Klaus Steger 1, Ines Aleithe 1, Hermann Behre 2 and Martin Bergmann 1,3

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1 Molecular Human Reproduction vol.4 no.3 pp , 1998 The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen Klaus Steger 1, Ines Aleithe 1, Hermann Behre 2 and Martin Bergmann 1,3 1 Institute of Anatomy and Cell Biology, University of Halle, Groβe Steinstraβe 52, D Halle (Saale) and 2 Institute of Reproductive Medicine, University of Münster, Germany 3 To whom correspondence should be addressed The quantitative distribution pattern of Ki-67 protein and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied in human testis biopsies. In normal seminiferous epithelium Ki-67 is expressed in nuclei of spermatogonia, while PCNA additionally occurs in nuclei of primary spermatocytes. The staining pattern of spermatogonia is as follows (Ki-67-positive/PCNA-positive): %/ %. No stage-dependent differences were found. Biopsies with mixed atrophy (score 7) showed a significant (P 0.05) decrease of immunopositive spermatogonia to %/ % (score 1) with minimal variation between different samples (score 7 to 1). Associated with defined histological defects such as hypospermatogenesis (hyp), spermatogenic arrest at the level of spermatids (sda), spermatocytes (sca) or spermatogonia (sga), however, there was a significant (P 0.05) decrease of Ki-67 staining in tubules showing hyp ( %), sda ( %), sca ( %) and sga ( %) and of PCNA staining in sca ( %) and sga ( %), respectively. The decrease of immunoreactive spermatogonia did not correspond to elevation of follicle stimulating hormone (FSH). These data demonstrate that the low spermatogenic efficiency in infertile men is not only due to postmeiotic events, but also to a decrease in the meiotic activity of spermatogonia, and is not related to serum FSH. Key words: human testis/immunohistochemistry/ki-67 protein/pcna/spermatogonial proliferation Introduction During normal spermatogenesis controlled cell proliferation is of fundamental importance, assuming highly coordinated mechanisms between the mitotically inactive Sertoli cells and the germ cells undergoing mitosis and meiosis. Efficiency of spermatogenesis depends on (i) the proliferative activity of spermatogonia and (ii) the loss of germ cells during meiosis and spermiogenesis. In the human, spermatogenic efficiency is low compared to other species and mainly due to the loss of spermatocytes (Johnson et al., 1992). In the human and primate testis, the population of type A spermatogonia can be differentiated into type A pale and type A dark depending on nuclear morphology (Clermont, 1963; Fouquet and Dadoune, 1986). It is still a matter of debate how these cells are involved in proliferative activity. On the basis of [ 3 H]thymidine labelling in humans (Chowdhury and Steinberger, 1977) and in nonhuman primates (Clermont and Antar, 1973), A dark spermatogonia were regarded as non-proliferating cells (for review see Meistrich and van Beek, 1993). For the primates this was later confirmed by Schlatt and Weinbauer (1994) immunohistochemically using antibodies against the proliferating cell nuclear antigen (PCNA). In the human, however, Paniagua et al. (1987) demonstrated that, in the normal seminiferous epithelium, A pale and A dark spermatogonia were able to replicate their DNA. In testicular biopsies from oligozoospermic men, various patterns of spermatogenic impairment are found in adjacent tubules, a phenomenon called mixed atrophy (Sigg, 1979). Because spermatogonia are the stem cells of spermatogenesis, this cell type is of special interest when studying spermatogenic impairment. Chaturvedi and Johnson (1993) suggested an involvement of differences of early spermatogonial divisions in men with low and high daily sperm production, and recent data from Werner et al. (1997) demonstrated that spermatogenic defects are associated with a decreasing cytoplasmic heat shock protein (hsp)60 immunoreactivity in spermatogonia. Because of the fact that hsp60 is necessary for normally functioning mitochondria, one can also assume negative effects on the mitotic proliferation of spermatogonia. Using monoclonal antibodies against the two proliferation markers PCNA and Ki-67 protein, we studied their quantitative distribution pattern in the seminiferous epithelium of men with normal spermatogenesis and oligozoospermic men in order to investigate the mitotic activity of the different types of spermatogonia and the possible involvement in the low spermatogenic efficiency in testes of infertile men. Materials and methods Testicular tissue used in this study was obtained from men attending the Infertility Clinic of the Institute of Reproductive Medicine in Münster. Patients were subjected to a thorough clinical investigation, including at least two semen analyses according to World Health Organization (1992) guidelines and hormone analyses. Serum follicle stimulating hormone (FSH) levels were measured by fluoroimmuno- European Society for Human Reproduction and Embryology 227

