1 Institute of Anatomy and Cell Biology, University of Halle (Saale), Germany, 2 Unité de Recherches sur l'Endocrinologie du Développement (INSERM), Ecole Normale Supérieure, Département de Biologie, Montrouge, France, 3 Department of Urology, University of Münster, Germany, 4 Institute of Reproductive Medicine, University of Münster, Germany, 5 Children's Hospital, University of Leipzig, Germany, 6 Department of Pathology, The Toronto Hospital, Toronto, Ontario, Canada, 7 Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, 8 Centro de Investigaciones Endocrinológicas, Hospital de Niflos, Buenos Aires, Argentina and 9 Institute of Veterinary Anatomy, University of Giessen, Germany
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Abstract |
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Key words:
anti-Müllerian hormone/cytokeratin/male infertility/M2A antigen/5-reductase deficiency
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Introduction |
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In pathological conditions, we have recently reported variable patterns of expression of CK18 and AMH in Sertoli cells in adult men exhibiting various degrees of spermatogenic dysfunction (Steger et al., 1996). In separate biopsies of individual testes with mixed tubular atrophy, we found either apparently normal adult-type Sertoli cells that did not express CK18 or AMH, those that expressed both markers, or those that expressed one of the two markers.
It is not known to what extent abnormal Sertoli cells associated with defective spermatogenesis are the consequence of the underlying pathological process or, because of their malfunction, a contributing factor to the spermatogenic dysfunction. In addition, the above results raise the question whether these Sertoli cells represent prepubertal Sertoli cells arrested in their normal maturation, or alternatively, adult-type Sertoli cells that have reverted to a less differentiated state. In order to address these issues, we have now examined testicular biopsies from a larger cohort of patients with defective spermatogenesis, including the testes of six patients with 5-reductase deficiency, aged 118 years, which are known to lack normal pubertal testicular development (Damjanov and Drobnjak, 1974
; Müller, 1984
; Aumüller and Peter, 1986
; Johnson et al., 1986
). We have also included an additional immunological marker of Sertoli cell maturation, the M2A antigen, defined by reactivity with the monoclonal antibodies M2A and D2-40. This antigen is expressed in prepubertal, but not adult, Sertoli cells (Bailey et al., 1986
; Baumal et al., 1989
).
Our results indicate that there is a reversion of the Sertoli cell phenotype to a less differentiated state as a heterogeneous process in which the expression of CK18, AMH and M2A antigen is not coordinately regulated. Furthermore, we provide evidence that, in adult subjects with 5-reductase deficiency and SCO syndrome, there is a persistence of immature Sertoli cells, which have apparently failed to progress to maturity at puberty. These non-functional Sertoli cells may well contribute to the spermatogenic dysfunction associated with this genetic defect.
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Materials and methods |
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Serum follicle stimulating hormone
Serum follicle stimulating hormone (FSH) concentrations were measured by fluoroimmunoassay on at least two occasions for each patient (Jockenhövel et al., 1989). Serum concentrations of >7 IU/l were regarded as elevated compared with normal men of proven fertility (Cooper et al., 1991
).
Immunohistochemistry
For cytokeratin 18 (CK 18) immunostaining, 5-µm sections were stained with monoclonal antibody to human CK 18 (Coulter Immunotech, Hamburg, Germany) using the avidinbiotinperoxidase complex (ABC) method (Vectastain Elite ABC Kit, Vector, Burlingame, CA, USA), as follows. After deparaffinization and rehydration, sections were digested with 1 mg pronase/ml Tris-buffered saline pH 7.4 (TBS) for 8 min, and treated with 3% H2O2 for 30 min, followed by 3% bovine serum albumin in TBS (TBS-3% BSA) for 30 min. Sections were then incubated with the primary antibody (1:50 in TBS-3% BSA) overnight, followed by the biotinylated secondary antibody for 30 min, and ABC also for 30 min. Following each incubation, sections were washed thoroughly with TBS. For colour development, the sections were incubated with DAB/H2O2 (Research Genetics, Huntsville, AL, USA). Finally, sections were mounted in DePeX for microscopic examination.
For CK18 and vimentin double-immunostaining, sections were first stained with the anti-CK 18 antibody, as described above, followed by the anti-vimentin antibody (Coulter Immunotech) using the alkaline phosphataseanti-alkaline phosphatase (APAAP) technique according to Cordell et al. (1984).
AMH immunohistochemistry was performed as previously described (Rey et al., 1996). Briefly, 5-µm sections were dehydrated, microwaved and incubated with rabbit anti-recombinant human AMH antibody followed by an alkaline phosphatase-conjugated goat anti-rabbit Ig antibody. For colour development, the sections were incubated with a solution of nitroblue-tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.
