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Regular Article |
and ß in the Reproductive Tractsof Adult Male Dogs and Cats1
a Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802
b MRC Human Reproductive Sciences Unit, Edinburgh EH3 9ET, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß were localized by immunohistochemistry using ER-specific antibodies. ER was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ER staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ER. In the cat, except for the initial region of caput epididymis, ER staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERß. In rete testis and efferent ductules, epithelial cells were weakly positive for ERß. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ER and ERß in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ER and ERß in male dog and cat reproductive tracts, demonstrating more intensive expression of ERß than ER. However, as in other species, the efferent ductules remained the region of highest concentration of ER.
epididymis, estradiol receptor, male reproductive tract, testis, vas deferens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although ER and ERß are similar in structure, they differ in C-terminal ligand binding and in N-terminal transactivation domains [15]. There are also major differences between male ER knockout (ERKO) and ERß knockout (ßERKO) mice. ERKO mice are infertile [3, 16, 17], whereas ßERKO males are fertile [18]. An expression of both receptors together in abundance is seen only in the head of the epididymis, whereas ERß is found throughout the male reproductive tract [8, 12, 19]. Thus, when evaluating estrogen function in a species for the first time, it is important to explore the distribution of both receptors in order to provide a basic foundation for future studies.
ER distribution has not been reported in the male reproductive system of dogs or cats. The dog is often selected as the nonrodent species of choice in studies of reproductive toxicity [20]. Both species exhibit worldwide overpopulation as stray animals, which raises the importance of developing a male contraceptive, in addition to the accepted practice of castration [21]. A potential target for male contraception is the ER. Therefore, the objective of the present study was to investigate the presence of ER and ERß in the male reproductive tract of normal adult dogs and cats, and to compare these domestic species with other mammals. Cellular localization was detected by immunohistochemistry in testis, rete testis, efferent ductules, epididymis, and vas deferens; and colocalization of ER was shown by double staining. Western blot analysis demonstrated that molecular weights of the respective ERs were similar to values reported for other species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An initial gross anatomical study of three dogs and three cats was made to determine the location of rete testis and efferent ductules. For immunohistochemistry, an additional three dogs and three cats were used, and for Western blot analysis four animals each were used. Testes were obtained after routine castration at local animal hospitals and Humane Society offices. All animals were sexually mature on the basis of age, breeding history, and examination of sperm in cauda epididymis. Animal experiments were approved by the Institutional Animal Care and Use Committees of the respective universities and were conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Immunohistochemistry
Immediately after castration, testes and associated epididymides were dissected into small pieces according to the anatomical regions of interest and immersion-fixed in 10% neutral buffered formalin (NBF) for 12 h at 4°C. The tissues were transferred to 70% ethanol and embedded in paraffin. Sections were cut at 5 µm thickness and then dried at 37°C overnight. Tissue types included testis, rete testis, efferent ductules, caput, corpus, and cauda epididymis, and vas deferens.
Single Staining
Tissues were stained for ERs as described previously [22]. To unmask the receptor protein, sections were microwaved in a 0.01 M citrate buffer solution (pH 6.0) for 20 min. Tissues were then incubated with either ER-specific antibody NCL-ER-LH2 (Novocastra, Newcastle upon Tyne, U.K.) at 1:50 dilution or ERß-specific antibody S-40 at 1:500 dilution for 12 h at 4°C. The generation of ERß antibody is described elsewhere [23]. Antibody bindings were visualized by using avidin-biotin complex (ABC Kit; Vector Laboratories, Burlingame, CA), and the diaminobenzidine (DAB) chromogen. Hematoxylin (Sigma, St. Louis, MO) was applied as a counterstain. Sections incubated without the primary antibody were used as the negative control. Images were captured with a Spot II digital camera (Diagnostic Instruments, Sterling Heights, MI) and compiled using Adobe Photoshop (Adobe Systems, San Jose, CA).
