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Regular Article |
a Department of Anatomy and Cell Biology
b Faculty of Dentistry, McGill University, Montreal, Quebec, Canada H3A 2B2
c Human Health Research Center, INRS-Institut Armand Frappier, University of Quebec, Pointe Claire, Quebec, Canada H9R 1G6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract, sperm, sperm maturation, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 kDa, OPN's reported SDS-PAGE migration varies due to extensive posttranslational modifications, polymorphism, and splicing variants [1–3]. Growth factors, hormones, and cytokines all affect the rate of gene transcription, mRNA processing, stability, translation, and the posttranslational modifications [1, 2]. Although rat OPN contains two active vitamin D-response elements upstream of the promoter, no androgen-responsive elements have yet been identified [1]. The ability to synthesize multiple, variably regulated forms of the OPN molecule suggests a means for creating tissue- and species-specific isoforms capable of executing various functions [2–5]. In addition to multiple sites for Ser and Thr phosphorylation and for N- and O-linked glycosylation, other motifs in the OPN molecule are conserved and thought to be important for its various actions. Osteopontin binds type I collagen, fibronectin, osteocalcin, and other OPN molecules [2, 3, 6]. In addition, conserved phosphoserines and a polyaspartic acid motif allow OPN to inhibit crystal growth by binding calcium ions and adsorbing onto crystal surfaces [2, 3]. Osteopontin also uses a conserved RGD (arginine, glycine, aspartic acid) sequence to bind to multiple integrin receptors and, thereby, triggers cell signals that stimulate cell activity, thus allowing OPN to promote cell adhesion, migration, flattening, intracellular calcium regulation, and cytoskeletal rearrangements [1–3, 6].
Osteopontin has been noted extensively on the surfaces of many epithelial cells communicating with an external luminal environment [2, 6, 7]. Osteopontin protein and/or mRNA have been localized in many soft tissues using immunocytochemistry, Northern blot analysis, and in situ hybridization [2]. Numerous studies have also identified OPN in various body fluids containing high levels of calcium [2, 3, 5]. It is postulated that the epithelium lining these environments secrete OPN into the lumen, where it acts to inhibit the formation of calcium stones via its extensive calcium ion- and crystal-binding capabilities, whereupon these complexes can be endocytosed or phagocytosed by the epithelial cells [2, 3, 6].
In the male reproductive tract, OPN has been immunolocalized in the rat to Sertoli cells of the testis and to the epithelium of the epididymis of all regions except the cauda [8]. In the bull, OPN has been noted in the ampulla of the vas deferens and the seminal plasma [9]. Thus, distinct differences in localization appear to exist between species.
The objective of the present study was to examine the expression of OPN in the testis, efferent ducts, and epididymis of adult rats using light- and electron-microscopic immunocytochemical analysis and Western blotting. In an effort to discern regulation of OPN by testicular factors, expression of OPN was examined in the efferent ducts and epididymides of adult rats who had been orchidectomized with or without immediate testosterone supplementation or whose efferent ducts were ligated. Studies on rats of different postnatal ages were also performed to determine whether a correlation exists between the onset of OPN expression in the different cell types of the testis, efferent ducts, and epididymis and the events known to occur in these tissues during postnatal development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague-Dawley rats (body weight, 350–450 g; age, 90 days) were obtained from Charles River Laboratory Ltd. (St. Constant, QC, Canada) and divided into five groups. Group 1 consisted of four normal, untreated rats. Group 2 consisted of rats with bilateral ligation of the efferent ducts. After an i.p. injection of sodium pentobarbital (Somnitol; MTC Pharmaceuticals, Hamilton, ON, Canada), the testes and epididymides were exposed through an incision of the anterior abdominal wall. Using a dissecting microscope, a ligature was placed around both the right and left efferent ducts at a site close to the rete testis while avoiding the adjacent blood vessels. The animals (n = 4 per interval) were killed at 3, 7, 14, and 21 days following surgery. Group 3 consisted of rats with bilateral orchidectomy. After anesthesia, both testes were removed after a ligature was placed around the efferent ducts and testicular blood vessels. The animals (n = 4 per interval) were killed at 3, 7, 14, and 21 days after surgery. Group 4 consisted of rats with bilateral orchidectomy receiving three 6.2-cm, testosterone-filled implants. Testosterone-filled, polydimethyl siloxane (silastic) implants with well-characterized steroid-release rates [10] were prepared according to the method of Stratton et al. [11]. Subsequent to anesthesia, both testes were removed, and the implants were placed subcutaneously immediately after orchidectomy. The rats (n = 4 per interval) were killed at 3, 7, and 14 days after surgery. Group 5 consisted of four sham-operated rats, two of which received three empty 6.2-cm implants. These rats were killed at 14 and 21 days after initiation of the experiment.
