 1 hr incubation with alkaline phosphatase-conjugated goat antirabbit IgG (Pro- toBlot; Bio/Can Scientific Inc.) and subsequent reaction with nitroblue tetrazolium and 5-bromo-4-chloro-in- dolylphosphate according to the supplier. As controls, a sperm extract prepared as described above for the original antigen was separated on SDS-PAGE, transferred to nitrocellulose, and labeled with the immune and preimmune sera. An SDS-PAGE pattern of decomplemented human serum was also probed with the immune serum.

Immunoprecipitation of Epididymal Proteins The method used to immunoprecipitate epididymal

proteins was a modification of that used by Wewer et al. (1987). A solution of 1 ml of the purified gamma- globulins (0.77 mg) was incubated for 2 hr a t room temperature in the presence of 1 ml of swelled protein A-Sepharose in PBS-D containing 0.05% Tween 20. Af- ter washing three times in the same buffer, the immu- nobeads were incubated overnight a t 4°C with 1 ml of radiolabeled epididymal proteins. The Sepharose beads were washed seven times with PBS-D/Tween 20, and bound material was eluted with the electrophoresis sample buffer (50 mM Tris HC1, 1.4 M glycerol, 5% 2-mercaptoethanol, 2% SDS, pH 6.8). The immunoprecipitated material was separated by electrophoresis, and labeled proteins were revealed by fluorography as described above.

RESULTS Incorporation of [35SlMethionine Into

Epididymal Proteins Radiolabeled methionine was incorporated into

newly synthesized acid-insoluble products in a time- dependent manner for a t least 7 hr (Fig. 2). Secreted radioactive proteins (5% of total r35Slmethionine incorporation) appeared in the medium only after a lag time of 1 hr (Fig. 2b). The rates of incorporation of radiola-

a) TISSUE

time (hours)

b) MEDIUM

lime (hours)

Fig. 2. Time course of L%lmethionine incorporation into tissue proteins (a) and their release into the medium (b). The corpus epididymal tissue of one subject was processed as described in Materials and Methods and 25 mg (in duplicate) were incubated with 25 pCi ["S~methionine (in duplicate) for the times indicated. Acid-insoluble radioactivity in the media or homogenized tissues was measured as described in Materials and Methods. Each point represents the mean of an experiment carried out in duplicate 2 standard deviation (some standard deviations were too small to be observed with the scale used).

beled methionine into tissue and medium proteins were low for the first 3 hr and more rapid between 3 and 7 hr (Fig. 2a,b). A 5 hr incubation period was cho- sen for subsequent incubations.

The corpus region appears to be the most active segment of the epididymis with regard to total incorporation of r35Slmethionine (tissue + media). It had a significantly higher rate of incorporation into tissue proteins compared to the cauda (Fig. 3a) (0.0005 < P 5 0.005). Conversely, the amount of acid-insoluble radioactivity found in the media was significantly different only between the corpus and the caput segments (Fig. 3b) (0.0005 < P I 0.005). Statistical analysis was done using paired Student's t test. Although the greatest amount of secreted radioactive proteins was found in the corpus, the highest percentage of secreted proteins

16 P. ROSS ET AL.

A TISSUE

I

C a p u t C o r p u s Cauda

B MEDIA -

C a p u t C o r p u s Cauda

Fig. 3. [“Slmethionine incorporation into different epididymal regions. The epididymides of six patients were subdivided into caput, corpus, and cauda segments (see Fig. 1). The acid-insoluble radioactivity in 25 mg of tissue (A) and corresponding media (B) was ex- pressed as a percentage of the segment that exhibited maximal incorporation. Each column is the mean of six experiments carried out in duplicate and is presented as the mean ? standard deviation. (*), Significant variation vs. the corpus segment (0.0005 < P 5 0.005).

versus total incorporation in a specific region was obtained in the cauda.