Double Staining
Double immunohistochemical staining using fluorescence-conjugated secondary antibody was modified based on a previous publication [24]. Dog and cat efferent ductules were examined for double staining. After antigen retrieval and blocking with 10% normal rabbit serum, sections were incubated sequentially in the following solutions, and sections were rinsed with PBS between each incubation: ER (NCL-ER-LH2) mouse antibody (1:25), fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG) (1:100; Sigma), ERß, S-40, sheep antibody (1:500), and Texas red-conjugated anti-sheep IgG (1:100; Vector Laboratories). Sections incubated without the primary antibody were used as the negative control. The sections were examined under a fluorescence microscope with a suitable filter for FITC and Texas red, and images were captured with a Spot II digital camera. In Adobe Photoshop, the individual image for ER (FITC-green) and ERß (Texas red) were combined using the overlay tool. Cells that contained both receptors were stained various shades of yellow-green to bright yellow.
Evaluation of Immunostaining
Scoring of staining intensity was done on the basis of nuclear staining, which was classified as negative (no staining), weak (+), moderate (++), or strong (+++). Nuclei were reported negative when the staining did not differ from the negative control sections. Staining for ER in efferent ductules was defined as a baseline strong staining.
Specificity of LH2 and S-40 in Dog and Cat Tissues
Recombinant human ER (Panvera, Madison, WI) and peptide P40 (the antigenic peptide for S40) were used to perform antibody competition. Briefly, 10- to 15-fold molar excess of proteins or peptides were incubated together with both ER and ERß antibodies overnight at 4°C, and were then used as the primary antibody, as described before for immunohistochemistry. Efferent ductules from both species were used for testing ER antibody LH2, and epididymides were used for testing ERß antibody S-40.
Western blot analysis was used to demonstrate that LH2 and S40 antibodies would recognize specific proteins of appropriate molecular weight in both dog and cat tissues. Dog efferent ductules, cat epididymis, and uterus (from both species as well as from mouse for comparison) were extracted for detection of ER protein. Epididymides from dogs and cats were extracted for examination of ERß protein. Human recombinant ERß was used as the standard protein (Panvera). The method was a modification of one described in a previous publication [25]. Protein samples were separated on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The filters were blocked with 5% dried milk in Tris-buffered saline (TBS; 50 mM Tris·Cl, pH 7.5/150 mM NaCl) containing 0.05% Tween-20 for 1.5 h, and then incubated overnight at room temperature with 1:50 diluted ER antibody NCL-ER-LH2 (Novocastra), and 1:2000 ERß antibody S-40 in TBS containing 1%–2% dried milk and 0.2% Tween-20. After washing the filter in TBS containing 0.05% Tween-20, the filters were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG diluted at 1:4000 [Pierce, Rockford, IL] and rabbit anti-sheep IgG diluted 1:40 000; Sigma) for 1 h in the same buffer that was used for primary incubation of antibodies. ER was visualized with DAB chromogen and ERß was visualized with x-ray film using a Supersignal West Femto system (Pierce).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of immunostaining are summarized in Table 1. The results clearly show species differences. Dog connective tissues are more positive for ER, whereas in the cat, the connective tissues are more positive for ERß. Also, epithelial cells in the dog epididymis are negative for ER, whereas the cat epithelium is moderately positive. Specific findings are presented by receptor and tissue type.
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Immunolocalization of ER
Testis
Within dog testis, intermittent positive staining for ER was seen in interstitial (Leydig cell-like cells and endothelial cells) and peritubular myoid cells (Fig. 1a). In the cat, interstitial cells were weakly positive for ER, but peritubular cells were negative (Fig. 1b). Some germ cells were easily stained by reagents but without primary antibody, which was a common problem in testis sections, but not in the other regions of reproductive tract. After comparing with the negative control (not shown), it was concluded that Sertoli and germ cells were negative in both species.
