ARTICLE |
Correspondence to: Louis Hermo, Dept. of Anatomy and Cell Biology, McGill University, 3640 University St., Montreal, QC, Canada H3A 2B2. E-mail: Lhermo@med.mcgill.ca
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Summary |
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The epithelial cells of the testis are involved in the production, differentiation, and sustenance of sperm, and those of the epididymis play a major role in sperm maturation, protection, and storage. These tissues express various proteins that respond differently to androgens. Cathepsin A is a multifunctional lysosomal carboxypeptidase that also functions as a protective and an activator protein for neuraminidase and ß-galactosidase. In this study, cathepsin A was immunolocalized by light and electron microscopy using a polyclonal affinity-purified antibody on the testis and epididymis of normal, orchidectomized with or without testosterone supplementation, efferent duct-ligated, and hypophysectomized adult rats. In normal rats, cathepsin A expression was noted in lysosomes of Sertoli and Leydig cells but not in germ cells of the testis, as well as nonciliated cells of the efferent ducts. In the epididymis, a cell- and region-specific distribution of cathepsin A was noted. In experimentally treated animals, no changes were noted in the expression of cathepsin A. Immunolabeling of tissues examined at the electron microscopic level revealed that lysosomes were reactive. These data indicate cell- and region-specific expression of cathepsin A in cells of the testis and epididymis and also indicate that cathepsin A expression is not regulated by testicular or pituitary factors. (J Histochem Cytochem 48:11311146, 2000)
Key Words: lysosomal enzymes, epididymis, testis, cathepsin A
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Introduction |
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The LYSOSOMAL PROTECTIVE PROTEIN/CATHEPSIN A (cathepsin A) is a serine acid carboxypeptidase present in lysosomes, where it associates with and forms a fully functional and stable high molecular weight multienzyme complex with ß-D-galactosidase and N-acetyl--neuraminidase (
Cathepsin A deficiency in humans causes a lysosomal storage disease called galactosialidosis, which is characterized by the deficiency of both neuraminidase and ß-galactosidase. The importance of lysosomal enzymes as constituents of lysosomes is emphasized in lysosomal storage diseases in which there is an increase in the number, volume, and distribution of lysosomes, resulting in abnormalities to cells affected and serious consequences to the health of the animal (
We have previously examined the differential expression of several lysosomal enzymes within lysosomes of various cell types of the testis and epididymis (
The purpose of the present study was to immunolocalize cathepsin A in the different cell types of the testis and epididymis of normal adult rats at the light and electron microscopic levels to determine its cell- and region-specific distribution in these tissues. Orchidectomy studies with and without testosterone replacement, ligation of the efferent ducts, and hypophysectomy were used to verify the role, if any, of testicular and pituitary factors in the expression of cathepsin A in these tissues.
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Materials and Methods |
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Animals and Protocols
Adult male SpragueDawley rats (350450 g) were obtained from Charles River Laboratory (St-Constant, QC, Canada). The animals were subdivided into six groups. The first group consisted of normal untreated animals. Bilateral ligation of the efferent ducts constituted the second group. After an IP injection of sodium pentobarbital (Somnitol; MTC Pharmaceuticals, Hamilton, ONT, Canada), the testes and epididymides of four rats were exposed through an incision of the anterior abdominal wall. A ligature was placed around both right and left efferent ducts at a site close to the rete testis. The animals were sacrificed at 3, 7, 14, or 21 days after surgery. Bilateral orchidectomy constituted the third group. After anesthesia, both testes of four rats were removed after a ligature was placed around the efferent ducts and testicular blood vessels. The animals were sacrificed at 3, 7, 14, or 21 days after surgery. Bilaterally orchidectomized rats that received three 6.2-cm testosterone-filled implants constituted the fourth group. Testosterone-filled polydimethyl-siloxane (silastic) implants were prepared according to the method of
The fifth group consisted of hypophysectomized rats with four rats per group being sacrificed at 7, 14 21, or 28 days after hypophysectomy. The sixth group consisted of four sham-operated animals, two of which received three empty 6.2-cm-long implants, with all rats being sacrificed 14 days after initiation of the experiment.
