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From the Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
| Correspondence to: Dr Louis Hermo, Department of Anatomy and Cell Biology, McGill University, 3640 University Street Room 1/33, Montréal, Québec, Canada, H3A 2B2 (e-mail: louishermo{at}mcgill.ca). |
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Abstract |
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Key words: Light microscopy, orchidectomy, ligation, hypophysectomy, immunocytochemistry
In the epididymis, the epithelial cells lining the duct include principal, narrow, clear, and basal cells, with each partaking in endocytosis in varying degrees (Moore and Bedford, 1979; Hermo and Robaire, 2002). In principal cells, lysosomes have a distinct morphological appearance and integral membrane proteins in the different epididymal regions (Hamilton, 1975; Robaire and Hermo, 1988; Suarez-Quian et al, 1992). In addition, various lysosomal enzymes are expressed in lysosomes of the epididymal epithelial cells, with some showing cell type-specific and region-specific variations, suggesting substrate specificity with regard to the turnover of proteins within the lysosomes of these cells (Hermo et al, 1992; Tomomasa et al, 1994; Abou-Haila et al, 1996).
The regulation of many epididymal epithelial functions has been documented over the years to be dependent on androgens, and recently, on estrogen, especially in efferent ducts, where it plays a role in the absorption of fluid from the lumen (Cornwall and Hann, 1995; Orgebin-Crist et al, 1996; Hess et al, 2002). Androgens also regulate the expression and activity of several lysosomal enzymes (Cornwall et al, 2002). However, studies of the regulation of lysosomal enzyme expression in individual cell types and regions of the epididymis have not been performed, with few exceptions (Luedtke et al, 2000).
Sulfated glycoprotein-1 (SGP-1), also referred to as prosaposin, has been noted in the epididymis by means of Northern blot analysis and in situ hybridization (Sylvester et al, 1989; Sun et al, 1994). Immunocytochemical studies with light and electron microscopy have localized SGP-1 to the lysosomes of epithelial cells (Hermo et al, 1992). SGP-1 is proteolytically cleaved in lysosomes into saposins A, B, C, and D, which are sphingolipid-binding proteins that function as activators for lysosomal enzymes involved in the hydrolysis of sphingolipids (Kretz et al, 1990; O'Brien and Kishimoto, 1991). On the other hand, cathepsins are lysosomal proteolytic enzymes present in cells of many tissues, and play a role in the intracellular degradation of exogenous and endogenous proteins (Kirschke et al, 1980; Kominami et al, 1991). Cathepsin D, an aspartyl endopeptidase, has a molecular weight of 42 kd and an optimal activity of pH 3.8 (Srivastava and Ninjoor, 1982). In humans, cathepsin D has been localized to lysosomes of epithelial cells of the corpus epididymidis (Raczek et al, 1995), whereas in rats it is expressed in an epididymal cell type and region-specific manner (Igdoura et al, 1995).
In a previous study we noted that SGP-1 expression in the efferent ducts was not dependent on luminal or circulating testicular factors, but was regulated by a pituitary factor (Rosenthal et al, 1995). Thus, it is of interest to determine whether this is also true for SGP-1 expression in the different cell types and regions of the epididymis. Likewise, it had been demonstrated that enzymatic activity of cathepsin D in the epididymis increased after orchidectomy but decreased after testosterone treatment (Mayorga and Bertini, 1982). However, the specific cell types and regions of the epididymis affected were not determined.
Thus, the purpose of the present study was to examine the regulation of SGP-1 and cathepsin D expression in the epididymis after various experimental protocols on adult rats. Both enzymes have been localized by us in control animals in earlier studies (Hermo et al, 1992; Igdoura et al, 1995). The protocols included orchidectomy with or without immediate testosterone replacement, efferent duct ligation, and hypophysectomy. The epididymides of rats were fixed with Bouin fixative, embedded in paraffin, and sections of the tissues were subsequently used for immunocytochemical analysis with light microscopy.
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Materials and Methods |
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The fifth group consisted of hypophysectomized rats with 4 rats per interval being killed at 7, 14, 21, and 28 days after hypophysectomy. The sixth group consisted of 4 sham-operated animals, 2 of which received 3 empty 6.2-cm-long implants, with all rats being killed 14 days after initiation of the experiment.
All experimentation was carried out with minimal stress and discomfort being placed on the animals both during and after surgery according to the guidelines and approval of the university animal care committee.