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Rete testis
In dogs and cats, rete testis forms a central channel within the testis, or mediastinum, and connects with the efferent ductules near the testicular capsule. In dogs, nuclei of cells that line the rete testis epithelium were strongly immunopositive for ER, but nuclei of cells in the surrounding connective tissue were weakly positive (Fig. 1c). In cats, rete testis epithelial cells were negative or weakly positive for ER, and no immunoreaction was presented within connective tissues (Fig. 1d).
Efferent ductules
The lining epithelium of dog efferent ductules was strongly ER positive in both ciliated and nonciliated cells (Fig. 1e). The efferent ductules were surrounded by smooth muscle cells and separated by connective tissue. Many nuclei of smooth muscle cells, endothelial cells, and other connective tissue cells were also positive for ER, but some cells were negative. In cat efferent ductules, strong positive staining for ER was detected in the nuclei of epithelial cells, but the ciliated cells were stained with lower intensity (Fig. 1f).
Epididymis
In the dog, from the initial segment to the cauda epididymis, epithelial cells were negative for ER (Fig. 1, g, i, and k), however, some peritubular smooth muscle cells and connective tissue cells were positive. In contrast, cat epididymis showed some positive staining for ER in selective areas. The basal cells of cat initial caput region and most of the principal cells from caput epididymis to cauda epididymis were positive for ER, whereas other epithelium cells of initial segment and most of connective tissues were negative for ER (Fig. 1, h, j, and l).
Vas deferens
In the vas deferens, the staining pattern was similar to that observed in the epididymis. In the dog, epithelial cells of the vas deferens were negative for ER, but cells in the lamina propria and smooth muscle layers were positive (Fig. 1m). In the cat, epithelial cells were positive for ER (Fig. 1n), however, some smooth muscle cells were also positive, whereas in the epididymis these cells were negative or weakly positive (Fig. 1, h, j, and l).
Immunolocalization of ERß
Testis Multiple cells were strongly positive for ERß in both dog and cat testes. In dog testis (Fig. 2a), round spermatids were strongly positive, and spermatogonia and spermatocytes were weakly positive, but spermatozoa were negative. Only the occasional Sertoli cell exhibited positive staining for ERß. Outside the seminiferous tubule, very few Leydig cells were positive, and only occasionally were peritubular cells positive. The cat testis was stained more intensely for ERß than the dog testis. Within the seminiferous tubule, round spermatid and pachytene spermatocyte nuclei were intensely positive for ERß (Fig. 2b). Spermatogonia and Sertoli cells were positive, but less intensely. Most peritubular and Leydig cells were positive for ERß.
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Rete testis In both dog and cat rete testes, epithelial cells were moderately to weakly positive for ERß, whereas connective tissues were intermittently positive (Fig. 2, c and d).
Efferent ductules In dogs, all efferent ductule epithelial cells stained positive for ERß, but some were more intensely stained than others, particularly ciliated cells (Fig. 2e). Similar to dogs, efferent ductule epithelium in cats was positive for ERß but appeared to be more intensely stained than epithelium in dogs. Connective tissue cells in both species were intermittently stained (Fig. 2f).
Epididymis In dogs and cats, all epithelial cells of the epididymis, from the initial region of the caput to the cauda, were moderately to strongly positive for ERß, with staining intensity increasing from the caput to the cauda (Fig. 2, g, i, and k). Connective tissues cells showed intermittent staining in both species throughout the epididymis, but it appeared that more cells in connective tissues of the cat were positive for ERß (compare Fig. 2, h, j, and l).
Vas deferens In both dogs and cats, all epithelial cells of the vas deferens were strongly positive for ERß, although cells in dogs appeared to be more intensely stained than those in cats (Fig. 2, m and n). Positive staining was seen in more connective tissue cells in cats than in dogs, but the peritubular smooth muscle layer in both species was positive.
