Goyal,2 ,3 Frank F. Bartol,4 Anne A. Wiley,4 and Christopher W. Neff3. Department of Anatomy ...... 3187-3199. 37. Greene GL, Nolan C, Engler JP, Jensen EV.
BIOLOGY OF REPRODUCTION 56, 90-101 (1997)
Immunolocalization of Receptors for Androgen and Estrogen in Male Caprine Reproductive Tissues: Unique Distribution of Estrogen Receptors in Efferent Ductule Epithelium' Hari 0. Goyal,2 ,3 Frank F. Bartol,4 Anne A. Wiley,4 and Christopher W. Neff3 Department of Anatomy, Tuskegee University, Tuskegee, Alabama 36088 Department of Animal and Dairy Science,4 Auburn University, Auburn, Alabama 36849 ABSTRACT Androgens and estrogens affect physiological processes inthe testis and male excurrent duct system. This study was designed to identify and characterize distribution of androgen receptors (AR) and estrogen receptors (ER) in the reproductive organs of the male goat. Tissues, including testis, efferent ductules, epididymis (regions I-V), and ductus deferens, were obtained from five mature Nubian goats, fixed in 4% paraformaldehyde, and embedded in paraplast. Antigenic sites for AR were unmasked by microwave treatment (four times, 5 min each) of tissue sections immersed in 10 mM citrate (pH 6) and were detected using the PG-21 rabbit anti-rat/human antibody. Antigenic sites for ER were identified using the H-222 rat anti-human monoclonal antibody after tissue sections were treated with pronase (0.5 mg/ml, 37°C, 8 min). Avidin-biotin horseradish peroxidase procedures were used to identify positive immunoreactivity. Irrelevant IgG was substituted for primary antibody in negative controls. Positive nuclear immunostaining for AR was observed in all types of epithelial cells, peritubular smooth muscle cells, and intertubular fibroblasts of the intratesticular rete, efferent ductules, epididymis (regions I-V), and ductus deferens, as well as in Sertoli, Leydig, and peritubular myoid cells and intertubular fibroblasts of the testis. In contrast, nuclear immunostaining for ER was confined to nonciliated cells of the efferent ductules. Thus, AR-positive cells are ubiquitously distributed in caprine testicular and excurrent ductular tissues, and ER-positive cells are unique to the efferent ductules. The caprine model should be useful in studies designed to determine mechanisms through which androgens and estrogens regulate development and function of the testes and excurrent ducts. INTRODUCTION Mammalian sperm mature functionally during their transit through the excurrent ducts (rete testis, efferent ductules, epididymis), acquiring the potential to move in a forward direction and fertilize ova by the time they reach the tail of the epididymis [1]. Mechanisms underlying sperm maturation are not fully understood. However, it is likely that this process depends upon the environment created within the excurrent ducts by absorptive and secretory activities of ductular epithelium. More than 90% of the testicular fluid is believed to be absorbed in the efferent ductules and initial segment of the epididymis [2-3]; numerous epididymal secretory proteins that coat sperm have been isolated and some have been sequenced [1,4-7]; and antibodies
produced against certain epididymal glycoproteins have been shown to render sperm infertile [8]. Virtually all epididymal functions, including absorption of testicular fluid, secretion of organic and inorganic molecules, and both transport and storage of sperm, have been shown to be androgen dependent [1]. However, epididymal responses to androgens are region specific and reflect the source and concentration of androgens. For example, 1) the caput has a higher androgen requirement than both the corpus and cauda [9-11]; 2) within the caput, it has been suggested that luminal androgens and other components of rete fluid are more important functionally than circulating androgens [12]; and 3) 5ot-dihydrotestosterone is more efficacious than testosterone in the initial segment of the epididymis of the rat [13] and in the middle caput and distal caput epididymis of the ram [14, 15]. In addition to androgens, estrogens may be important for maintenance of structural and functional integrity of specific segments of the male reproductive tract. In this respect, epithelium of the efferent ductule has been shown to contain more estrogen receptor (ER) than any other part of the male reproductive tract [16, 17]. Because of the implied significance of androgens and estrogens in mediation of sperm maturation, and because these steroids may have region-specific effects in the male reproductive tract, several laboratories have investigated AR and/or ER in various male reproductive organs of rats [18-21], mice [16, 17, 22], rabbits [23, 24], monkeys [25, 26], humans [27-31], and rams [15]. In the present study, immunocytochemical methods were used to determine the spatial pattern of distribution for both AR and ER in reproductive tissues of the male goat, a large animal species that has been the subject of extensive microanatomical study [32, 33] but for which no information on steroid receptor distribution is available. MATERIALS AND METHODS Animals Five mature Nubian bucks, approximately 2-3 yr of age and with a satisfactory spermiogram (scrotal circumference - 26 cm; sperm abnormalities - 20%) were used. They were kept in a covered shelter, allowed to walk freely, and given hay and water at libitum. A general management schedule for deworming, disease prevention, and hoof trimming was followed.