Electrophoretic Patterns of Epididymal Proteins Proteins from the different regions of the epididymis

(see Fig. 1) were run on electrophoresis gels to compare the patterns obtained with tissues originat- ing from different subjects. There were no significant differences between each subject, and only minor variations in the relative intensity of certain bands were visible (data not shown). Samples of tissue and media proteins obtained from different subjects are shown in Figures 4a and 4b, respectively. The major cytosolic proteins having molecular weights of 80, 75, 70,65, 45, 32, and 31 kDa are present throughout the epididymis. Their relative concentration in each segment tends to be very similar along the different regions (Fig. 4a).

In contrast, the pattern of proteins in the medium varies along the epididymis (Fig. 4b). The major bands differ from those of the cytosols both with regard to their molecular weights as well as their relative intensities. The major bands are proteins of 23-24 kDa present mainly from the distal caput to the proximal cauda, the doublet of 28-29 kDa that appears mostly in the caput and distal segment of the cauda, bands of about 38 kDa that are present in higher proportion in the corpus, a protein of 56 kDa present almost exclu- sively in the proximal caput, and a 58 kDa component that appears to be secreted mostly in the distal corpus and proximal cauda region. Proteins of 90 and 135 kDa are present throughout the epididymis.

Antisperm Antibodies It is well known that sperm components that

recognize and bind to the zona pellucida are localized on the head region of the spermatozoa (for review, Fournier-Delpech and Courot, 1987). In view of this functional property of the sperm head, an antiserum was raised against a Triton extract of ejaculated human sperm heads to characterize those proteins that bind to the sperm plasma membrane during their passage through the epididymis. A preparation similar to the original antigen has been separated by SDS-PAGE and is shown in Figure 5 (lane 1). Most proteins obtained by the Triton X-100 extraction have molecular weights ranging between 35 and 94 kDa. When probed with the antiserum (lane 21, these proteins show different relative intensities compared to the silver stain. The bands of higher molecular weights (266 kDa) appear to be less intensive when probed with the antibodies compared with their corresponding silver stain, with the exception of a band at 170 kDa, which represents a very minor component of the sperm extract. However, the lower- molecular-weight proteins were found to react more strongly. The preimmune serum was used to probe the antigen preparation and did not react with the exception of a weak reaction in the region of 66 kDa (lane 3). Decomplemented human serum also reacts with components of similar molecular weights (lane 4).

The distribution of antigenic sites on the surface of ejaculated spermatozoa was examined by immunof luorescence studies. Antibody labeling was restricted to the head and was more intense in the equatorial region (Fig. 6a). The preimmune serum produced no labeling (Fig. 6b), nor did a control using the second layer antibody alone (data not shown). Electron microscopic examination after protein A-gold labeling of freeze-fracture sections revealed the distribution of gold particles on the external leaflet of the plasma membrane with an increase in particle concentration in the equatorial region (Fig. 6c). Control sections labeled with the preimmune serum did not show any reaction.


stds

4 200

4 116 4 92

4 66

4 45

4 31

4 21

Fig. 4. Fluorogram showing the SDS-PAGE separation of tissue (a) and media (b) proteins labeled with [”Slmethionine. The pattern of bands shown here (from one patient) is typical of all others obtained, 40,000 cpm of labeled proteins were added to each lane. Major bands are indicated on the left and standards of known molecular weight on the right. The letters p and d refer to the proximal and distal regions, respectively.

Immunodetection of Secreted Epididymal Proteins With Antisperm Antibodies

Figure 7a shows the SDS-PAGE pattern of secreted proteinsobtained from the different regions ofthe epididymis following their exposure to antiserum. Major immunoreactive bands of 80-76 (doublet), 66,48,22, and 17 kDa were found to be present in specific regions. A doublet at -80-76 kDa showed increased immunoreactivity from the proximal corpus to the distal cauda. A similar increase in the intensity of a band of 48 kDa was observed in those regions downstream from the distal corpus. Certain proteins (27, 29, and 32 kDa) reacted faintly in all the regions, and two antigens of 22 and 17 kDa were localized only in the distal part of the cauda epididymis. A preimmune serum was tested on dot blotted epididymal media proteins and was found to be negative (data not shown).

