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a Station de Physiologie de la Reproduction des Mammifères Domestiques, URA INRA-CNRS 1291, 37380 Monnaie, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions.
The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidase (train I), retinoic acid binding protein (train N), and unidentified train E characterized the proximal corpus. Two unknown trains, M and H, were specific to the distal corpus, while only two minor proteins appeared in the cauda. This specificity of epididymal secretion is progressively established with age [10, 11]. However, most studies on the postnatal development of the epididymis have been conducted on rodents [12]. In the mouse, the differentiation of the epithelium begins in the distal region (2 wk after birth), followed by the middle (3 wk) and then the proximal regions (4 wk) [12]. No similar studies have been performed in the boar, apart from one study concerning epididymal sperm maturation according to age [13].
The differentiation and function of the epididymal epithelium is controlled by androgens (for review see [14, 15]). However, other factors such as testicular factors from the testicular fluid or associated with spermatozoa may be involved in the epididymal secretion [16–20].
The aims of the present study were 1) to analyze the secretory activity of epididymal cells during postnatal development and 2) to determine the regulation of the major regionalized proteins secreted in the adult boar epididymis using various physiological experimental conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty-nine adult Large White boars were used in this study: 3 normal animals; 12 animals castrated at 3, 8, 15, and 75 days before slaughter (3 boars in each group); and 2 hemicastrated at 15 and 60 days before slaughter (one animal each time). The testes were removed through a scrotal incision, and the epididymis was returned to the scrotum. For 2 animals, the ductus efferens of one testis were ligated for 4 mo (close to the extratesticular rete testis with sterile braided silk), the ipsilateral epididymis of these animals being used as controls. Twelve other animals were castrated at 75 days before slaughter and then given testosterone propionate replacement for 3, 8, 15, and 30 days at a dose of 90 mg/animal injected i.m. every 3 days (Interteston DC; Intervet, Angers, France). Animals aged 1, 2, 3, 4, 5, and 6 mo were used for the postnatal development study (3 per group). A sample of blood was collected and used for RIA testosterone assay [21] for all animals.
Reagents and Chemicals
Dulbecco's modified Eagle's medium without methionine and cysteine (DMEM-), complete Dulbecco's modified Eagle's medium (DMEM+), x-ray films (X-OMAT, XAR5; Eastman Kodak, Rochester, NY), and CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) were purchased from Sigma Chemical Company (St. Louis, MO); [35S]methionine and [35S]cysteine (35S-protein labeling mix, EXPRE35S35S) from NEN (Les Ulis, France); acrylamide (30% acrylamide, 0.8% N,N-methylenebisacrylamide) from Millipore (St. Quentin, France); ampholytes (Servalytes) from Serva (Heidelberg, Germany); Coomassie brilliant blue (Phastgel Blue R) and an electrophoresis calibration kit (standard protein) from Pharmacia (Orsay, France); Amplify from Amersham (Les Ulis, France); and Bradford assay from Bio-Rad (Paris, France). Other chemicals were from Prolabo (Paris, France) or Sigma in the best available grade.
In Vitro Secretion of [35S]Methionine-Cysteine-Labeled Proteins from Tissue Pieces
The epididymides were surgically removed from freshly killed animals at a local slaughterhouse. The epididymis was subdivided into 10 segments (E0 to E9) (Fig. 1) as previously described by Dacheux and Dacheux [22]. In vitro secretion of [35S]methionine-cysteine-labeled proteins was analyzed from tissue pieces as described by Syntin et al. [9]. Briefly, tissue from various epididymal regions (about 30 mg) was minced into small pieces (1–2 mm3) and washed with 0.5 ml of DMEM at 32°C under 95% O2 and 5% CO2 for 1 h. The washed tissue pieces were incubated in 0.5 ml of DMEM in the presence of 100 µCi 35S-protein labeling mix with agitation for 5 h at 32°C. The incubation medium was then removed and centrifuged (18 000 x g for 10 min). All supernatants were used directly or stored until use at -20°C.