Tissue Preparation for Light Microscopy Immunocytochemistry At the end of each treatment described above, the epididymides of each Sprague-Dawley rat were fixed by perfusion with Bouin fixative via the abdominal aorta for 10 minutes. Following perfusion, the epididymides were removed and cut so that sections would include all the major regions of the epididymis (ie, initial segment, intermediate zone, caput, corpus, and cauda; Hermo et al, 1991b). The tissue was then immersed in Bouin fixative for 72 hours, after which it was dehydrated and embedded in paraffin.
Light Microscopy Immunostaining
Sections (5 µm thick) were cut and mounted on glass slides. They were
then deparaffined with xylene and hydrated in graded concentrations of ethanol
(from 100% to 50%). During hydration, immersing the tissues in 70% ethanol
containing 1% lithium carbonate for 5 minutes neutralized residual picric
acid. Inactivation of any endogenous peroxidase activity, use of glycine
solution in order to block free aldehyde groups, blocking with goat serum, and
washing with Tween buffer solution was performed as described previously
(Hermo et al, 1992).
A dilution factor of 1:20 and 1:100 in Tris-buffered saline (TBS) was used for the polyclonal anti-cathepsin D and anti-SGP-1 antibody, respectively. Dr M.D. Griswold initially provided us with the anti-SGP-1 antibody, whereas Dr C.R. Morales provided us with anti-SGP-1 antibody more recently. Both antibodies are well-characterized and described by Sylvester et al (1989) and Morales et al (2000). The anti-cathepsin D antibodies were purchased from Calbiochem (La Jolla, CA) and DAKO (Carpinteria, CA), and we had used them in a previous study of the epididymis (Igdoura et al, 1995). Each tissue section was incubated in the primary antibody for 1.5 hours. After incubation, the sections were immersed in Tween, blocked with goat serum, and subsequently incubated with goat anti-rabbit immunoglobulin G (IgG) conjugated to peroxidase (Sigma, St Louis MO) at a dilution of 1:250 in TBS and incubated for 30 minutes at 35°C in a humidified incubator. After incubation with a secondary antibody, the tissue was washed by immersion in 4 wells of Tween buffer solution for 2 minutes each.
The final reaction product was obtained by incubating the slides for 10 minutes 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 counter-stained with 0.1% methylene blue (2 minutes) and then dehydrated in a graded series of ethanol solutions (30 seconds each) and xylene (3 minutes). Cover slips were mounted onto glass slides using Permount. Incubation with normal rabbit serum at a dilution of 1:100 in TBS and incubation of tissues in secondary antibody alone, without primary antibody, served as controls.
Routine Electron Microscopy Analysis
An additional 9 adult male Sprague-Dawley rats (350–450 g) were used
in this study for routine electron microscopy analysis. Three of the animals
served as controls, and whereas another 3 were bilaterally orchidectomized,
the others had their efferent ducts ligated on both sides; all animals were
killed 14 days later. The epididymides of these animals were fixed by
perfusion with 2.5% glutaraldehyde in sodium cacodylate buffer via the
abdominal aorta for 10 minutes. Thereafter, the tissue was removed, cut into
the appropriate epididymal regions, and remained in buffer overnight. On the
following day, the tissue was postfixed in potassium
ferrocyanide–reduced osmium tetroxide
(Karnovsky, 1971), dehydrated
in alcohol and propylene oxide, and embedded in Epon; thick and thin sections
were cut and treated as described in our previous studies, with thin sections
being examined with a Philips electron microscope
(Hermo et al, 1991a).
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Basal cells also continued to express SGP-1 at all time points after orchidectomy, with the reaction product being seen as small, punctate granules at the base of the epithelium or as thin, dense bands encompassing the nucleus of the hemispherical basal cells and stretching along their thin, elongated processes (Fig. 1a–c). In the initial segment, narrow cells also displayed intense reactivity for anti-SGP-1 antibody (Fig. 1a), as did clear cells of the caput, corpus (Fig. 1b), and cauda (Fig. 1c) regions. The reaction product in these cells often appeared evenly distributed throughout their cytoplasm.
Orchidectomy followed by immediate testosterone supplementation (up to 21 days) also revealed no major changes to the expression of SGP-1 in epithelial cells of the entire epididymis. Principal cells displayed small, intensely reactive spherical lysosomes in all epididymal regions, and narrow, basal, and clear cells remained intensely reactive (Fig. 2a–c). The addition of testosterone to orchidectomized rats restored the epididymal tubule and size of the epithelial cells of the different regions to normal size. However, principal cells, in addition to small spherical lysosomes, continued to show large, spherical supranuclear and infranuclear lysosomal elements next to the nucleus (Fig. 2b and c).