When the antiserum was used to immunoprecipitate the radiolabeled proteins present in the medium, only a few bands were revealed by fluorography (Fig. 7b). The most prominent bands were the radioactive antigens at 80-76 kDa (doublet) and at -37 kDa that ap-

pear mostly in the corpus and proximal cauda regions. A faint and diffuse band of 48 kDa was also present throughout the caput, corpus, and proximal cauda segments; another at 60 kDa in the proximal region of the cauda was also found to react.

DISCUSSION The present study examines the incorporation of ra-

diolabeled precursors into proteins synthesized and secreted by different segments of the human epididymis and the interaction of certain of these proteins with antisperm antibodies. The term “secreted” has been ap- plied to those radioactive proteins that were found in the media. This is partly based on the fact that the lag time of 60 min observed before the appearance of these radiolabeled proteins in the medium (Fig. 2) could correspond to the time required for their synthesis and release (Flickinger, 1981; Fain-Maurel et al., 1981). Furthermore, the major protein bands present in the media (Fig. 4b) are not the same as those found in the tissue (Fig. 4a). The latter figures also show a significantly different pattern of media proteins derived from

18 P. ROSS ET AL.

Fig. 5. Sperm heads were extracted in a manner identical to the antigen preparation (see Materials and Methods), and the extracts were run on SDS-PAGE and silver stained to reveal the protein bands (lane 1). After Western transfer, they were labeled with a gamma- globulin fraction purified from immune (lane 2) and preimmune (lane 3) sera. The bands were revealed with a n alkaline phosphatase- conjugated antibody as described in Materials and Methods. Each lane contains the equivalent of 50 x 10" spermatozoa. Human serum (5 p.8) was also assayed and was found to react with our immunoglobulins (lane 4). Lanes 1 and 2 are from 10-15% linear gradient electrophoresis; lanes 3 and 4 are 12% acrylamide gels.

the various epididymal segments, whereas tissue proteins are very similar. Therefore, we consider that the radioactive products found in the media are selectively released rather than the product of cell lysis. These data are consistent with results obtained in the rat (Brooks, 1981), the mouse (Holland and Orgebin-Crist, 19881, and the rabbit (Jones et al., 1981).

Protein synthesis, as judged by ["Slmethionine incorporation, has a tendency to be higher in the corpus region of the epididymis (Fig. 3). However, perhaps be- cause of the limited number of subjects, we were able to show a significant difference only between the caput and corpus secretions and between the corpus and cauda cytosolic proteins. The human epididymis would appear t o be different in this aspect from that of the rat (Brooks, 1981) and the rabbit (Jones et al., 1981),

where the caudal region is the most active in protein synthesis, and also from that of the mouse (Murphy and Carroll, 1987; Holland and Orgebin-Crist, 1988), where it has been shown that those segments proximal to the testis are the most active.

[36S]Methionine Incorporation Into Proteins From Different Epididymal Regions

As judged by SDS-PAGE, the human epididymis does not exhibit qualitative differences in the overall synthesis of its major cytosolic proteins along the various segments (Fig. 4a). This observation has also been reported for rat (Brooks, 1981) and mouse (Holland and Orgebin-Crist, 1988) epididymis. The epididymal fluid, on the other hand, is distinguished by regional differences in the selective release of proteins into the lumen (Fig. 4b) as reported for other species (see Cooper, 1986, for review). Those proteins of 21, 29, and 38 kDa that were detected mainly in the caput and corpus media could correspond to three androgen-dependent proteins of similar molecular weight that were shown by Tezon et al. (1985a) to be secreted into the lumen of human epididymal tissues in culture. The pattern of proteins obtained from all patients was constant except for mi- nor variations in the relative intensities of certain faint bands.

Antisperm Antibodies To characterize the antibody used in this study, the

localization of the antigen was determined by fluorescence and electron microscopy. The antiserum produced by immunizing rabbits with a Triton extract of spermatozoa (Fig. 5 , lane 1) reacts only in the head region of ejaculated sperm as determined by indirect immunofluorescence (Fig. 6a). The equatorial region is labeled more intensely. Accordingly, examination of label-fractured specimens immediately reveals the regionalization of antigenic sites on the sperm head surface (Fig. 6c). Label-fracture images show a sparse but unclustered labeling of the acrosomal region. However, intense labeling by gold particles is found over the equatorial segment of the sperm head. The polyclonal antibody does not label an intramembrane particle-free zone at the base of the sperm head. These results indi- cate regionalization of antigenic sites on the plasma membrane of human spermatozoa. Thus this antigen preparation consists of plasma membrane proteins, and this is confirmed by subsequent observations (Fig. 5).

