Human Reproduction, Vol. 15, No. 7, 1537-1542,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men
1 Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center and 2 Sackler Faculty of Medicine, Tel Aviv University, Israel and 3 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
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Abstract |
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The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.
Key words: microdeletions/cytokeratin-18/immunohistochemistry/infertile men/Sertoli cells
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Introduction |
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Human seminiferous epithelium contains both somatic cells (Sertoli and myoid) and germ cells (from the early stem cells to the spermatozoa). Sertoli cells play a key role in triggering and regulating the process of spermatogenesis (Russell, 1993
; de Kretser et al., 1998). They contain numerous intermediate filaments which form a meshwork extending throughout the whole cytoplasm.
Cytokeratin intermediate filaments represent an excellent marker for epithelial differentiation since all epithelial cells, whether of ectodermal, endodermal, or mesodermal origin, contain cytokeratins, and several types are ordinarily present in any given cell (Moll et al., 1982; Miettinen et al., 1985). Cytokeratin-18 (CK-18) and vimentin are typically co-expressed in the Sertoli cell cytoplasm of the male fetus as well as throughout childhood (Stosiek et al., 1990; Aumüller et al., 1992). The normal matured `germ epithelium' differs from other true epithelia by the absence of cytokeratin filaments and typical desmosomes. The predominant intermediate filament in mature Sertoli cells of the adult is of the vimentin type, indicating the mesenchymal origin of these cells (Franke et al., 1979). Expression of cytokeratin in adult seminiferous epithelium is regarded as a sign of either maintaining or regaining undifferentiated immature features (Miettinen et al., 1985; Stosiek et al., 1990; Aumüller et al., 1992; Bergmann and Kliesch, 1994; Steger et al., 1996, 1999).
Much information has recently accumulated on the genes which might control spermatogenesis and germ cell differentiation (Ma et al., 1993; Reijo et al., 1995; Lahn and Page, 1997), including the identification of genes on the long arm of the Y chromosome. Macrodeletion of the long arm of the Y chromosome results in azoospermia, suggesting the existence of a gene called the `azoospermia factor' or AZF (Tiepolo and Zuffardi, 1976). Microdeletions of the long arm of the Y chromosome are also associated with infertility and were found in azoospermic or oligozoospermic men (Ma et al., 1992; Reijo et al., 1995, 1996; Qureshi et al., 1996). Recent data suggest that there are likely to be at least three AZF regions which can be individually deleted, i.e. AZF(a–c) (Vogt et al., 1996). Genes encoding two distinct RNA-binding proteins are encoded by the AZFb and AZFc regions, i.e. the RNA-binding motif (RBM) and deleted in azoospermia (DAZ), respectively (Ma et al., 1993; Reijo et al., 1995). These proteins are expressed exclusively in the male germ line (Elliott et al., 1997; Haberman et al., 1998).
A common histological finding of testicular biopsies taken from non-obstructive azoospermic men is a phenomenon described as mixed atrophy (Sigg, 1979). This involves different degrees of spermatogenic impairment in adjacent seminiferous tubules, ranging from qualitatively normal spermatogenesis to Sertoli cell-only (SCO) tubules or total atrophy of adjacent tubules. Since the aetiological mechanism for explaining the underlying pathogenesis of the mixed atrophy has yet to be elucidated, we studied the expression of CK-18 (as a marker of maturation state) in order to evaluate the status of the differentiation of the Sertoli cells in different spermatogenic impairments. In addition, we tested whether Sertoli cell differentiation was affected in men with AZF deletions, in order to evaluate the way Sertoli cells contribute to the various spermatogenic dysfunctions present in the biopsies of men carrying Y chromosome deletions. Morphological and functional lines of evidence on Sertoli cell maturation in men carrying AZF deletions have not been reported previously.
