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Identification and Characterization of an Antigen Recognized by Monoclonal Antibody TRA 54 in Mouse Epididymis and Vas Deferens

HIROMITSU TANAKA

YOSHITAKE NISHIMUNE

From the ^* Laboratory of Cytochemistry and Immunocytochemistry, Department of Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil; and the Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Correspondence to: Dr Luís Antonio Violin Pereira, Department of Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), PO Box 6109, 13083-970, Campinas, SP, Brazil (e-mail: lviolin{at}unicamp.br).

Received for publication April 7, 2004; accepted for publication July 9, 2004.

	Abstract

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Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.

     Key words: Testis, sperm antigens, cryptorchidism, castration, testosterone replacement

The alterations that occur in spermatozoa during epididymal maturation are essential for the success of fertilization (Yanagimachi, 1994). However, relatively little is known about the role of the epididymis and the modification of sperm in this process (Moore, 1998). The study of antigens expressed by cells of the male reproductive organs with the use of monoclonal antibodies (mAb) has contributed to our understanding of the role of these proteins in sperm maturation and the formation of functional sperm (Watanabe et al, 1992; Enders and May, 1994; Tsuchida et al, 1995; Kirchhoff et al, 1998). The mAb TRA 54, a monoclonal antibody raised by immunizing rats with mouse testicular germ cells, recognizes an antigen expressed by spermatocytes and spermatids of the seminiferous tubules of C57 BL/6 mice (Pereira et al, 1998). This antibody has been used to assess the dynamics of cytoplasmic bridges between spermatids (Ventelä et al, 2003) and to characterize the principal proteins involved in spermatogenesis (Deng and Lin, 2002).

In this study, we investigated some of the properties and the ontogenic expression of the antigen recognized by mAb TRA 54 in mouse epididymis and vas deferens. We also examined whether the antigen expressed in these organs was testosterone dependent and differed from the antigen molecule expressed in testicular germ cells.

	Materials and Methods

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Preparation of Monoclonal Antibody

The monoclonal antibody TRA 54 was obtained as described elsewhere (Pereira et al, 1998).

Animals

Male C57 BL/6 mice were housed under standard conditions of temperature and light with free access to water and food. The mice were sacrificed by cervical dislocation, and the organs were processed immediately for subsequent immunohistochemical or immunoblotting analysis. All of the animal experiments were approved by the Committee for Ethics in Animal Experimentation of the Institute of Biology, UNICAMP.

Ontogeny

To study the ontogenic development of the antigen recognized by mAb TRA 54, an immunohistochemistry analysis was performed in the paraffin-embedded testes, epididymis, and vas deferens collected from mice 1, 5, 12, 16, 24, 30, and 60 days old. Testes, epididymis (caput, corpus and cauda segments), vasa deferentia, brain, liver, kidneys, spleen, and seminal vesicles from adult C57 BL/6 (60 days old) mice were collected for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The freshly collected organs were homogenized in 10 mM Tris-HCl, pH 7.4, containing 10 mM EDTA (Mallinckrodt, Paris, Ky), 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium orthavonadate, 2 mM Phenylmethylsulfonylfluoride (PMSF), and 0.1 mg of aprotinin/mL (Sigma Aldrich, St Louis, Mo).

Castration and Experimental Cryptorchidism

To verify the effect of testis on epididymal TRA 54 antigen expression, 15-day-old mice were uni- or bilaterally orchidectomized by the abdominal route under anesthesia (Keenan and Thomas, 1975; Mirosevich et al, 1999), and the epididymis was replaced close to the scrotum. To distinguish the TRA 54 antigens produced by the differentiated germ cells (Pereira et al, 1998) and to eliminate the influence of sperm on antigen expression in the epididymis and vas deferens, 15-day-old mice underwent experimental uni- or bilateral cryptorchidism (Nishimune et al, 1978; Pereira et al, 1998). The mice were sacrificed 3 months after the operations, and the epididymis and vasa deferentia were removed, fixed in Bouin solution, and embedded in paraffin for immunohistochemistry analysis. In addition, epididymis from cryptorchid mice were collected for SDS-PAGE and immunoblotting.