Accepted August 16, 1996. Received February 5, 1996. 'This work was supported by grants NIH-MBRS-5-S06-GM08091 (H.O.G.), USDA-CSR-EES-ALX-TU-CT1, and NIH-RCMI-5-G12RR0305908. 'Correspondence: H.O. Goyal, Department of Anatomy, School of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088. FAX: (334) 727-81 77.
Tissues and Fixation The testes and excurrent ducts were collected under general anesthesia and were fixed by vascular perfusion through the testicular artery with precooled 4% (w:v) paraformaldehyde in PBS (pH 7.4). Tissue samples collected from the testis, rete testis, three areas of the ductuli effer90
ANDROGEN AND ESTROGEN RECEPTORS IN MALE GOATS entes (proximal, closer to the extratesticular rete; middle; and distal, nearer the beginning part of the epididymis), five regions of the epididymis (region I, proximal caput; region II, middle caput; region III, distal caput; region IV, body; and region V, tail), and ductus deferens were further fixed in the same fixative overnight at 4°C. After fixing, tissues were dehydrated through graded concentrations of ethanol, cleared in xylene, embedded in Paraplast-plus, cut at 5-jim thickness, and mounted on poly-L-lysine (Sigma Chemical Co., St. Louis, MO)-coated slides. Antigen Retrieval and Immunolocalization of AR and ER Antigen retrieval. Sets of slides containing two sections per slide were deparaffinized, rehydrated, and subjected to antigen retrieval procedures. Conditions for antigen retrieval were determined empirically. To unmask AR, tissue sections were subjected to microwave irradiation [34]. Slides were immersed in 10 mM citrate solution (pH 6.0) and irradiated in a standard microwave oven (500 W) at full power for 20 min. Buffer level was checked at 5-min intervals, and lost fluid was replaced with distilled water. After heating in the microwave, the container holding the slides was removed and allowed to cool for 15 min at room temperature. Slides were then washed in PBS at room temperature for 5 min before initiation of immunolocalization procedures. Microwave irradiation in citrate solution for 10 min, or in distilled water for 10, 20, or 30 min, was ineffective. To unmask ER, tissue sections were incubated in PBS for 5 min at room temperature and then submerged in a prewarmed (37°C) solution of protease type XIV (0.5% w: v, Pronase E; Sigma, type XIV) in PBS for 8 min [35]. To stop protease digestion, slides were rinsed in cold tap water and placed in PBS for 5 min. Preliminary trials indicated that nuclear staining intensity for ER increased in association with protease digestion times from 1 to 8 min. Omission of the protease digestion step prevented development of positive nuclear staining for ER. Immunolocalization procedures. Androgen receptor (AR) protein was immunolocalized using the PG-21 rabbit anti-rat/human AR polyclonal antibody (PG-21) [36], at a concentration of 3 jig/ml in PBS containing 1% (w:v) BSA, and the Biostain (anti-rabbit IgG) Super ABC immunoassay detection system (Biomeda, Foster City, CA). One section on each slide served as a negative control and received irrelevant rabbit IgG (Jackson Immunological Research Laboratories, West Grove, PA) in place of PG-21. In preliminary studies, PBS was also substituted for PG-21 to establish that nonspecific binding of the secondary antibody was negligible. Groups of slides representing tissues from each animal were processed together. Procedures were essentially identical to those suggested in kit instructions. Briefly, individual sections were first encircled with a peroxidase antiperoxidase complex pen (Research Products International Corp., Mount Prospect, IL). After application of the diluted protein blocker reagent, slides were incubated in a humidified chamber for 20 min at 37°C. Excess blocker was removed and replaced immediately with PG-21 (test sections) or irrelevant rabbit IgG (negative control sections). Slides were incubated overnight at 4C in a humidified container, brought to room temperature, and rinsed in PBS for 5 min. Next, biotinylated secondary antibody was applied to each section, and slides were incubated for 40 min at 37°C in a humidified chamber and rinsed in fresh PBS for 5 min at room temperature. Slides were then placed