Monoclonal antibodies M2A and D2-40 were produced by immunizing mice with the human ovarian adenocarcinoma cell line HEY (Bailey et al., 1986) and a dysgerminom tumour (A.Marks, unpublished results), respectively. Both antibodies are directed to the same antigen and react with tissues fixed in Bouin's fixative. In addition, D2-40 (but not M2A) reacts with tissues fixed in formalin. The antibodies were purified from ascitic fluid of mice by chromatography on a protein ASepharose (Pharmacia, Uppsala, Sweden) column and concentrated to 1.6 mg/ml in phosphate-buffered saline (PBS; 140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 pH 7.2). For immunostaining, the sections were incubated with M2A (2 µg/ml) or D2-40 (0.1 µg/ml) for 1 h at room temperature. The sections were then incubated with biotinylated goat anti-mouse IgG antibody (DAKO, Carpinteria, CA, USA), at a 1:400 dilution, followed by a horseradish peroxidaseavidin conjugate (Zymed, San Francisco, CA, USA) at a 1:600 dilution. For colour development, the sections were incubated with 0.05% diaminobenzidine and 0.03% H2O2 for 1 min, rinsed in water, and counterstained with haematoxylin.
For each immunoreaction, control incubations were performed by substituting buffer for the primary antibody. These sections were completely immunonegative.
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Results |
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Serum FSH concentrations were <1 IU/l in the three children and <7 IU/l in the five adults with OAZ. Patients with NOAZ and OAT exhibited elevated serum FSH concentrations (NOAZ, 19.8 ± 11.4 IU/l; OAT, 11.2 ± 7.0 IU/l).
The histological appearance of the seminiferous tubules varied from qualitatively normal prepubertal seminiferous epithelium in ectopic testes (Figures 13) and normal adult spermatogenic progression (NSPG) in OAZ, to SCO in NOAZ. Biopsies from patients with OAT exhibited a range of histological appearances, including NSPG, arrest at various stages of spermatogenic development, and SCO (Figures 46).
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The results of analysis of a total of 10 testicular biopsies from six patients with 5-reductase deficiency are shown in Table II
.
Serum FSH concentrations were <1 IU/l in prepubertal patients, aged 1, 2 and 10 years (mean 0.8 ± 0.1 IU/l). The 16-year-old patient, and the two 18-year-old patients exhibited elevated serum FSH concentrations (20.3 ± 12.8 IU/l).
Biopsies from all of these patients were examined (Figures 712). The histological examination of the prepubertal patients revealed the presence of prepubertal seminiferous cords with Sertoli cells and prespermatogonia in two patients, aged 1 and 2 years, and in addition to the former cell types, spermatogonia were also seen in the patient aged 10 years. Testicular tissue of the 16-year-old patient (Figures 7
9) exhibited a wide range of histological appearances, with a small fraction of prepubertal seminiferous cords, a majority of tubules with SCO, and some tubules showing arrest in spermatogenic progression at either the level of spermatogonia or spermatocytes. A small proportion of the tubules of the two adult patients (Figures 1012) had evidence of remnants of prepubertal seminiferous cords. However, the predominant histology of the tubules in these patients was SCO.
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Discussion |
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At puberty, Sertoli cells undergo marked morphological and physiological changes in response to hormonal and paracrine effectors (for review see Gondos and Berndston, 1996). This maturation of Sertoli cells is essential for the normal development of prepubertal seminiferous cords to functional adult tubules exhibiting normal spermatogenic progression. Neither the mechanisms through which Sertoli cells influence this process, nor the reciprocal effects of spermatogenesis on the maintenance of Sertoli cells are understood. However, it is certain that the definition of sequential differentiation markers to follow the timely maturation of Sertoli cells would lead to important insights into these questions. Our results have a direct bearing on this issue.
First, we confirm the validity of the three previously reported markers (Steger et al., 1996) of immature Sertoli cells, CK18, AMH and M2A antigen. These three markers were coordinately expressed in normal prepubertal testes, but were absent in normal adult testes. Second, we found that in various pathological disorders of the adult testis associated with infertility and histological abnormalities in spermatogenic progression, CK18 and/or AMH were expressed sporadically. In keeping with previous reports (Rey et al., 1996
; Al-Attar et al., 1997
), AMH was expressed only in premeiotic seminiferous tubules. Remarkably, M2A antigen was never expressed in this large cohort of patients, including 65 testicular biopsies from 50 adults.
The expression of cytokeratin (Miettinen et al., 1985; Stosiek et al., 1990
; Soosay et al., 1991
; Aumüller et al., 1992
; Bergmann and Kliesch, 1994
; Rogatsch et al., 1996
; Steger et al., 1996
, deMiguel et al., 1997) and AMH (Steger et al., 1996
) in postpubertal Sertoli cells in pathological conditions associated with infertility has already been reported. The expression of these markers may indicate a de-differentiation of adult-type Sertoli cells in the context of testicular tubular pathology. This suggests that normal spermatogenic progression may be required to maintain Sertoli cells in their fully differentiated adult stage. In contrast, the absence of expression of M2A antigen in Sertoli cells in these pathological tubules is a novel finding, suggesting that loss of expression of M2A antigen is an irreversible marker of transition of prepubertal Sertoli cells to a differentiated adult state. Therefore, it is likely that the observed abnormalities in Sertoli cells in adult infertility associated with tubular dysfunction are not a primary defect, but may be interpreted as a partial de-differentiation as a consequence of the underlying derangement in spermatogenesis resulting in the re-expression of CK18 and AMH.