2 K.Steger et al. assay on at least two occasions for each patient (Jockenhövel et al., 1989). Serum levels of 7 IU/l were regarded as elevated compared to normal men with proven fertility (Cooper et al., 1991). Biopsies were performed in azoospermic patients in order to distinguish between obstructive and non-obstructive azoospermia, and in patients with oligozoospermia, when neither medical history, clinical investigation, semen analysis or hormone levels had provided an explanation for their infertility. The testicular biopsy specimens, each about the size of a grain of rice, were routinely cut into halves and fixed by immersion in either 5.5% glutaraldehyde and embedded in Epon or in Bouin s fixative and embedded in paraffin wax using standard techniques. For histological evaluation semithin sections (1 µm) were stained with Toluidine Blue and paraffin sections (5 µm) were stained with haematoxylin. Both were scored according to the method of Holstein and Schirren (1983), which describes the percentage of seminiferous tubules bearing elongated spermatids, i.e. score 10 means that 100% of the seminiferous tubules contain elongated spermatids. For immunohistochemistry, paraffin sections were mounted on slides coated with 3-aminopropyltriethoxysilane (Sigma, München, Germany). After deparaffinization and rehydration, sections were stained with the monoclonal antibodies PC10 (Dakopatts, Hamburg, Germany) against the human proliferating cell nuclear antigen (PCNA) and MIB-1 (Dianova, Hamburg, Germany) against the human Ki-67 protein using the alkaline phosphatase anti-alkaline phosphatase (APAAP) method (Cordell et al., 1984). In the case of the Ki-67 protein, sections were pretreated by the antigen retrieval technique using microwaves (Bremner et al., 1994). In brief, sections were microwaved for 25 min at 1000 W in sodium citrate buffer (ph 6.0), and after cooling sections were incubated with the undiluted primary antibody for 1 h. Sections were then exposed twice to the secondary antibody (alkaline phosphataseconjugated rabbit anti-mouse IgG, 1:25; Dakopatts, Hamburg, Germany) followed by APAAP (1:50; Dakopatts) for 30 min each (first incubation) and for 10 min each (second incubation). After each incubation sections were thoroughly washed with Tris-buffered saline (TBS; ph 7.4). For PCNA immunohistochemistry sections were treated with normal rabbit serum (1:5; Dakopatts) for 20 min followed by incubation with the primary antibody (1:20) for 3 h. The biotinylated second antibody (rabbit anti-mouse, 1:400; Dakopatts) was applied for 30 min followed by incubation with alkaline phosphatase-conjugated streptavidin (1:200; Dakopatts) for 30 min. After each incubation sections were thoroughly washed with TBS (ph 7.4). In both cases the immunoreaction was visualized by developing sections with Fast Red TR/Naphthol AS-MX (Sigma, München, Germany). Finally, sections were mounted in Dako Glycergel. For each test, control incubations were performed by substituting buffer for the primary antibody. The tissue sections were completely immunonegative. Spermatogonial proliferation is thought to be stimulated by FSH (van Alphen et al., 1988; Arslan et al., 1993). Oligozoospermic men often show elevated FSH concentrations, probably due to the occurrence of focal Sertoli cell-only syndrome (SCO) (Bergmann et al., 1994; Martin-du-Pan and Bischof, 1995). In order to evaluate a possible relationship between spermatogonial proliferative activity and FSH, we therefore analysed the percentage of labelled spermatogonia and FSH values depending on the occurrence of focal SCO. A total of 139 biopsies from 79 patients were investigated. Statistics Data were analysed using Student s t-test. P 0.05 was regarded as significant. 