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Original Studies |
Department of Cell Biology, Georgetown University Medical Center (C.A.S.-Q.), Washington, D.C. 20007; and the Department of Morphology, University Autonoma of Madrid (F.M.-G., M.N., J.R.), and the Department of Pathology, La Paz Hospital (M.N.), Madrid, Spain 28029
Address all correspondence and requests for reprints to: Dr. Carlos A. Suárez-Quian, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20007. E-mail: suarezc{at}gunet.georgetown.edu
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Abstract |
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Introduction |
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The majority of immunostaining studies cited above employed a version of the biotin-streptavidin-immunoperoxidase histochemistry method to identify AR-positive cells in testes. For the most part, however, immunoperoxidase assays are difficult to quantify, and in addition, most investigators (including our own work cited above) perform the immunostaining reaction at saturating concentrations of the primary antibody. That is, at the concentration that renders a robust, specific signal capable of being readily detected by the investigator. Unfortunately, at saturating primary antibody concentrations it is not possible to distinguish the relative immunostaining intensities of AR in distinct populations of the testis. Presumably, distinct AR immunostaining intensities reflect the relative protein concentrations of the AR protein present in the varying AR-positive testicular cell types and/or the availability of the epitope to the primary antibody. Thus, it is possible that subtle concentration differences may exist in immunoreactive AR protein levels in testicular cell types, but have not yet been ascertained by conventional biotin-streptavidin-immunoperoxidase methods.
In the present investigation the distribution of AR immunoreactive protein in normal adult human testis from an archival tissue collection was examined. Immunocytochemistry was performed using conventional biotin-streptavidin-immunoperoxidase methods, but in addition a biotin amplification step was incorporated into the immunostaining procedure to enhance the sensitivity of the assay (15). By increasing the sensitivity of the assay it was possible to determine the relative AR immunostaining intensity in distinct testicular cell types, including Sertoli cells, as a function of the cycle of the seminiferous epithelium.
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Materials and Methods |
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Five testes were collected from men (57–69 yr old) undergoing therapeutic orchidectomy as treatment for malignant prostate carcinoma at La Paz Hospital (Madrid, Spain). Patients did not receive antiandrogens before surgery. For AR immunostaining, the testicular tissue was fixed with formalin and processed for routine paraffin embedding. This tissue forms part of tissue collection of the Pathology Department at La Paz Hospital.
Histological/pathological examination of tissue
Normal spermatogenesis was used to define tissue lacking any detectable pathological conditions meeting the following criteria. First, testicular tissue was selected that exhibited a homogeneous histological appearance with respect to the absence of pathological conditions. At least six paraffin blocks from each of the testes were examined histologically. In the five cases selected for our report, the majority of seminiferous tubules and interstitial spaces appeared normal. Only rarely were tubules observed that displayed characteristics of hypoplasia, and only one sclerotic tubule was ever detected in one of the testis included in the study.
Normal testicular parameters included all of the following. Tubules
diameters were large (
225 µm) and exhibited complete
spermatogenesis consisting of abundant round and elongated spermatids
and lacking immature germ cell sloughing. In addition, lack of tubule
lumen dilatation and tubule walls circumscribed by only one or two
layers of myoid cells were evident. The appearance of collagen
structures within the interstitial tissue appeared normal, and no
evidence of edema in the interstitial space was noted. Leydig cells
were present as either single cells or nests, without any evidence of
abnormal morphology. All arteries and arterioles appeared normal, and
venules did not exhibit angiectasia or dilatation. The walls of veins
were thin and without varicosities. Further, no sites of inflammation
were ever detected. The rete testis appeared normal without dilation
and contained normal levels of spermatozoa (excessive spermatozoa would
have been interpreted as evidence of an obstructive pathological
condition, but none was ever observed).
Given the strict selection criteria required to meet our definition of the normal spermatogenesis group, only testes from 5 patients were selected for AR immunohistochemistry analysis from over 50 archival samples examined. In addition, we limited our study to testes from patients who were relatively young and had received no antiandrogen, chemotherapy, or other drug treatment known to exert an effect on testicular histology. Lack of drug therapy was confirmed by examining the clinical history and afterward by questioning the patients. All 5 cases fulfilled these requirements. This collection of tissue is unique with respect to uniformity and absence of the pathological conditions so often encountered when working with human material.
Antibody and immunostaining supplies
All immunocytochemistry procedures were performed using the PG21 antibody. This is an affinity-purified rabbit polyclonal antibody made to a synthetic peptide corresponding to the first 21 amino acids of the androgen receptor. Its use as a valid immunological probe for AR from a variety of species, including rat and human, has been previously established (5, 14, 16, 17).