Immunodetection of Secreted Epididymal Proteins With Antisperm Antibodies

The absorption of secreted epididymal proteins by the sperm surface has been reported in the following species: cattle (Killian and Amann, 19731, hamster (Moore, 1980, 1981b; Sullivan and Bleau, 1985; Ro- bitaille et al., 1986), mouse (Fox et al., 1982; Vernon et al., 1982), rabbit (Moore, 1980, 1981a; Thomas et al., 1984), rat (Lea et al., 1978; Kohane et al., 1980; Drav-


land and Joshi, 1981; Faye et al., 1980; White et al., 1982; Wong and Tsang, 1982; Brooks and Tiver, 1983; Klinefelter and Hamilton, 1985), chimpanzee (Young et al., 19871, and human (Tezon et al., 1985b; Dacheux et al., 1987). In the present paper, we provide evidence for the interaction of epididymal fluid proteins with antibodies raised against antigenic Triton extracts of human sperm heads. Thus those proteins found in the epididymal fluid exhibit antigenic similarities with sperm proteins (Fig. 5). However, it is recognized that

Fig. 6. a: Indirect immunofluorescence labeling of washed ejaculated human spermatozoa with antisperm antibodies. x 280. b Same as A, using the preimmune serum. x 280. c: Protein A-gold labeling showing the surface distribution of the antigen on freeze-fracture images of a human sperm cell. The E-face of the sperm head plavma membrane is shown. X 17,100.

those proteins of the media from the different regions that immunoreact do not necessarily originate from the segments where they are ultimately found. Some of these proteins may be synthesized and secreted in up- stream segments and transported within the lumen through the subsequent segments. The majority of these unlabeled proteins are removed by the preliminary washings (see Materials and Methods); however, it is recognized that during the 5 hr incubation, some residual proteins could leak into the media along with

20 P. ROSS ET AL.

80 kDa

66 kDa

48 kDa

37 kDa

32 kDa 29 kDa 27 kDa

22 kDa

17 kDa

Fig. 7. a: Western blot of the media proteins obtained from the different epididymal regions labeled with the antiserum. Approxi- mately 100 pg of protein was added to each lane. The molecular weights of the major immunoreactive bands are indicated. b: Media proteins (-20,000 cpm) from the different epididymal regions were

immunoprecipitated with the sperm Triton-extract antibodies a s described in Materials and Methods and separated on SDS-PACE. Ka- diolabeled proteins were revealed by fluorography using Fuji RX film. The molecular weights of major immunoprecipitated bands are indicated a s values x 10 ' I .

the newly synthesized (radioactive) products and thereby react with the antibody.

The appearance of a highly immunoreactive protein at 66 kDa in all the epididymal regions (Fig. 7a) and in the sperm extract (Fig. 5, lane 1) is noteworthy and could be the result of the secretion of this protein by the testis and/or the subsequent epididymal segments. However, the absence of a corresponding radioactive protein in the immunoprecipitates of the epididymal fluids (Fig. 7b) would suggest that it is synthesized and secreted by the testis or by the ductuli efferentes. It is also possible that this protein is serum albumin, since our antiserum recognizes a band in human serum with a molecular weight corresponding to that of albumin (Fig. 5 ) .

The proteins of 76 and 80 kDa that first appear in the proximal corpus and exhibit an increasing immunoreaction on western blot in the subsequent segments (Fig. 7a) are precipitated mostly in the corpus and proximal caudal regions (Fig. 7b). Proteins of similar molecular weights are also present as major components on the silver stain of the SDS-PAGE-separated

sperm extract (Fig. 5, lane 1). It is thus conceivable that these proteins are secreted by the corpus and proximal cauda segments and bind to the sperm plasma membrane. They may also correspond to the 70 kDa protein described by Tezon et al. (1987) that is present on cauda but not caput epididymal spermatozoa. The differences in molecular weights in the two studies may be attributed to different concentrations of polyacrylamide used, which would result in differences in the resolution of proteins with these molecular weights.