With the above inferences in mind, the interpretation of our results in patients with 5-reductase deficiency provides an additional insight into the relationship between Sertoli cell maturation and spermatogenesis. Again, the three markers of immature Sertoli cells, CK18, AMH and M2A antigen, were uniformly expressed in prepubertal spermatogenic cords in testis from young children with this genetic defect. Remarkably, Sertoli cells in the tubules of the prepubertal child, aged 10 years, the 16-year-old patient and the two 18-year-old patients displayed a gradation of expression of these markers, suggesting that Sertoli cells in this disease are arrested at various stages of transition between the immature and adult state. Furthermore, since these stages of transition of Sertoli cells were associated with various degrees of aborted spermatogenic progression, it is possible that 5
-reductase deficiency interferes with the maturation of Sertoli cells, as a primary defect. In turn, these Sertoli cells at various stages of functional maturation would allow only partial progression of spermatogenesis to sequential end-points.
The loss of expression of M2A antigen appears to coincide with a mandatory step in the functional maturation of Sertoli cells. In the 16-year-old patient, loss of expression of M2A antigen in Sertoli cells coincided with the presence of tubules exhibiting spermatogenic progression to the level of spermatogonia and spermatocytes. In contrast, in the two 18-year-old patients, whose Sertoli cells expressed M2A antigen, indicating that they were functionally immature, there was no histological evidence of spermatogenesis, only the presence of prepubertal seminiferous cords and SCO.
The loss of expression of the other two markers of Sertoli cell maturation, CK18 and AMH, appear to bracket temporally the loss of expression of M2A antigen. Loss of CK18 expression occurs very early during maturation of Sertoli cells. The loss of this marker may signal the earliest transition of these cells to a functional state which is, therefore, manifested inconsistently. Thus, in the 10-year-old prepubertal patient whose Sertoli cells showed loss of CK18 expression, prepubertal cords contained spermatogonia in addition to prespermatogonia. However, in one 18-year-old adult whose Sertoli cells also did not express CK18, there was no evidence of spermatogenic progression.
In contrast, loss of AMH expression appears to be a late maturation marker. It never preceded the loss of expression of M2A antigen. In fact, in the 16-year-old patient, AMH continued to be expressed in Sertoli cells even after the loss of expression of M2A antigen. Only in those tubules in which spermatogenesis progressed to the stage of spermatocytes was there loss of AMH expression. This suggests that loss of AMH expression coincides with a further functional maturation of Sertoli cells, allowing the progression of spermatogenesis to a more advanced stage. This is in keeping with results derived from mice (Al-Attar et al., 1997).
The notion (Damjanov and Drobnjak, 1974; Müller, 1984
; Aumüller and Peter, 1986
; Johnson et al., 1986
) that 5
-reductase deficiency affects Sertoli cell maturation as a primary defect with resulting effects on spermatogenesis is consistent with our data. However, this does not exclude the possibility of a more complex phenotype with spermatogenic impairment as a direct consequence of this genetic defect and, in addition, secondary effects of impaired spermatogenesis on Sertoli cell differentiation. For example, the observed expression of CK18 in Sertoli cells that no longer expressed M2A antigen (patient 4) may be an indication of de-differentiation of these cells, following an earlier loss of CK18 expression during early maturation. It is also likely that the penetration of the genetic defect varies among individual patients restricting the maturation of Sertoli cells to different degrees, and allowing partial spermatogenic progression in some patients, but not others.
Our combined results provide evidence for a reciprocal regulation between Sertoli cells and spermatogenesis. Functional Sertoli cells are required for normal spermatogenic progression, which in turn is necessary to maintain Sertoli cells in their fully differentiated state. We suggest that Sertoli cells with partially reverted maturation markers in adult testis, associated with some forms of spermatogenic dysfunction, acquire this phenotype through de-differentiation. Conversely, in 5-reductase deficiency, the failure of Sertoli cell to progress fully to a mature functional state at puberty is a primary defect which, in turn, does not allow normal spermatogenic progression. In this respect, our description of immature Sertoli cells in two adults with this genetic defect represents the first report of the persistence of immature, rather than de-differentiated Sertoli cells in adult testis.
We also suggest a temporal sequence for the three markers of Sertoli cell maturation, with the loss of CK18 expression being the earliest, followed by the loss of M2A antigen expression, and finally the loss of AMH expression. Each of these maturation steps is associated with an expanded capability of Sertoli cells to allow spermatogenesis to progress to a further end-point. The loss of expression of M2A antigen appears to coincide with the most important transition of Sertoli cells to a functional state. Furthermore, the loss of expression of this antigen may be irreversible, in the sense that it does not revert in de-differentiated Sertoli cells associated with impaired spermatogenesis. The elucidation of the molecular mechanisms which control the sequential loss of expression of CK18, AMH and M2A antigen, and the definition of the effector functions which are acquired by Sertoli cells at these maturation steps will contribute to a further understanding of the relationship between Sertoli cells and spermatogenesis.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 27, 1998; accepted on October 7, 1998.