Immunocytochemistry
Paraffin sections (6–8 µm thick) were dewaxed and hydrated, first in two (5-min) rinses in xylene, followed by 5-min rinses in descending grades of ethanol (100%, 90%, and 70%). Epitope retrieval entailed bathing the hydrated sections in 0.01 mol/L citrate buffer, pH 6.0, and microwaving the sections for 10 min using a 600-watt microwave oven. The sections were allowed to come to room temperature before continuing with the immunostaining procedure.
Streptavidin-biotin-peroxidase immunostaining was carried out using Histostain-SP kits (Zymed Laboratories, San Francisco, CA) as described previously (18). Endogenous peroxidase activity was blocked with a 10-min incubation in 3% H2O2 in methanol, and endogenous avidin and biotin were blocked using the commercially available avidin and biotin blocking solutions according to the manufacturer’s instructions, followed by blocking nonspecific antibody binding with 10% nonimmune goat serum. Next, tissues were incubated at 37 C for 1 h in a moist chamber with PG21, followed by a 10-min incubation with biotinylated goat antirabbit antibody, and then a 5-min incubation in streptavidin-peroxidase. At this point the biotin amplification was incorporated into the immunostaining procedure as follows. Biotinyl-tyramide was prepared as previously described (15), and sections were treated with the biotinyl-tyramide plus H2O2 for 20 min as described by Berghorn et al. (19), creating a biotin "cloud" at the site of the PG21-epitope complex. Streptavidin-peroxidase was again added to the sections to react with the newly formed biotin cloud, followed by the addition of the substrate-chromogen mixture. The sections were counterstained for approximately 30 s with hematoxylin and then examined with a Zeiss Axiophot light microscope fitted with Planapo x20 N.A. or x100 1.4 N.A. objectives using an 80A blue filter. A positive reaction using this protocol is characterized by the deposition of a reddish reaction product at the site of the antibody-antigen reaction. Nuclei not immunostained for AR appear blue from the hematoxylin counterstain. Unfortunately, the hematoxylin counterstain does not discriminate between AR-positive and -negative nuclei, and at times it is difficult to discern the presence of faint deposition of reaction product in the nuclei. To minimize this problem, images were recorded on Kodacolor 100 ASA film using a Pale Gold filter from Bogen Cine (Ramsey, NJ) that helped to quench the blue of the AR-stained nuclei.
PG21 was used at concentrations ranging from 3–50 µg/mL. Controls included preadsorption of the primary antibody with a 10-fold excess of the antigenic peptide or with an unrelated peptide of the AR and then using the preadsorbed antibody as the primary antibody. In addition, the primary antibody was omitted to test background staining of the sections with the secondary antibody and/or the incorporation of biotinylated tyramine into the staining protocol.
Scoring of immunostaining intensity
Immunostaining intensities of Sertoli cell nuclei as a function of the cycle of the seminiferous epithelium were scored as intense (++), moderate (+), or negative (-) by visual inspection. However, for a nuclei to be evaluated as intense, all of the nuclear area identified with a x100 objective had to contain intense deposition of immunostaining reaction product. If any of the nuclear area could be found to lack immunostaining reaction product by focusing through the nuclei, then it was scored as moderate only, even if the reaction product observed within the nucleus was robust. Similarly, a moderate score included all Sertoli cell nuclei that contained only a trivial deposition of immunostaining reaction product detected by focusing up and down on the immunostained tissue section; even if detection of the reaction product was questionable, that particular nuclei would be scored as moderate. Thus, bias in the scoring was designed to err against scoring Sertoli cell nuclei as moderate or negative; any nuclei that could not be interpreted as true intense or negative would be scored as moderate.
Data acquisition and analysis
Staging of the seminiferous epithelium was based on the scheme described by Clermont (20), but was significantly aided by using the cell map published by Johnson (21). After confidence in the immunostaining protocol was established, one section from each of the five testes examined were pooled as groups and immunostained as a batch using the PG21 antibody at varying concentrations. Care was taken to ensure that each of the sections was treated in an identical fashion and that they were subjected to identical times in all of the immunostaining steps for each antibody dilution. At the completion of the immunostaining protocol, sections were evaluated and scored as a group. Within each batch of slides and using identical PG 21 concentrations, immunostaining intensity did not vary. Further, immunostaining intensity was reproducible between sections taken from the same testis, but processed at different times. To minimize edge artifacts possibly interfering with the scoring of the immunostaining, only tubules at the center of the testes were evaluated. However, before scoring of the immunostained Sertoli cell nuclei, the tubules to be examined were carefully screened to ensure that they appeared well fixed. That is, the epithelium exhibited only minimal evidence of sloughing, and within the epithelium no presence of holes was discerned. At least five tubules (range, 5–12) were counted from each of the testicular sections that met these criteria. Because it was difficult to consistently distinguish between stages I and II and between stages IV and V, data from these stages were pooled. In addition, results from stage VI were pooled with those from stages IV and V.