Western blotting of proteins present in caudal fluid revealed two additional bands of 17 and 22 kDa that were not immunoreactive in other segments of the epididymis and not precipitated as radiolabeled products (compare lanes corresponding to the distal cauda segment in Figure 7a and b). Although unsubstanti- ated, this could be due to any one of the following: the chemical modification (in the distal caudal region) of proteins synthesized in previous segments, which would reveal or create new antigenic sites; the release by the sperm plasmalemma of some of these proteins


spermatozoa. The identification and physiological role of certain of these epididymal proteins is also under investigation. Special attention will be focused on the bands of 37 and 60 kDa, since proteins of similar molecular weights (35 and 60 kDa) have been reported to bind to solubilized radiolabeled human zona pellucida proteins (Shabanowitz and O’Rand, 1988).

into the caudal lumen; the degradation of antigenic products of higher molecular weights; or simply that these proteins did not incorporate radiolabeled methionine as is evident in Figure 4b, or finally that the immune complexes formed with these two proteins were not immunoprecipitable. Similarly, the bands of 27,29, and 32 kDa observed on the western blot (Fig. 7a) were not significantly revealed when subjected to immunoprecipitation.

In addition, the radioactive band around 38 kDa secreted from the distal caput to the proximal cauda (Fig. 4b) also appears to be immunoprecipitated in the same regions (37 kDa, Fig. 7b) but reacts only slightly in the western blot analysis (Fig. 7a). This discrepancy is not understood and is presently under investigation. A protein of similar molecular weight was shown to be secreted mostly in the caput in an androgen-dependent manner and was found to immunoreact with antiserum raised against human spermatozoa (Tezon et al., 1985a,b). Dacheux et al. (1987) also reported the acqui- sition of a 37 kDa sialoprotein on the membrane of human spermatozoa during their transit through the epididymis. Moreover, a 36 kDa glycoprotein is labeled by the galactose oxidase/Na,BH, method on the surface of human sperm treated with neuraminidase to remove sialic acid residues (Kallajoki et al., 1986). A doublet of proteins corresponding to this molecular weight (35-37 kDa) is present in our sperm head extract (Fig. 5 , line 1). A high-molecular-weight component of -170 kDa (Fig. 7b) was also detected by immunoprecipitation and, on western blot analysis, appeared as a faint band in the same epididymal region, namely, the distal part of the caput segment.

Antigenic membrane proteins of similar molecular weights have been reported to be present on human spermatozoa (Wolf et al., 1983; Aitken et al., 1987). Interestingly, Aitken et al. (19871, using a gammaglob- ulin fraction of antisera obtained from patients exhib- iting idiopathic autoimmunity against sperm antigens, showed an immune reaction of these antibodies with surface-labeled human sperm proteins of 35,45,66,90, and 150 kDa, which could represent those proteins of similar molecular weights discussed above.

In this study, we have demonstrated protein synthesis and secretion in the different regions of the epididymis obtained from patients of different ages and patho- logical conditions. Previous investigators (Oshima et al., 1984) have reported age-relatedmorphological variations of epididymal tissues. However, we did not ob- serve any significant differences in our results obtained from each patient, although it is recognized that only a small population has been reported in this study. The interaction of certain of the secreted proteins with spermatozoa suggests the implication of the epididymis in sperm surface modifications. A more detailed study of these immunoreactive proteins is warranted in order to determine their site of synthesis, since the accessory sex organs may also secrete proteins that interact with

ACKNOWLEDGMENTS The authors are deeply indebted to Drs. Gilles Bleau

and Robert Sullivan as well as Mr. Pierre Boulanger, Mr. Michel Desaulniers, and Mr. Pierre Guerette for helpful discussions. We are very grateful to Ms. Clau- dette Boudreault for her patience and secretarial assis- tance in typing the manuscript. This research was sup- ported by the Medical Research Council of Canada and a Studentship from the Fonds de Recherche en Sante du Quebec (P.R.).

