1 Physiology and Biophysics Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, PO Box 70005, Postal Code 6530499, and 2 Las Condes Clinic, Lo Fontecilla 441, Santiago, Chile
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Abstract |
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Key words: androgens/antiandrogens/cell culture/glycosidases/human epididymis
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Introduction |
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A number of epididymal secretory products have been associated with sperm motility, binding to zona pellucida and gamete membrane fusion (Blaquier et al., 1980, Cooper, 1993
; Ben Ali et al., 1994
, Boue and Sullivan, 1996
). However, the specific mechanism involved in the action of these molecules on sperm maturation and its regulation by androgens are not completely understood. Secretion of specific glycosidases by epididymal cells has been found in several species including man (Skudlarek and Orgebin-Crist, 1986
; Guerín et al., 1990
; Barbieri et al., 1994
; Miranda et al., 1995
; Raczek et al., 1995
; Abou-Haila et al., 1996
). These enzymes may be responsible for changes in surface glycoproteins of the spermatozoon (Tulsiani et al., 1995a
,b
). Also, binding of some of these enzymes to specific glycosidic receptors on the human and rat sperm membranes have been reported (Barbieri et al., 1994
; Belmonte et al., 1998
).
In humans, as in other species, the maturation process begins in the proximal part of the organ (caput) and ends at the distal regions (corpus and cauda) (Mathieu et al., 1992). Also, there is evidence that epididymal function is under androgen regulation (Brooks, 1981a
). Development and maintenance of sperm fertilizing ability is not achieved unless androgens are present (Orgebin-Crist, 1975
) and specific protein and mRNA synthesis in the epididymis are influenced by androgen (D'Agostino et al., 1980
; Jones et al., 1980
). The epididymis receives testicular androgens from the rete testis and the blood circulation. In this organ, testosterone is converted to dihydrotestosterone (DHT) by the enzyme 5-
-reductase. DHT is the main androgen in epididymal tissue.
In the present work, we studied the influence of different concentrations of testosterone, DHT and cyproterone acetate (CA) on the secretion of -glucosidase (
-GLI), N-acetyl-glucosaminidase (NAG), ß-glucuronidase (ß-GLU) and
-mannosidase (
-MAN) by epithelial cell cultures obtained from human caput, corpus and cauda epididymides.
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Materials and methods |
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Cell isolation and culture
Briefly, epididymides were cut into three segments corresponding to caput, corpus and cauda. A small piece of each segment was processed for histological control. For this purpose, samples were fixed in Bouin, dehydrated in ethanol, embedded in Paraplast and stained with haematoxylin/eosin. The epididymal segments were freed of the surrounding connective tissue with fine forceps and scissors. Then, the segments were separately incubated in 30 ml of culture medium containing 0.35% trypsin, 0.2% hyaluronidase, 0.1% bovine serum albumin (BSA) and 0.01% DNase for 23 h at 35°C, in a water bath. Then, the remaining connective tissue was removed with fine forceps. Resulting segments were washed with culture medium containing 5% fetal bovine serum (FBS). Then, segments were separately incubated in 30 ml of culture medium containing 0.35% collagenase, 0.2% hyaluronidase, 0.2% elastase, 0.1% BSA, 0.01% DNase and 5% FBS, for 810 h at 35°C. After this treatment, large tubule fragments were obtained. These fragments were cut into small fragments with fine scissors and resuspended in culture medium containing 5% FBS. The fragments were dispersed by gently pipetting until cell aggregates (2030 cells) were obtained. The resulting aggregates of each epididymal segment were washed with culture medium without FBS. After washing, aggregates were resuspended in 4045 ml (~106 cells/ml) of culture medium containing human transferrin 5 mg/l, insulin 2 mg/l, epidermal growth factor 10 µg/l, vitamin A and E 200 µg/l, progesterone 108 mol/l, hydrocortisone 108 mol/l, sodium selenite 2 µg/l, and cytosine arabinoside 3 mg/l to inhibit growth of fibroblasts. Depending on the experiment 0, 1, 10 or 100 nmol/l of testosterone, DHT or CA (the latter together with 10 nmol/l of DHT) were added to culture medium. Finally, aggregates were plated on Matrigel-coated multi-well culture plates (24-well) at an approximate density of 106 cells/ml (1.5 ml/well). Cells were counted before plating and, in some wells, at the end of each experiment. In these conditions the cell loss was estimated to be 1015%. After 3 days, most aggregates were attached to the Matrigel surface and the medium was changed. After a week, cells have started to move away from the fragments and spread out through the culture plate. After that, media were collected every 48 h, centrifuged at 3000 g and stored at 25°C until assayed. The percentage of epithelial cells was evaluated using an immunocytochemical assay for cytokeratin and cell viability was determined by the trypan blue exclusion test.