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Gel Electrophoresis
The neosynthetic proteins present in the incubating medium were separated by two-dimensional (2D) gel electrophoresis. Isoelectric focusing was performed using O'Farrell's technique [23] modified as described by Syntin et al. [9]. Slab gels were made on glass tubes of 14-cm length (1.5 mm i.d.) that were filled with 4% acrylamide, 9.2 M urea, 2% ampholytes (1% pH 3–10 [Pharmacia] and 1% pH 2–11 [Servalytes]), and 2% CHAPS. The gels were allowed to polymerize for at least 3 h. Samples were prepared by boiling the protein solution for 5 min with 4% (v:v) of a denaturing solution (0.15 M dithioerythritol, 10% SDS). After cooling, the samples were mixed with an equal volume of a solution containing 9.2 M urea, 0.1 M dithioerythritol, and 2% CHAPS. Sample preparations of 20 µl were loaded on the top of the gel tube. The first step of the 2D gel migration was run at 20 mA, 0.1 W/tube, 700 V for a total of 10 000 V/h followed by 20 mA, 0.1 W/tube, 3000 V, and a total of 2000 V/h. The anode and cathode buffers were 25 mM H3PO4 and 0.1 M NaOH, respectively. Isoelectric points of the proteins were deduced directly from the pH pattern obtained by measuring the pH of 0.5-cm pieces of control gel incubated in 1 ml solution of deionized water for at least 1 h. The 2D separation was performed by laying the focusing gel on the top of a 6–16% acrylamide gel (1.5 mm thick) and running it at 20 mA, 1W, 300 V. Gels were stained either with Coomassie brilliant blue or through use of the silver staining technique [24]. After staining, the gels were impregnated with a fluorography enhancer (Amplify; Amersham, Arlington Heights, IL; now Pharmacia Biotech, Piscataway, NJ) and dried. Radioactive proteins were detected by autoradiography on preflashed x-ray film after several days' exposure at -80°C.
Fluorography Analysis
The fluorography analysis was performed using two different methods as previously described [9], a manual method in which the fluorographs are visually superimposed and a 2D electrophoresis computer analysis system. The gels were digitalized with an Eikonix 1412 scanner camera (Eastman Kodak), and patterns were compared using Kepler software (Large Scale Biology Corporation, Rockville, MD) on a DEC Alpha 3000–2000 (Digital Equipment Corp., Maynard, MA). The software integrated the surface and the intensity of each spot and automatically eliminated background and streaks. In this process, spots were modelized as Gaussian least squares, so that each gel pattern was reduced to a spot file. Each pattern was matched initially by interactive hand marking and then by an automatic process. A master gel was generated from three fluorographs of the same zone for each region, and only spots present in all three samples were taken into account. The final master pattern is a theoretic representation including the position and the relative intensity of each specific epididymal isoform. Numeration of spots begins with the highest molecular mass proteins and proceeds from left to right on the gel. The estimation of each spot intensity (i.e., radioactivity incorporated into each component) is given by the volume of the spot (calculated using the height and the surface of the modelized Gaussian spot).
Protein Enumeration and Protein Detection
Spot numbers on the 2D gels were the same as those previously obtained in the 2D map of the boar epididymal secreted proteins [9]. The 33 nonspecific spots previously described as secreted by external tissues [9] were not taken into account in this study. They were detectable in all animals, irrespective of their physiological condition and age, and were used as internal markers for the gel electrophoresis analysis (see Fig. 4a). The disappearance of a spot according to the physiological stage of the animals was taken in account only when the protein disappeared in all three animals studied. The proteins considered to be absent were the proteins that disappeared in all three animals. If the protein was present in one animal and absent in the other two, it was considered to be present.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Puberty occurs in Large White boars between 5 and 6 mo of age. In the present experiment, the plasma testosterone concentration was 3.5 ± 0.2 ng/ml at 1 mo; it decreased to 0.5 ± 0.2 ng/ml at 2 mo and then increased progressively to reach 12.7 ± 2.9 ng/ml at 6 mo of age (Fig. 2). For these animals aged from 1 to 6 mo, the secretory activities of the various epididymal regions were estimated by in vitro biosynthesis, and the number of proteins secreted was compared to those obtained in the same epididymal regions of adult animals.
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Total proteins secreted by the epididymis As early as 1 mo of age, 11 specific spots were secreted in the lumen of the tubule, representing several proteins including their isoforms (Figs. 2 and 3). The number of isoforms increased regularly to reach 47, 62, 80, 135, and 187 spots at 2, 3, 4, 5, and 6 mo, respectively (Fig. 2). At 6 mo of age, the number of proteins secreted in the epididymal lumen was nearly identical to that observed in the adult (Table 1). However, the secretory activity of each region varied according to the stages of postnatal differentiation.