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Results |
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In previous studies the concentration of PG21 antibody determined
to render a robust immunostaining signal in adult rat prostatic and
testicular tissue ranged between 10–25 µg/mL, respectively (4, 16).
Inclusion of the biotin amplification step in the immunoperoxidase
method increased the sensitivity of the immunostaining approximately
25- to 100-fold. Thus, it became possible to use PG21 between the range
of 0.4–0.8 µg/mL for immunostaining rat prostate or testis,
respectively (data not shown). In contrast, the results of initial
experiments performed to determine the limiting concentration of PG21
necessary to render a robust immunostaining signal in human testicular
archival tissues were interpreted to suggest that below 2–3 µg/mL,
no positive immunostaining was present. At concentrations higher than 6
µg/mL, a much stronger reaction became evident in the testicular
section (Fig. 1
). These results led us to begin screening the archival,
testicular human tissue at 3 µg/mL.
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At a limiting PG21 concentration (3 µg/mL) not all seminiferous
tubules from human testes exhibited AR-immunopositive staining in
Sertoli cell nuclei (Fig. 1D
). In
addition, at this concentration, it was evident that not all testicular
somatic cells predicted to be AR immunopositive exhibited positive
immunostaining. Taken together, these results were interpreted to
suggest that 1) different somatic cells of the testis contain different
concentrations of AR; and 2) the intensity of AR immunostaining in
Sertoli cell nuclei varied as a function of the cycle of the
seminiferous epithelium. These two assumptions formed the basis of
subsequent analysis of the immunostaining results.
As the concentration of the PG21 antibody was increased, more
AR-immunopositive cells were detected in the testes sections (Fig. 1, A–C). At 6 µg/mL, for example, the AR immunostaining intensity of
some Sertoli cell nuclei was often scored as moderate, and some tubules
even exhibited intense scoring. In contrast, AR immunostaining of
peritubular cell nuclei was not consistent at this concentration, and
interstitial cell immunostaining was negligible, if not completely
absent. AR immunostaining of peritubular cell nuclei became more
consistent at 9 µg/mL, and at 12 µg/mL, all peritubular cell nuclei
noted were AR immunopositive. Leydig cell nuclei were not discerned to
be AR immunopositive until the PG21 antibody was used at 9 µg/mL, and
even at 12 µg/mL, there was a significant disparity in AR
immunostaining between Leydig cells.
AR immunostaining using varying concentrations of the PG21 antibody was
examined next in the rete testis (Fig. 2, A–C). As with the rest of the testis, by increasing the concentration
of the PG21 antibody, the number of epithelial cell nuclei of the rete
exhibiting positive AR immunostaining was increased. A similar
immunostaining patter was observed in the cells found in the adjacent
interstitium. Even at the highest concentration of PG21 antibody used
(12 µg/mL), however, not all principal cells forming the epithelium
of the rete were AR positive (Fig. 2A).
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and 4, respectively, and the results are
presented graphically in Figs. 5 and 6.
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). Only by focusing through the section,
however, was it possible to discern that the robust reaction product
was not deposited homogeneously throughout the nucleus and that not
every Sertoli cell should be scored as intense. Indeed, even
AR-negative Sertoli cell nuclei could be discerned within the
epithelium (Fig. 4, B and B'). Sertoli cell nuclei exhibiting no
detectable AR immunostaining was more readily apparent when the
concentration of PG21 was decreased to 9 µg/mL (Fig. 4A). Within a
tubule cross-section, Sertoli cell nuclei would present as moderate,
for example, whereas the remaining nuclei were completely devoid of any
visible AR immunostaining (Fig. 4, A and C). Again, focus through the
section was required to confirm both a negative and an intense score
assigned to a particular Sertoli cell nuclei (Fig. 4, B and B').
As expected, increasing the concentration of the PG21 antibody
increased the total number of Sertoli cell nuclei scored as intense or
moderate and diminished the number of AR-negative Sertoli cell nuclei.