Double Staining for ER and ERß
Colocalization of ER and ERß was examined in dog and cat efferent ductules. Both ER (green) and ERß (red) were expressed in epithelial nuclei of dog efferent ductules (Fig. 3, a and b). Colocalization of the two receptors in the same cell was detected by a yellowish color. Staining intensities, with variations from yellow-green to intense yellow, indicated differences in the proportion of the two receptors in individual cell nuclear (Fig. 3c). Both receptor types were also colocalized in epithelial nuclei of cat efferent ductules (Fig. 3, d–f). A transition area from the efferent ductules to the head of the epididymis was observed in a section from a cat (Fig. 3, g–i). One tubule belonging to an efferent ductule was stained strongly positive for ER (Fig. 3g), but both the efferent ductule and epididymal ducts were stained for ERß (Fig. 3h). The combined photo showed that only epithelial cells of the efferent ductules coexpressed both ER and ERß, and that epithelial cells of the head of the epididymis expressed only ERß, and not ER (Fig. 3i). In the transition area, basal cells were rarely positive for ER.
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Specificity of LH2 and S-40 in Dog and Cat Tissues
Antibody competition
Recombinant human ER protein warded off all nuclear staining given by LH2 in both dog and cat efferent ductules (Fig. 4, a' and b'). The peptide P40 was not able to ward off or reduce the staining of LH2 (Fig. 4, a and b). On the other hand, peptide P40 completely warded off the staining given by S40 in both dog and cat epididymis (Fig. 4, c' and d'), but not the recombinant human ER protein (Fig. 4, c and d).
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Western blot analysis
ER was detected as a single dominant protein band of approximately 66 kDa in efferent ductules of dogs; epididymis of cats; and uterus of dogs, cats, and mice (Fig. 5a). The mouse uterus was used as a positive control. One or two smaller weak staining bands were considered to be products of partial degradation (Fig. 5a). ERß antibody in particular recognized human recombinant ERß, with a molecular size of 53.4 kDa. A dominant single protein band was detected in both dog and cat epididymis, with the comigration of the recombinant protein (Fig. 5b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ERß are localized in the reproductive tracts of adult male dogs and cats, and the first of colocalizing both receptors in male reproductive tissues. Using double staining, both receptors were colocalized in the same nucleus of efferent ductule epithelial cells in dogs and cats. Using single staining, both receptors showed exclusive staining in the nucleus in both epithelial and stromal cells, but with region and species differential expression. Antibody specificity was confirmed by Western blot analysis. As in other species, ER was abundant in the epithelium of efferent ductules of both species, but significant differences were found in ER localization in rete testis and epididymis. In contrast to dogs and other large mammals, including humans [7, 12, 13, 26], cats showed moderate to strong epithelial staining for ER in portions of the epididymis. ERß was more widely distributed than ER and was abundant in testis, rete testis, efferent ductules, epididymis, and vas deferens. In some regions of the epididymis, the staining intensity of ERß was as strong as it was in efferent ductules for ER.
ER
In the testis, ER staining was exclusive to the interstitium, as seminiferous epithelium was negative in dogs and cats, which is similar to most reports in the literature [7, 27]. Dog testis showed a stronger staining than cat testis. However, inconsistent results have been reported for ER in testis of other species. For example, in monkeys, ER was positive in the Leydig cells [7], but negative in a later study [12], which was in agreement with the results of West and Brenner [13]. The variability of these studies may be due to different procedures, fixation methods, antibodies, or a combination of these. In situ hybridization showed that ER mRNA was expressed in testis, efferent ductules, and epididymis of the goat [19]. However, with immunohistochemistry, goat testis and epididymis were negative for ER [19, 26]. Only two reports have shown immunostaining of the germinal epithelium for ER [27, 28], and the function of ER in testis is still unknown. Although ERKO males are infertile, testicular ER is not required for germ cell development [29]. The presence of ER in the efferent ductules, and possibly other regions of the epididymis, appears to be essential for sperm concentration, maturation, and fertilization [1].
