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ABSTRACT: In addition to the maturation of sperm, the epididymis also serves to protect sperm from harmful reactive oxygen species. To this end, various antioxidant enzymes are produced by the epididymis, such as glutathione S-transferases (GSTs), a family of dimeric proteins that catalyze the conjugation of glutathione to various electrophilic compounds, thus providing cellular detoxification. In the present study, the regulation of the Yb1 subunit of GST was examined in Bouin-fixed epididymides of adult control, orchidectomized (O) rats with or without testosterone (T) supplementation and efferent duct—ligated (EDL) rats using light microscope immunocytochemistry with an anti-Yb1—GST antibody. The intensely reactive ciliated cells of the efferent ducts and principal cells of the epididymis showing a checkerboard staining pattern were unaltered in their expression of Yb1-GST after all experimental procedures, suggesting their regulation by factors other than of testicular origin. On the other hand, the intense reaction of narrow/apical cells and moderate reaction of basal cells of the proximal initial segment of control animals became negligible in O rats and was not restored with T supplementation. As staining was also absent after EDL, the data suggest that a luminal testicular factor(s), other than androgens, regulates expression of Yb1-GST in narrow/apical and basal cells of the proximal initial segment. Although basal cells of the caput and cauda epididymidis were unreactive after all experimental protocols, as also noted in controls, the intensely reactive basal cells of the corpus epididymidis of control animals became unreactive in O animals. However, Yb1-GST expression was restored to these cells with T supplementation, and as there was no effect on Yb1-GST expression after EDL, the data suggest that circulating testosterone or one of its metabolites regulates expression of Yb1-GST in basal cells of the corpus region. Taken together, these data indicate a differential regulation with respect to the expression of Yb1-GST in the various cell types and regions of the epididymis.
The epididymis, in addition to its function in the maturation of sperm, plays an important role in protecting them from harmful substances while they traverse and are stored in this duct (Robaire and Hermo, 1988; Hinton et al, 1995; Aitken, 2002). In many species, the low temperature of the epididymis, especially the cauda region, facilitates sperm storage by enhancing oxygen availability to cells (Djakiew and Cardullo, 1986). However, oxygen implies the increased presence of reactive oxygen species, which are implicated in inducing oxidative damage to DNA, proteins, and lipids. Sperm are particularly susceptible to reactive oxygen species, as they contain high levels of polyunsaturated phospholipids in their membranes. As a result, sperm must be protected as these reactive oxygen species can result in lipid peroxidation of their membranes, membrane fragility, and impaired fertility (Jones and Mann, 1977; Alvarez and Storey, 1989). Thus it has been noted that the epididymis contains various antioxidant molecules, such as glutathione, that prevent oxygen radical injury, in addition to the presence of numerous antioxidant enzymes such as gamma glutamyl transpeptidase, glutathione peroxidase, superoxide dismutase, and glutathione S-transferase (GST; Hinton et al, 1995; Aitken, 2002; Cornwall et al, 2002; Hermo and Robaire, 2002).
GSTs are a family of soluble isozymes involved in cellular detoxification. They prevent the build up of potentially toxic substances by catalyzing the conjugation of reduced glutathione with various electrophilic substances, thereby protecting cellular components such as DNA, proteins, and lipids. The electrophilic substances can be of cellular origin, such as free radicals formed during normal metabolism or exogenous chemical compounds (Daniel, 1993). In addition to their main function in detoxification, they also play an important role in steroid isomerization (Benson et al, 1977); glutathione peroxidation (Prohaska and Ganther, 1976); leukotriene C biosynthesis (Pemble et al, 1986); and binding noncovalently to a number of nonsubstrate ligands, including steroids (Homma et al, 1986).
Cytosolic, microsomal, and nuclear forms of GSTs have been identified (Hayes and Strange, 2000). They are dimeric proteins from a family of 16 genes grouped into five classes (α, μ, π, θ, and σ) according to the amino acid homology of their subunits (Mannervik and Danielson, 1988; Buetler and Eaton, 1992; Daniel, 1993). GSTs form homo- or heterodimers from subunits that are members of 1 gene family but not between gene families (Mantle et al, 1990). GSTs have similar molecular weights but differ with respect to isoelectric point and substrate specificities (Jakoby et al, 1976). The vast number of different GST subunits implies their importance in the protection of cells against a wide variety of potentially harmful substances that they can encounter.