For postnatal developmental studies, timed pregnant female Sprague-Dawley rats were obtained from Charles River Laboratory Ltd. A number of litters, out of which 36 male pups were utilized, were maintained on a 10L:14D photoperiod. They were provided with food and water ad libitum. After birth, the normal development of the male pups was monitored by assessing gain in body weight and by palpating their testes and epididymides. Only those pups showing normal trends in development, as reported by Hermo et al. [12], were used. Six rats at each of the following intervals were used; Postnatal Days 7, 21, 29, 39, 49, and 56. At each age, two rats were used to obtain the weights of the paired testes and epididymides, whereas the other four were used to prepare the tissue for light-microscopic immunocytochemical analysis. The body weights of all animals used, as well as the testicular and epididymal weights of the two unfixed animals, were within the normal range obtained previously [12]. The size and appearance of the testes and epididymides of the four chemically fixed animals (see below) were similar to those of the two unfixed animals.
All protocols and experiments performed on animals utilized in this study were done in accordance with the guidelines set up by the Animal Care Committee of McGill University.
Tissue Preparation and Light-Microscopic Immunostaining
The testes and epididymides of all animals were fixed with Bouin fixative through the heart (Days 7 and 21) or via retrograde perfusion of fixative through the abdominal aorta (all other ages) for 10 min. For the normal adult group, two additional rats were fixed by perfusion with Sainte-Marie fixative (95% methanol:glacial acetic acid, 99:1 [v/v]) in place of the Bouin fixative. Following perfusion with either fixative, the testes and epididymides were removed; the latter were cut so that given sections would include all major regions of the epididymis (i.e., the initial segment, intermediate zone, caput, corpus, and cauda) [13]. The tissues were then immersed in either Bouin or Sainte-Marie fixative for an additional 72 h, after which they were dehydrated and embedded in paraffin.
Sections (thickness, 5 µm) were cut and mounted on glass slides. They were then deparaffinized with xylene and hydrated in graded concentrations of ethanol (from 100% to 50%). During hydration, the residual picric acid was neutralized by immersing the tissues in 70% (v/v) ethanol containing 1% (v/v) lithium carbonate for 5 min. To inactivate any endogenous peroxidase activity, the tissue sections were incubated for 5 min in 70% ethanol containing 1% (v/v) hydrogen peroxide. Following hydration, the sections were incubated (5 min) in a 300 mM glycine solution to block free aldehyde groups. The tissues were then blocked again with 40 ml of 5% (v/v) BSA diluted in 20 mM Tris-HCL-buffered saline containing 0.1% BSA (TBS) at pH 7.4 for 25 min at room temperature. The slides were then washed with Tween buffer solution (TWBS; TBS with 0.1% Tween-20).
The LF-7 and LF-123 antibodies, generously supplied by Dr. Larry W. Fisher (National Institutes of Health, Bethesda, MD), were used with the normal and experimental groups as well as for the postnatal studies. The antibodies have been characterized and shown to be specific for OPN [14, 15].