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Secretory activity according to epididymal region At 1 mo of age, the secretory activity was the same at all points throughout the epididymis (Fig. 3, A and B), essentially represented by three trains, J, K, and L (Fig. 4a), which were those found in the caudal region of the adult (see Fig. 7h). Very few specific proteins were detected at this age, except train D, spots 26, 41, and 146 (Table 1). Differences between the regions appeared at 2 mo of age (Table 1, Fig. 3) when major components began to be secreted in the distal caput, i.e., hexosaminidase (train C), clusterin (train F/G), and lactoferrin (spot 43), and in the corpus, i.e., trains O and Q (Fig. 4b and see Fig. 6). Two other transitory proteins of 31 kDa and 72 kDa appeared at 2 mo and remained until 4 mo of age (indicated by an asterisk in Fig. 4, b and c, and Fig. 5, a and c) but were absent in the adult boar. At 3 mo, the secretory activity increased dramatically in the middle and distal caput (Fig. 3A), especially due to the appearance of numerous minor proteins (spots 190–193, 147, 164, 167, 170, 171, 186, 208) (Table 1, Fig. 4c); and at 4 mo, two major trains, A and B (GPX) (Table 1, Fig. 5a and Fig. 6), appeared in the proximal caput and trains E and M in the corpus (Table 1, Fig. 5c). At 5 mo, protein secretion increased significantly in the anterior part of the epididymis (Fig. 3) with secretion of procathepsin L (train P) in the middle caput and mannosidase (train I) and E-RABP (train N) in the distal caput (Table 1), and the emergence of numerous minor components in the proximal caput (Table 1, Fig. 5d, spots 118, 142, 132, 133, 175, 176, 177). At 6 mo, and until the adult period, the number of minor proteins secreted increased in the distal caput and in the corpus (Fig. 3) with the appearance of train H (Table 1, and see Fig. 7g). In the proximal caput, the secretory activity (number of proteins secreted and volume of secretion) increased regularly from 1 mo to the adult period (Fig. 3).
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Sites of secretion of the major proteins Some major proteins such as GPX, mannosidase, and E-RABP (Fig. 6) appeared in the same regions as in the adult. It is interesting to note that these proteins were secreted in the latter part of postnatal development (4, 5, or 6 mo). Thus, GPX, which appeared at 4 mo (maximum secretion in E1), and mannosidase (train I) and E-RABP (train N), which appeared at 5 mo (maximum secretion in E4), maintained their secretion in the same regions until the adult period (Fig. 6). For the other proteins, i.e, hexosaminidase, clusterin, lactoferrin, and train O, which appeared early during postnatal development (as early as 2 mo), secretion began in a more posterior region than in the adult. Hexosaminidase (train C) began to be secreted at 2 mo with maximal secretion in E4; but at 3 mo the maximum secretion appeared in E3, and then in the adult boar in E2. Clusterin (train F/G) and lactoferrin (spot 43) were secreted at 2 mo, with a maximum in E4 and then in E3 until the adult period for clusterin, while lactoferrin shifted to E4 in adulthood. The secretion of the four polypeptides of train O (spots 187, 182, 179, and 183, Table 1) began at 2 mo (Fig. 4b and Fig. 6) from E4 to E9, with a maximum in E5 that shifted progressively in E4 and then in E3 in adulthood. For this polymorphic protein, the secretion of spot 187 was detected in regions E0–E1 at 4 mo (Table 1).
Effect of Efferent Duct Ligation on Protein Secretion
A total of 39 spots disappeared in the ligated epididymis, including the 8 major compounds of the anterior region (Table 2), while the protein secretion appeared normal in the contralateral epididymis (Fig. 7). Ligation affected the various epididymal regions differently: 18 spots disappeared in regions E0–E1, 17 in regions E2–E3, 4 in regions E4–E5, and none in the other distal regions.
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In regions E0–E1, major train A disappeared completely (Fig. 7a), while it represented 87.5% of the total secretion of this region on the normal side (Fig. 7e). Other minor proteins specific to these regions also disappeared, such as spots 118, 142, and 176 (Table 2; Fig. 7, a and e). Some proteins, such as spots 26, 41, and 208 secreted in these regions and in other parts or at all points throughout the organ, disappeared only in E0–E1 (Table 2; Fig. 7, a and e). GPX (train B) and hexosaminidase (train C) were still secreted in these regions but were greatly reduced (Fig. 7, a and e). Clusterin (trains F/G) specifically secreted from E2 to E6 in normal boars appeared to be the major protein in E0–E1 after efferent duct ligation (Fig. 7a). In the other regions (E3 to E8/9) (Fig. 7), the patterns of secretion were similar to those of the normal side except for some proteins such as procathepsin L (train P), which had disappeared (Table 2).