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Materials and methods |
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Testicular biopsies
Testicular biopsies were obtained from 43 azoospermic men attending the Institute for the Study of Fertility in the Israeli institution. The patients (aged 26–46 years) underwent clinical investigation, including at least two semen analyses according to World Health Organization (1992) guidelines and serum hormone (FSH) analysis (Hauser et al., 1998
). Bilateral biopsies were performed following an attempt to extract and isolate testicular sperm cells to be used in our testicular sperm extraction (TESE), IVF, intracytoplasmic sperm injection (ICSI) programme (Hauser et al., 1998). The biopsies were fixed in Bouin's solution (for ~2–4 h). The specimens were embedded in Paraplast. Histological analysis of spermatogenesis was performed on haematoxylin and eosin-stained sections. Forty men were divided into three groups according to the histopathology diagnosis: 12 with SCO syndrome, 22 with mixed atrophy, and six with obstructive azoospermia characterized by the presence of normal spermatogenesis in the biopsy.
Three of the mixed atrophy group had cryptorchidism that had been surgically corrected in their childhood (one of them CK-18 positive in biopsy). One of the SCO group had undergone ligation of a varicocele (CK-18 positive in biopsy).
No additional clinical findings which might shed light on the aetiological factor of the infertility were observed in those men.
Microdeletions in the AZF region were detected in three patients in the Israeli study group and histological sections of six testicular biopsies of patients with microdeletions in the Y chromosome were obtained from the MRC Human Genetics Unit in Scotland. Histological preparation was performed according to a published protocol (Elliott et al., 1997).
All the men with detected AZF deletion comprised the AZF-deleted group: one man with a deletion of AZFb region which included the active RBM gene(s) and four men with a deletion of the AZFc region which included the DAZ gene(s). The remaining four patients had a deletion of the entire AZF(a–c) region.
PCR Y chromosome analysis
The 43 Israeli patients were screened for deletions of the Y chromosome AZF region by multiplex polymeras chain reaction (PCR) analysis using different sequence-tagged sites (STS) (Kleiman et al., 1999a). Analysis of the additional six patients for the detection of AZF microdeletion were performed according to a published protocol (Elliott et al., 1997).
Cytogenetic evaluation
Chromosome analysis was performed on peripheral blood lymphocytes using G-banding staining. An average of 27 metaphases were analysed. All 49 study patients had a 46 XY complement of chromosomes.
Immunohistochemistry
Immunohistochemistry was performed using monoclonal antibodies against CK-18 (Clone:DC10; Zymed, San Francisco, CA, USA) and vimentin (Clone:V9; Dako, Copenhagen, Denmark). Sections of 3 µm were mounted on Super Frost/Plus glass (Menzel–Glaser, Braunschweig, Germany) and processed by labelled (Strept) avidin–biotin (LAB-SA) method using a HistostainTM plus kit (Zymed). Heat-induced antigen retrieval was performed by controlled microwave treatment using an H2800 model processor (Energy Beam Sciences Inc., Agawam, MA, USA) in 10 mmol/l citrate buffer, pH 6.0, for 10 min at 97°C. The sections were treated with 3% H2O2 for 5 min, followed by incubation with normal serum for 10 min and subsequently by incubation for 1 h with ready-for-use CK-18 antibody. Sertoli cell detection was performed using a 1:400 dilution of anti-vimentin antibody. Negative control incubations were performed by substituting non-immune serum for the primary antibody. Biotinylated second antibody was applied for 10 min, followed by incubation with horseradish peroxidase-conjugated streptavidin (HRP-SA) for 10 min. Following each incubation, the slides were washed thoroughly with Optimax wash buffer (Biogenix, San Ramon, CA, USA). The immunoreaction was visualized by an HRP-based chromogen/substrate system, including diaminobenzidene (DAB) (brown) chromogen (Liquid DAB substrate kit; Zymed). The sections were then counterstained with Mayer's haematoxylin, dehydrated and mounted for microscopic examination.
Histomorphometric analysis
Quantitative microscopic analysis was performed using an Olympus BX50 microscope at x200 magnification. A systemic grid with a square density of 100 squares/mm2 was superimposed on the measured field. The area of seminiferous tubules and the area of interstitial tissue were then determined. The number of squares within each compartment is an estimate of the area (Bellhouse, 1981). The ratio of the area of CK-18 immunopositive cells compared to the interstitial area and the rest of the tissue (representing the reference area) was expressed as the immunopositive area fraction. Calculations were made as described by Williams (Williams, 1977) and presented as mean ± SEM. Student's t-test was used to analyse the data. P < 0.05 was considered significant.
