Changes in the specific activity of N-acetyl-beta-D-hexosaminidase in the rat epididymis with age.

The specific activity of the lysosomal enzyme N-acetyl-beta-D-hexosaminidase was determined in the caput and cauda of the epididymis of rats as a function of age. The activity peaks at six weeks of age in both parts of the epididymis but is higher in the cauda. The results indicate a relationship between changes occurring in sperm in the epididymis and the lysosomal enzymes.     

Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.

A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.     

Developmental changes in and hormonal regulation of estrogen and androgen receptors present in the rabbit epididymis

Both androgen and estrogen receptors (AR and ER) are present in the rabbit epididymis. We have used the sucrose gradient method to examine receptor sedimentation properties, receptor concentration, and distribution of receptors among the caput, corpus, and cauda of the epididymis to determine changes that occur in these parameters as the animals age. The 9S form of the ER is present in all three epididymal segments of the immature rabbit, with the highest concentration occurring in the cauda. The 8.2S form of the AR is also present in all three segments of the immature epididymis, with the highest concentration occurring in the caput. Short-term castration (3 days) leads to an increase in the amount of both AR and ER detected. ER are present in all segments of the immature epididymis at higher concentrations than AR. The functional 9S form of the ER disappears as the animals mature, the result of a tissue-specific protease that our laboratory previously has shown proteolyzes ER to a non-DNA-binding 3.8S form. Long-term castration (3 mo) of adult rabbits results in the reappearance of the 9S form of the ER in all segments of the epididymis. The reappearance of the 9S form of the ER is also seen in animals castrated for 1 mo, but not in those castrated for 2 wk. Administration of testosterone once daily for 2 wk to adult animals castrated for 6 wk results in the disappearance of the 9S form of the ER and the reappearance of the 3.8S form, suggesting that the tissue-specific protease is androgen-dependent. In this way, circulating androgens may play a role in regulating the concentration and form of the ER in the rabbit epididymis. There is little change in the concentration or distribution of AR in the epididymis of adult rabbits castrated for 3 mo as compared to those castrated for 3 days. This implies that circulating androgens are not required for maintenance of AR in the epididymis. Our data demonstrate that there are temporal differences in the presence and concentration of ER and AR in the epididymis and suggest that there is a differential, age-dependent regulation of the development and function of the epididymis by androgens and estrogens.     

Characterization of cell- and region-specific abnormalities in the epididymis of cathepsin A deficient mice

Cathepsin A (PPCA), a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multi-enzyme complex, has been shown to be expressed in the epithelial cells of the epididymis. In the present study, the epididymis of PPCA-/- mice from 2 to 10 months of age was compared with those of their wild-type counterparts. Major accumulations of pale vacuoles, corresponding to lysosomes, were noted in principal and narrow/apical cells in PPCA-/- mice, and clear cells also appearing highly vacuolated, were grossly enlarged in size. This was especially evident in the caput and corpus regions, where quantitative analyses confirmed that the epithelium of the tubules in these regions was expanding in profile area. In addition, the base of the epithelium in these regions was often greatly vacuolated, corresponding to cells that presented no identifiable features and appeared to be degenerating. Halo cells dispersed at various levels in the epithelium also appeared to be abnormal, accumulating pale lysosomes. Furthermore, numerous macrophages were observed in the intertubular space of the entire duct, presenting a large size and plethora of pale lysosomes. Taken together, the present data indicate major lysosomal abnormalities in the epididymis of PPCA-/- mice in a cell type and region specific manner. In addition, it is suggested that the compromised halo cells, due to PPCA deficiency within their lysosomes, cannot function properly and as a result there is a recruitment of macrophages in the intertubular space.     

Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors.

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 beta-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.     

Histological and histochemical studies on testis, epididymis and ductus defrens of the rooster (Gallus domesticus)]

Testis, epididymis and ductus deferens of the adult domestic fowl and male gonads of juvenile roosters have been studied by means of histochemical and histological methods. Testicular interstitial cells: According to enzyme-histochemical results oxidative energy production seems to be of minor importance. An extraordinarily high activity of lysosomal enzymes (acid phosphatase and esterases in the adult, esterases only in the immature) is observed. Positive reactions of 3beta-steroid-dehydrogenase and enzymes of the pentose phosphate cycle indicate steroid hormone production; the pathways of the steroid synthesis, however, are probably different in adult and immature testes. A remarkable LAP content of juvenile Leydig cells is a parameter of an increased protein metabolism. Areas of reserve cells: Focal accumulation of these cell types are observed in testis and epididymis of the immature and in the epididymis of the adult fowl. Reserve cells reveal distinct activities of LAP (prospective growth ability) and 3beta-HstDH (reserve capacity for steriod synthesis). All other enzymes studied react weakly, thus pointing to a generally low metabolic activity. Seminiferous tubules: The strong reaction of alkaline phosphatase in the peritubular cells may play a part in energy disposition for contractions. Sertoli cells of adult animals are rich in lysosomal enzymes and enzymes of the glycolytic chain but oxidoreductases react weaker than in mammalian Sertoli cells. This indicates that nutritive interactions between germ and Sertoli cells in birds are different from those in mammals: The basally orientated germ cells of birds contain strong activities of diaphorases, LDH, SDH, Cyto-Ox and seem to be metabolically rather indipendent from Sertoli cells.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Morphological features of the epididymal epithelium of gerbil, Meriones unguiculatus