Hormonal Replacement

The effect of androgens on the expression of epididymal and vas deferens antigens recognized by mAb TRA 54 was assessed in bilaterally orchidectomized mice administered replacement testosterone. Thirty days after orchidectomy, a 3 mg/kg daily dosage of testosterone (Deposteron–testosterone cipionate; Novaquímica, São Paulo, Brazil) in mineral oil (Sigma Aldrich) was injected intraperitoneally for 30 days. The epididymis and vasa deferentia were subsequently collected, fixed in Bouin solution, and embedded in paraffin for immunohistochemical analysis. The sham-operated control mice received mineral oil alone.

Epididymal Sperm Isolation

Epididymis from 60-day-old mice were carefully dissected, and sperm was allowed to disperse in 200 µL of phosphate-buffered saline (PBS; Dulbecco 1x PBS solution; Irvine Scientific, Santa Ana, Calif) at 37°C by cutting the epididymal cauda. The suspension of sperm was washed 3 times in PBS by centrifugation for 10 minutes at 200 x g. For immunohistochemical analysis, the sperm pellet was resuspended in 50 µL of PBS, and 5-µL samples were allowed to dry on the glass slide. The samples were fixed with iced methanol for immunostaining.

For SDS-PAGE and immunoblotting, the sperm pellet was resuspended and homogenized in 100 µL of 10 mM Tris-HCl buffer, pH 7.4, as described in the section "Ontogeny."

Immunohistochemistry

Immunohistochemistry was performed as described elsewhere (Pereira et al, 1998). All the reagent dilutions were made in PBS containing 1% bovine serum albumin (Sigma Aldrich). Sections were blocked with 20% normal goat serum and incubated overnight at 4°C with ascites fluid containing mAb TRA 54 (diluted 1:4000), followed by incubation for 1 hour with goat anti-rat biotinylated secondary antibody (Dako A/S, Glostrup, Denmark) diluted 1:200 and then with Strept ABC kit (Dako A/S) for 45 minutes. The reaction was developed by incubating the sections with hydrogen peroxide and diaminobenzidine (Sigma Aldrich); the sections were counterstained with hematoxylin.

SDS-PAGE and Immunoblotting

The homogenates of tissues and sperm were centrifuged (10 000 x g), and the protein concentrations of the supernatants were determined with a Bradford protein assay kit (Bio-Rad, Richmond, Calif). Aliquots of each homogenate (100 µg per lane) were diluted in sample buffer to perform the SDS-PAGE with 7.5% gels prepared according to Laemmli (1970). The proteins were transferred electrophoretically to polyvinylidenedifluoride (PVDF) membranes (Millipore, Bedford, Mass), which were then blocked with 5% low-fat dry milk for 1 hour and washed with 0.05% Tween–Tris buffer–saline (TBS-T). The membranes were subsequently incubated with mAb TRA 54 (diluted 1:2500 in TBS-T) overnight, washed in TBS-T, and finally incubated with peroxidase anti-rat immunoglobulin (Dako A/S) for 1 hour. Immunoreactive bands were detected by incubating the membranes with 0.03% hydrogen peroxide and 0.05% diaminobenzidine in 50 mM Tris-HCl, pH 7.2. To determine whether the epitope of the antigen detected by mAb TRA 54 resided in sugar moieties, one half of the blotted membrane filter was treated with 20 mM of sodium m-periodate (Sigma Aldrich) for 10 minutes before immunoreaction, as described by Woodward et al (1985).