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Results |
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Elevated FSH concentrations (upper range value of 10 IU/l) were detected in all the men in the SCO group and in most of the men in the mixed atrophy group as shown in Table I
. The testosterone concentrations were within physiological ranges. The expression of CK-18 in the patients with an elevated FSH concentration was variable, being present in a fraction of Sertoli cells in some patients and absent in others (Table I).
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Histomorphometric evaluation revealed the highest quantity of CK-18 immunopositive seminiferous tubules in the group classified as mixed atrophy (Figure 1
). A lower expression of CK-18 was demonstrated in the SCO group, while there was only residual staining for CK-18 in the group with normal spermatogenesis and in the AZF-deleted group. CK-18 expression in the mixed atrophy group differed significantly (P < 0.001) from all other groups, while the SCO group also differed significantly (P < 0.05) from the one with normal spermatogenesis and the AZF-deleted group. No differences in CK-18 expression were found between the normal spermatogenesis and the AZF-deleted groups.
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CK-18 immunopositive Sertoli cells were found in SCO tubules, both in biopsies with mixed atrophy and in SCO biopsies. In addition, cytokeratin immunoreactive Sertoli cells were found in tubules, characterized by arrest of spermatogenesis at the level of spermatogonia or primary spermatocytes (Figure 2A
). Tubules showing elongated spermatids or qualitatively complete spermatogenesis were almost negative for CK-18, both in the normal spermatogenesis group and in the mixed atrophy group.
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The cytoplasm of Sertoli cells was uniformly immunopositive for vimentin in all the biopsies, independent of spermatogenic impairment or the state of Sertoli cell differentiation (Figure 2B
and data not shown). This applied to all the studied groups without exception.
Sertoli cells were negatively stained for CK-18 in all nine patients with Y chromosome microdeletions (Figure 2C–E). A single SCO tubule was identified in one patient with AZFc deletion: this tubule showed weak residual staining for CK-18 (Figure 2D).
In the patient carrying a deletion exclusive to the AZFb region, germ cell maturation was arrested at the spermatocyte level. In the four patients with a deletion of the AZFc region, germ cell development was arrested at the spermatocyte level or at the round spermatid level, while few spermatozoa were present in single seminiferous tubule. The four men who had a deletion of the entire AZF region (AZFa–c) were classified as having SCO syndrome.
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Discussion |
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The involvement of Sertoli cells in spermatogenic dysfunctions of patients with detected Y chromosome deletions was studied for the first time. In the testis of men with deletions in the AZF region, the Sertoli cells were found to be mature, as indicated by the absence of CK-18 expression. In these cases, there is no association between spermatogenesis and the maturation state of the Sertoli cells. This suggests that the aetiology of the defective spermatogenesis in such cases is intrinsic to germ cells and not due to a malfunction of the Sertoli cells. The defect in germ cells may be a direct result of the Y chromosome deletions (Reijo et al., 1995
, 1996; Elliott et al., 1997).
Microdeletion of the AZFb gene(s) which removes RBM protein expression results in spermatocyte arrest (Elliott et al., 1997; Brandell et al., 1998), while microdeletion of the AZFc regions which removes the DAZ protein expression, results in spermatogenic impairments. Spermatozoa can usually be retrieved by testicular sperm extraction in AZFc-deleted men (Mulhall et al., 1997; Kleiman et al., 1999b; Page et al., 1999). Deletion of the entire AZF(a–c) region results in SCO syndrome (Foresta et al., 1998). In the Simoni et al. compilation of the literature on the frequency of Y chromosome deletions among infertile men, the reports generally agreed that it is low, with deletions having been found in 12% of the azoospermic men and in 3% of the oligozoospermic men (Simoni et al., 1998).
Normal maturation of the somatic Sertoli cells in the AZF-deleted patients was not impaired by an inter-relation with anomalous germ cells or by the lack of germ cells. This result suggests that in these cases Sertoli cell maturation is independent of a fully functional germ line.