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in 3% hydrogen peroxide at room temperature for 5 min to block endogenous peroxidase activity, rinsed in tap water, rinsed in fresh PBS for 5 min, incubated with the streptavidin-labeled peroxidase complex for 40 min at 37C, rinsed in fresh PBS for 5 min, and then placed in a freshly prepared peroxidase substrate solution (0.1% diaminobenzidine tetrachloride in 0.1 M Tris buffer, pH 7.2, and an equal volume of 0.02% hydrogen peroxide prepared from 30% stock in distilled water) until a brown color developed (510 min). Developed sections were rinsed in tap water, dehydrated in ethanol, and cleared in xylene. Coverslips were applied using Permount. Immunolocalization of ER was accomplished using the monoclonal rat anti-human ER antibody H-222 [37] at a working concentration of 7 jIg/ml in PBS, containing 1% (w:v) BSA, and the Biostain anti-mouse/rat IgG Super ABC immunoassay detection system. Negative control sections received irrelevant rat IgG at the same concentration as used for H-222. Otherwise, procedures were identical to those described above for AR. Evaluation of immunostaining. Sections representing each tissue and region from each animal were observed and scored for presence and intensity of nuclear staining characteristic of AR or ER. On the basis of visual examination, nuclear staining intensity was designated as negative (absent), weak, or strong. Nuclei were declared negative if receptor staining intensity did not differ visibly from that of negative control sections on a within-slide basis. In contrast, staining observed consistently for AR in principal cells of the epididymis was designated as strong. Specificity of PG-2 1 and H-222 in Goat Tissues Western blots were performed to confirm that the PG-21 and H-222 antibodies would recognize specific proteins of the appropriate molecular weight in goat tissues. To this end, proteins were extracted from the efferent ductules according to the procedure of Glatstein and Yeh [38]. Briefly, the frozen tissue (0.5 g) was minced on an ice-cold glass plate, homogenized in 300 il of extraction buffer (1 mM Tris-EDTA, 1 mM dithiothreitol, 10% glycerol, and 100 VIg/ml PMSF) on ice using a Tissue-Tearor (Biospec Products, Bartlesville, OK), and sonicated using a Sonifier Cell Disruptor (Heat Systems-Ultrasonics, Inc., Plainview, NY). The sonicated sample was centrifuged at 10 000 x g for 10 min at 4C. The supernatant was assayed for protein concentration using the Pierce Coomassie Protein Reagent (Pierce, Rockford, IL) and then stored at - 80 0C until needed. Multiple aliquots of tissue extracts (150 jig total protein per lane) were subjected to SDS-PAGE [39], using a 7.5% separating gel [40] and conditions recommended for the Bio-Rad Mini-Protein II Dual Slab Electrophoresis Cell system (Bio-Rad, Richmond, CA). The separated proteins were transferred to nitrocellulose membrane according to procedures recommended for the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell. After transfer, the nitrocellulose membrane was stained with 0.1% (w:v) Ponceau S in 1% acetic acid to visualize protein bands and facilitate identification of individual lanes, which were cut into strips. Strips were destained in Tween 20 (0.5% w:v)/Tris-buffered saline (TTBS). Destained strips of blotted proteins were blocked for 30 min in 1% (w:v) nonfat dry milk in TTBS at room temperature, incubated with PG-21 or H-222 (1.0 ig/ml) overnight at 4°C, washed four times (5 min each) in 1% nonfat
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TABLE 1. A comparison of immunolocalization of AR and ER in the male reproductive tract of goats with that of published data in other species.* AR Rat Organ A. Testis Sertoli cells Germ cells Peritubular myoid cell Leydig cells
Goat
+ -
+
Human
1201
l19]
ER
118]
1311
+ +
+ + + +
+
+
[29]
Monkey [261
Ram [15]
Goat
+ +
+ + +
ne ne ne ne
ne ne ne ne
-
1301
[28]
+ + +
+
Mouse [431
Monkey [251
-
--
+ +
+ +
+ +
B. Tubulus rectus Sertoli cells
+
ne
ne
ne
ne
ne
ne
ne
ne
ne
-
ne
ne
C. Rete testis Epithelium Stromal cells
+ +
ne ne
ne ne
ne ne
+ ne
ne ne
ne ne
ne ne
ne ne
ne ne
-
ne ne
ne ne
D. Efferent ductules Epithelium Stromal cells
+ +
ne ne
ne ne
ne ne
ne ne
ne ne
ne ne
ne ne
+ +
+(nc)
+
+Inc)
E. Epididymis Epithelium Stromal cells
+ +
ne
ne +
+
ne ne
+ +
+ +
ne ne
+ +-
-
+
+
F. Ductus deferens Epithelium Stromal cells
+ +
ne ne
ne ne
ne ne
ne ne
+ +
+ +
ne ne
ne ne
ne ne
* Symbols are as follows: + positive;
+
_+
+
-
faintly present; - negative; ne, not examined; nc, nonciliated cells. Numbers in brackets indicate references.