REFERENCES Ahluwalia B, Holman RT (1969): Fatty acid composition of lipids of

bull, boar, rabbit, and human semen. J Reprod Fertil 18:431-437. Aitken RJ, Hulme MJ, Henderson CJ, Hargreave TB, Ross A (1987):

Analysis of the surface labelling characteristics of human spermatozoa and the interaction with anti-sperm antibodies. J Reprod Fer- ti1 80:473-485.

Antaki P, Bleau G, Chapdelaine A, Roberts KD (1984): Determina- tion du poids moleculaire de l’acrosine in situ dans le spermatozolde humain. Club de Recherches Cliniques du Quebec; Union MBdicale du Canada, abstract 11.

Baumgarten HG, Holstein AF, Rosengren E (1971): Arrangement, ultrastructure, and adrenergic innervation of smooth musculature of the ductuli efferentes, ductus epididymidis and ductus deferens of man. Z Zellforsch 120:37-79.

Blaquier JA, Cameo MS, Stephany D, Piazza A, Tezon JG, Sherins RJ (1987): Abdormal distribution of epididymal antigens on spermatozoa from infertile men. Fertil Steril 47:302-309.

Brooks DE (1981): Secretion of proteins and glycoproteins by the rat epididymis: Regional differences, androgen-dependence, and effects of protease inhibitors, procaine, and tunicamycin. Biol Reprod 25: 1099-1117.

Brooks DE, Tiver K (1983): Localization of epididymal secretory proteins on rat spermatozoa. J Reprod Fertil69651-657.

Cameo MS, Blaquier JA (1976): Androgen-controlled specific proteins in the rat epididymis. J Endocrinol 69:47-55.

Cooper TG (1986): “The Epididymis, Sperm Maturation and Fertili- zation.” Berlin: Springer-Verlag.

Dacheux JL, Chevrier C, Lanson Y (1987): Motility and surface trans- formations of human spermatozoa during epididymal transit. In M-C Orgebin-Crist and Danzo BJ (eds): “Cell Biology of the Testis and Epididymis.” Ann NY Acad Sci 513; 560-563.

Dravland E, Joshi MS (1981): Sperm-coating antigens secreted by the epididymis and seminal vesicle of the rat. Biol Reprod 25:649-658.

Fain-Maurel MA, Dadoune JP , Alfonsi MF (1981): High-resolution autoradiography of newly formed proteins in the epididymis after incorporation of tritiated amino acids. Arch Androl 6:249-266.

Faye JC, Duguet L, Mazzuca M, Bayard F (1980): Purification, radio- immunoassay, and immunohistochemical localization of a glycopro- k i n produced by the rat epididymis. Biol Reprod 23:423-432.

Flaherty SP, Breed WG (1983): The sperm head of the plains mouse. Pseudomys australis: Ultrastructure and effects of chemical treat- ments. Gamete Res 8231-244.

Flickinger CJ (1981): Regional differences in synthesis, intracellular transport, and secretion of protein in the mouse epididymis. Biol Reprod 25:871-883.

22 P. ROSS ET AL. Fournier-Delpech S, Courot M ( 1987): Sperm-zona pellucida binding

activity. Oxford Rev Reprod Biol 9294-321. Fox N, Damjanov I, Knowles BB, Solter D (1982): Teratocarcinoma

antigen is secreted by epididymal cells and coupled to maturing sperm. Exp Cell Res 137:485-486.

Gonzalez Echeverria FM, Cuasnicu PS, Blaquier JA (1982): Identifi- cation of androgen-dependent glycoproteins in the hamster epididymis and their association with spermatozoa. J Reprod Fertil 64: 1-7.

Hinrichsen MJ, Blaquier J A (1980): Evidence supporting the exis- tence of sperm maturation in the human epididymis. J Reprod Fer- ti1 60991-294.

Holland MK, Orgebin-Crist M-C ( 1988): Characterization and hormonal regulation of protein synthesis by the murine epididymis. Biol Reprod 38:487-496.