Effect of Castration on Protein Secretion
Four main effects were observed on protein secretion after castration: a) increase in some proteins, b) decrease in and disappearance of other proteins, c) unaffected proteins, and d) appearance of new polypeptides. The testosterone level was 0.13 ± 0.01 ng/ml for the three 3-day-castrated animals and < 0.1 ng/ml for the 8- and 15-day-castrated animals.
a) Increase in protein secretion The clusterin secretion (train F/G) localized in regions E2–E6 in normal boars (Fig. 7f) became the major compound secreted at all points throughout the epididymis (E0–E7) after 3 days of castration (Table 2). Its secretion increased after 15 days of castration to reach about 10–20 times the concentration observed in the normal boar, depending on epididymal region (Fig. 8, and see Fig. 12). The secretion of other proteins such as spots 26 and 41 (Fig. 8) also increased after castration, or appeared at all points throughout the epididymis (E0–E8), e.g., train D (Table 2; Fig. 8, a and b), secreted only in E3 in the normal boar.
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b) Decrease in and disappearance of proteins The secretory activity estimated by the number of spots identified in the protein secretion in the whole epididymis decreased gradually, representing 51.3%, 37.5%, 33.3%, and 19.8% of the normal secretion after 3, 8, 15, and 75 days of castration, respectively (Table 2, Fig. 9).
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After 3 days of castration, the most important modification was the sharp reduction in train A in regions E0–E1 (Table 2), composed only of two isoforms secreted among the six forms identified in the normal boar (Fig. 7e). Spot 171 disappeared in the middle caput, and mannosidase (train I) was no longer secreted in the distal caput (Table 2). Spots 147, 148, and 208, specific to the caput and corpus, had also disappeared (Table 2).
After 8 days of castration, train O, lactoferrin (spot 43), E-RABP (train N), and trains H and M disappeared (Table 2). GPX (train B) disappeared after 15 days of castration (Fig. 8a), and hexosaminidase (train C) and spots 146, 190, 27, and 29 were absent after 75 days of castration (Table 2).
c) Proteins unaffected by castration After a long period of castration, only 37 spots (Fig. 9) were identified among the proteins secreted at all points throughout the epididymis (representing 19.8%) (Table 2). These secretions were composed of proteins with increased secretion after castration (described above) and also of minor spots (10 spots) unaffected by castration or any other physiological conditions (Fig. 10, black spots; Table 3).
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d) Secretion of new proteins Some proteins absent in the normal boar appeared after castration. One train at 35 kDa (pI 5.5–7) was secreted from E1 to E6 after 15 days of castration (Fig. 8, a and b, asterisks). Another train at 53 kDa (same level as train E in the normal animal) appeared in the medium from the caput to the corpus after 8 and 15 days of castration, and disappeared after 75 days of castration. It had the same molecular mass as train E (Fig. 7f) but with new basic spots from 7.3 to 9.1 (Fig. 8a). These new spots could represent new forms of train E or a new train composed of basic proteins. A similar result was obtained for train K of 30 kDa, which was found from the distal corpus to the cauda in the normal animal (Fig. 7, g and h). It was strongly secreted after 15 and 75 days of castration at all points throughout the epididymis with new basic spots between 5.8 and 7.5 (Fig. 8, a and b).
Effect of Androgen Supplementation on Epididymal Secretion
After androgen treatment in castrated animals, blood testosterone levels were 7.6 ± 0.7, 10.1 ± 0.8, and 9.3 ± 1.4 ng/ml after 8, 15, and 30 days of injection, respectively. These values were not significantly different from those obtained on the same animals before castration (10.2 ± 3.6 ng/ml).
Three effects were obtained on the protein secretion: a) restoration of specific proteins, b) absence of restoration, and c) repression of proteins.
a) Restoration of specific proteins The proportion of proteins secreted after 3, 8, and 15 days of supplementation (number of spots) increased progressively to reach 28.8%, 40.6%, and 52.9%, respectively (Table 2, Fig. 9). After 30 days of treatment, the secretion reached a plateau at which no more proteins were detected. We characterized a total of 62 spots (33.15%) (Table 3) such as GPX, hexosaminidase, E-RABP, train O, and lactoferrin (spot 43) (Fig. 10, blue spots) that reappeared after androgen supplementation.