	Results

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Ontogenic Expression of Antigen Molecules Recognized by mAb TRA 54

No mAb TRA 54 immunohistochemical staining was seen in sections of the epididymis and vas deferens of mice younger than 24 days old (Figure 1a through e). At 24 days of age, a specific positive reaction was seen in epididymal caput and corpus segments (Figure 1f). Control reactions without TRA 54 showed no positive labeling (Figure 1g). Positive staining was seen on the epithelial cell surfaces of the caput and corpus (Figure 1f through h), but not in initial and caudal segments of the epididymis (Figure 1f, h, and i). The stereocilia of vas deferens epithelial cells were strongly stained, whereas the apical cytoplasm of these cells showed weak labeling with mAb TRA 54 (Figure 1j). Epididymis of adult (60 days old) mice showed strong specific staining in the whole epididymal extension, except the initial segment (Figure 1k). Control reactions without TRA 54 showed no positive labeling (Figure 1l). The staining in the caput epithelial cells was seen in the supranuclear cytoplasm (Figure 1m), whereas in the corpus and caudal segments, only the surface of the epithelia were stained (Figure 1n). Luminal contents and sperm present in all the segments were labeled (Figure 1m and n). The stereocilia and cytoplasm of the epithelial cells in vas deferens were also strongly stained (Figure 1o).

View larger version (130K): [in this window] [in a new window]   Figure 1. Immunohistochemical staining with TRA 54 in cross sections of epididymis and vas deferens taken from mice (a–e) 16, (f–j) 24, and (k–o) 60 days old. (a, f, k) Whole epididymis (initial segment [IS], caput, corpus, and cauda segments); (b, g, l) epididymal caput segment without TRA 54 (negative control); (c, h, m) epididymal caput; (d, i, n) epididymal cauda; (insert) isolated sperm from epididymal cauda; (e, j, o) vas deferens. No staining was verified on organs from 16-day-old mice. The initial segment of the epididymis showed no sign of the antigen expression at any stage. At 24 days of age, the immunochemical staining was verified in the surface of the epididymal epithelial cells of the caput and in the cytoplasm and surface of the vas deferens epithelial cells. At 60 days of age, immunochemical staining was verified in the apical cytoplasm, or the surface of epithelial cells of the epididymis and vas deferens, or both, as well as in luminal contents and sperm. Scale bars: (a) 460 µm; (b–e) 50 µm; (f) 770 µm; (g–j) 50 µm; (k) 1250 µm; (l–o) 50 µm.

Antigen Expression in Epididymis and Vas Deferens of Cryptorchid Testis

To determine whether the antigenic molecule recognized by mAb TRA 54 in epididymis and vas deferens was expressed by the epithelium per se, we induced cryptorchidism at 15 days of age to suppress the differentiation of germ cells. Thirty days later, antigen expression in epididymis and vas deferens was assessed. The morphological evaluation of cryptorchid testis confirmed the classical description; that is, no differentiated sperm cell was present in the seminiferous tubules.

The pattern of antigen expression in epididymis of either bilaterally or unilaterally cryptorchid testis (Figure 2a) was the same in normal adult mice epididymis, except that no sperm was observed in the lumen of the epididymis with cryptorchid testis (Figure 2b through d). In the same way, the stereocilia and cytoplasm of the epithelial cells in vas deferens were strongly stained (Figure 2e), similar to those seen in normal mice. Control reactions without TRA 54 showed no positive labeling (Figure 2b).

View larger version (118K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining with mAb TRA 54 in a cross section of ipsilateral epididymis and vas deferens. (a–e) Unilaterally cryptorchid mice; (f–j) bilaterally castrated mice; (k–o) bilaterally castrated mice with testosterone replacement. (a) Whole epididymis; (f, k) whole epididymis and vas deferens section; (b, g, l) epididymal caput segment without TRA 54 (negative control); (c, h, m) epididymal caput; (d, i, n) epididymal cauda; (e, j, o) vas deferens. Immunohistochemical pattern of the antigen expression in epididymis and vas deferens from cryptorchid mice was similar to the adult (60 days old) mice, except that there was no sperm in the lumen of the ducts. The antigen was not detected in epididymis from bilaterally castrated mice, and vas deferens showed a remainder antigen in the surface of the epithelial cells. Testosterone replacement recovered the immunohistochemical pattern of the antigen expression in both epididymis and vas deferens. Scale bars (a) 1500 µm; (b–e) 50 µm; (f) 620 µm; (g–j) 50 µm; (k) 1100 µm; (l–o) 50 µm.