dry milk in TTBS, and then incubated for 1 h at 37C with a 1:2000 dilution of horseradish peroxidase goat anti-rabbit secondary antibody (Jackson Immunological Research Laboratories) in the case of PG-21 and with a 1:5000 dilution of horseradish peroxidase goat anti-rat secondary antibody in the case of H-222. After incubation with the secondary antibody, blots were washed four times (5 min each) in 1% nonfat dry milk in TTBS and then washed a fifth time in TTBS only. Both AR and ER proteins were visualized using the ECL chemiluminescence system (Amersham Life Science, Cleveland, OH) and Kodak XOMAT LS film (Eastman Kodak, Rochester, NY). Negative control strips representing identical tissue extracts received rabbit IgG (1.0 pxg/ml) in place of the PG-21 or rat IgG (1.0 pxg/ml) in place of the H-222. Additional controls for the PG-21 included incubation of strips with a mixture of PG-21 and a 15-fold molar excess of AR21 (the antigenic peptide) or AR 462, an unrelated peptide [36]. RESULTS In Table 1, regional differences in the localization of AR and ER in the male reproductive tract of the goat are summarized and compared with those represented by previously published data in other species. AR Testis. Examination of the testis at low magnification revealed positive immunostaining in the periphery of each seminiferous tubule and in a few intertubular cells (Fig. , a and b). These positive areas, when examined at high magnification and compared with glycol methacrylate sections of the testis that were stained with hematoxylin and eosin, were found to be the nuclei of Sertoli cells, peritubular myoid cells, Leydig cells, fibroblasts, and smooth muscle cells surrounding the blood vessels (Fig. 1, c and d). Of all these cell types, the nuclei of Sertoli cells were most heavily stained. Immunostaining patterns did not differ between
seminiferous tubules containing different germ cell lineages, indicating that the presence and staining intensity of AR in Sertoli cells may not be dependent upon the stage of the seminiferous epithelial cycle. However, nuclei of Sertoli cells in the straight part of the seminiferous tubule, where germ cells are absent and cytoplasmic extensions of Sertoli cells form the so-called Sertoli cell plug [41], took a lighter stain than those in the convoluted part (Fig. I e). Neither the nuclei of germ cells nor the cytoplasm of somatic cells or germ cells showed immunostaining for AR. However, spermatids did display positive staining in the Golgi area that was significantly greater in intensity than that observed in negative control sections (Fig. I a). Not all intertubular cells were positive or equally stained. Within a group of Leydig cells, some nuclei were darker than others. Endothelial nuclei were negative for AR (Fig. f). Rete testis. The rete testis is a maze of tubules lined by simple cuboidal epithelial cells. All epithelial nuclei and most stromal nuclei were positive (Fig. 1, g and h). Generally, AR staining intensity was stronger in epithelial nuclei than in stromal nuclei underlying the epithelium. Efferent ductules. The simple columnar epithelium consists of two main cell types, ciliated cells and nonciliated cells. The latter can be further classified into three subtypes. While type I cells contain neither granules nor vacuoles in the cytoplasm, type II cells are characterized by dense gran-
FIG. 1. a-h) Immunocytochemical localization of AR in the testis and intratesticular rete. a) This low-magnification micrograph of testis shows AR-positive immunostaining in many nuclei located in the periphery of the seminiferous epithelium (arrows) and in some nuclei located in the intertubular tissue (arrowheads). b) Control section of testis incubated with an irrelevant IgG in place of the primary antibody. Note lack of positive immunostaining. c) An enlargement of a part of the section shown in Figure 1a. Note that nuclei of Sertoli cells (arrows) and peritubular myoid cells (arrowheads) are positive for AR, while those of adjoining germ cells are negative. Open arrows indicate positive immunostaining in the Golgi area of spermatids. d) Testicular tissue in this micrograph was embedded
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in glycol methacrylate and stained with hematoxylin and eosin. High resolution achieved by this procedure allows a clear identification of nuclei of Sertoli cells (arrows). e) Nuclei of Sertoli cells in the straight part of the seminiferous tubules take a lighter immunostain than those in the convoluted part (compare with c). An abundance of cytoplasmic vacuoles (v) is a characteristic feature of Sertoli cells from this part of the seminiferous tubule. f) Leydig cells (L) within a group show a varying intensity of immunostaining in their nuclei. Note the absence of immunostaining in the nuclei of endothelial cells lining the blood vessel (bv). g) In this micrograph of intratesticular rete, note positive immunostaining in all nuclei of epithelial cells and some nuclei of connective tissue cells underlying the epithelium. h) Control section of intratesticular rete incubated with an irrelevant IgG in place of the primary antibody. Note lack of positive immunostaining. a-c and e-h, paraplast sections; d) glycol methacrylate section. a-b, x90; c-h, x370.