All experimentation was carried out with minimal stress and discomfort to the animals both during and after surgery, as set up by the guidelines and approval of the University Animal Care Committee.
Tissue Preparation for LM Immunocytochemistry
At the end of each experiment, the testes (normal and hypophysectomized animals) and epididymides (all procedures) of each rat were fixed by perfusion with Bouin's fixative via the abdominal aorta for 10 min. After perfusion, the testes and epididymides were removed. The latter were cut so that given sections would include all of the major regions of the epididymis, i.e., the initial segment, intermediate zone, caput, corpus, and cauda (
LM Immunostaining
Sections 5 µm thick were cut and mounted on glass slides. They were then deparaffinized with xylene and hydrated in graded concentrations of ethanol (from 100% to 50%). During hydration, residual picric acid was neutralized by immersing the tissues in 70% ethanol containing 1% lithium carbonate for 5 min. To inactivate any endogenous peroxidase activity, the tissue sections were incubated for 5 min in 70% ethanol containing 1% (v/v) hydrogen peroxide. After hydration, the sections were incubated (5 min) in a 300 mM glycine solution to block free aldehyde groups. The tissue was then blocked with 40 ml of 10% goat serum diluted in TBS (20 mM Tris-HClsaline containing 0.1% bovine serum albumin) at pH 7.4 for 25 min at room temperature. The slides were then washed with Tween buffer solution (TBS with 0.1% Tween-20) (TWBS).
A dilution factor of 1:100 in TBS was used for the polyclonal anti-cathepsin A antibody. The antibody was provided by Dr. Y. Suzuki (Tokyo Metropolitan Institute of Medical Science; Tokyo, Japan), and its characterization and specificity have been documented in
The final reaction product was obtained by incubating the slides for 10 min in 250 ml of TBS containing 0.03% hydrogen peroxide, 0.1 M imidazole, and 0.05% diaminobenzidine tetrahydrochloride (DAB), pH 7.4. The sections were counterstained with 0.1% methylene blue (2 min) and then dehydrated in a graded series of ethanol solutions (30 sec each) and xylene (3 min). Coverslips were mounted on glass slides with Permount. Incubation with normal rabbit serum at a dilution of 1:100 in TBS and incubation of tissues in secondary antibody alone served as controls.
EM Immunocytochemistry
Four additional adult male SpragueDawley rats (350450 g) obtained from Charles River Laboratories were anesthetized with an IP injection of sodium pentobarbital (Somnitol). The testes and epididymides of the rats were fixed by perfusion through the abdominal aorta for 10 min with 0.5% glutaraldehye in 0.1 M phosphate buffer containing 50 mM lysine at pH 7.4. After fixation, the testes and epididymides were removed, cut into small pieces (1 mm3), immersed for 2 hr in the same fixative at 4C, washed with 0.15 M PBS containing 4% sucrose (pH 7.4) at 4C, and then treated with PBS containing 4% sucrose and 50 mM NH4Cl for 1 hr at 4C. The tissue was then washed, dehydrated in graded methanol up to 90%, and embedded in LR White.
Ultrathin sections of selected testicular and epididymal regions were mounted on 300-mesh formvar-coated nickel grids. Each section was floated for 15 min on a drop of 20 mM Tris-HCl-buffered saline containing 5% bovine serum albumin (TBS) and then incubated for 1 hr on 15- µl drops of a polyclonal anti-cathepsin A antibody diluted 1:5 in TBS. Sections were washed four times for 5 min each in TWBS, transferred for 15 min to drops of TBS containing 5% bovine serum albumin, and incubated for 30 min on 20-µl drops of goat anti-rabbit IgG antibodies conjugated to 15-nm colloidal gold particles. The sections were subjected to three 5-min washes in TWBS followed by 5-min washes in distilled water. Sections were counterstained with uranyl acetate in 30% ethanol and lead citrate. Photographs were taken on a Philips 400 electron microscope. Normal rabbit serum at a dilution of 1:5 served as a control.