In previous studies, Hales et al (1980) had shown that the epididymis—vas deferens contains high GST activity, approximately 50% of that found in the liver on a protein weight basis, and that there are a large number of different GSTs in the epididymis—vas deferens, which are differentially localized along the length of this tissue. Recently, we examined with light microscopic immunocytochemistry the distribution of different GST subunits along the epididymis and vas deferens of adult rats. Our studies revealed that the expression of the different GSTs in the epididymis and vas deferens was often cell type– and region-specific (Veri et al, 1993, 1994; Papp et al, 1995; Andonian and Hermo, 1999). The absence of reactivity in 1 cell type of a given region was usually compensated for by the reactivity in another cell type of that region. The varied GST expression in the different cell types was thought to ensure that sperm would be protected from a wide variety of blood-borne electrophiles as they traversed the epididymis and during storage in the cauda region and vas deferens (Papp et al, 1995; Andonian and Hermo, 1999).
Although the activity of GSTs in the epididymis has been shown to be androgen-dependent and at times region-specific (Hales et al, 1980; Robaire and Hales, 1982), the regulation of expression of the different GST subunits has not been examined in a cell-type and region-specific manner, with only one exception. Indeed, in the epididymis, although basal cells were shown to be unaffected in their expression of Yf-GST by the absence of testicular and pituitary factors, the expression of Yf-GST by principal cells was shown to be dependent on testosterone (Hermo and Papp, 1996). However, little is known about the regulation of other GST subunits according to the different cell types and regions of the epididymis.
The purpose of the present study was, therefore, to examine the regulation of the Yb1-GST subunit in the epididymis of adult rats following orchidectomy with or without testosterone supplementation and efferent duct ligation using light microscope immunocytochemistry in conjunction with an anti-Yb1—GST antibody. The experimental procedures were designed to evaluate the role of androgens on Yb1-GST expression, well known regulators of many epididymal functions (Cornwall et al, 2002), as well as luminal testicular factors, other than androgens, which enter the epididymis via the lumen of the seminiferous tubules of the testis and efferent ducts and which have recently been defined as lumicrine factors (Hinton et al, 1998). The data from experimentally treated animals were qualitatively compared with those obtained for control adult rats based on numerous images generated from the 4 animals of each treatment group.
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Principal cells are the major epithelial cell type lining the epididymis, which has been shown to be active in secretion and endocytosis of various substances, among other functions (Cooper, 1986; Robaire and Hermo, 1988; Hermo et al, 1994). In the present study, in the case of Yb1-GST expression, principal cells of control animals showed no reaction in the proximal initial segment and a checkerboard-staining pattern in all other epididymal regions. The latter is a term reflecting the fact that although some cells showed high levels of Yb1-GST immunoreactivity, others showed moderate, little, or no reactivity. This suggests that some principal cells are synthesizing Yb1-GST, whereas others are not, and/or that these cells are out of synchrony with respect to their synthesis of Yb1-GST at any given moment of time. Such a staining pattern has been already recognized for many other proteins, including secretory and lysosomal (Rankin et al, 1992; Hermo et al, 1994; Igdoura et al, 1995). Nevertheless, no noticeable differences in Yb1-GST expression were observed in principal cells of any epididymal region after orchidectomy with or without testosterone administration at the different time points examined (Figure 5, Table). The staining pattern and intensities were comparable in both control and experimentally treated animals. Thus Yb1-GST expression in principal cells does not appear to be regulated by androgens.