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FIG. 2. a-h) Immunocytochemical localization of AR in the efferent ductules. a) This low-magnification micrograph shows AR-positive immunostaining in nuclei of epithelial cells (arrow), smooth muscle cells surrounding the epithelium (arrowhead), and intertubular connective tissue cells (open arrowhead). b) Control section of efferent ductules incubated with an irrelevant IgG in place of the primary antibody. Note lack of positive immunostaining. c, e, g) Efferent ductules lined predominantly by type I (c), type 11(e), and type III (g) nonciliated cells. Note positive nuclear immunostaining for AR in both ciliated (arrows) and nonciliated cells (arrowheads). d, f, h) Complementary regions of efferent ductules stained with toluidine blue to show the morphology of type I (d), type II (f), and type III (h) nonciliated cells. While type I cells lack both granules and vacuoles, type II cells contain dense granules, and type III cells contain empty-appearing vacuoles. a-c, e, and g, paraplast sections; d, f, and h, Epon sections. a-b, x75; c-h, x300.
ANDROGEN AND ESTROGEN RECEPTORS IN MALE GOATS ules positive for periodic-acid Schiff, and type III cells by empty-appearing, vacuoles negative for periodic-acid Schiff [32]. All nuclei of both ciliated and nonciliated cells, and most nuclei of smooth muscle cells and fibroblasts, were AR positive. The intensity of stain differed among nonciliated cell types. Nuclei of type II cells were comparatively lighter than those of type III or type I cells (Fig. 2, a-h). Epididymis. The goat epididymis can be divided into five regions: regions I, II, and III constitute the head; region IV the body; and region V the tail (Fig. 3a; for details see [33]). Regardless of the region, the pseudostratified epithelium consists of two major cell types (principal cells and basal cells) and one minor cell type (apical cells). In addition, two connective tissue cells, intraepithelial lymphocytes and macrophages, can be seen at all levels of the epithelium. The nuclei of all epithelial cells, as well as those of most of the surrounding smooth muscle cells and intertubular connective tissue cells, were positive for AR. By comparison, nuclei of principal cells were more strongly stained than those of any other cell type in the epididymis or in other male reproductive organs examined here. Furthermore, among epididymal regions, nuclear AR staining was darker in regions II, III, and IV than in regions I and V (Fig. 3, b-g). Ductus deferens. The ductus deferens is a cord-like structure whose pseudostratified epithelium, containing principal cells, basal cells, and intraepithelial lymphocytes and macrophages, is surrounded by a thick coat of concentrically arranged layers of smooth muscle cells. Positive immunostaining for AR in epithelial cells as well as in smooth muscle cells of the ductus deferens was similar to that described for the epididymis in general and for region V of the epididymis in particular (Fig. 3h). ER Among all male reproductive organs examined, including the testis, rete testis, efferent ductules, epididymis (regions I-V), and ductus deferens, only epithelial nuclei of efferent ductules displayed positive immunostaining for ER. No staining above background was observed in intertubular connective tissue cells or in smooth muscle cells surrounding the epithelium. Within this epithelium, staining was observed in nonciliated cells, but was absent in ciliated cells whose nuclei were located nearer the lumen. Among the three types of nonciliated cells, type II cells showed lighter staining intensity than type I or type III cells (Fig. 4, a-h; Fig. 5, a-h). Immunoblot Analyses Immunoblot analyses of proteins extracted from efferent ductules are shown in Figures 6 and 7. Using the PG-21 anti-AR antibody, a single specific band was identified with an estimated molecular mass of 110 kDa (Fig. 6). This band was not seen when irrelevant rabbit IgG was substituted for PG-21 or when a 15-fold molar excess of the antigenic peptide AR-21 [36] was coincubated with PG-21; however, this band was observed when PG-21 was coincubated with a 15-fold molar excess of unrelated peptide AR-462 (Fig. 6). Using the monoclonal H-222 anti-ER antibody, a single specific immunoreactive protein was identified with an estimated molecular mass of 65 kDa (Fig. 7). This band was not seen when irrelevant IgG was substituted for primary antibody or when blots were incubated with secondary antibody alone.