Each slide-mounted tissue section was incubated in the primary antibody at a dilution of 1:100 in TBS for 1.5 h at 35°C in a humidified incubator. After incubation, the sections were washed by immersing them in four consecutive wells of TWBS for 2 min each. The sections were then blocked with 90 ml of 5% BSA and subsequently incubated with goat anti-rabbit IgG conjugated to horse radish peroxidase (Sigma, St. Louis, MO) at a dilution of 1:250 in TBS for 30 min at 35°C in the humidified incubator. After the incubation with secondary antibody, the tissue was washed by immersion in four wells of TWBS for 2 min each.
The final reaction product was obtained by incubating the slides for 10 min in 250 ml of TBS containing 0.03% hydrogen peroxide, 0.1 M imidazole, and 0.05% diaminobenzidine tetrahydrochloride (pH 7.4). The sections were counterstained with 0.1% methylene blue (2 min) and then dehydrated in a graded series of ethanol solutions (30 sec each) and xylene (3 min). Cover slips were mounted onto glass slides using Permount.
Incubation with preimmune rabbit serum at a dilution of 1:100 in TBS and incubation of tissues in secondary antibody alone served as negative controls. Positive controls consisted of antibody incubation with Sainte-Marie-fixed kidney tissue, for which staining of the distal tubules and collecting ducts has already been reported [7].
Electron-Microscopic Immunocytochemistry
Four adult male Sprague-Dawley rats (body weight, 350–400 g) were anesthetized with sodium pentobarbital and their epididymides fixed by perfusion through the abdominal aorta with a fixative containing 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After their removal, the tissue was trimmed into small pieces (0.5 mm3), immersed for 2 h in the above fixative at 4°C, washed two or three times in 0.15 M PBS (pH 7.4), and then treated with PBS containing 1.5 M sucrose. The tissue was then frozen in liquid nitrogen until sectioned.
Ultrathin sections of selected epididymal regions were mounted on 300-mesh, formvar-coated nickel grids (Canemco, Montreal, QC, Canada). Each grid was floated for 15 min on a drop of 2% bovine serum albumin, 2% casein, 0.5% ovalbumin (BCO) and then incubated for 1 h on 15-µl drops of LF-7 OPN antibody diluted 1:5 in BCO. Sections were washed six times for 5 min each in Dulbecco phosphate-buffered saline (DPBS), transferred for 15 min to drops of BCO, and incubated for 30 min on 20-µl drops of goat anti-rabbit IgG antibodies conjugated to 10-nm colloidal-gold particles. The sections were subjected to six 5-min washes in DPBS, followed by six 5-min washes in distilled water. Sections were counterstained with uranyl acetate oxalate stain (pH 7) in water. Sections were protected with a layer of 2% methyl cellulose (Anachemia, Montreal, QC, Canada). Photographs were taken on a Philips 400 electron microscope (Philips, Eindhoven, The Netherlands). Preimmune rabbit serum at a dilution of 1:5 served as a control.
