1 Service de Cytogénétique-Immunocytologie-Biologie du Développement et de la Reproduction and 2 Service dUrologie-Andrologie, CHU et EA MENRT 3185, Faculté de Médecine et de Pharmacie, Place Saint-Jacques, 25030 Besancion Cedex, France
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Abstract |
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Key words: AMH/azoospermia factor/M2A/male infertility/Sertoli cells
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Introduction |
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Thus, the immunohistological phenotype of gonads could be different in infertile patients with AZF deletions. A recent study using the cytokeratin-18 antibody did not show any differences in immunoreactivity in AZF deleted patients (Bar-Shira Maymon et al., 2000).
However, Foresta et al. report that the inhibin B production in patients with Yq deletions was 70% higher than in non-deleted patients, and the functional relationship between FSH and inhibin B was normally preserved (Foresta et al., 2001
).
Other markers may also allow us to detect modifications of gonadal immunohistological phenotype in infertile patients with AZF deletions.
Two potential markers of Sertoli cell functional status [anti-Müllerian hormone (AMH) and M2A] attracted our attention; these markers seem to be expressed independently of cytokeratin-18.
AMH is a functional marker of Sertoli cell differentiation (Tran et al., 1987; Josso et al., 1993
). The role of AMH during postnatal gonadal maturation and after puberty remains uncertain. Its implication in germ cell proliferation (Cazorla et al., 1998
; Fénichel et al., 1999
), as well as its possible effect on the differentiation and function of Leydig cells, were recently suggested (Racine et al., 1998
). Conventional immunohistochemical methods cannot detect AMH immunoreactivity in testes of adult patients with normal spermatogenesis, but a renewal of AMH immunoreactivity was described in the Sertoli cells of patients with spermatogenesis arrest or Sertoli cell-only syndrome (Steger et al., 1996
).
M2A is an oncofetal marker corresponding to a surface sialoprotein expressed in fetal gonocytes and Sertoli cells (Baumal et al., 1989; Jorgensen et al., 1993
, 1995
; Marks et al., 1999
). This antigen was also identified in several testicular tumours such as seminomas, carcinomas in situ and teratomas (Bailey et al., 1986
; Giwercman et al., 1988
; Marks et al., 1999
).
AMH and M2A are sequential markers of immature or de-differentiated Sertoli cell status: AMH expression seems to be arrested later than that of M2A (Steger et al., 1999). AZF microdeletions are supposed to appear at very early ontogenetic developmental stages, so that Sertoli cells may keep some immature characteristics. At the same time, progressive decrease of spermatogenesis marked by partial reversion of Sertoli cell immunophenotype may be observed in infertile patients. To verify these hypotheses, we tested the two immunohistological markers (AMH and M2A) of immature Sertoli cells and examined the possibility of differential AMH and/or M2A immunoexpression in patients with two well characterized spermatogenic disorders associated or not with AZF microdeletions.
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Materials and methods |
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Two fetal testes (18 and 20 weeks) and two postnatal testicular biopsies (2 months and 4 years) were used as positive controls. Two testicular biopsies of patients with active spermatogenesis and without AZF microdeletions were used as a reference.
Immunohistochemistry was performed using D2-40 mouse monoclonal antibodies against the M2A antigen (courtesy of Professor A.Marks, Department of Medical Research, University of Toronto, Ontario, Canada) and rabbit polyclonal antibodies against recombinant human AMH (courtesy of Dr R.Rey, Unité de Recherches sur lEndocrinologie du Développement, Montrouge, France).
M2A immunohistochemistry
All biopsy samples were fixed in a Bouin mixture, paraffin embedded. The sections (7 µm thick) were deparaffinized and rehydrated. The slides were incubated in Target Retrieval Solution (Dako, Glostrup, Denmark) at 80°C for 20 min. They were then left in the above solution for 20 min at room temperature (RT) and immersed in phosphate-buffered saline solution (PBS), pH 7.4, for 10 min. Aspecific sites were blocked with PBS containing 10% bovine serum albumin (BSA) for 30 min at RT. The sections were incubated overnight at 4°C with the primary antibody diluted in PBS containing 0.3% Triton X-100, 10% lactoproteins and 1% BSA. The titre of the M2A antibody was 1:4000. After being washed three times for 5 min each in PBS, the sections were incubated for 1 h at RT with CYTM3-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immuno Research, West Grove, USA).