Jones R, Brown CR, VonGlos KI, Parker MG (1980): Hormonal regulation of protein synthesis in the rat epididymis. Characterization of androgen-dependent and testicular fluid-dependent proteins. Biochem J 188:667-676.

Jones R, VonGlos KI, Brown CR (1981): Characterization of hormon- ally regulated secretory proteins from the caput epididymidis of the rabbit. Biochem J 196:105-114.

Kallajoki M, Virtanen I, Suominen J (1986): Surface glycoproteins of human spermatozoa. J Cell Sci 8211-22.

Kan FWK, Pinto da Silva P ( 1989): Label-fracture cytochemistry. In MA Hayat (ed): “Colloidal Gold: Principles, Methods and Applica- tions.” Orlando, FL: Academic Press, Vol 2, pp 175-201.

Killian GJ, Amann RP (1973): Immunoelectrophoretic characterization of fluid and sperm entering and leaving the bovine epididymis. Biol Reprod 9:489-499.

Klinefelter GR, Hamilton DW (1985): Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa. Biol Reprod 33:1017-1027.

Kohane AC, Gonzalez Echeverria FMC, Pineiro L, Blaquier J A (1980): Interaction of proteins of epididymal origin with spermatozoa. Biol Reprod 23:737-742.

Laemmli UK (1970): Cleavage of structural proteins during the as- sembly of the head of bacteriophage T,. Nature 227580-685.

Langlais J (1985): Un modele membranaire moleculaire de la capac- itation et de la reaction de l’acrosome du spermatozoide hurnain. Doctoral thesis, Biochemistry Department, University of Montreal.

Lea OA, Petrusz P, French FS (1978): Purification and localization of acidic epididymal glycoprotein (AEGI: A sperm coating protein secreted by the rat epididymis. Int J Androl Suppl 2592-607.

Legault J 91979): Etudes des sterols sulfoconjuges dans la biochimie de la reproduction. Doctoral thesis, Biochemistry Department, Uni- versity of Montreal.

Moore HDM (1980): Localization of specific glycoproteins secreted by the rabbit and hamster epididymis. Biol Reprod 22705-718.

Moore HDM (1981a): Effects of castration on specific glycoprotein secretions of the epididymis in the rabbit and hamster. J Reprod Fertil 61:347-354.

Moore HDM (1981b): Glycoprotein secretion of the epididymis in the rabbit and hamster: Localization on epididymal spermatozoa and the effect of specific antibodies on fertilization in vivo. J Exp Zoo1 21577-85.

Moore HDM, Hartman TD, Pryor JP 11983): Development of the oocyte-penetrating capacity of spermatozoa in the human epididymis. Int J Androl 6310-318.

Morrissey J H (1981 ): Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal Bio- chem 117:307-310.

Murphy GJP, Carroll J (1987): Detection and localization of antigens on the surface of mouse spermatozoa. Biochem J 241:379-387.

Orgebin-Crist M-C, Hoffman LH, Olson GE, Skudlarek MD (1987): Secretion of proteins and glycoproteins by perifused rabbit corpus epididymal tubules: effect of castration. Am J Anat 180:49-68.

Oshima S, Okayasu 1, Uchima H, Hatakeyama S (1984): Histopatho- logical and morphometrical study of the human epididymis and testis. Acta Pathol Jpn 34:1327-1342.

Pinto da Silva P, Kan FWK (1984): Label-fracture: A method for high resolution labeling of cell surfaces. J Cell Biol 99:1156- 1161.

Rankin T1, Holland MK, Orgebin-Crist M-C (1987): Mouse epididy- ma1 fluid glycoproteins. In M-C Orgebin-Crist and Danzo BJ leds): “Cell Biology of the Testis and Epididymis.” Ann NY Acad Sci 513: 557-558.

Robaire B, Hermo L (1988): Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In E Knobil, JD Neill, et al. (eds): “The Physiology of Reproduction.” New York Raven Press Ltd, vol 1, pp 999-1080.

Robitaille G, Ross P, Sullivan R, Chevalier S, Bleau G (1986): The caput epididymis secretes a protein involved in sperm-egg interaction. Biol Reprod 341Suppl 1 I Abstract 147.