228 Results In the normal seminiferous epithelium in biopsies from patients showing obstructive azoospermia both antibodies provided a nuclear staining of spermatogonia. In the case of the PC10 antibody, primary spermatocytes up to the pachytene stage were additionally stained (Figures 1, 2). In our material %/ % of all spermatogonia were found to be positive for Ki-67/PCNA respectively. During normal human spermatogenesis, three different types of spermatogonia can be differentiated: A pale, A dark and B spermatogonia. Compared to type A spermatogonia (Ki-67- positive: %; PCNA-positive: %) the type B spermatogonia revealed a significantly higher immunostaining for both Ki-67 ( %) and PCNA ( %) (Table I). According to the different stages of spermatogenesis (Clermont 1963), there were no significant differences between stages I III (Ki-67: %; PCNA: %) and IV VI (Ki-67: %; PCNA: %) (Table II). In testes of oligozoospermic men showing mixed atrophy and a score of 8, there was a significant (P 0.05) reduction of the percentage of labelled spermatogonia to % (Ki-67) and % (PCNA) at score 1 (Table III). However, significant differences between score 7 and score 1 could not be found. The score value principally represents the percentage of tubules showing elongated spermatids, but disregards the distribution pattern of defined spermatogenic defects. When summarizing the data according to defined defects such as hypospermatogenesis (only qualitatively intact spermatogenesis) or spermatogenic arrest at the level of early round spermatids, spermatocytes or spermatogonia, there was a significant (P 0.05) reduction of PCNA-labelled spermatogonia in tubules showing spermatocytic ( %) or spermatogonial ( %) arrest compared to normal biopsies. For the MIB-1 antibody this significant decrease (sca: %; sga: %) applied in addition to spermatogenic arrest at the level of spermatids ( %) and hypospermatogenesis ( %) (Table IV). FSH values of oligozoospermic patients revealed a significant increase up to 28.3 IU/l in patients showing unilateral and bilateral focal SCO. The proliferative activity of spermatogonia was significantly (P 0.05) reduced as was found after score dependent evaluation regarding both Ki-67 and PCNA values, but was not related to FSH elevation (Table V). Discussion Mitotic activity of cells can be sufficiently evaluated by different methods, i.e. the counting of mitotic figures, application of labelled nucleotides such as [ 3 H]thymidine or bromodeoxyuridine (BrdU) or by detecting specific S-phase related proteins such as PCNA (Miyachi et al., 1978) or Ki-67 antigen (Gerdes et al., 1984). Using human material, only the latter approach is possible because of ethical reasons. PCNA is an auxiliary protein to DNA polymerase-δ, being involved in nucleotide excision repair mechanisms (Miyachi et al., 1978; Hall et al., 1993). With a biological half-life of

3 The proliferation of spermatogonia in the human testis Figures 1 and 2. Immunostaining of the nuclei of spermatogonia (arrowheads) with the MIB-1 (Figure 1) and the PC10 (Figure 2) antibody. In addition, proliferating cell nuclear antigen is present in the nuclei of primary spermatocytes (arrows) up to the pachytene stage of meiosis (Figure 2). Bar 1 µm. Table I. Percentage of type A and type B spermatogonia immunopositive for MIB-1 and PC10 in normal human seminiferous tubules. n number of patients Type of spermatogonia A B Table II. Percentage of MIB-1 and PC10 immunopositive spermatogonia in normal human seminiferous tubules of different stages of the seminiferous epithelial cycle. n number of patients Stages of the seminiferous epithelium I III IV VI Table III. Percentage of MIB-1 and PC10 immunopositive spermatogonia in human testicular biopsies of different scores. n number of biopsies Score 10, 9 (Normal) * * * * * * * * * * 5.7 *Indicates values which are significantly different (P 0.05) from normal values (score 9, 10). 229