AMH immunohistochemistry
The sections were treated as previously described before blocking endogenous peroxidase activity by treatment with 3% H2O2 for 10 min. The slides were then incubated overnight at 4°C with the primary antibody diluted 1:1000 in PBS. The sections were then incubated for 1 h at RT with the secondary antibody, unlabelled goat anti-rabbit IgG (P.A.R.I.S, Compiègne, France) diluted 1:50 in the same solution that was used for the primary antibody. Finally, the sections were incubated with rabbit PAP (peroxidase-anti-peroxidase) (Dako). The reaction was revealed by diaminobenzidin (DAB).
Controls
All tests were performed on fetal, postnatal and adult control testicular tissue. The negative control was not treated with the primary antibody.
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Results |
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We observed membrane and sometimes perinuclear M2A immunostaining in spermatogonia and in the Sertoli cells of fetal and 2 month old male gonads (Figure 1). However, M2A immunostaining was not observed in the testes of a 4 year old boy and in two adult reference tissues.
The use of anti-AMH antibodies yielded intense cytoplasmic immunostaining in the Sertoli cells of both fetal and child testes (Figure 1). On the other hand, the Sertoli cells were AMH negative in adult reference tissues.
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Discussion |
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M2A negativity could mean either an irreversible loss of the expression of this marker after puberty, or a very low immunoreactivity level. Steger et al. suggest an abolition of M2A immunoexpression at puberty with a possible resumption in the case of a neoplastic process (Steger et al, 1999).
The situation with AMH is more complex or even contradictory. Fetal testes present important AMH immunoreactivity, but the production of this substance decreases successively after birth and especially at puberty. In adult subjects with qualitatively normal spermatogenesis, immunohistochemistry methods cannot detect the presence of AMH in Sertoli cells (Steger et al., 1996, 1999
). A possible renewal of AMH immunoreactivity in cases of seriously impaired spermatogenesis (Steger et al., 1996
, 1999
) seems to be in apparent contradiction with the recent work, suggesting that the lowest spermatic concentrations of AMH correspond to particular severity of the spermatogenic disorder (Fenichel et al., 1999
). Immunoreactivity to AMH in situ does not necessarily correspond to an increased level of this hormone in seminal fluid, but perhaps could be explained by retention (altered release) of the AMH by altered Sertoli cells. On the other hand, the absence of AMH in-situ immunoreactivity (without taking into account the sensitivity of the technique) may be explained by either a complete release of this hormone into the seminiferous lumen, or a general decrease of the hormone production level.
Our study and similar ones include patients having histological syndromes whose aetiology is not clear. The results of the studies mentioned above are difficult to compare because of possible aetiological heterogeneity and the different sensitivity of the immunodetection techniques applied. Combined use of both techniques (biopsy immunostaining and ELISA) may be useful in assessing the data available. An absence of AMH immunoreactivity as in our case proves that the immature and/or dedifferentiated phenotype of Sertoli cells is rather rare in infertile patients.
A recent study attempting to evaluate cytokeratine-18 expression in the Sertoli cells of patients with non-obstructive infertility did not demonstrate a difference between the immunoreactivity pattern in subjects with and without AZF microdeletions (Bar-Shira Maymon et al., 2000). In our study, we did not observe any immunohistological differences between samples with and those without Y deletion. In the cases of deletions in the AZF region, neither tested marker was discriminating. AZF microdeletions do not appear to interfere with immunoexpression of AMH and/or oncofetal M2A antigen.
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Acknowledgements |
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Notes |
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References |
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Bar-Shira Maymon, B., Paz, G., Elliott, D.J., Hammel, I., Kleiman, S.E., Yogev, L., Hauser, R., Botchan, A. and Yavetz, H. (2000) Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men. Hum. Reprod., 15, 15371542.
Baumal, R., Bailey, D., Giwercman, A., Skakkebaek, N., Stratis, M. and Marks, A. (1989) A novel maturation marker for human Sertoli cells. Int. J. Androl., 12, 354359.[ISI][Medline]
Blagosklonova, O., Fellmann, F., Clavequin, M.C., Roux, C. and Bresson, J.L. (2000) AZFa deletions in Sertoli cell-only syndrome: a retrospective study. Mol. Hum. Reprod., 6, 795799.