Enzyme assays
Enzymatic activities were measured by previously described spectrophotometric methods using 150 µl of conditioned medium and 1 mg/ml of the p-nitrophenyl derivates as substrates and p-nitrophenol as standard (Barratt and Heath, 1977). Enzyme activity was expressed as U/106 cells. One unit of enzyme activity hydrolyses 1 nmol/h of substrate.
Statistical analysis
Data were evaluated using the KruskalWallis non-parametric analysis of variance (ANOVA) test followed by Dunn's test. P < 0.05 was considered significant.
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Results |
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Effect of testosterone, dihydrotestosterone and cyproterone acetate on secretion of glycosidase activities
The activities of ß-GLU, ß-MAN, -GLI and NAG were measured in conditioned culture media from caput, corpus and cauda epididymides in the presence of 0, 1, 10 or 100 nmol/l of testosterone, DHT or CA (the last in the presence of 10 nmol/l of DHT). Media for these assays were collected every 48 h starting from day 6 of culture. The hormone effect was evaluated between 1012 or 1214 days, depending when enzyme secretion was maximal.
Testosterone (Figures 1 and 2), and DHT (Figures 3 and 4
) increased glycosidase secretion in a concentration-dependent manner. This increase was higher in corpus and cauda than in caput. In most cases, DHT effect was more pronounced than the testosterone effect.
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Discussion |
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In humans, arguments concerning the necessity of the epididymis have been discussed (Silber et al., 1989; Cooper, 1990
). Suggestions that the epididymis may not be relevant for sperm maturation are based on observations that spermatozoa from patients undergoing surgical bypass of a major part of the epididymides still have the ability to fertilize oocytes (Silber, 1989
). Nevertheless, most authors agree that pathological tissue may have different functional capacities compared to normal (Cooper, 1990
, 1993
; Rajalakshmi et al., 1993
). On the other hand, there is consistent evidence, in most mammal species, that epididymal function is androgen regulated (Gupta et al. 1974
; Orgebin-Crist et al, 1975
; Brooks, 1981a
). Testicular testosterone reaches epididymal tissue from the rete testis and the blood and lymphatic supply. Testosterone may be found free, conjugated or bound to androgen binding protein. The latter form is probably predominant in the proximal segment. In epididymal epithelium, 5-
-reductase converts testosterone to DHT which is the main androgen in this organ (Inano et al., 1969
). Secretion of specific epididymal proteins is regulated by androgen (Jones et al., 1980
). Also, in rat caput epididymides mRNA synthesis was found to be influenced by the androgen status (D'Agostino et al., 1980
). Considering that an important event in sperm maturation is the modification of the molecular composition of the sperm surface and a number of epididymal glycosidases have been reported (Mayorga and Bertini, 1985
; Skudlarek and Orgebin-Crist, 1986
; Cooper et al., 1988
; Raczek et al., 1995
), it seems reasonable to suggest that these enzymes might be involved in digesting residues of side chains of existing glycoproteins, unmasking new domains on the sperm surface. In addition, some of these glycosidases have shown a regionalized secretion pattern (Skudlarek and Orgebin-Crist, 1986
; E.Castellón, J.Balbontin and C.Huidobro, submitted) and androgen dependence (Mayorga and Bertini, 1982
). Interestingly, differences in androgen concentration along the epididymis have been reported in rat (Turner et al., 1984
). Also, differences in testosterone metabolism between human corpus epididymis and efferent ducts have been established using a cell culture system (Raczek et al., 1994
). In the present work, we have studied the androgen influence on glycosidase secretion in an in-vitro model consisting of separated cultures of epithelial cells from human caput, corpus and cauda epididymides. All glycosidases studied showed an androgen stimulation in a concentration-dependent manner. This stimulation showed different degrees depending on enzyme, epididymal region and androgen used, suggesting that these glycosidases are differentially regulated. Whether this androgen dependence is due to a specific stimulation of gene expression, secretion rate or changes in cell volume or androgen receptors, remains to be elucidated.