Figure 5. . Diagrammatic representation of the staining pattern for Yb1-GST in the proximal initial segment (PIS), distal initial segment/intermediate zone (DIS), caput (Cap), corpus (Cor) and cauda (Cau) epididymidis of control animals, orchidectomized animals without (O) or with testosterone (O+T) supplementation, and efferent duct—ligated (EDL) animals. Principal cells are represented as columnar cells with a microvillar brush border and are present in all epididymal regions. Narrow cells, found in the PIS and DIS, are represented as thin attenuated cells at the right margin of each epithelial section, whereas apical cells also present in these regions are shown as cup-shaped cells, which do not reach the basement membrane. Clear cells, present only in the Cap, Cor, and Cau epididymidis, are represented as columnar cells without microvilli. Basal cells, seen as hemispherical cells, do not contact the lumen and are noted in all epididymal regions. After all experimental treatments at the different time points, principal cells maintain their staining pattern comparable with that seen in control animals. Although unreactive in the PIS, their nuclei are intensely reactive in the DIS. They show a prominent checkerboard-staining pattern of their cytoplasm and nucleus only in the Cap, Cor, and Cau regions. Narrow/apical cells and basal cells, reactive in control animals in the PIS region, become unreactive after all experimental treatments at the different time points, suggesting that they are regulated by testicular lumicrine factors. In the Cap and Cau regions in control animals, basal cells are unreactive and remain so after all experimental treatments. However, in the Cor region, basal cells are intensely reactive in control and EDL animals, become unreactive after O, but regain their intense reactivity after T administration. This suggests that Yb1-GST expression in the case of basal cells of the Cor is regulated by testosterone or 1 of its metabolites. Clear cells are consistently unreactive in control and all experimental animals.
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This data contrasts with data obtained for Yf-GST expression, where principal cells noted to be reactive for Yf GST in control animals became unreactive after orchidectomy. The administration of testosterone to these animals restored Yf-GST expression, indicating that androgens were responsible for regulating Yf-GST expression in principal cells (Hermo and Papp, 1996). Clearly differences in the regulation of the various isoforms of GST appear to occur in the case of principal cells. Although it is well established that many epididymal functions are under the control of androgens (Robaire and Hermo, 1988; Orgebin-Crist, 1996; Cornwall et al, 2002; Ezer and Robaire, 2002), several proteins have been shown to be unaffected by androgen withdrawal in a cell-specific manner, such as SGP-1, cystatin c, SGP-2, and cathepsins A and D (Hermo et al, 2000b; Luedtke et al, 2000; Cornwall et al, 2002; Hermo and Andonian, in press). This also appears to be the case for Yb1-GST expression by principal cells.
However, in addition to the regulation mediated by androgens, factors emanating from the testis that enter the epididymis via the lumen of the duct, defined as lumicrine factors, also play a role in regulating epididymal functions (Hinton et al, 1998; Cornwall et al, 2002). In the present study, efferent duct ligation also had no effect on Yb1-GST expression in principal cells, eliminating a role for lumicrine factors on its expression (Figure 5, Table), and this was also observed to be the case for Yf-GST (Hermo and Papp, 1996) and several other proteins expressed by the epididymis (Ezer and Robaire, 2002).
In the present study, the anti-Yb1 antibody often showed a nuclear reaction in principal cells that was more intense than the reaction seen in the cytoplasm (Figure 5, Table), and this was noted throughout most of the epididymis. It has been demonstrated that Yb1 proteins exist in a soluble form in both the nucleus and cytoplasm; however, they also exist in a bound form in the nucleus, where they bind to DNA (Bennett et al, 1986; Hayes and Mantle, 1986; Ketterer et al, 1990). It has also been shown that Yb1 in carcinoma cells migrates into the nucleus, providing further evidence for their strong nuclear affinity (Bennett et al, 1986; Bennett and Yeoman, 1987). In the epididymis, the Yb1 protein may protect DNA and RNA from harmful electrophiles that may be encountered by principal cells. In the present study, at no time was the expression of Yb1-GST noted to be different in experimentally treated animals as compared with controls, suggesting that testicular factors did not regulate Yb1-GST expression in the nucleus of principal cells.