Brown, G.M., Furlong, R.A., Sargent, C.A., Erickson, R.P., Longepied, G., Mitchell, M., Jones, M.H., Hargreave, T.B., Cooke, H.J. and Affara, N.A. (1998) Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene. Hum. Mol. Genet., 7, 97107.
Cazorla, O., Seck, M., Pisselet, C., Perreau, C., Saumande, J., Fontaine, J., de Reviers, M. and Hochereau-de Reviers, M.T. (1998) Anti-Müllerian hormone (AMH) secretion in prepubertal and adult rams. J. Reprod. Fertil., 112, 259266.[Abstract]
Chandley, A.C. and Cooke, H.J. (1994) Human male fertility-Y-linked genes and spermatogenesis. Hum. Mol. Genet., 3,14491452.[Abstract]
Fenichel, P., Rey, R., Poggioli, S., Donzeau, M., Chevallier, D. and Pointis, G. (1999) Anti-Müllerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia. Hum. Reprod., 14, 20202024.
Foresta, C., Bettella, A., Moro, E., Roverato, A., Merico, M. and Ferlin, A. (2001) Sertoli cell function in infertile patients with and without microdeletions of the azoospermia factors on the Y chromosome long arm. J. Clin. Endocrinol. Metab., 6, 24142419.
Giwercman, A., Marks, A., Bailey, D., Baumal, R. and Skakkebaek, N.E. (1988) A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis. APMIS, 96, 667670.[ISI][Medline]
Jorgensen, N., Giwercman, A., Muller, J. and Skakkebaek, N.E. (1993) Immunohistochemical markers of carcinoma in situ of the testis also expressed in normal infantile germ cells. Histopathology, 22, 373378.[ISI][Medline]
Jorgensen, N., Rajpert-De Meyts, E., Graem, N., Muller, J., Giwercman, A. and Skakkebaek, N.E. (1995) Expression of immunohistochemical markers for testicular carcinoma in situ by normal human fetal germ cells. Lab. Invest., 72, 223231.[ISI][Medline]
Josso, N., Lamarre, I., Picard, J.Y. Berta, P., Davies, N., Morichon, N., Peschanski, M. and Jeny, R. (1993) Anti-Müllerian hormone in early human development. Early Hum. Dev., 33, 9199.[ISI][Medline]
Marks, A., Sutherland, D.R., Bailey, D., Iglesias, J., Law, J., Lei, M., Yeger, H., Banerjee, D. and Baumal, R. (1999) Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours. Br. J. Cancer, 80, 569578.[ISI][Medline]
Mazeyrat, S., Saut, N., Sargent, C.A., Grimmond, S., Longepied, G., Ehrmann, I.E., Ellis, P.S., Greenfield, A., Affara, N.A. and Mitchell, M.J. (1998) The mouse Y chromosome interval necessary for spermatogonial proliferation is gene dense with syntenic homology to the human AZFa region. Hum. Mol. Genet., 7, 17131724.
Menke, D.B., Mutter, G.L. and Page, D.C. (1997) Expression of DAZ, an azoospermia factor candidate, in human spermatogonia. Am. J. Hum. Genet., 1, 237241.
Racine, C., Rey, R., Forest, M.G., Louis, F., Ferre, A., Huhtaniemi, I., Josso, N. and di Clemente, N. (1998) Receptors for anti-Müllerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiation. Proc. Natl Acad. Sci. USA, 95, 594599.
Steger, K., Rey, R., Kliesch, S., Louis, F., Schleicher, G. and Bergmann, M. (1996) Immunohistochemical detection of immature Sertoli cell markers in testicular tissue of infertile adult men: a preliminary study. Int. J. Androl., 19, 122128.[ISI][Medline]
Steger, K., Rey, R., Louis, F., Kliesch, S., Behre, H.M., Nieschlag, E., Hoepffner, W., Bailey, D., Marks, A. and Bergmann, M. (1999) Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis. Hum. Reprod., 14, 136143.
Tran, D., Picard, J.Y., Campargue, J. and Josso, N. (1987) Immunocytochemical detection of anti-Müllerian hormone in Sertoli cells of various mammalian species including human. J. Histochem. Cytochem., 35, 733743.[Abstract]
Submitted on October 10, 2001; resubmitted on January 30, 2002; accepted on April 10, 2002.