Immunoblots
Adult male Sprague-Dawley rats were killed with CO2 and their epididymides isolated and sectioned into four different regions (i.e., initial segment, caput, corpus, and cauda) as previously described [13] and placed in liquid nitrogen. Frozen tissues were homogenized in buffer (pH 6.8) (60 mM Tris-Cl, 2 mM CaCl2, 1 mM phenylmethylsulfonate, 40 mM ß-octylglucopyranoside, 20 µg/ml of aprotinin, 20 µg/ml of trypsin inhibitor, 20 µg/ml of pepstatin A, 40 µg/ml of antipain, and 20 µg/ml of leupeptin) using a Teflon pestle on ice. The protein content was determined using a colorimetric assay (Bio-Rad Protein Assay Kit; Bio-Rad Laboratories, Inc., Mississauga, ON, Canada). Protein samples (50 µg) were diluted in Laemmli [16] buffer, boiled for 5 min, and electrophoresed on 10% polyacrylamide gel at 200 V. Separated proteins were subsequently transferred onto a polyvinylidene fluoride membrane (Bio-Rad) at 100 V for 1 h. Transfer efficiency was confirmed using prestained molecular weight markers (Bio-Rad). After transfer, the membrane was blocked for 1 h with enhanced chemiluminescence (ECL) blocking agent (Amersham Pharmacia Biotech Inc., Piscataway, NJ) dissolved in 1% Tween 20/50 mM TBS (pH 7.4). Osteopontin was immunodetected with anti-human OPN antiserum LF-123 for 2 h at room temperature at a dilution of 1:1000. Membranes were washed with TBS-Tween and subjected to anti-rabbit-horse radish peroxidase conjugate (Caltag Laboratories, Burlingame, CA) for 1 h. After washing, OPN was visualized with chemiluminescence using an ECL Plus kit (Amersham) according to the manufacturer's instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the testis of normal adult animals, OPN was expressed in germ cells of the seminiferous epithelium, where it was restricted to spermatogonia, preleptotene, leptotene, zygotene, and early pachytene spermatocytes (Fig. 1). An absence of reaction was found over mid- and late-pachytene spermatocytes, spermatids, and Sertoli cells. In the interstitial space, a reaction was limited to a small population of cells, presumably macrophages, judging from their low numbers compared to the more numerous Leydig cells (Fig. 1B). In the efferent ducts, a distinct reaction was observed over the apical region of nonciliated cells (Fig. 2).
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In the epididymis, principal cells showed a variable staining pattern, ranging from a supranuclear granular reaction in the proximal initial segment (Fig. 3) to a distinct apical band of reaction in all other regions (Fig. 4). The strongest reaction was noted in the distal caput epididymidis (Fig. 4, A and B), followed by the corpus (Fig. 4C) and intermediate zone (Fig. 4A, inset) and then the distal initial segment, proximal caput, and cauda (Fig. 4D) regions.
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The apical region of narrow cells of the initial segment and intermediate zone was intensely reactive (Fig. 3). Clear cells of the proximal caput and cauda (Fig. 4D) regions showed an intense apical band of reaction, whereas those of the distal caput (Fig. 4, A and B) and corpus regions appeared to be unreactive. Basal cells were unreactive in all regions, as were the luminal contents, including spermatozoa (Figs. 3 and 4).
Use of preimmune serum or incubation without primary antibody failed to show any reaction over cells of the testis and epididymis, including their luminal contents, with these images being similar to those already published by us in previous studies [13, 17].
Electron-Microscopic Immunocytochemistry
Electron-microscopic immunocytochemistry revealed gold particles representing OPN antigenic sites over microvilli, small endocytic vesicles, and large endosomes of the apical region of nonciliated cells of the efferent ducts as well as principal (Fig. 5) and clear cells of the epididymis in regions corresponding to those with moderate to intense light-microscopic immunostaining. No labeling was noted over sperm in the lumen or over the Golgi apparatus, endoplasmic reticulum, or lysosomes of nonciliated, principal, and clear cells. Use of preimmune serum or incubation with secondary antibody alone showed only an occasional gold particle in a given field (1–3 particles/field) as compared to more than 30–50 gold particles in a comparable field of similar magnification when primary antibody was utilized. Such background levels of immunolabeling are characteristic of the controls used and are similar to those already published by us in previous studies [18].
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Postnatal Studies
At Postnatal Day 7, a reaction was observed in the testis and was restricted to spermatogonia of the seminiferous epithelium and a few cells in the interstitial space (Fig. 6A). Epithelial cells of the efferent ducts already showed an intense apical reaction (Fig. 6B); however, epithelial cells of all epididymal regions appeared to be unreactive (Fig. 6C). By Day 21, reaction in the testis was most intense in spermatogonia and early spermatocytes, but not in pachytene spermatocytes (Fig. 7). In the efferent ducts, the epithelial cells showed an intense apical band of reaction product similar to that seen in adult animals. In the epididymis, principal cells of all regions were unreactive or only weakly reactive. By Day 21, narrow cells of the initial segment and intermediate zone were stained, as were clear cells of the proximal caput and cauda regions (not shown).