At the various time points after efferent duct ligation (up to 21 days), numerous intensely reactive spherical lysosomes of principal cells were observed in all epididymal regions; narrow, clear, and basal cells also remained intensely reactive (Fig. 3a and b). As with orchidectomy, the large, spherical lysosomal elements in the supranuclear and infranuclear regions of principal cells of the epididymis were still prominent at the later time points after efferent duct ligation in the caput, corpus, and cauda regions of the epididymis (Fig. 3b).
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After hypophysectomy (4 weeks), changes in SGP-1 expression were not noted in any cell type of any given epididymal region (Fig. 4a and b). The presence of supranuclear and infranuclear large, spherical lysosomal elements was, however, evident in principal cells of the caput, corpus, and cauda epididymal regions (Fig. 4).
Expression of Cathepsin D in Epithelial Cells of the Epididymis After
Experimental Treatments
After orchidectomy, no major changes in cathepsin D expression were noted
in principal cells of the entire epididymis. There were few small reactive
lysosomes in the proximal initial segment
(Fig. 5a) and intermediate zone
(Fig. 5b) and more in the caput
(Fig. 5c), corpus
(Fig. 5d), and cauda
(Fig. 6a) regions, as noted in
control animals (Table).
However, with SGP-1 after various treatments, large, spherical lysosomal
elements were noted in the supranuclear and infranuclear regions of the
cytoplasm of principal cells in the caput, corpus, and cauda regions (Fig.
5c and
d;
Fig. 6a). Comparable to control
animals, narrow cells were intensely reactive for anti-cathepsin D antibody in
the initial segment (Fig. 5a)
and intermediate zone (Fig 5b)
at all time points after orchidectomy
(Table). Basal cells were
intensely reactive and prominent in the intermediate zone
(Fig. 5b) and moderately
reactive in the initial segment (Fig.
5a) and caput (Fig.
5c) regions, but unreactive in the corpus
(Fig. 5d) and cauda
(Fig. 6a) epididymidis
(Table). Clear cells continued
to be intensely reactive in the caput region
(Fig. 5c), comparable to that
noted in control animals. However, after orchidectomy, at various time points
clear cells became intensely reactive in the corpus
(Fig. 5d) and cauda
(Fig. 6a) regions, where they
were consistently unreactive in control animals
(Table).
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After hypophysectomy, cathepsin D expression was un-altered and comparable to that of control animals in principal, narrow, and basal cells (Fig. 6b, Table). As noted after orchidectomy, large, spherical supranuclear and infranuclear lysosomal elements were observed in principal cells (Fig. 6b). In addition, clear cells that were unreactive in control animals were intensely reactive after all time points after hypophysectomy (Fig. 6b, Table).
Orchidectomized animals supplemented immediately with testosterone (Fig. 6c), as well as efferent duct-ligated animals (Fig. 6d) also revealed no changes in cathepsin D expression in principal, narrow, and basal cells compared with that of control animals at all time points examined (Table). However, with both treatments, clear cells in corpus and cauda epididymides were completely unreactive (Fig. 6c and d), as was the case in control animals (Table).
When tissue sections were treated with normal rabbit serum or without the primary antibody, there was a complete absence of reaction over the epithelium or intertubular space. We have already published photographs of controls for SGP-1 and cathepsin D (Hermo et al, 1992; Igdoura et al, 1995), and similar images were consistently noted in all our many control preparations.
Electron Microscopic Observations of Principal Cells After
Orchidectomy and Efferent Duct Ligation
To further understand the nature of the large supranuclear and infranuclear
lysosomes, electron microscopy was used. At 14 days after efferent duct
ligation or orchidectomy, principal cells of the caput, corpus, and cauda
epididymidis revealed large membrane-bound electron-lucent, spherical
structures (Figs. 7 and
8). Such structures contained
flattened membranous profiles and whorls of various sizes and shapes embedded
in a relatively electron-dense, granular material (Figs.
7 and
8). Occupying a position next
to the nucleus, these structures were large, at times approximating that of
the nucleus. They were not that abundant and whereas 1 or 2 could be seen in
some principal cells, other cells showed none. Adjacent to these structures
were typical, electron-dense lysosomes, as well as multivesicular bodies
(Figs. 7 and
8); their size and relative
numbers did not differ from those noted in control animals. These large
supranuclear and infranuclear structures were immunostained with both
anti-SGP-1 and cathepsin D antibodies as seen with light microscopy,
indicating that they corresponded to lysosomal elements.