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DISCUSSION This study describes the distribution of AR and ER in the testis and excurrent ducts of the goat through use of immunocytochemistry. AR were found in all reproductive organs examined, including the testis, intratesticular rete, efferent ductules, epididymis (regions I-V), and ductus deferens. In contrast, ER were confined to epithelium of the efferent ductules. Technically, the immunocytochemical method employed here using formalin-fixed, paraffin-embedded tissues, as compared to previous methods employing immunocytochemistry and/or autoradiography in frozen section, provides better resolution and clearer localization of receptors at cellular and subcellular levels [34]. However, in our hands, this method required that antigens be unmasked with microwave irradiation for AR and with pronase digestion for ER. The best results were achieved when sections were irradiated for 20 min in 10 mM citrate solution or were digested with pronase for 8 min at 37C. Without these treatments, receptor staining was negative. The utility and efficacy of such antigen unmasking procedures has been demonstrated in a variety of other tissues [34, 35]. Using the PG-21 rabbit anti-rat/human polyclonal antibody against AR [36] and the H-222 rat anti-human monoclonal antibody against ER [37], detectable receptor proteins were shown to be confined to the nuclei of all receptor-positive cells except spermatids, in which the Golgi stained positively for AR. Nuclear localization observed for AR and ER in the male goat agrees with the widely accepted hypothesis that functionally these ligand-dependent transcriptional regulators reside primarily, if not exclusively, in the nucleus of steroid-sensitive target cells [42]. Our observations are generally consistent with data reported for nuclear immunolocalization of AR and/or ER in certain cells of the testis, efferent ductules, epididymis, and/or vas deferens in several species including humans [28-31], monkeys [25], rats [19], and mice [43]. Cytoplasmic staining for AR in caprine spermatids was not anticipated, but it did not appear to be nonspecific and was observed consistently. In this regard, it should be noted that Vornberger and coworkers [20], using immunoperoxidase and immunofluorescence detection systems, reported positive staining for AR in the cytoplasm of elongated spermatids at steps 12-19 of the seminiferous epithelial cycle in rats. Cytoplasmic localization of AR in developing germ cells is difficult to explain. It has been suggested that such staining might reflect loss of the ability of developing germ cells to degrade cytoplasmic proteins, including AR proteins that might otherwise have been degraded prior to nuclear translocation [20]. Potential developmental and functional implications of these data remain to be resolved. Although AR-positive cells were observed in all reproductive organs of the male goats examined in the present study, nuclear staining intensity for AR differed between cell types within organ as well as between organs. In the testis, for example, staining intensity observed for Sertoli cells was greater than that observed for other AR-positive testicular cells, including peritubular myoid cells, interstitial fibroblasts, and Leydig cells (Fig. lc). In contrast, all epithelial nuclei of the intratesticular rete (Fig. Ig) exhibited strong immunostaining that was greater in intensity than immunostaining observed for Sertoli cells. Differences in staining intensity can reflect relative differences in receptor numbers and/or concentrations. The latter could vary easily with changes in nuclear volume among various cell
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types. Receptor protein was not quantitated here. Consequently, inferences related to quantitative differences in the distribution of AR must be drawn with caution. The presence of AR in Sertoli cells confirms the role of these cells as targets for androgen actions in support of spermatogenesis. More advanced germ cells (spermatocytes and cells at later stages), because of the formation of bloodtestis barrier near the basal area of the seminiferous epithelium, depend upon Sertoli cells for their nutritive needs [44]. Many functions of Sertoli cells, including secretion of certain proteins [45], formation of testicular fluid [46], and release of sperm from the testis [41], are believed to be dependent upon the supply of androgens. A recent study in rats not only identified stage-dependent secretion of certain proteins by the seminiferous tubules but also noted stagespecific effects of androgen withdrawal on these proteins as well as on total proteins. It was hypothesized that the distribution of AR in Sertoli cells may change in relation to the stage of the seminiferous epithelial cycle [47]. Further studies from the same laboratory [19] and another independent study appearing at about the same time [20] confirmed this hypothesis. Reports describing AR distribution in the efferent ductules are limited to an immunocytochemical study in monkeys [26] and two autoradiographic studies in mice [16, 22]. Present results in the goat agree with findings of these previous studies in that nuclei of both ciliated and nonciliated cells, as well as those of stromal cells underlying the epithelium, stained positively for AR. Actually, in developing mice, efferent ductule epithelium was found to be the first site to express AR [22]. Since nonciliated cells of the caprine efferent ductules can be further classified into three subtypes, our observations that immunostaining was lighter in nuclei of type II cells than in those of type III or type I cells are new, though the functional significance