ER staining of the rete testis was also stronger in dogs than in cats. Only a few studies have examined this region for ER staining, and the results are also variable. The rat rete testis was positive for ER in two studies [7, 8], but negative in another [30]. The rete testis in monkeys was also negative for ER [7]. The function of rete testis is not known, but it does serve as a channel for accommodating sperm release from the seminiferous tubules [31]. There have been numerous reports of abnormalities associated with rete testis development following diethylstilbestrol (DES) and estradiol treatments [32–36], and also after antiestrogen treatment in the adult male [37, 38]. In the ERKO male, the rete forms cystic dilations that occupy nearly one-third of the testis [1, 16, 37]. These data suggest that estrogen may be more important in rete testis function during development than in the adult.
The most abundant staining for ER was found in efferent ductule epithelium of both dogs and cats. This is similar to all other species reported, including rats, mice, roosters, goats, monkeys, and humans [7, 8, 10–14, 30, 39]. Although the efferent ductule epithelium repeatedly has been shown to contain an abundance of ER [40], variations have been noted in the presence of the receptor in ciliated cells. In goats, monkeys, and humans, ciliated cells have been shown to be negative for ER [5–8, 11, 13, 26] by using antibodies that are different from the antibody that was used in the present study, whereas in rats, both ciliated and nonciliated cells were strongly positive [8]. In the current study, both ciliated and nonciliated cells of efferent ductules were positive for ER, but in the cat the ciliated cell nucleus was only slightly positive. In the female, ciliated cells of the oviduct are regulated by estrogen [41], therefore, a potential exists for estrogen regulation of ciliary function in the male. In the absence of a functional ER (ERKO and antiestrogen-treated mice), a dramatic reduction in the number of cilia per cell in efferent ductules has been reported [37, 42].
The epididymis has been shown to express ER mRNA in several species [8, 14, 19, 43], but there have been mixed results with ER immunohistochemistry, with some species being completely negative [5–8, 11, 13, 44–46], whereas other species have specific ER-positive cells in the epididymis, depending upon the antibody used [5, 8, 10, 14, 30, 47, 48]. In the present study, epididymal staining was located primarily in stromal cells, but in cats, the epithelium was also positive in certain regions. In the initial region of the caput epididymis of cats, strong immunostaining was seen only in the basal cells, which was interesting because the same cell type was ER-positive in macaques [12]. Principal cells of the caput, corpus, and cauda epithelium were also strongly positive in cats, but those in dogs were negative to slightly positive. Currently, there is no known function for ER in the epididymis, but based on morphological changes seen in the ERKO male, it is possible that estrogen is important in several of the epithelial cell types [42].
Although it is unclear whether ER has an essential function in the adult epididymis, there is considerable evidence that estrogen has a major effect on epididymal as well as rete testis development [49]. Recently, DES treatment in the neonate was shown to cause underdevelopment of the epididymis, a decrease in androgen receptor expression, abnormal expression of progesterone receptor in the epididymis, and failure to express androgen receptor [32, 45, 46]. It is interesting that coadministration of testosterone prevented the loss of androgen receptors and reduced the histopathological response to DES [32]. A precise biochemical mechanism to account for DES-induced abnormal development is lacking, but the ability of in utero exposure to DES to alter the expression of steroid hormone receptors suggests that adult function of the epididymis is dependent upon an exposure to normal concentrations of estrogens during development.
ER is not present in the vas deferens epithelium in most species [8, 12, 45, 50], including the dog, as reported here. However, in contrast, epithelium of the cat vas deferens is strongly positive for ER. In most species, the muscle layer of vas deferens was consistently positive for ER. In rat vas deferens, the outer muscle layer responds to estradiol treatment [51]. Estradiol has also been shown to regulate zinc concentrations in vas deferens, although the subtypes of estrogen receptor were not known when that research occurred [52].