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Discussion |
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-dihydrotestosterone, are considered to
be the prime modulators of epididymal functions
(Robaire and Viger, 1995). However, in addition to the endocrine regulation mediated by circulating
hormones, lumicrine factors derived from the testis and entering the
epididymal lumen directly from the efferent ducts also must be considered
(Cornwall et al, 2002). In the present study, we examined the regulation of SGP-1 and cathepsin D in the various epithelial cell types of the epididymis. For SGP-1, no noticeable effect on expression was observed in any cell type of any region of the epididymis after the different experimental procedures. Principal cells revealed numerous small, intensely reactive spherical structures in their cytoplasm, which have been shown by electron microscopy immunocytochemistry to represent lysosomes (Hermo et al, 1992). Also evident in these cells after the various treatments was that some lysosomes were larger in size and occupied both the supranuclear and infranuclear cytoplasm, the significance of which will be addressed later. Narrow, clear, and basal cells also remained as intensely reactive as noted in control animals, with expression being evident throughout their cytoplasm. Such a reaction pattern has been shown to be due to the abundance of lysosomes present in the large clear cells and their relative abundance in the smaller-sized narrow and basal cells (Hermo et al, 1988, 1994; Adamali and Hermo, 1996).
Thus in the epididymis, SGP-1 expression does not appear to be regulated by androgens. This is in contrast to a number of proteins, including lysosomal enzymes, which are markedly reduced after orchidectomy (Mayorga and Bertini, 1982; Gupta and Setty, 1995; Abou-Haila et al, 1996). In addition, no effect on expression was noted after efferent duct ligation, eliminating a role for lumicrine factors on SGP-1 expression. Hypophysectomy also had no effect on SGP-1 expression in the epididymis, as was shown by Garrett et al (1991) for the caput epididymidis. This was also the case for SGP-2 (Apo J/clusterin), cystatin C, and cathepsin A, all of which were demonstrated to be unaffected in their expression after orchidectomy or hypophysectomy (Hermo et al, 2000; Luedtke et al, 2000; Cornwall et al, 2002).
In the efferent ducts, expression of SGP-1 within lysosomes was not dependent on luminal or circulating androgens, nor was it dependent on a luminal factor entering the ducts from the testis (Rosenthal et al, 1995). However, the absence of SGP-1 expression after hypophysectomy suggested that a pituitary factor, which may have a direct or indirect effect, is involved in its regulation in nonciliated cells of the efferent ducts (Rosenthal et al, 1995). Thus, different regulatory factors appear to come into play between the nonciliated cells of the efferent ducts and epithelial cells of the epididymis in the case of SGP-1. That androgens or hormones did not regulate SGP-1 expression in lysosomes of the epididymal epithelial cells suggests that it is constitutively expressed, independent of stimulatory or inhibitory regulatory factors. This is consistent with its highly conserved protein structure and ubiquitous expression (Collard et al, 1988; Morales et al, 1996, 1998).
Regulation of Cathepsin D in the Epididymis
The regulation of cathepsin D was markedly different from that of SGP-1.
Although there was no noticeable effect on cathepsin D expression after the
various experimental procedures in principal, narrow, and basal cells of the
entire epididymis, clear cells showed region-specific responses to the absence
of androgens (Table;
Figure 9). In control animals,
clear cells of the caput epididymidis were intensely reactive, but they were
unreactive in the corpus and cauda epididymidis
(Igdoura et al, 1995). In the
present study, clear cells of the caput region were unaffected in their
staining intensity by the various experimental procedures, however, those of
the corpus and cauda regions became intensely reactive after orchidectomy
(Table;
Figure 9). Administration of
testosterone to orchidectomized animals abolished the staining of clear cells,
which together with their absence of staining in efferent duct-ligated
animals, suggests that testosterone or one of its metabolites inhibits the
expression of cathepsin D in clear cells of these regions
(Table;
Figure 9). It is interesting
that the enzymatic activity of cathepsin D in the epididymis has been shown to
be increased above normal levels after orchidectomy and decreased after
testosterone treatment (Mayorga and
Bertini, 1982), coinciding with our present immunocytochemical
data. However, our study indicates that the specific cell type affected is the
clear cell and that it is affected in a region-specific manner. Other proteins
that demonstrated greater enzymatic activity in the epididymis after androgen
withdrawal, often in a region-specific manner, include glucuronidase, RNase
II, transforming growth factor ß-1, gamma glutamyl-transpeptidase III,
and SGP-2 (Mayorga and Bertini,
1982; Cyr and Robaire,
1992; Palladino and Hinton,
1994; Gupta and Setty,
1995; Desai and Kondaiah,
2000).