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Results |
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Light Microscopy
In the testis, expression of cathepsin A was noted in Sertoli cells at all stages of the cycle of the seminiferous epithelium. Whereas the immunoperoxidase reaction was intense in the basal region of Sertoli cells at Stages IVIII and XIIIXIV, it was weak at Stages IXXII (Fig 1a1d). There was no apparent staining of germ cells at any stage of the cycle. Late residual bodies, as judged by their position near the base of seminiferous epithelium at Stages IXXII, were intensely reactive (Fig 1c and Fig 1d), but early residual bodies situated closer to the lumen were unreactive. In the interstitial space, Leydig cells were identified as those cells showing several intensely reactive lysosomes, whereas macrophages known to contain many lysosomes appeared to be represented by those cells showing intense cytoplasmic staining (Fig 1a and Fig 1d).
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In the efferent ducts, the supranuclear lysosomes of nonciliated cells were intensely reactive (Fig 2a and Fig 2b). In the epididymis, expression of cathepsin A in principal cells was region-specific. Many reactive lysosomes were noted in the proximal and distal initial segment, where they occupied the principal cell's supranuclear region (Fig 3a, Fig 3b, Fig 4, and Fig 5). The intermediate zone, characterized by the presence of giant endosomes located in the apical region of principal cells, revealed immunostaining that differed between its proximal and distal regions (Fig 4 and Fig 6). The former showed many reactive lysosomes that occupied a distinct region over the nucleus of the principal cell (Fig 6), whereas few reactive lysosomes were noted in the distal region (Fig 4). A difference was also noted between the proximal and distal caput regions. In the proximal region, many reactive lysosomes were noted in the majority of principal cells (Fig 7). In the distal region, many lysosomes of some principal cells were intensely reactive, whereas others showed few reactive lysosomes or none at all (Fig 8). Principal cells of the corpus and proximal cauda epididymidis revealed many intensely reactive lysosomes (Fig 9 and Fig 10).
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Narrow cells of the initial segment and intermediate zone were intensely reactive (Fig 3 Fig 4 Fig 5 Fig 6), as were clear cells of the caput, corpus, and cauda regions (Fig 7 and Fig 8). In contrast, basal cells showed a region-specific expression. Many intensely reactive basal cells were noted in the distal initial segment (Fig 5) and proximal intermediate zone (Fig 6), followed by a progressive decrease from the caput to the cauda region (Fig 7, Fig 8, Fig 9a, Fig 9b, and Fig 10).
Electron Microscopy
Gold particles were noted over the lysosomes of those cells that were reactive as seen in the light microscope, examples of which are Sertoli cells of the testis (Fig 11), principal cells of the initial segment (Fig 12) and corpus (Fig 13), epididymidis, and clear cells of the caput region (Fig 14). Not illustrated are nonciliated and Leydig cells. There was no labeling of the coated pits or vesicles, endosomes, and multivesicular bodies (MVBs) of these cells. In general, the pattern of immunogold labeling of lysosomes mimicked that noted for the immunoperoxidase reaction, because corresponding cells and regions that were intensely or weakly reactive in the light microscope revealed many or few gold particles in the electron microscope, respectively, further attesting to the specificity of the reaction because different fixatives and embedding media were utilized in each case.
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In an attempt to determine whether or not circulating or luminal testicular factors or pituitary factors regulated the expression of cathepsin A in the epididymis, various experimental protocols were performed. After ligation of the efferent ducts for up to 21 days, no difference was noted in the level of expression of cathepsin A in lysosomes of all cell types and in all the different regions (Fig 15 and Fig 16). This was also the case after 21 days of orchidectomy (Fig 17 and Fig 18). Twenty-eight days after hypophysectomy, no difference in cathepsin A expression was noted in Sertoli or Leydig cells of the testis (Fig 19 and Fig 20) or in epithelial cells of the epididymis (Fig 21 and Fig 22).