Schmidt SS, Schoysman R, Stewart BH (1976): Surgical approaches to male infertility. In ESE Hafez (ed): “Human Semen and Fertility Regulation in Man.” St. Louis: C.V. Mosby Co, pp 476-493.

Schoysman RJ, Bedford JM (1986): The role of the human epididymis in sperm maturation and sperm storage as reflected in the conse- quences of epididymovasostomy. Fertil Steril 46293-299.

Setchell BP, Brooks DE (1988): Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In E Knobil, J D Neill, et al. (eds): “The Physiology of Reproduction.” New York: Raven Press Ltd, Vol 1, pp 753-836.

Shabanowitz RB, ORand MG (1988): Molecular changes in the human zona pellucida associated with fertilization and human sperm/ zona interactions. In HW Jones and C Schrader [eds): “In Vitro Fertilization and Other Assisted Reproduction.” Ann NY Acad Sci 541:621-632.

Silber SJ 11987): Apparent fertility of human sperm from the caput epididymis. 43rd Annual Meeting of the American Fertility Society, Reno, Nevada. Abstract 039.

Silber SJ (1988): Pregnancy caused by sperm from vasa efferentia. Fertil Steril 49:373-375.

Slot JW, Geuze HJ (1985): A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38:87-93.

Sullivan R, Bleau G (1985): Interaction of isolated components from mammalian sperm and egg. Gamete Res 12:lOl-116.

Sutherland PD, Matson PL, Moore HDM, Goswamy R, Parsons JH, Vaid P, Pryor JP (1985): Clinical evaluation of the heterologous oocyte penetration (HOP) test. Br J Urol 57233-236.

Sylvester SR, Skinner MK, Griswold MD (1984): A sulfated glycoprotein synthesized by sertoli cells and by epididymal cells is a component of the sperm membrane. Biol Reprod 31:1087-1101.

Tezon JG, Herme C, Dawidowsky A, Scorticatti C, Blaquier JA (1987): Characterization of secretory proteins in the human epididymis. In M-C Orgebin-Crist and BJ Danzo BJ (ed): “Cell Biology of the Testis and Epididymis.” Ann NY Acad Sci 513:611-613.

Tezon JG, Ramella E, Cameo MS, Vazquez MH, Blaquier JA (1985a): Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa. Biol Reprod 32: 591-597.

Tezon JG, Vazquez MH, Pineiro L, DeLarminat MA, Blaquier J A (1985b3: Identification of androgen-induced proteins in human epididymis. Biol Reprod 32584-590.

Thomas TS, Reynolds AB, Oliphant G (1984): Evaluation of the site of synthesis of rabbit sperm acrosome stabilizing factor using immu- nocytochemical and metabolic labeling techniques. Biol Reprod 30: 693-705.

Towbin H, Staehelin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Proce- dure and some applications. Proc Natl Acad Sci USA 764350- 4354.

Vernon RB, Mueller CH, Herr JC, Feuchter FA, Eddy EM (1982): Epididymal secretion of a mouse sperm surface component recog- nixed by a monoclonal antibody. Biol Reprod 26:523-535.

Wewer UM, Tichy D, Damjanov A, Paulsson M, Damjanov 1 11987): Distinct antigenic characteristics of murine parietal yolk sac lami- nin. Dev Biol 121:397-407.


White MG, Huang YS, Tres LL, Kierszenbaum AL (1982): Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats. J Reprod Fertil 66475-484.

Wolf DP, Sokoloski JE, Dandekar P, Bechtol KB (1983): Character- ization of human sperm surface antigens with monoclonal antibodies. Biol Reprod 29:713-723.

Wong PYD, Tsang AYF (1982): Studies of the binding of a 32 K rat

epididymal protein to rat epididymal spermatozoa. Biol Reprod 27: 1239-1246.

Wooding FBP (1973): The effect of Triton X-100 on ultrastructure of ejaculated bovine sperm. J Ultrastruct Res 42:502-516.

Young LG, Gould Kg, Hinton BT (1987): Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm. Gamete Res 18:163-178.

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

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