ERß
ERß is abundant in the male reproductive system [8, 43], however, its presence in the adult male reproductive tract has been localized in only four species: rats, mice, monkeys, and humans [8, 12, 46, 53]. In the present study, this receptor was present in every region examined from testis to vas deferens, in both dogs and cats. In testis, ERß-positive cells included spermatogonia, spermatocytes, and round spermatids; and weakly positive cells included Leydig and peritubular myoid cells. Others have also reported that Sertoli and Leydig cells and various germ cells are positive for ERß in rats, mice, monkeys, and humans [12, 23, 39, 53–55]. The differential expression of ERß in testis suggests the potential for estrogen in the regulation of spermatogenesis. However, ßERKO mice have normal male reproductive tract development and are fertile [18].
In the efferent ductules, ERß coexisted with ER in the same cell, in both dogs and cats. In vitro, ER and ERß have been shown to form heterodimers, with retained DNA-binding ability and specificity [56]. In vivo, the expression of both receptors in the same tissues has been reported, especially in male efferent ductules [8, 12]. However, colocalization of both receptors in the same cell has been shown only in mammary gland [24]. This is the first report of coexpression of ER and ERß in the same cells of the male. However, the importance of simultaneous expression of the two receptors in a cell remains to be determined. One hypothesis suggests that one of the physiological roles of ERß is to act as a negative regulatory partner of ER [57].
Staining for ERß in the epididymis and vas deferens was strong in all epithelial cells of dogs and cats, which is consistent with other species, including rats, mice, monkeys, and humans [12, 30, 46, 53]. However, stromal cells were selectively stained in dogs and cats, compared to a wider expression of ERß among stromal cells of rats, monkeys, and humans [12, 30, 46].
In conclusion, ER and ERß are abundant in the male reproductive tract of dogs and cats, but are differentially expressed in a species-specific manner. However, a copious expression of ER in the efferent ductule epithelium is the one common observation that continues to hold firm across all species studied to date. This finding is important in light of the response observed after targeted disruption of ER in the ERKO male and treatment of mice and rats with the antiestrogen ICI 182,780 [1, 37, 40, 58, 59]. The dominant effect of ER inhibition is the accumulation of luminal fluid in the efferent ductules due to the inhibition of Na+ transport by the epithelium [1, 37, 40, 58, 59]. These ductules are unique in that they reabsorb more than 90% of the rete testis fluid, as they concentrate sperm for epididymal storage [60, 61], a function that is apparently essential for fertility [58].
On the basis of common morphological appearance, function, and abundance of ER in the efferent ductule epithelium among all species [62], it is reasonable to predict that estrogen will have a common function across species. The reproductive tracts of male dogs and cats are organized similar to that found in humans, with numerous efferent ductules independently entering the epididymis [63]. Because of this similarity, dogs and cats are better models than rodents for studying estrogen function in human efferent ductules. The rodent ductules merge to form a single tubule that enters the epididymis. This funnel-like design has a greater risk for fluid accumulation and subsequent testicular atrophy than multiple-entry design found in larger mammals [40, 62–65]. If fluid reabsorption can be inhibited using antiestrogen chemicals, without testicular atrophy, then the development of a reversible male contraceptive that targets estrogen receptor function in efferent ductules and decreases sperm concentrations becomes a feasible goal. On the basis of data presented here, dogs and cats could serve as excellent models for testing this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 R.A.H. was supported by the Kenneth A. Scott Charitable Trust and by grant HD35126 from the National Institutes of Health. 
2 Correspondence: Rex A. Hess, Department of Veterinary Biosciences, University of Illinois, 2001 S. Lincoln, Urbana, IL 61802. FAX: 217 244 1652; r-hess{at}uiuc.edu
Accepted: November 7, 2001.
Received: August 16, 2001.
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& ß) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 1997; 18:602-611 knockout (ERKO) male mouse. Anat Rec 2001; 263:10-18[CrossRef][Medline]This article has been cited by other articles:
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C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
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