However, the secretion of these proteins was differently reinduced as a function of time of treatment. A small secretion of hexosaminidase appeared as early as after 3 days of treatment, whereas this occurred only after 8 days for GPX (train B), train O, and lactoferrin (spot 43) (Fig. 11, a and b). E-RABP (train N) was reinduced after 15 days of testosterone treatment only in E4 (Table 2, Fig. 11e); and among the nine spots, only four spots were restored (spots 143, 144, 145, and 149) with a level of secretion lower than in the normal epididymis (Fig. 11e). For train O and lactoferrin, the intensity of secretion was restored to a normal level after 15 days of supplementation (Fig. 12), although secretion of lactoferrin was not reinduced in the distal part of the epididymis (E6–E7) (Fig. 12). The secretion of GPX was restored in the middle caput, while restoration was not complete in the proximal caput (E0–E1) (Fig. 12). However, after testosterone treatment, GPX secretion appeared in some distal regions (E4–E5) where the protein was absent in the normal animal (Fig. 12). Secretion of hexosaminidase localized in E0–E4 in the normal boar did not return to normal levels, and after 15 days of supplementation only a small intensity of secretion was detected at all points throughout the epididymis (E0–E7) (Fig. 12). Secretion of mannosidase (train I) reappeared only after 1 mo of supplementation. However, this secretion, which was revealed by in vitro biosynthesis, was just at the level of detection. Using an immunoperoxidase technique in tissue sections and immunoblotting in caudal fluid after 1 mo of testosterone replacement, an important localization was seen both in tissue and in caudal fluid (not shown).
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b) Proteins not reinduced by androgen supplementation Some specific proteins were not reinduced, irrespective of the time of androgen supplementation, especially in the proximal and middle caput. Secretion of train A was not restored in the proximal caput; nor were 12 other spots, including 118, 142, and 176. Procathepsin L (train P) and spots 132, 133, 175, 177, and 178 were not restored in the middle caput (Table 2). Secretion of trains M and H was not restored in the corpus (Table 2).
c) Decreased secretion of some proteins All the proteins with increased secretion after castration were reduced by testosterone administration. The greatest decrease was observed for clusterin (trains F/G) (Fig. 11). After 3 days of supplementation, clusterin secretion decreased at all points throughout the epididymis (E0–E7) (Table 2, Figs. 11 and 12). However, 30 days of supplementation was necessary to restore normal regionalization. Secretion of other proteins (spots 26, 41, trains D and K) also decreased as early as 3 days after supplementation without a change in their localization; and for trains D and K, 30 days of supplementation was unable to restore normal regionalization (Table 2).
Effect of Hemicastration on Protein Secretion
The proteins secreted by various regions of the intact and castrated sides of the epididymis are shown in Figure 13 (A and B). None of the specific proteins secreted in the various regions of the normal epididymis were visible on the castrated side after 2 mo of hemicastration, except for mannosidase (train I) in E4 (Fig. 13A) The reduction in protein secretion on the castrated side was visible at 15 days of hemicastration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When castrated animals had plasma levels of testosterone similar to those of normal animals, we found that 47.6% of all the epididymal proteins secreted were under androgenic control (stimulated or repressed); 47% were modulated by other factors and 5.3% remained unchanged, irrespective of the physiological conditions.
The epididymis has always been described as being essentially dependent upon androgens and principally upon 5-dihydrotestosterone [25]. However, protein secretion is greatly influenced by other factors. Several studies have indicated that certain testicular factors in the proximal caput of the epididymis are necessary to maintain morphological structure [16] and protein secretion in the rat [17] and mouse [18–20]. We have shown in the boar that 50% of proteins specific to this region, such as train A, the major protein of region E0-E1, seemed to be regulated by factors coming from the testis. These proteins that disappeared after castration or efferent duct ligation were not reinduced after testosterone supplementation. It was interesting to note that secretion began in the latter part of postnatal development (4–5 mo) when spermatozoa and testicular fluid enter the epididymal lumen. However, the nature of the factors involved is unknown—spermatozoa, testicular proteins, or testosterone from the fluid. It has recently been shown in the rat that spermatozoa or spermatozoa-associated factors [17] and some testicular proteins that are actively endocyted in the proximal part of the epididymis can modulate the protein secretion of this segment [25].