of these observations remains to be determined. The presence of AR in nonciliated cells establishes them clearly as targets for androgen actions. Although studies pertaining to hormone regulation of the efferent ductules are limited, androgen deprivation resulted in a number of morphological and/or biochemical changes in efferent ductule epithelium in bulls [48], goats [49], and rats [50]. Unlike nuclei in the testis, but similar to those in the rete testis and efferent ductules, the nuclei of all epididymal epithelial cells (principal cells and basal cells) and smooth muscle cells surrounding the epithelium showed a strong to mild immunostaining for AR in the goat. Of all cell types examined in this study, principal cells showed strongest immunostaining for AR in their nuclei. These results are in general agreement with those reported for humans [28, 30], rats [18], and monkeys [26]. However, all these authors either did not report AR staining in basal cells or did not make any distinction between basal and principal cells. Strong AR immunostaining in principal cells supports the well-founded fact of their dependence on androgens for maintenance of their structure and function. Furthermore, differences in immunostaining intensity observed in principal cells from different regions of the goat epididymis corroborate our previous observations in the goat [11], as well as those of others in other species [1, 13], that the response of principal cells to androgen deprivation differs among regions of this organ. The presence of AR in the basal cells of the goat epididymal epithelium is a novel finding. Because of their basal location in the epithelium and also because of the paucity of cell organelles in their cytoplasm, no definite role
in sperm maturation has been ascribed to these cells. However, it has been postulated that they replace old principal cells [51], support the epithelium [52], protect the epithelium from oxygen stress [53], and phagocytose antigenic products endocytosed by principal cells [54]. Whether androgens play an important role in regulating the structure and functions of basal cells remains unknown at this time, since studies on the effects of androgen withdrawal either have not been undertaken or are fragmentary [55]. Immunohistochemical localization of AR in the ductus deferens has been reported only in humans [28, 30]. Our observations, showing AR in the nuclei of epithelial cells (principal cells and basal cells) and smooth muscle cells, are in agreement with those of the authors cited above and signify the role of androgens in the ductus deferens. Although morphological studies on the effects of androgen deprivation are limited in this organ [1], androgen-dependent epithelial secretory proteins, as well as mRNAs for proteins, have been identified in the mouse vas deferens [56-581. The most notable finding of the present study was the exclusive localization of ER in the efferent ductules of the goat. All other male reproductive organs including the testis, rete testis, epididymis, and ductus deferens were ER negative. Within the efferent ductules, staining was found only in the nuclei of nonciliated cells. Nuclei of adjoining ciliated cells were negative. These observations are in exact accord with those previously reported in the monkey [25]. The authors of that investigation studied testes, efferent ductules, and caput, corpus, and cauda regions of the epididymis using immunocytochemistry on frozen sections and sucrose gradient assay; they found ER only in the nonciliated cells of the efferent ductules. Similarly, in studies using immunocytochemistry on frozen sections in mice [43] and using autoradiography on frozen sections in mice [16, 17], very intense ER staining was found in efferent ductule epithelium. However, in all of these cases, authors encountered varying degrees of nuclear labeling or staining of epithelial and muscle cells of the epididymis. Likewise, the biochemical studies showed binding of [3 H]estradiol with cytosolic and/or nuclear extracts of epididymides in various species including humans [27], rats [21], rabbits [23, 24], and rams [14]. The binding was higher in the cauda than in the caput or corpus [14, 23] and was higher in immature animals than in mature animals [23]. Reasons for differences between our results and those of others may be procedural. However, taken together, these data provide
FIG. 3. a-h) Immunocytochemical localization of AR in the epididymis and ductus deferens. a) A schematic drawing depicting histological boundaries of epididymal regions (I-V). Efferent ductules (ED) occupy the proximal one third of the first limb of the head of the epididymis; appendix epididymis (AE) forms a promontory-like structure and lies against the spermatic cord (SC); and ductus deferens (VD) continues from the tail of the epididymis (V) as a cord-like structure. b) Control section from region III of the epididymis incubated with an irrelevant IgG in place of the primary antibody. Note lack of positive immunostaining, which was also the case in other epididymal regions. c-h) All nuclei of epithelial cells, smooth muscle cells, and intertubular connective tissue cells show positive staining for AR in regions I (c), 11(d), III (e), IV (f), and V (g) of the epididymis and in the ductus deferens (h). Note that nuclei of principal cells (p) in regions II, Ill, and IV of the epididymis are more darkly stained than those of regions I and V of the epididymis or the ductus deferens. Nuclei of basal cells (arrows), nuclei of apical cells (arrowheads), nucleus of intra-epithelial macrophage (open arrowhead), and nucleus of principal cell undergoing mitosis (triangle). b-h, paraplast sections. b, 90; c-h, x300.