Hormonal Control of Antigen Expression in Epididymis and Vas Deferens

Immunohistochemical staining of the epididymis and vas deferens in bilaterally castrated mice showed no positive reaction at any region of the ducts (Figure 2f). Control reactions without TRA 54 showed no positive labeling (Figure 2g). Epithelia of the epididymis was atrophic in all segments (Figure 2g through i). Vas deferens was also atrophic (Figure 2j). In contrast, unilateral orchidectomy had no effect on the expression of the antigen or morphology either in epididymides or in the vas deferens of both sides. The daily administration of testosterone to bilaterally castrated mice for 30 days restored the normal expression of TRA 54 antigen and morphology of the epididymis and vas deferens (Figure 2k), as seen in the animals of the control group. Control reactions without TRA 54 showed no positive labeling (Figure 2l). In the epididymis, antigen was strongly expressed at the supranuclear cytoplasm and surface of the epididymal epithelial cells, as well as in the luminal content of the epididymis (Figure 2m and n). Vas deferens showed that normal morphology and antigen expression was seen on the surface or stereocilia of epithelial cells; thereafter, sperm were absent (Figure 2o).

Western Blotting of Antigen Molecules Expressed by the Testis and Epididymis

In testis, a few immunoreactive bands from approximately 90 to 260 kd were detected with mAb TRA 54, whereas no positive bands were observed in blots of brain, liver, kidney, and spleen segments (Figure 3a). Immunoreactive bands from about 200 to 260 kd were detected in isolated sperm (Figure 3a) and epididymis, even though the signal was weak in isolated corpus and cauda (Figure 3a and b). In vas deferens, only a weak positive band at about 260 kd was observed (data not shown). Blotted bands treated with sodium metaperiodate inhibited immunoreactivity, indicating that the antigen epitope probably involved a sugar moiety (Figure 3b). Epididymal extracts from cryptorchid mice showed immunoreactive bands similar to those found in control epididymides (data not shown), despite the complete absence of the antigen in testis.

View larger version (63K): [in this window] [in a new window]   Figure 3. Western blot analysis with mAb TRA 54. Protein samples were electrophoresed and transferred to a nitrocellulose membrane and immunoblotted with mAb TRA 54. The positions of the molecular mass markers are indicated on the left. Control immunoblot without TRA 54 showed no bands (cont). (a) Testis (te), whole epididymis (epi), sperm isolated from epididymal cauda (sp), brain (br), liver (lv), kidney (kd), and spleen (spl). Immunoreactive bands of approximately 90 to 260 kd were detected in testicular extract, whereas bands of about 200 to 260 kd were detected in epididymal and sperm extracts. Arrows indicate the endpoints of the main band complexes. (b) Caput (cap), corpus (cor), and cauda of epididymis (cau). The right half of the membrane was treated with sodium metaperiodate (*) and immunoblotted with TRA 54 antibody. Treatment of the membrane suppressed immunoreactivity.

	Discussion

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Spermatozoa in testicular fluid is known to have weak forward motility and cannot fertilize eggs, but the period spent in the epididymis leads to further maturation of the sperm and provides the ability to fertilize eggs because of the surface modifications by enzymes secreted by different regions of the epididymis (Dacheux et al, 1987; Hamil et al, 2000). In this study, we characterized the new pattern of expression of an antigen previously identified in germ cells by mAb TRA 54 (Pereira et al, 1998; Ventelä et al, 2003). This antigen is possibly secreted and released by mouse epididymis and vas deferens to the luminal sperm.

Expression of the TRA 54 antigen evaluated by immunohistochemistry was limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. This observation is in accordance with the concept that epithelial cells along the epididymis and vas deferens should have domains of specific functions regulated by cell-specific gene expression (Robaire et al, 2000; Rodriguez et al, 2001). This experiment confirmed the previous data showing TRA 54 antigen expression by spermatocytes and spermatids, but not by spermatozoa in the seminiferous tubules (Pereira et al, 1998). Intriguingly, the TRA 54 antigen was detected in the epididymis and vas deferens luminal spermatozoa, suggesting that molecules containing antigen epitopes recognized by mAb TRA 54 could be released from epididymis and vas deferens epithelial cells and could bind to the surfaces of spermatozoa during their transit through these ducts.