Regardless of the concentration at which PG21 was used, the trend of
the data indicated that fewer Sertoli cell nuclei from stages IV–VI
were scored as intense or moderate than at the earlier stages (Figs. 5 and 6). Conversely, using PG21 at 9 µg/mL led to the scoring of a
greater number of nuclei as negative at stages IV–VI than at stages
I–III.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented herein are consistent with the interpretation that androgen regulation of spermatogenesis in humans occurs solely via somatic cells, but do not provide definitive proof that germ cells lack immunopositive AR. In the rat and mouse, immunopositive AR was detected first in the nuclei of elongated spermatids, just before the time that nuclear condensation occurs, followed by its appearance in the cytoplasm of elongated spermatids once nuclear condensation had been completed (4). Unfortunately, the cytoplasm of elongated spermatids in the archival human testis could not be discerned at an identical level of resolution. Thus, the present investigation cannot exclude the possibility, albeit remote, that immunopositive AR would similarly be detected in nuclei of human elongated spermatids if these were readily detected in tissue sections. However, one significant difference observed in the AR immunostaining pattern between human and rodent was that at no time was immunopositive AR detected in the cytoplasm of elongated spermatids.
Incorporating the biotin amplification step into the biotin-streptavidin-immunoperoxidase assay increased the sensitivity for PG21 antibodies nearly 10-fold when archival human testis tissue was used. In other reports as well as in on-going investigations in our laboratory, the biotin amplification step has increased immunostaining sensitivity by nearly 100-fold for various primary antibodies (19, 22). The increase in the immunostaining sensitivity was exploited to determine relative concentration levels in immunopositive AR in the somatic cells of the testes. The findings of the present study may be interpreted to suggest that there is a hierarchy in immunopositive AR levels among the somatic cells of the human testis, an observation consistent with prior reports in rodents (4, 5, 14, 17). Specifically, the relative order of immunopositive AR in the human testis, going from higher to lower, appears to be Sertoli cells, peritubular myoid cells, smooth muscle cells of blood vessels, and finally Leydig cells. That levels of immunopositive AR in human Sertoli cell nuclei appear to be greater than those in the nuclei of peritubular myoid cells is in marked contrast to the situation reported in rodents (4, 10). Thus, it is possible to speculate that specific androgen regulation of spermatogenesis occurring via Sertoli and peritubular myoid cells may vary between species.
Alternatively, it is possible that there is no net effect in the specific regulation of androgen control of spermatogenesis between species, even though there is relatively less immunopositive AR per individual peritubular myoid cell than per individual Sertoli cell when cells from human and rodent testes are compared. In man, the seminiferous tubule is surrounded by several layers of peritubular myoid cells. In contrast, the rodent tubules are circumscribed by a single layer of myoid cells, distal to which are found the lymphatic endothelial cells forming a wall of the lymphatic sinusoids. Thus, if the peritubular myoid cells mediate in part androgen regulation of spermatogenesis via a paracrine mechanism (23), then the lack of immunopositive AR per cell found in the human condition may be compensated for by increasing the number of peritubular myoid cells participating in the process. That is, the increased number of peritubular myoid cells may exert a local cumulative effect in the human seminiferous tubules that is comparable to the effect exerted by individual peritubular myoid cells in rodents. Indeed, the results presented herein provide further evidence, albeit circumstantial, for the paracrine regulation of spermatogenesis in humans involving the androgen-AR system.
The finding that immunopositive AR in human Sertoli cell nuclei may vary as a function of the cycle is at variance with prior reports (11, 13). It is also at variance with results obtained in the goat (12) and our own observations in the degu, a rodent-like, seasonal breeder from Chile (14, 17). These observations, however, are consistent with the findings made in rodents (4, 5, 10) that AR immunostaining of Sertoli cell nuclei is a function of the cycle of the seminiferous epithelium. The principal explanation that may reconcile these putative discrepancies is that the method used in the present investigation to detect stage-specific changes in AR immunostaining intensities varied significantly from those used in the prior studies. Specifically, as discussed above, the primary antibody was not used at saturating concentrations, permitting information regarding the relative immunopositive AR in Sertoli cell nuclei as a function of the cycle to be "teased" out of the results.