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FIG. 4. a-h) Immunocytochemical localization of ER in the testis (a), intratesticular rete (c), and efferent ductules (e-g). Corresponding negative control sections (b, d, and h), in which irrelevant IgG was substituted for primary antibody, are also shown. No positive staining for ER is observed in cells of the testis (a) or intratesticular rete (c). In contrast, positive ER staining is observed in nuclei of type I (e), type 11(f), and type III (g) nonciliated cells (arrowheads) of efferent ductules. The nuclei of adjoining ciliated cells (arrows) do not show immunostaining. a-h, paraplast sections. a-h, x300.
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FIG. 5. a-h) Immunocytochemical localization of ER in epididymal regions I (a), II (b), III (c), IV (d), and V (e) and in the ductus deferens (f). Negative control sections from region II of the epididymis (g) and the ductus deferens (h) are also shown. No ER-positive staining is observed in these organs. a-h, paraplast sections. a-h, x300.
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organ express themselves similarly or differently in response to androgen deprivation and during postnatal differentiation. In conclusion, while AR are ubiquitously distributed in reproductive organs of the male goat, ER are confined to the efferent ductules-a finding consistent with that reported for primates [25]. Results set the stage for further investigations of the role of androgens and estrogens in mediation of reproductive tract function in the goat, and reinforce the importance of this large animal as a relevant and viable model system for such studies. ACKNOWLEDGMENTS
FIG. 6. Immunoblot characterization of AR protein from solubilized caprine efferent ductules. The PG-21 anti-AR antibody recognized a specific band of approximately 110 kDa (first lane). This band was not seen when irrelevant rabbit IgG (second lane) was substituted for PG-21 or when a 15-fold molar excess of the antigenic peptide AR-21 was coincubated with PG-21 (third lane). However, the 110-kDa band was seen when PG-21 was coincubated with a 15-fold molar excess of unrelated peptide AR-462 (fourth lane). Protein bands with molecular mass estimates of less than 97.4 kDa, observed consistently in all lanes, reflected nonspecific interactions of rabbit IgG and/or secondary IgG (Ab2; fifth lane) with extracted proteins.
strong support for the idea that estrogens have a role in mediation of the function of epithelial cells in efferent ductules. It will be important to determine this role, especially in light of recent data indicating that sperm aspirated from the efferent ductules can cause pregnancy in humans [59] and that male mice lacking functional ER are infertile [60]. Along with ER, the presence of AR in the nonciliated cells of the efferent ductules raises the possibility that an interrelationship exists between androgens and estrogens in regulating the structure and function of these cells. Hence
it will be interesting to know whether both receptors in this
FIG. 7. Immunoblot characterization of ER protein from solubilized caprine efferent ductules. The monoclonal H-222 anti-ER antibody recognized a specific band of approximately 65 kDa (first lane). This band was not seen when irrelevant rat IgG was substituted for H-222 (second lane), or when blots were incubated with the secondary antibody alone (Ab2; third lane). Protein bands greater than 65 kDa and minor bands with molecular mass estimates of less than 65 kDa reflected nonspecific interactions of rat IgG and/or secondary IgG with extracted proteins.
The authors thank Dr. G.S. Prins (University of Illinois) for her gift of the PG-21 antibody, Dr. G.L. Greene (University of Chicago) for his gift of the H-222 antibody, V. Ingram and T Martin for providing technical assistance, S.M. Bryant for typing the manuscript, and C.S. Williams for editing the manuscript.
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