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Use of normal rabbit serum for LM immunostaining showed no staining over cells of the testis or epididymis, and EM immunolabeling revealed only background levels of gold particles over the cells, examples of which can be seen in
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Discussion |
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In this study, lysosomes of Sertoli cells were reactive for cathepsin A antibody (Table 1), as suggested by
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Sertoli cells of the testis are actively involved in endocytosis, which has been shown to account for the formation of lysosomes, whereby endosomes, formed by fusion of coated vesicles, progressively transform into multivesicular bodies (MVBs) which in turn evolve into lysosomes (
The absence of cathepsin A expression in lysosomes of germ cells (Table 1) is not surprising because several other lysosomal enzymes, such as ß-hexosaminidase A and cathepsin B, were not noted in germ cells (
Lysosomal enzymes have been localized within lysosomes of Leydig cells (
The nonciliated cells of the efferent ducts express cathepsin A (Table 1), and other enzymes (
Epididymis
In the present study, the lysosomes of principal cells showed a region-specific distribution for cathepsin A along the length of the epididymis with most regions showing many reactive lysosomes except for the intermediate zone (Table 2; Fig 23). Furthermore, in the distal caput region, whereas some principal cells showed many reactive lysosomes, others showed few or none, representing a checkerboard-like staining pattern also noted for expression of various other synthesized proteins (
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Clear cells are noted for their role in the endocytosis of various substances from the epididymal lumen (
Narrow cells are involved in the acidification of the epididymal lumen of the initial segment and intermediate zone (
Basal cells are present along the entire length of the epididymis, where they form an incomplete mesh-like barrier at the periphery of the tubule, and by expressing various GSTs may protect sperm from harmful electrophils (
In the present study, the cathepsin A-reactive structures in nonciliated, principal, and clear cells of normal animals were confirmed to be lysosomes rather than endosomes or MVBs, on the basis of similarities in their shape, position, and appearance to similar structures confirmed to be lysosomes from earlier studies employing morphology, tracer studies, and acid phosphatase cytochemistry (
In nonciliated and various epididymal epithelial cells (
Region-specificity has also been noted for other lysosomal enzymes in the case of nonciliated, principal, clear, and narrow cells (
In the present study, no noticeable difference was observed in the expression of cathepsin A in the different cell types of the epididymis after efferent duct ligation, hypophysectomy, or orchidectomy, with or without testosterone supplementation. These data indicate that cathepsin A is not regulated in these cell types by testicular or pituitary factors. This is in direct contrast to many proteins synthesized by the epididymal epithelial cells that have been shown to be regulated by luminal or circulating testicular factors as well as by pituitary factors (
In the present study, electron microscopic analysis on experimental animals was not performed because we assumed that cathepsin A was still localized to lysosomes, as evidenced from the light microscopic analysis of a reaction over punctate supranuclear structures corresponding to the position and shape of lysosomes in cells of control animals.
Genetic lesions in the cathepsin A gene cause severe loss of neuraminidase activity and render ß-galactosidase susceptible to rapid intralysosomal proteolysis, resulting in a combined enzyme deficiency that is the basis of the lysosomal storage disorder galactosialidosis. The latter is characterized by the lysosomal storage of sialylated oligosaccharides and glycopeptides in affected tissues (
In summary, cathepsin A was localized to Sertoli and Leydig cells but not to germ cells in the testis and to epididymal epithelial cells in a cell- and region-specific manner. In addition, cathepsin A expression does not appear to be regulated by testicular or pituitary factors.
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Acknowledgments |
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Supported by the Medical Research Council of Canada.
We thank Dr Y. Suzuki for the cathepsin A antibody, which he generously provided to us. We thank Jeannie Mui, Mathilda Cheung, P.B. Mukopadhyay, and Jodi Fox for expert technical assistance.
Received for publication October 20, 1999; accepted March 15, 2000.
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