-MAN was differentially secreted by cultures from different epididymal regions and presented the highest dependence on androgen. This enzyme has been associated with spermegg interaction (Okamura et al., 1995
). In addition, a rat sperm membrane
-MAN immunologically homologous to epididymal
-MAN and a sperm binding site for this enzyme have been recently reported (Tulsiani et al., 1995a
; Belmonte et al., 1998
).
ß-GLU also presented a slight regionalized secretion pattern and showed a higher androgen-dependent increase in corpus and cauda than in caput, suggesting that this enzyme may have more relevance in those epididymal segments. In addition, different isoforms of this enzyme have been reported in mouse epididymis. These isoforms are differentially distributed along the epididymis and are regulated by androgens (Abou-Haila et al., 1996).
Neutral -GLI showed a less pronounced dependence on androgens and the increase was slightly higher in corpus and cauda than in caput. This may be correlated with the removal of glucosidic residues from sperm glycoproteins which would take place mainly in more distal segments of the epididymis. This enzyme has been extensively used as clinical marker of epididymal function (Cooper et al., 1988
), and recently, it has been associated with zona pellucida binding capacity (Ben Ali et al., 1994
). However, the actual role of this glycosidase in sperm maturation is still uncertain.
NAG is secreted mainly in cauda cell cultures and showed a moderate dependence on androgens compared with others glycosidases studied. Also, the extent of the increase was similar in cultures obtained from the three epididymal regions. Affinity sites for NAG have been found on the surface of rat epididymal spermatozoa (Barbieri et al., 1994) and bidirectional secretion of this enzyme has been reported using a similar system of human epididymal cell culture (Cooper et al., 1990
). Also, different NAG isoforms were found in human epididymal fluid and the specific cauda isoform was found to be associated with the sperm surface (Miranda et al., 1995
).
DHT showed higher stimulatory effect than testosterone for most glycosidases studied. The androgen antagonist, CA, consistently inhibited the androgen-induced stimulation of glycosidase secretion in all epididymal regions showing that receptor availability is necessary for glycosidase stimulation. Interestingly, a rapid decline of androgen responsiveness has been reported in cultures of Percoll-purified epithelial cell from rat epididymis. This responsiveness was retained when cells were co-cultured with epididymal fibroblast (Carballada and Saling, 1997). In our system, we had ~70% epithelial cells (cytokeratin-positive), the remaining percentage corresponded mainly to epididymal fibroblasts which may explain, in part, the maintenance of androgen responsiveness in our cultures. On the other hand, the presence of androgen receptor and its mRNA have been detected throughout the human epididymis, albeit in different quantities, being maximal in corpus epididymis (Ungefroren et al., 1997
). We have found androgen responsiveness in cultures from all epididymal regions, mostly in corpus and cauda. It might be possible that the distribution of androgen receptor changes in culture conditions. Also, the different concentrations of androgen added to the cultures may differentially modify androgen receptor expression in vitro. At present, we are determining androgen receptors in our culture conditions.
It is concluded that specific glycosidases which are differentially secreted by epithelial cell cultures obtained from different epidydimal regions are also differentially regulated by androgens. The inhibition of glycosidase secretion by the antiandrogen CA may represent a valuable tool for influencing human sperm maturation.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 14, 1998; accepted on February 8, 1999.