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By Postnatal Day 39, spermatogonia and spermatocytes up to early pachytene spermatocytes were reactive. In the epididymis, principal cells of most regions were still, for the most part, weakly reactive or unreactive. However, these cells showed comparable staining intensities to those of adults by Day 49. The exception was the distal caput region, where principal cells revealed a distinct apical band of reaction by Day 39 (Fig. 8A) that was maintained, and was more prominent, at Day 49. Clear cells of the proximal caput and cauda (Fig. 8B) regions were intensely reactive by Day 39, whereas clear cells of other regions remained unreactive, as seen in normal, 90-day-old adult animals.
Experimental Animals
Three days after orchidectomy, no difference was noted in the level or pattern of reaction in any epithelial cell type of each epididymal region (e.g., Fig. 9) as compared to 90-day-old, control adult animals. However, 7 days after orchidectomy, principal cells of the initial segment, intermediate zone, proximal caput, and cauda regions became unreactive, whereas those of the distal caput and corpus regions showed a weaker reaction compared to control adult animals. At 14 and 21 days after orchidectomy, principal cells of all regions became unreactive (e.g., Fig. 10). Nonciliated cells of the efferent ducts and narrow and clear cells of the epididymis maintained their intense reactivity, as seen in control animals, at all time points after treatment (e.g., Fig. 10B).
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At 3, 7, and 14 days after orchidectomy with immediate testosterone replacement, no change was observed in the pattern or level of reactivity over any of the epithelial cells of each region (e.g., Fig. 11), with reactivity being comparable to that noted in control animals. Bilateral ligation of the efferent ducts of animals examined at 3, 7, 14, and 21 days postoperative also revealed no change in reaction as compared to control animals over any epithelial cell of any region (e.g., Fig. 12).
Osteopontin was detected in the epididymis by Western blot analysis as a 30-kDa form (Fig. 13), corresponding to the previously reported molecular weight of OPN found in epididymal fluid and sperm [8] In the testis, OPN was present as a 60-kDa form (Fig. 13), corresponding to OPN normally found in fluids (e.g., milk) where OPN is commonly more heavily phosphorylated [19]. In the epididymis, a 30-kDa form of OPN was found in caput, corpus, and cauda epididymides, but not in the initial segment. Small quantities of the 60-kDa form were also present in all epididymal regions (Fig. 13).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, OPN was expressed during the early phases of germ cell differentiation, from spermatogonia up to early pachytene spermatocytes, but not in other germ cells. Because spermatogonia and preleptotene spermatocytes establish contact with the basement membrane on one hand and with Sertoli cells on another, OPN may anchor early germ cells to extracellular matrix components as well as to Sertoli cells via its cell surface-binding capabilities, a function that has been well documented for this protein [1–3]. Also prominent during early germ cell development is the migration of early spermatocytes from the basal compartment of the seminiferous epithelium to the adluminal compartment above the Sertoli-Sertoli blood testis barrier [20]. The ability of OPN to promote transient cell attachment and cell migration [1–3] may contribute to this phenomenon by binding to integrin receptors on the Sertoli cell surface and inducing intracellular signals necessary for moving early spermatocytes from one compartment to the next. Identification of the subcellular distribution of OPN in germ cells using electron microscopy was not possible due to the poor preservation of the seminiferous epithelium as a result of the weak fixative used, which was required for maintaining adequate levels of antigenicity.
