Glycosidases in cultured rat epididymal cells: enzyme activity, synthesis and secretion

During transit through the epididymis, spermatozoa acquire fertilizing the cell surface exhibits an altered glycoprotein pattern. Epididymal cells and their secretions contribute to these sperm-surface changes. To examine this process, epithelial cells from rat caput and cauda epididymidis were cultured and examined for the synthesis, processing and secretion of two glycoprotein-modifying enzymes, beta-galactosidase and beta-glucuronidase. Cells were cultured four days, incubated with D-2-[3H] mannose and L-[35S] methionine, and placed in isotope-free media. Levels of both cellular and secreted beta-galactosidase and beta-glucuronidase were determined by immunoprecipitation of cell homogenates or medium, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scintillation counting of bands. During a 1-h pulse, both caput and cauda cells synthesize two precursor forms of beta-galactosidase (Mr = 84,000 and 87,000), which are processed to the mature (Mr = 63,000) enzyme during a 24-h chase. Caput cells release a high molecular weight (HMW) form (Mr = 90-100,000) and mature beta-galactosidase into the media, but not the Mr = 84-87,000 precursor. On the other hand, cauda cells release mostly mature beta-galactosidase. Ratios of radiolabeled mannose/methionine demonstrate a 7-fold greater mannose content in the cellular precursor of beta-galactosidase than in total protein. Another glycosidase, beta-glucuronidase, is synthesized as a Mr = 78,000-precursor which is processed to the mature Mr = 72,000 form. Medium in which caput and cauda cells were cultured contains both mature enzyme and a Mr = 94,000 form, but no 78,000-precursor form. Ratios of radiolabeled mannose/methionine in the cellular precursor of beta-glucuronidase are 2-fold greater than ratios in the total glycoprotein. Secretion is the major pathway of turnover for several epididymal glycosidases, since more than 50% of the total is secreted/day. These results indicate that cultured epithelial cells from the epididymis synthesize glycosidases and that processing and release differ, depending on the enzyme and the epididymal segment from which the epithelial cells were isolated.     

Functional and morphological effects of hypophysectomy and androgen replacement in the rabbit epididymis

Summary The fertilizing ability of spermatozoa from the distal corpus epididymidis of 55 male rabbits of proven fertility has been determined after artificial insemination 1, 2, 3, 5, 7, and 11 days after hypophysectomy. A significant decrease in fertility was noted 3, 5, and 7 days after hypophysectomy. Testosterone propionate (2 mg/kg/day) given for 7 days to hypophysectomized rabbits maintained the fertilizing ability of spermatozoa from the corpus epididymidis when injections were started from the day of hypophysectomy or delayed by a week. The effect of hypophysectomy on the histological features of the epididymal epithelium were studied with light and electron microscopy. Regressive changes were observed: reduction in size of the stereocilia border and the endoplasmic reticulum 3 days after hypophysectomy followed by a decrease in tubular diameter and cell height, a reduction in size of the Golgi apparatus and an increase in number of lysosome-like figures and a vacuolization of the epithelium.These results are discussed in relation to the physiological requirements for sperm maturation.     

Lectin binding characterstics of mouse epididymal fluid and sperm extracts

Glycoproteins from luminal fluid of the mouse cauda epiciidymidis have been compared with glycoproteins from Triton X-100 extracts of mouse spermatozoa from varying regions of the epididymis, using lectins with specific affinity for different sugar residues. Concanavalin A recognizes 11 glycocomponents on Western blots of fractionated caudal fluid; wheat germ agglutinin (WGA) binds 12 proteins; Ulex europaeus agglutinin (UEA) binds seven; and Dolichos biflorus agglutinin (DBA) recognizes nine. Several of these glycoproteins display an affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues; whereas other proteins bind only one of the four lectins used. The results also show that some glycoproteins exhibit a higher affinity for particular lectins. Eight glycoproteins of similar mobility and lectin-binding characteristics are detected in Triton X-100 extracts of spermatozoa from different regions of the epididymis and in caudal fluid. The lectin affinity of some proteins appears or increases in spermatozoa from distal epididymal regions (54 kD, 32 kD), whereas the lectin affinity of others decreases (29 kD, 40 kD). There are differences in lectin affinities between proteins in sperm extracts and in caudal fluid. Some proteins show an affinity for three or four lectins in caudal fluid, but proteins of similar electrophoretic mobility in sperm extracts bind only one or two of the lectins. These data show that glycoproteins of similar mobility are present in caudal fluid and in Triton-X-100 sperm extracts, implying a potential interaction between caudal fluid components and epididymal sperm.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of an endogenous murine leukemia virus-related proviral sequence is androgen regulated and primarily restricted to the epididymis/vas deferens and oviduct/uterus.

A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.     

Swainsonine induces the production of hybrid glycoproteins and accumulation of oligosaccharides in male reproductive tissues of the rat

Early studies have shown that spotted locoweed (Astragalus lentiginosus) has an adverse effect on male reproduction. Rams fed locoweed showed a reduced number of primary and secondary spermatocytes and spermatids in the testis, and of spermatozoa in the epididymis and vas deferens. In addition, the Sertoli cells and other epithelial cells were severely vacuolated. Swainsonine, an indolizidine alkaloid, has been identified as the sole or principal toxin in locoweed and perhaps in the plants of genus Swainsona. The toxin is an inhibitor of lysosomal alpha-D-mannosidase, cytosolic alpha-D-mannosidase, and Golgi mannosidase II. The in vitro and in vivo inhibition of Golgi mannosidase II induces the production of abnormal glycoproteins. Since epididymis-mediated modifications of sperm-surface glycoproteins are believed to be important for sperm-egg interactions, we initiated studies to determine effects of swainsonine on processing and catabolism of N-linked glycoproteins in male reproductive tissues. The results presented in this report indicate that feeding of the alkaloid led to accumulation of mannose-rich oligosaccharides (OS) in the testis and epididymis of rats. The major OS was purified from the reproductive tissues of swainsonine-fed rats, and its structure was deduced by comparison of the size of the OS before and after treatment with jack bean alpha-D-mannosidase, and by affinity column chromatography. In addition, the rat epididymal epithelial cells produced abnormal glycoproteins when cultured in the presence of the toxin. This result provides indirect evidence for the presence of a swainsonine-sensitive mannosidase II-like processing enzyme in the epididymal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.     

The effects of testosterone, 5α-dihydrotestosterone, 3α-androstanediol,and 3β-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture

Summary The fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3–4 days when testosterone (0.5 g/ml) or 5-dihydrotestosterone (0.5 g/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5-dihydrotestosterone, 3-androstanediol, or 3-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 g/ml. Only with 5-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.     

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.