Narrow/apical cells are found only in the initial segment and intermediate zone of the epididymis (Adamali and Hermo, 1996). Aside from a function in endocytosis, these cells also express GSTs in a region-specific manner (Papp et al, 1995; Hermo et al, 2000a). Apical cells express Yf-GST in the initial segment and intermediate zone, whereas narrow cells do not. In the proximal initial segment only, both apical and narrow cells express Yo GST (Adamali and Hermo, 1996). In the present study, Yb1-GST was expressed in narrow/apical cells of the proximal initial segment only. We did not separate these cells from each other, as both the thin narrow cells as well as the cup-shaped apical cells, which did not reach the basement membrane, were reactive (Adamali and Hermo, 1996). Although little is known about the regulation of the functions of these cells, the present data indicate that Yb1-GST expression is not regulated by androgens. Indeed, expression was lost after orchidotomy and not restored following testosterone supplementation. However, since a lack of expression was also noted after efferent duct ligation (Figure 5, Table), it was concluded that regulation of Yb1-GST in narrow/apical cells of the proximal initial segment was via a lumicrine factor. Lumicrine factors derived from the testis have been shown to regulate several proteins synthesized by the epididymis. Indeed, ligation of the efferent ducts induces changes in epididymal gene and protein expression (Cornwall et al, 2002). The luminal testicular factors that may regulate gene expression in the initial segment include ions, solutes, proteins, steroids, and even germ cells. These factors can up- or down-regulate gene expression after efferent duct ligation (Brooks, 1983). Expression of cystatin-related lipocalin and proenkephalin are among the proteins that have already been shown to be regulated by lumicrine factors (Garrett et al, 1991; Lareyre et al, 2001; Cornwall et al, 2002), and to this list may be now added Yb1-GST expression by narrow/apical cells of the proximal initial segment.
Basal cells are small hemispherical cells that reside on the basement membrane and do not reach the lumen of the duct. They also send thin, footlike processes along the basement membrane that collectively encompass a large portion of the circumference of each tubule (Veri et al, 1993). In this way they form a barrier, albeit an incomplete one, between the blood vessels and other contents of the intertubular space and the epididymal lumen. They can, therefore, to a degree effectively eliminate potentially harmful substances emanating from the blood that are trying to access the sperm in the lumen. In this context, basal cells express various isoforms of GSTs, such as the Yf, Ya, Yb2, and Yc subunits (Papp et al, 1995), as well as superoxide dismutase (Nonogaki et al, 1992).
Basal cells of control animals express Yb1-GST, but only in the proximal initial segment and corpus regions (Figure 5, Table). After orchidectomy and efferent duct ligation, these cells showed a differential response to androgens and lumicrine factors, dependent on their location along the duct. In the proximal initial segment, Yb1-GST expression was not restored to orchidectomized animals supplemented with testosterone, and expression was also absent after efferent duct ligation (Figure 5, Table). However, in the corpus region, expression was restored to control levels in orchidectomized animals that received testosterone, and there was no effect on expression in efferent duct—ligated animals (Figure 5, Table). Taken together, the data suggest that in the corpus region, Yb1-GST expression in basal cells is regulated by testosterone or one of its metabolites, but in the proximal initial segment, expression is regulated by a lumicrine factor. These data differ dramatically from that obtained for the Yf-GST subunit, where its expression in basal cells was unaltered after orchidectomy and efferent duct ligation, as well as hypophysectomy, indicating that neither testicular nor pituitary factors governed Yf-GST expression in basal cells (Hermo and Papp, 1996). Thus basal cells of different regions show differential responses to the absence of androgens or testicular lumicrine factors in their expression of a given GST as well as between different GSTs. Very little is known about what regulates basal cell functions, as data on the functions of these cells is only slowly being gathered. However, the expression of metallothionein by basal cells, although detectable in all epididymal regions, was shown to be androgen-dependent according to specific regions (Cyr et al, 2001).
Aside from the family of GSTs, the expression of glutathione peroxidase at the message and protein levels has also been noted to be androgen-dependent (Vernet et al, 1997; Schwaab et al, 1998). At the mRNA level, gamma glutamyl-transpeptidases, which show multiple transcripts, are also differentially regulated by androgens and/or lumicrine factors in the different epididymal regions (Palladino and Hinton, 1994), as is their secretion and activity (Agrawal and Vanha-Perttula, 1988). Thus various antioxidant enzymes show a differential regional response in their expression to the presence or absence of testicular factors. However, their regulation has not as yet been determined on a cell-type and region-specific manner. In summary, the present study has revealed that the expression of Yb1-GST is regulated along the epididymis in a cell-type and region-specific manner.