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Whereas androgens play a prominent role in regulating a variety of
epididymal functions, a role for estrogens has also been implicated in the
efferent ducts and epididymis, where estrogen receptors (ERs) and
ß have been located (Meistrich et al,
1975; Orgebin-Crist et al,
1983; Fisher et al,
1997; Hess et al,
1997). In fact, in mice, the corpus and cauda epididymidis
revealed a much denser labeling of the nuclei of clear cells with 3H-estradiol
than with testosterone, suggesting that estradiol may serve to modulate clear
cell function in the epididymis
(Schleicher et al, 1984).
In the present study, cathepsin D expression in clear cells after
orchidectomy became intense, but supplementing testosterone to these animals
resulted in a decrease in staining, as was observed in control animals. It
could be argued that estrogen rather than testosterone may suppress the
expression of cathepsin D in clear cells of the corpus and cauda epididymidis.
With acid phosphatase activity I, Nikkanen and Vanha-Perttula
(1977) also noted that there
was an increase in activity with estrogen treatment in the epididymis. Future
studies employing ER and ß mouse knockout models in conjunction
with immunocytochemistry would help resolve the role of estrogen on cathepsin
D expression in clear cells, studies that are beyond the scope of the present
investigation.
In the present study, we acknowledge that the immunostaining pattern of SGP-1 and cathepsin D reflects steady state protein levels and that our methods do not indicate differences in protein production. In addition, it is possible that protein degradation occurs following our different treatments, but that the proteins (degradation fragments) remain immunoreactive. If this is the case, biological function of each protein could be affected but based on immunocytochemistry, no changes in expression would be detected. Another approach such as Western blot analysis would reveal protein degradation and could be addressed in future studies along with quantitative electron microscopy immunocytochemistry. However, both of these approaches are beyond the scope of the present study.
Electron Microscopic Observations on Lysosomes of Principal Cells
After Various Experimental Procedures
Orchidectomy and hypophysectomy causes the epididymal weight to decrease
(Robaire et al, 1977),
accompanied by a decrease in luminal diameter of the tubules and decrease in
epithelial cell height (Orgebin-Crist et
al, 1975; Delongeas et al,
1987 Hermo and Papp,
1996). Principal cells are particularly sensitive to androgen
levels, showing morphological changes such as accumulation of lysosomes,
unlike that noted for the other epithelial cells, which appear to be
unaffected (Moore and Bedford,
1979).
For SGP-1 and cathepsin D alike, after all procedures, large spherical structures became evident in the supranuclear and infranuclear regions of principal cells. Such structures were highly reactive for anti-SGP-1 and anti-cathepsin D antibodies, suggesting that they corresponded to lysosomal elements. Using electron microscopy, these structures corresponded to large, membrane-bound spherical structures with electron-lucent content in which membranous profiles of various shapes and sizes and a granular material were evident. Such structures were not noted in control animals.
Although it is not fully clear as to why such structures become prominent, one hypothesis that could be proposed is the adverse effect that orchidectomy, efferent duct ligation, and hypophysectomy have on expression of a variety of proteins and genes in the absence of androgens, luminal factors, or both emanating from the testis (Robaire and Viger, 1995; Cornwall et al, 2002; Ezer and Robaire, 2002). In fact, several lysosomal enzymes such as acid phosphatase, N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, N-acetylhexosaminidase, and arylsul-phatase, show a decreased enzymatic activity after orchidectomy (Mayorga and Bertini, 1982; Gupta and Setty, 1995; Abou-Haila et al, 1996). Thus the reduction in this activity could result in the accumulation of substrates normally acted on by these enzymes and result in a phenotype as noted for various lysosomal storage diseases (Neufeld et al, 1975; Hammel and Alroy, 1995; Trasler et al, 1998). The latter have been shown to result in a dramatic increase in number and size of lysosomes and a change in their appearance and location within principal cells (Adamali et al, 1999a,b). However, in the present study, the effect was not noted to be as dramatic as it was for a given lysosomal gene knockout. This may be explained by enzymatic activities of these enzymes being merely reduced after the various procedures and not completely abolished (Mayorga and Bertini, 1982). Nevertheless, a somewhat similar phenotype for lysosomes in principal cells appears to occur as a result of the adverse effects of orchidectomy, efferent duct ligation, and hypophysectomy on expression of different proteins and genes in the epididymis. Finally, while the significance for up-regulation of cathepsin D after orchidectomy and hypophysectomy is not known, it may be suggested that cathepsin D expression compensates for the reduction in activity and expression of other lysosomal enzymes reported to occur after orchidectomy (Mayorga and Bertini, 1982; Gupta and Setty, 1995; Abou-Haila et al, 1996).
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Acknowledgments |
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Footnotes |
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