The quantitative data in the current study regarding the CK-18 expression in the mixed atrophy and SCO groups are in accordance with previous studies which demonstrated, either qualitatively or semiquantitatively, the expression of CK-18 as a marker of immature or undifferentiated Sertoli cells in the seminiferous epithelium of infertile men (Miettinen et al., 1985; Stosiek et al., 1990; Bergmann and Kliesch, 1994; Steger et al., 1996, 1999). Since there is wide variability in CK-18 expression in testicular biopsies, the advantage of the histomorphometric analysis that we performed is that it provided an additional uniform method for defining the amount of undifferentiated seminiferous epithelium present in a biopsy.
In the present study, CK-18 expression differed significantly in the mixed atrophy and SCO groups compared to the group representing normal spermatogenesis. These results support those of previous studies in which Sertoli cell morphology in pathologies associated with defective spermatogenesis, such as Klinefelter's syndrome, cryptorchidism, oestrogen treatment and SCO syndrome, differs from that of matured Sertoli cells of normal testicular tissue (Nistal et al., 1990). The highest level of CK-18 expression found in our mixed atrophy group indicates that the cause that determined the interrupted spermatogenesis had damaged both the Sertoli and the germ cells. The CK-18 immunoreactive tubules showed either arrest of spermatogenesis at pre-meiotic stages or SCO.
Some variability was observed among the SCO biopsies related to CK-18 expression, with some of the tubules having been positively stained while others lacked CK-18 immunoreactivity (although CK-18 was found occasionally in single Sertoli cells in some of the tubules). Therefore, there is coexistence of different types of Sertoli cells within the seminiferous epithelium of SCO cases. This phenomenon has been described by Nistal et al., who classified the Sertoli cells in SCO biopsies from infertile men as immatured, dysgenetic or involuting (Nistal et al., 1990). The existence of several types of Sertoli cells may be the end result of different aetiological factors that determine the pattern of SCO tubules (Nistal et al., 1990; Terada and Hatekayama, 1991).
In the present study, high serum FSH concentrations were exhibited in the non-obstructive azoospermia patients, while variable patterns of CK-18 expression were observed in their dysfunctional tubular epithelium. It is well known that non-obstructive azoospermic men demonstrate elevated serum FSH concentrations (Steger et al., 1999). Sertoli cells are the only testicular cells that express FSH receptors (Böckers et al., 1994). Accordingly, these high FSH concentrations in non-obstructive patients are due to the failure of Sertoli cell differentiation, indicating a normal development pattern of the hypothalamic–pituitary–gonadal axis (Steger et al., 1999).
The coexpression of anti-Müllerian hormone (AMH) and CK-18 in testicular biopsies of infertile patients had been reported (Steger et al., 1996, 1999). AMH is considered to be an early Sertoli cell marker in gonadal embryogenesis (Josso et al., 1993). This co-expression confirms the validity of CK-18 as a marker of an undifferentiated Sertoli cell state (Steger et al., 1996, 1999).
The current study contributes to the understanding of the multifactorial mechanism underlying the complicated process of spermatogenesis. Functional Sertoli cells are required for progression of normal spermatogenesis which, in turn, is necessary to maintain Sertoli cells in their fully differentiated state. However, when the primary defect is intrinsic to the germinal line, there is no effect on the Sertoli cells' differentiated phenotype once these cells have reached maturity.
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Acknowledgments |
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The authors express their gratitude to Prof. H.J.Cooke from the MRC Human Genetics Unit for his help with this project. Mr N.Davidson from the Photography Department MRC Unit is thanked for the excellent preparation of the histological coloured plate. The laboratory technicians in the Institute for the Study of Fertility, Tel Aviv Sourasky Medical Center, are thanked for their excellent work. Dr R.Maymon, Tel Aviv University Faculty of Medicine, is thanked for revising the manuscript and his constructive remarks. The skilful assistance of Dr A.Eisenthal and Dr B.Lifshitz-Mercer from the Institute of Pathology of the Israeli hospital is gratefully acknowledged. Ms Esther Eshkol, MA, is thanked for editorial assistance.
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Notes |
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4 To whom correspondence should be addressed at: Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. E-mail: paz{at}tasmc.health.gov.il
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Submitted on November 23, 1999; accepted on April 20, 2000.
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