Distribution of OPN in the Efferent Ducts and Epididymis
In the present study, OPN was expressed in nonciliated cells of the efferent ducts and in principal cells of the epididymis in a region-specific manner, with the most intense expression occurring in the distal caput region, followed by the corpus, and with weaker expression occurring in all other regions (Table 1 and Fig. 14). The apical band of reaction product was noted by electron-microscopic immunocytochemistry to be localized over apical spherical vesicles of nonciliated and principal cells. The latter represent early endocytic vesicles, as deduced from previous electron-microscopic studies employing routine morphology and electron-dense tracers [21–23]. In the absence of OPN expression from Sertoli cells and lack of staining of the luminal contents, including sperm, of the efferent ducts and epididymis, it is unlikely that OPN is being internalized by these cells from the lumen, or that OPN is being secreted into the lumen, because no reaction was noted over the Golgi apparatus or endoplasmic reticulum (which is not the case for well-known secretory proteins of principal cells [13, 24]). The apical band of reaction for OPN is, however, similar to that shown for LRP-2, a cell surface receptor localized in nonciliated and principal cells [25–27]. Thus, we propose that OPN may function in a receptor-like manner, as will be discussed below.
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The exception to the apical band of reaction is the proximal initial segment. Here, principal cells showed a prominent supranuclear granular reaction (Table 1 and Fig. 14). The punctate reaction suggests lysosomal staining, as noted for several lysosomal markers [17, 28–30]; however, in the absence of electron-microscopic localization of OPN in the proximal initial segment, due to the small area of the epididymis occupied by this region, this fact could not be determined in the present study.
Osteopontin was also expressed as a distinct apical band in narrow and clear cells (Table 1 and Fig. 14). Narrow cells are endocytic in nature, and apart from pumping protons into the lumen to acidify it, not much is known regarding the functions of these cells [23, 31, 32]. In clear cells, OPN expression was region specific, with the highest expression occurring in the proximal caput and distal cauda regions (Table 1 and Fig. 14). In fact, a region-specific reciprocating pattern of OPN expression in clear cells was noted, whereby the most intensely stained regions were those in which principal cells were least intensely stained. The apical reaction in narrow and clear cells is suggestive of labeling of early endocytic vesicles, which are prominent in these highly endocytic cells [22, 32].
Immunoblot analysis of adult epididymal tissue revealed a prominent band at a molecular weight of 30 kDa, corresponding to that reported in the literature for OPN in various tissues [1–3] and confirming OPN expression in the epididymis. The more intense band noted in the caput, corpus, and cauda regions was consistent with the high levels of expression noted in these regions during light-microscopic immunocytochemistry.
Our results differ from those of Siiteri et al. [8], who reported localization of OPN to Sertoli cells of the rat testis, and from those of Cancel et al. [9], who reported that OPN was not present in the testis of the bull. In addition, Siiteri et al. [8] reported a confluent cytoplasmic reaction in all epididymal cells, excluding the cauda, as well as in the lumen and on the sperm surface, whereas Cancel et al. [9] reported no OPN expression in the epididymis or on the sperm surface in the ampulla and seminal fluid. In addressing this problem, we utilized 2 well-characterized OPN antibodies and fixed our tissues in both Sainte-Marie and Bouin fixative. Under these conditions, a consistent staining pattern could be observed. However, certain combinations of tissue preparation and choice of antibody, including 5 additional OPN antibodies not presented here, revealed only slight to no visible staining (data not shown). Nevertheless, when reactive, the staining pattern was consistent with that which we obtained for the LF-7 and LF-123 used in the present study. Thus, in summary, the data indicate that expression can vary between studies, between species, and even within a species. These differences likely are attributable to incubation protocol, tissue preparation, or variations in antibody specificity and sensitivity for OPN's reputed isoforms.
OPN as a Cell Surface Receptor
Calcium ions are established components of the epididymal lumen, in which, at appropriate levels, they have been shown to be essential for optimal sperm motility [33, 34]. It has been reported that OPN has calcium-binding properties, and that as many as 50 calcium ion-binding sites occur in the bone variant of OPN [35]. The ability of OPN to bind and promote the internalization of calcium microcrystals in cultured kidney cells has been reported as well [36–38]. In the efferent ducts of the rooster, cysts containing calcium have been reported to result in a thin, eroded epithelial layer, with few luminal sperm and reduced fertility levels [39]. Accumulation of calcium crystal deposits have also been noted in the lumen of the rete testis, efferent ducts, and epididymis of the human [40, 41]. The formation of these crystals often occurs in conjunction with other pathological conditions, including stone formation in tissues such as the kidney [42].