Additional factors may regulate locally restricted function in more distal regions of the epididymis, leading to such region-specific protein secretion. Thus, a protein secreted from a specific part of the epididymis may regulate protein secretion in the following segment. In the boar, procathepsin L (train P), specific to the distal caput, disappeared after castration and was not reinduced after testosterone treatment. We have recently postulated that platelet-derived growth factor, whose mRNA is highest in the middle caput just preceding the site of procathepsin L secretion, can directly regulate its synthesis and secretion in the distal caput [26]. Similarly, trains H and M, which are specific to the corpus and persist after efferent duct ligation, disappear after castration and are not reinduced after testosterone treatment, also controlled by epididymal proteins from previous segments. However, the nature of the proteins involved remains to be determined.
We have shown that 47.6% of epididymal proteins in the boar are dependent on androgens, of which 33.1% are stimulated. However, the degree of androgen dependence varies. Some proteins seemed to be regulated essentially by testosterone, like train O, which was reinduced after testosterone treatment in exactly the same secretory regions. For other proteins, control of their secretion seemed multifactorial, depending not only on testosterone but also on other factors, probably epididymal factors, in anterior and posterior regions. The two proteins hexosaminidase (train C) and GPX (train B), which were secreted in the caput (E0–E3 and E0–E4, respectively), presented a different level of sensitivity to androgen. GPX, which completely disappeared after 15 days of castration, was more sensitive to testosterone than hexosaminidase, which persisted even after 1 mo of castration. Testosterone supplementation was unable to restore normal secretion of these proteins in the proximal caput, indicating that other factors were necessary to modulate their secretion in this segment, as has been suggested in the mouse epididymis for GPX mRNA expression [27]. It has also been speculated that PEA3 (-domain of the Py enhancer), a transcriptional factor expressed in the initial segment of the rat epididymis [28], may control GPX gene expression. However, some testicular factors (testosterone or testicular proteins) may also be involved, since the secretion of GPX was strongly reduced after efferent duct ligation. It has recently been shown that A-raf kinase, a protein that is highly expressed in the initial segment of the mouse epididymis and is influenced by testicular factors [17], could regulate the activity of PEA3 [29].
It is interesting to note that more distal regions, which do not secrete these proteins (trains B and C) in the normal boar, become secretory zones in testosterone-replaced boars, suggesting that these regions are inhibited by certain factors under normal conditions. This result has previously been observed for hexosaminidase (antiagglutinin) using immunohistochemical techniques, demonstrating that the distal regions of the epididymis are potentially secretory under certain conditions, especially in supplemented animals [22].
Secretion of train N (E-RABP), which was reinduced only in region E4 after testosterone replacement, suggested that some epididymal factors may also be involved in the maintenance of secretion in distal regions (E5–E7), as has previously been shown for cellular retinol-binding protein [30]. Similarly, secretion of lactoferrin, which was not restored in the distal regions (E6–E7), probably required epididymal proteins emanating from some anterior regions.
We have shown in the boar that, among the 14.4% of proteins repressed by androgens, clusterin, which is specific to regions E2–E6 in the normal boar, became the major secretory protein at all points in the epididymis after castration except in the cauda, while testosterone replacement restored normal regionalization. Such repression by androgens has been previously observed in the prostate [31, 32]. However, the unusual secretion of clusterin in the proximal caput and the distal corpus of the epididymis suggested different segment-specific androgen control, as has been shown for its mRNA level in various regions of the rat epididymis [33]. This restricted clusterin mRNA expression raises questions regarding the control mechanisms that regulate such specific gene expression. The different levels of clusterin secretion at all points throughout the epididymis may be correlated with different rates of promoter methylation, as has been shown for several organs secreting different levels of clusterin [34].
In conclusion, regulation of such region-specific protein secretion in the epididymis is a complex system that depends on the region of expression and its variable level of sensitivity to testosterone and the action of other factors originating from the testis or the epididymis. However, further studies are required to identify these factors to achieve greater understanding of the molecular mechanisms that control the secretory activity of epididymal cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 47 42 77 43; dacheux{at}tours.inra.fr
Accepted: August 10, 1999.
Received: August 10, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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