Biosynthesis of two lysosomal enzymes in macrophages. Evidence for a precursor of beta-galactosidase.

We have purified beta-galactosidase and beta-glucuronidase from macrophages of thioglycollate-treated mice using concanavalin A chromatography and immunoprecipitation. The apparent molecular weight of the beta-galactosidase subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changed during a long term pulse-chase experiment. Following a 1-h pulse with [3H]leucine, radiolabel was present exclusively in an Mr = 82,000 form. However, after a 3-h chase in medium containing unlabeled leucine, most label migrated at Mr = 63,000, and at 24 h, all label was in the Mr = 63,000 form. Electrophoresis of peptides produced by cyanogen bromide cleavage of immunoprecipitates demonstrated structural similarities between precursor and mature forms. A mutation in the mouse, which is known to depress the rate of synthesis of beta-galactosidase in many cell types, proportionately decreased incorporation of [3H]leucine into both the Mr = 82,000 and 63,000 forms. Therefore, by kinetic, structural, and genetic evidence, the large molecular weight beta-galactosidase is a precursor of mature macrophage enzyme. No precursor of the Mr = 75,000 subunit of beta-glucuronidase was detected.     

Effect of swainsonine on rat epididymal glycosidases

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

Changes in rat sperm membrane glycosidase activities and carbohydrate and protein contents associated with epididymal transit

Rat spermatozoa were recovered from the caput, corpus, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for beta-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex

The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.     

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis. Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.     

Immunochemical localization and association with spermatozoa of androgen-regulated proteins of MR 24000 secreted by the mouse epididymis

A polyclonal monospecific immune serum was raised against androgen-regulated proteins with Mr 24000 secreted by the mouse caput epididymidis (6). Sections of frozen tissues from the different regions of the epididymis have been studied by indirect immuno- fluorescence. Results indicate that the antigens are secretory proteins produced by the epithelial cells of the caput epididymidis, essentially in the medial and distal segments. Accumulation of the antigens was observed in the lumen of the caput and the corpus epididymal duct. Subsequently, their association with the sperm surface occurred and persisted down to the cauda epididymidis.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of swainsonine on the processing and turnover of lysosomal beta-galactosidase and beta-glucuronidase from mouse peritoneal macrophages

The effect of swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase, on the synthesis, processing, and turnover of two glycoproteins, lysosomal beta-galactosidase and lysosomal beta-glucuronidase, has been studied in cultured mouse peritoneal macrophages. No effect of the inhibitor on the relative rates of synthesis of the precursor form of either enzyme was observed. On the other hand, carbohydrate processing of beta-galactosidase and beta-glucuronidase was markedly altered by swainsonine, consistent with a blockage by the inhibitor of the removal of the alpha-1,3- and alpha-1,6-linked mannose residues which occurs in normal processing. In homogenates of both normal and swainsonine-treated cells, the precursor forms of the enzymes were found exclusively in the light membrane fraction on Percoll gradients and the mature forms exclusively in the lysosomal fractions indicating that translocation from Golgi to lysosomes and proteolytic processing in the lysosome were not impaired by the presence of abnormal oligosaccharide side chains. There was no detectable effect of swainsonine during a 4-day chase period on the total cellular turnover of these enzymes which involves two processes, secretion and degradation. In the absence of swainsonine, secretion represented about 40% of the total turnover of beta-galactosidase and about 50% with beta-glucuronidase. The presence of swainsonine increased these proportions to about 60 and 70%, respectively.     

Effects of castration, 5 alpha-dihydrotestosterone and cyproterone acetate on enzyme activity in the mouse epididymis

The influence of castration, androgen replacement therapy and cyproterone acetate on the activity of beta-glucuronidase, acid and alkaline phosphatases and glucose-6-phosphate dehydrogenase was studied in the caput, corpus and cauda epididymidis of the mouse. The results add further evidence that the epididymis is not uniform but has regional differences in activity. Thus beta-glucuronidase was found to be androgen-dependent only in the cauda epididymidis, whereas glucose-6-phosphate dehydrogenase was under androgenic control in the caput epididymidis. The response of alkaline and acid phosphatases to castration and to androgen replacement was different in different segments.     