In accordance with OPN's various binding properties, OPN localization on the apical cell surface of nonciliated, principal, narrow, and clear cells may allow it to act as an adhesive, receptor-like protein that sequesters calcium ions or binds microcrystals in the epididymal lumen for subsequent endocytosis. Cell surface receptors such as transferrin and LRP-2 (megalin/gp330), which are present in the efferent ducts and epididymis [25, 27, 43, 44], are continuously recycled after internalization by coated pits to the cell surface in tubules emanating from endosomes, a system known as CURL or compartment for uncoupling receptors from ligands, and thus escape transport to the lysosomes [45]. Such a system is also present in epithelial cells of the rete testis, efferent ducts, and epididymis [23, 46, 47] and may explain why OPN is localized mainly to the apical cell surface. Thus, under normal conditions, OPN may remove small calcium-containing crystals that undergo dissolution at the low pH of the endosome [36], thus ensuring that they do not form stones in the efferent ducts and epididymal lumen and, subsequently, decrease sperm fertility levels.
Regional variations in OPN staining pattern as noted in the present study could be accounted for by a corresponding regional distribution of calcium microcrystals along the epididymis. By using in situ precipitation, Li et al. [48] localized Ca2+ granules along the microvilli of epididymal cells in a region-specific intensity distribution. This distribution corresponded to our reported OPN distribution in principal cells along the epididymis. Specifically, these investigators reported a peak intensity of the Ca2+ crystals in the distal caput/corpus region, with intensity decreasing bidirectionally. Taken together, these data reflect an important means by which the epididymis may regulate Ca2+ ions in the epididymal lumen.
Regulation of OPN Expression
In the efferent ducts, OPN expression in nonciliated cells was unaltered after either orchidectomy or efferent duct ligation. Thus, testosterone does not appear to regulate OPN expression in these ducts. It has been demonstrated that estrogen regulates several functions of nonciliated cells, and that estrogen receptors are expressed at high levels in the efferent ducts [49]. However, in the absence of testosterone from orchidectomized rats, it would appear that this is not the case for OPN expression.
In the epididymis, many attributes of principal cell functions are disrupted by the absence of testosterone [50–53]. In the present study, the disappearance of OPN expression in principal cells was noted after orchidectomy (Table 2). Restoration of OPN expression with testosterone replacement and continued expression in efferent duct-ligated animals indicates a role for circulating androgens in the regulation of OPN in principal cells. However, the lack of any reported androgen-responsive elements in the promoter region of OPN [1] suggests that other, perhaps more general transcription factors are involved in OPN expression, and that these factors are being regulated by testosterone. It is also unlikely that estrogen regulates OPN expression in principal cells of the epididymis, because estrogen receptors have been shown to be expressed at high levels only in the initial segment of the rat epididymis [49] and not in the corpus and cauda regions, where maximal levels of OPN expression were noted during the present study.
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In the case of narrow and clear cells, OPN was not affected by orchidectomy, suggesting a lack of dependence on testosterone or estrogen for OPN expression by these cells. Little is known regarding what regulates the functions of either of these cells, but clearly, they are regulated differently from principal cells.