One limitation of the assay used to determine whether immunopositive AR in Sertoli cell nuclei did indeed vary as a function of the cycle is that it does not permit a rigorous statistical analysis of the data; at best, it is only possible to report a trend for the results. The reason for this is as follows. 1) Identification of the different stages in the human seminiferous epithelium is a difficult task, requiring a trained investigator highly familiar with the field of spermatogenesis. Thus, by necessity, scoring of the AR immunostaining intensity in Sertoli cell nuclei as a function of the cycle cannot be carried out using "blind" methodology; there is investigator bias incorporated into the analysis. 2) Scoring of the Sertoli cell nuclei required focusing through the stained sections, a most tedious and difficult method to assign a reliable value to the immunostained cells. 3) Tissue preservation and limitation of its availability to test the reproducibility of the assay did not permit the examination of vast numbers of cases. Therefore, given these concerns, an assay was designed that biased the collection of data toward the average; any doubt in the investigators mind as to what value to assign to a particular Sertoli cell nuclei would lead to a moderate scoring. Incorporation of this bias into the scoring analysis compensates for investigator bias and the limitation of through focusing of the section to assign it a value, ensuring that the trend observed is likely to be real. Indeed, even with this scoring bias, two significant results were obtained at the two primary antibody concentrations used. At 12 µg/mL, intense scoring decreased in Sertoli cell nuclei found in the latter stages of the cycle. At 9 µg/mL, negative scoring increased in Sertoli cells residing in the latter stages of the cycle. We speculate that these two trends are a functional manifestation of AR protein levels in Sertoli cell nuclei. The trends observed in the levels of immunopositive AR in human Sertoli cell nuclei are reminiscent in part of the pattern detected in rodents. Thus, it is attractive to speculate that although there are distinct morphological differences in the stages after sperm release between humans and rodents (only three stages are defined in humans, whereas in the rat the process is divided into six stages), these stages may be functionally equivalent nevertheless.
An elegant cautionary warning was raised by Saunders and colleagues against overinterpreting the absence of AR immunostaining of Sertoli cell nuclei during the latter stages of the rodent testis (13). The logic of this argument was based on the fact that the two antibodies used to detect AR were raised against peptide-specific sequences of the NH2-terminus. As it is possible that proteins present in Sertoli cell nuclei may bind to the NH2-terminus and mask the epitope recognized by the antibody, the absence of AR immunostaining is not definitive proof that AR is truly lacking. Instead, Saunders et al. (13) correctly suggested that antibodies to other peptide sequences of AR should be employed to resolve this issue. Unfortunately, to our knowledge, other antibodies made to different peptides of AR or to the intact receptor have not been suitable for immunocytochemistry in the rat. However, we report that immunoreactive AR in Sertoli cell nuclei present in later stages of the cycle of the seminiferous epithelium was never detected, even when the concentration of the PG21 antibody was increased to render background staining of the Sertoli cell cytoplasm (data not shown). One plausible explanation for this observation is that, indeed, the absence of immunoreactive AR in late stage Sertoli cell nuclei truly represents the lack of protein.
Given that the data presented herein may be interpreted to suggest that there is a functional variation in the AR concentration in Sertoli cell nuclei during the different stages of the cycle of the seminiferous epithelium, it is now necessary to address the relevance of this observation to androgen regulation of spermatogenesis in the human. In the rodent, the greatest AR immunostaining intensity in Sertoli cell nuclei was observed in those stages known to be most susceptible to androgen deprivation (4, 10). AR immunostaining in Sertoli cell nuclei of rodent testes was also observed in other stages, but it was the intense AR immunostaining at stages VII–VIII, correlating with marked germ cell loss at these stages after androgen deprivation that suggest that not only is the presence of AR in Sertoli cell nuclei a requirement for normal completion of spermatogenesis, but that there is a critical AR concentration per nuclei requirement as well. Thus, the present finding that there appears to be a change in the intensity of immunopositive AR in Sertoli cell nuclei is interpreted to suggest that the human seminiferous epithelium functions in a similar fashion with respect to androgen regulation as does the rodent seminiferous epithelium. To our knowledge, this is the first report in the human testis providing evidence that a Sertoli cell protein changes as a function of the cycle of the seminiferous epithelium.
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Acknowledgments |
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Footnotes |
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Received May 28, 1998.
Revised September 30, 1998.
Accepted October 13, 1998.
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References |
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J. Regadera, F. MartÍnez-GarcÍa, P. González-Peramato, A. Serrano, M. Nistal, and C. Suárez-Quian Androgen Receptor Expression in Sertoli Cells as a Function of Seminiferous Tubule Maturation in the Human Cryptorchid Testis J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 413 - 421. [Abstract] [Full Text]
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T. J. Murray, P. A. Fowler, D. R. Abramovich, N. Haites, and R. G. Lea Human Fetal Testis: Second Trimester Proliferative and Steroidogenic Capacities J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4812 - 4817. [Abstract] [Full Text]
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V. Pentikäinen, K. Erkkilä, L. Suomalainen, M. Parvinen, and L. Dunkel Estradiol Acts as a Germ Cell Survival Factor in the Human Testis in Vitro J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 2057 - 2067. [Abstract] [Full Text]
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