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.     

beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum

The post-translational processing of beta-glucuronidase in BW5147 mouse lymphoma cells is slow relative to other newly synthesized lysosomal enzymes. To characterize this slow maturation the acid hydrolase was immunoprecipitated from cells pulse-labeled with [2-3H]mannose. Radiolabeled beta-glucuronidase migrated as the precursor form of the enzyme for up to 4 h of chase, whereas another acid hydrolase, beta-galactosidase, was processed completely to its mature form within this same time period. Both beta-glucuronidase and beta-galactosidase obtained high levels of mannose 6-phosphate (Man 6-P) within 60 min of their biosynthesis. The Man 6-P content of beta-galactosidase declined rapidly during a subsequent chase while that of beta-glucuronidase remained high during the first 4 h of chase and then slowly declined. 3H-Labeled phosphorylated high mannose-type oligosaccharides isolated from beta-glucuronidase after 1 h of chase were composed primarily of species with one or two phosphodiester groups, but oligosaccharides with one and two phosphomonoesters became the predominant phosphorylated species with longer chase times. The phosphorylated oligosaccharides attached to other newly synthesized acid hydrolases, on the other hand, contained primarily phosphodiester species at all chase times. When BW5147 cells were pulsed with [3H]mannose and chased in the presence of monensin to disrupt transport, the number of phosphorylated oligosaccharides recovered from beta-glucuronidase was comparable to the quantity recovered from the enzyme produced by non-drug-treated cells. The number of phosphorylated units recovered from all other newly synthesized acid hydrolases, however, was greater in the presence of the ionophore than in its absence. Nondenaturing gel electrophoresis studies indicated that beta-glucuronidase existed in two forms at steady state within BW5147 cells and, as such, was similar to liver beta-glucuronidase in which a large percentage of the enzyme was present as a complex bound to egasyn. These data suggest that newly synthesized beta-glucuronidase produced by BW5147 cells complexes with an egasyn-like protein within the endoplasmic reticulum. This interaction retards the enzyme's migration through the secretory apparatus but does not prevent its access to Golgi-associated processing enzymes.     

The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency

In our previous studies, we showed that a finasteride-induced DHT deficiency may cause changes in the morphology of the seminiferous epithelium without any morphological alteration of the epididymis. In this study, we demonstrated the constitutive immunoexpression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of Wistar rats treated with finasteride for 28 days (the duration of two cycles of the seminiferous epithelium) and 56 days (the duration of one spermatogenesis). We noted that a 56-day finasteride treatment mainly caused a decrease in the level of circulating DHT, as well as a statistically insignificant decrease in the level of T. The hormone deficiency also led to a change in the iNOS immnoexpression in the testis and epididymis of the finasteride-treated rats. In vitro, DHT did not modify NO production by the epithelial cells of the caput epididymis even when stimulated with LPS and IFNγ, but it did give rise to an increase in NO production by the epithelial cells of the cauda epididymis without the stimulation. DHT did not have a statistically significant influence on estradiol production by cultured, LPS- and IFNγ-stimulated epithelial cells from the caput and cauda epididymis. In conclusion, our data clearly indicates that a finasterideinduced DHT deficiency intensifies the constitutive expression of iNOS in most rat testicular and epididymal cells, so it can be expected that the expression of inducible nitric oxide synthase (iNOS) could be regulated by DHT. On the other hand, the profile of the circulating DHT and T levels strongly suggests that the regulation of constitutive iNOS expression is complex and needs more detailed study.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

Trichloroethylene exposure elicits damage in epididymal epithelium and spermatozoa in mice

We have investigated the toxic effects of trichloroethylene (TCE) on the epididymis and epididymal sperm in mice. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for 1 to 4 weeks. Segments of the epididymis (caput, corpus and cauda) were examined by light and electron microscopy. At the light microscopic level, degeneration and sloughing of epithelial cells were evident as early as 1 week after TCE exposure, and were most pronounced after 4 weeks. Such epithelial damage was observed in the caput, corpus and cauda regions of the epididymis. Ultrastructural observations revealed vesiculation in the cytoplasm, disintegration of basolateral cell membranes, and sloughing of epithelial cells. Sperm were found in situ in the cytoplasm of degenerated epididymal cells. Additionally, a large number of sperm in the epididymal lumen exhibited abnormalities including malformation of head and tail components. Our results demonstrated that exposure to TCE by inhalation causes damage to the epididymal epithelium and sperm.