Postnatal Studies
By Postnatal Day 7, spermatogonia of the testis already express OPN, suggesting an early role for this protein during germ cell development (Table 3). At later ages, as subsequent generations of germ cells evolve, OPN is expressed, but only up to early pachytene spermatocytes, as noted in the adult. In the case of nonciliated cells of the efferent ducts, OPN expression was also present by Postnatal Day 7 and, for narrow and clear cells of the epididymis, by Day 21 (Table 3). Thus, OPN's early expression in these cell types does not coincide with the high androgen levels already noted by Day 39 [54], supporting our findings that OPN expression in nonciliated, narrow, and clear cells is not regulated by testosterone. However, the postnatal expression of OPN in principal cells, beginning by Day 21 and progressively increasing up to Day 49, correlates well with the increasing levels of androgens that are known to occur during development (Table 4). This finding also concurs with those of our orchidectomy studies employing testosterone implants, which indicated the dependence of principal cells on testosterone for OPN expression.
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Summary
Osteopontin was localized in the testis, efferent ducts, and epididymis. In the testis, OPN appears in early germ cells from spermatogonia to early pachytene spermatocytes, whereas nonciliated cells express OPN in the efferent ducts. In the epididymis, OPN is expressed in a cell- and region-specific manner. Data from postnatal and experimentally treated adult animals suggest that OPN is regulated in principal cells by circulating testosterone, but that this is not the case for nonciliated, narrow, or clear cells. In the efferent ducts and epididymis, OPN appearing at the the cell surface and in endosomes of the epithelial cells may serve to bind to Ca2+ crystals, thus preventing luminal calcium-containing crystal aggregation that would eventually reduce sperm fertilizing capability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Canadian Institutes of Health Research to L.H. and M.D.M. 
2 Correspondence: Louis Hermo, Department of Anatomy and Cell Biology, McGill University, 3640 University St., Montreal, QC, Canada H3A 2B2. FAX: 514 398 5047; lhermo{at}med.mcgill.ca
Accepted: December 10, 2001.
Received: October 12, 2001.
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  F. Yasuhara, G. R. O. Gomes, E. R. Siu, C. I. Suenaga, E. Marostica, C. S. Porto, and M. F. M. Lazari Effects of the Antiestrogen Fulvestrant (ICI 182,780) on Gene Expression of the Rat Efferent Ductules Biol Reprod, September 1, 2008; 79(3): 432 - 441. [Abstract] [Full Text] [PDF]
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 D. W Erikson, A. L Way, D. A Chapman, and G. J Killian Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization Reproduction, May 1, 2007; 133(5): 909 - 917. [Abstract] [Full Text] [PDF]
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 S. Seenundun and B. Robaire Time-Dependent Rescue of Gene Expression by Androgens in the Mouse Proximal Caput Epididymidis-1 Cell Line after Androgen Withdrawal Endocrinology, January 1, 2007; 148(1): 173 - 188. [Abstract] [Full Text] [PDF]
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Y. Hao, N. Mathialagan, E. Walters, J. Mao, L. Lai, D. Becker, W. Li, J. Critser, and R. S. Prather Osteopontin Reduces Polyspermy During In Vitro Fertilization of Porcine Oocytes Biol Reprod, November 1, 2006; 75(5): 726 - 733. [Abstract] [Full Text] [PDF]
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T. R. Chauvin and M. D. Griswold Androgen-Regulated Genes in the Murine Epididymis Biol Reprod, August 1, 2004; 71(2): 560 - 569. [Abstract] [Full Text] [PDF]
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 L. Hermo, H. I. Adamali, and J. M. Trasler Postnatal Development and Regulation of {beta}-Hexosaminidase in Epithelial Cells of the Rat Epididymis J Androl, January 1, 2004; 25(1): 69 - 81. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer Osteopontin: Roles in Implantation and Placentation Biol Reprod, November 1, 2003; 69(5): 1458 - 1471. [Abstract] [Full Text] [PDF]
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G. A. Johnson, R. C. Burghardt, M. M. Joyce, T. E. Spencer, F. W. Bazer, C. A. Gray, and C. Pfarrer Osteopontin Is Synthesized by Uterine Glands and a 45-kDa Cleavage Fragment Is Localized at the Uterine-Placental Interface Throughout Ovine Pregnancy Biol Reprod, July 1, 2003; 69(1): 92 - 98. [Abstract] [Full Text] [PDF]
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