Many reports show that molecules involved in sperm-egg fusion (Boué et al, 1995; Cornwall and Hann, 1995; Poulton et al, 1996; Légaré et al, 1999; Syntin and Cornwall, 1999; Chu et al, 2000; Cohen et al, 2000, 2001; Liu et al, 2000; Gaudreault et al, 2001; Pauls et al, 2003), which differ in molecular mass from the TRA 54 antigen, are produced and released by columnar epithelia of the epididymis into the lumen and bind to the surface of spermatozoa during maturation in the duct. Many of them are also expressed by testicular germ cells (Syntin and Cornwall, 1999; Gaudreault et al, 2001; Pauls et al, 2003).

The epitope of TRA 54 antigen probably involves carbohydrate domains because it was sensitive to periodate treatment (Pereira et al, 1998). Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar. However, the presence of various immunoreactive bands might indicate that mAb TRA 54 recognizes a common epitope of different isoforms of the same molecule modified by different-sized carbohydrate chains. Similar variation within a given molecule has been reported (Hall et al, 1996; Syntin and Cornwall, 1999), and changes in the carbohydrate chains of glycoconjugates during spermatogenesis could explain the loss of immunoreactivity of some proteins in the acrosome of late testicular spermatids as a result of antigen masking (Toshimori et al, 1988; Pereira et al, 1998).

On the other hand, the antigens recognized by mAb TRA 54 identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells because epididymis from cryptorchid mice showed the normal pattern of antigen expression. Whereas antigen expression and immunoreactive band patterns were unaffected by cryptorchidism, bilateral castration of adult mice fully inhibited the expression of the antigen in epididymis and vas deferens from both sides but not in the unilateral orchidectomy. These findings, together with the testosterone replacement experiment in bilaterally castrated mice, confirmed that molecules recognized by mAb TRA 54 expressed in the epithelial cells of the epididymis and vas deferens is testosterone dependent. Approximately 48% of proteins released by the epididymal caput are produced in an androgen-dependent manner (Syntin et al, 1999; Robaire et al, 2000), and the molecules recognized by mAb TRA 54 synthesized in epididymal epithelial cells belongs to this androgen-dependent category.

Furthermore, immunohistochemistry showed expression of the TRA 54 antigen in the cytoplasm of epididymal epithelial cells on 24-day-old and adult mice, but not during younger developmental stages. It means that the TRA 54 molecule in the epididymal epithelial cells is essential for functional activity of the epididymis as male reproductive organ, rather than as structural or intrinsic molecules committed to epithelial cell survival.

The results described here clearly show that the molecules expressed by the epithelia of the epididymal caput and vas deferens detected by mAb TRA 54 are expressed independently by those found in testicular germ cells. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind to spermatozoa moving down through the epididymis and vas deferens ducts. Expression of this antigen in vas deferens suggests that this duct could participate in maturation, as well as storage and conduction to the spermatozoa residing in the lumen. Whether the molecules expressed during sperm cell differentiation in the seminiferous tubules are different isoforms of the same molecules expressed in the epididymis and vas deferens epithelial cells or are different molecules showing the same antigen epitopes recognized by mAb TRA 54 is being investigated, although these molecules seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertile.

	Acknowledgments

 
This work is part of a thesis submitted by K.F.A. to the Institute of Biology, State University of Campinas, in partial fulfillment of the requirements for a master's degree and was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank Mr Baltazar Pereira de Paula for helping to care for the animals and Dr Stephen Hyslop for revising the English.

	Footnotes

 
^? Supported by grants 1150/01 and 1359/02 from FAEP-UNICAMP, grants 2001/12773-7 and 2002/04336-9 from The State of São Paulo Research Foundation (FAPESP), and grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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