Principal cells of the ducts epididymis of the Mongolian gerbil showed ultrastructural characteristics of lining epithelium cells close related to processes of protein secretion, and transcytosis occurring between adjacent principal cells which were mainly verified in the initial segment. Principal cells also presented roles of fluid phase and adsorptive endocytoses, as well as autophagic and heterophagic lysosomal activities mainly observed in the caput epididymis. Columnar (principal) cells of the corpus epididymidis presented great number of variable vesicles and vacuoles distributed in all the cytoplasmic levels occurring a progressive coalescence pattern among them, which help to guarantee formation of cytoplasmic channels for fluid phase transport between the tubular lumen and epididymal interstitium. Clear cells were presented in the initial segment and predominately in the cauda epididymis epithelium of the gerbil and showed marked ultrastructural characteristics of endocytosis activities occurrence, perhaps directly related to the turnover of fluid phase of spermatozoa stored into the lumen of the distal tail. Other epididymal epithelium cells were verified and described such as basal, halo, apical and dark cells, but they did not presented special ultrastructural features.     

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.     

The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.     

β-hexosaminidase immunolocalization and α- and β-subunit gene expression in the rat testis and epididymis

β-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G_M2 gangliosidoses. β-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for β-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the α- and β-subunits of β-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. β-hexosaminidase α-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, β-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7–91 postnatal days of age, testis levels of α-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, β-subunit mRNA was expressed at low levels throughout testis development. In isolated male germ cells, β-hexosaminidase α-subunit expression was most abundant in haploid round spermatids, whereas the β-subunit mRNA was not detected in germ cells. Within the epididymis both α- and β-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that β-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, β-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput, and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that β-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of β-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions. Mol. Reprod. Dev. 46:227–242, 1997. © 1997 Wiley-Liss, Inc.     

Male reproductive toxicity of vincristine: ultrastructural changes in the epididymal epithelial apical cell

The toxic effect of vincristine on the apical cells of the rat caput epididymis was investigated. The drug was administered at 20 and 40 microg/kg body weight daily for 15 days. Light microscopy using semithin sections, and transmission electron microscopy, of the caput epididymis were undertaken. The results revealed that the basal region of the apical cell was in contact with the basement membrane and the luminal end took part in endocytosis. The apical cells reflected a dose-dependent response to vincristine (VCR) treatment. In general the changes included protrusion of the apical ends deep into the lumen, with the nucleus of the cell located in such protruded ends, and an increase in the abundance of lysosomal bodies and multivesicular bodies. These changes reflected the physiological response of the apical cell to VCR treatment rather than toxic manifestations.     

Castration induces changes in the cation‐dependent mannose‐6‐phosphate receptor in rat epididymis: Possible implications in secretion of lysosomal enzymes

It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen‐dependent fashion. In this study, we attempted to discern whether mannose‐6‐phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation‐dependent MPR (CD‐MPR) and cation‐independent MPR (CI‐MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti‐androgenic drug flutamide. Furthermore, we observed that the CD‐MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD‐MPR in the secretion, we observed an increase in pro‐cathepsin D levels in epididymal fluid after castration. We conclude that the CD‐MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis. J. Cell. Biochem. 110: 1101–1110, 2010. Published 2010 Wiley‐Liss, Inc.     

beta-N-acetylglucosaminidase in the reproductive organs and seminal plasma of the bull

The highest specific activity of beta-N-acetylglucosaminidase (beta-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate beta-NAG activity. The beta-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8.0-4.0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high beta-NAG activity in bull seminal plasma. The major secretory forms of beta-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the beta-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all beta-NAG isoenzymes was observed at pH 5.0. They were almost totally inactivated at 60 degrees C and about 75-80% of the activity was lost at 55 degrees C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents. Histochemical studies showed a strong granular (lysosomal) reaction for beta-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a beta-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.     

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.