4 K.Steger et al. Table IV. Percentage of MIB-1 and PC10 immunopositive spermatogonia in human seminiferous tubules with hypospermatogenesis (hyp) and spermatogenic defects such as arrest of spermatogenesis at the level of round spermatids (sda), spermatocytes (sca) and spermatogonia (sga). n number of seminiferous tubules Spermatogenic defect Normal hyp * sda * sca * * 11.8 sga * * 9.5 *Indicates values which are significantly different (P 0.05) from normal values. Table V. Percentage of MIB-1 and PC10 immunopositive spermatogonia depending on the serum follicle stimulating hormone (FSH) from men with obstructive azoospermia and oligozoospermic men without Sertoli cell-only syndrome (SCO), with unilateral SCO and with bilateral SCO Normal Without SCO Unilateral SCO Bilateral SCO FSH (IU/l) n Mean (IU/l) * 16.1* SD (IU/l) Ki-67 n Mean (%) * 24.3* 26.1* SD (%) PCNA n Mean (%) * 36.2* 33.6* SD (%) *Indicates values which are significantly different (P 0.05) from normal values. ~20 h (Bravo and MacDonald-Bravo, 1987), PCNA is expressed in the nuclear matrix of cells during all phases of the cell cycle with a maximum in S- and G 2 -phases (Hall et al., 1990; Casasco et al., 1993). The proliferation-associated antigen Ki-67 is expressed in the nuclear matrix of cells during late G 1 -, S-, G 2 - and M- phases of the cell cycle with a maximum in G 2 - and early M- phases (Gerdes et al., 1984; Sasaki et al., 1987). Its biological half-life is ~1 h (Duchrow et al., 1994). The Ki-67 protein is absent in resting cells (G 0 -phase of the cell cycle) and in cells during early G 1 -phase, nor is it detectable during DNA repair processes (Hall et al., 1993; Kubbutat et al., 1994). Becker et al. (1992) developed and characterized the monoclonal antibody MIB-1, which after microwave treatment of the tissue sections recognizes the Ki-67 antigen even in formalin-fixed and paraffin-embedded tissues (Cattoretti et al., 1992). The specific characteristics of both proteins are summarized in Table VI. Although the immunohistochemical detectability of Ki-67 protein and PCNA is highly influenced by the fixation procedure (Bruno et al., 1992; Kujat et al., 1996), it has been demonstrated that these techniques yield labelling indices comparable to nucleotide incorporation (Böswald et al., 1990), giving results which are generally in accordance with the proliferative pattern obtained by counting of mitotic figures (Miething, 1993). However, the more widely spreading the Ki-67 protein and PCNA within the cell cycle, the greater will 230 be the increase in the proliferative pattern obtained by MIB- 1 and PC10 immunohistochemistry compared to nucleotide incorporation calculations of S-phase cells. In the mouse an average of ~25% of the spermatogonia show radiolabelling with [ 3 H]thymidine (Oakberg, 1970). In the normal human seminiferous epithelium, Lamont et al. (1981) found an average of ~10% of the spermatogonia at the metaphase of mitosis by counting mitotic figures. Our material revealed %/ % positive spermatogonia for Ki-67/PCNA respectively. The higher percentage of PCNA staining as well as the labelling of primary spermatocytes corresponds with results from bull (Wrobel et al., 1996), rodent and primate testes (Schlatt and Weinbauer 1994), but it cannot be explained simply by the long half-life of the PCNA. It is probably due to the involvement of PCNA in nucleotide excision repair mechanisms (Hall et al., 1993; Kubbutat et al., 1994). With the MIB-1 antibody there is no immunostaining of postspermatogonial germ cells in the human testis. This is in accordance with the fact that the amount of the Ki-67 protein is at its lowest level immediately after mitosis (Du Manoir et al., 1991) and that cell nuclei are generally MIB-1 immunonegative during early G 1 - and G 0 -phase of the cell cycle (Gerdes et al., 1984). Thus, the change of spermatogonial immunoreactive pattern obtained with both antibodies reflects the differences associated

5 The proliferation of spermatogonia in the human testis Table VI. Comparison of Ki-67 antigen and proliferating cell nuclear antigen (PCNA) Antigen Ki-67 PCNA Monoclonal antibody MIB-1 (Dianova) PC10 (Dakopatts) Cellular localization Nuclear matrix Nuclear matrix Biological half-life ~1 h ~20 h Expression during cell cycle Late G 1 -, S-, G 2 -, M-phase G 1 -, S-, G 2 -, M-phase Maximum G 2 -, early M-phase S-, G 2 -phase Absent G 0 -, early G 1 -phase G 0 -phase Additional characteristics Auxiliary protein to polymerase-δ Involvement in DNA repair processes with spermatogenic impairment, but these data have to be considered in respect of the specific methodological conditions. Comparing the different types of spermatogonia occurring during normal human spermatogenesis, a significantly higher percentage of the B spermatogonia were immunoreactive for both Ki-67 and PCNA. In men the cycle of the seminiferous epithelium can be divided into six stages (Clermont, 1963). Both the Ki-67 protein and the PCNA immunoreactive spermatogonia reveal no significant stage-dependent differences. As shown by Chaturvedi and Johnson (1993) and Johnson et al. (1996) the architectural make-up of stages within seminiferous tubules and the presence of atypical cell types within stages depend on the efficiency of spermatogenesis. These variations cause difficulties concerning the exact classification of the stages and stage specific quantification. Testis biopsies with score 7 to score 1 obtained from oligozoospermic men reveal a significant decrease of immunopositive spermatogonia compared to normal control testes. However, there is no clear score-related decrease of spermatogonial labelling. This was also found by Lamont et al. (1981) who counted metaphase spermatogonia in testes with normal spermatogenesis and in subfertile testes with mixed atrophy using the method of Johnsen (1970). In both cases immunopositive spermatogonia were counted in whole biopsies comprising a variety of spermatogenic defects in adjacent seminiferous tubules. Considering the phenomenon of mixed atrophy we also looked at single seminiferous tubules instead of whole biopsies comprising immunopositive spermatogonia in tubules with the same spermatogenic defect: there was a decrease of the spermatogonial proliferation dependent on the defined spermatogenic defect including an arrest at the levels of spermatogonia, primary spermatocytes, round spermatids or even hypospermatogenesis. This confirms earlier studies from Werner et al. (1997) who found a low level of hsp60 expression in spermatogonia, a mitochondrial chaperone having essential importance for the mitochondrial protein import machinery in dividing cells in order to generate further mitochondria for the daughter cells. The data indicate that the low spermatogenic efficiency in oligozoospermic men is not only due to meiotic and postmeiotic defects, but that these defects are also associated with a population of functionally impaired spermatogonia. The proliferation of spermatogonia was shown to be stimulated by FSH in the mouse (Haneji and Nishimune, 1982) and in the primate testis (Van Alphen et al., 1988; Weinbauer et al., 1991; Arslan et al., 1993). In the human, low efficiency of spermatogenesis is often found to be correlated with high levels of FSH (de Kretser et al., 1974; Francavilla et al., 1990; Johnson et al., 1990; Kula, 1991) including reduced numbers of spermatogonia (Baker et al., 1976). Our data show that the decreased proliferative activity of spermatogonia does not correspond with increasing FSH values. The high FSH levels are believed to be due to the occurrence of focal SCO (Bergmann and Kliesch, 1994; Martin-du-Pan and Bischof, 1995). This can be explained by deficits in Sertoli cell differentiation and function in SCO tubules (Bergh and Cajander, 1990; Terada and Hatekayama, 1991; Bruning et al., 1993; Steger et al., 1996) leading to the observed changes of the neuroendocrine axis. In addition, there is now sufficient evidence that Sertoli cells are the only target cells for FSH (Kliesch et al., 1992; Böckers et al., 1994). In mammals, the existence of a Sertoli cell-derived mitogenic factor acting on spermatogonia has been suggested (Feig et al., 1980; Kancheva et al., 1990), and in the newt testis model, FSH-induced spermatogonial proliferation only occurs in contact with Sertoli cells assuming that mediator molecules stimulate the proliferation of spermatogonia (Maekawa et al., 1995). Using fetal and prepubertal markers such as nuclear structure (Bruning et al., 1993), intermediate filament expression (Bergmann and Kliesch, 1994), or the persistence of anti- Muellerian hormone protein (Steger et al., 1996), it was recently shown that spermatogenic arrest and hypospermatogenesis are also associated with a population of Sertoli cells lacking full differentiation. Therefore, we conclude that the observed high levels of FSH cannot trigger spermatogonial proliferation because of the functionally impaired population of Sertoli cells in these seminiferous tubules. Acknowledgements We are grateful to Prof. Dr E.Nieschlag, Institute of Reproductive Medicine, and Prof. Dr L.Hertle, Department of Urology of the University, Münster, for providing the biopsies. The skilful technical assistance of I.Strotbaum, I.Ehrling, H.Mrusek and R.Rappold is gratefully acknowledged, as is Ms Susan Luginsland for checking the English of the manuscript. Funding of this research programme was provided by DFG grant BE 1016/4-1. Data were partly presented at the Symposium The fate of the male germ cells, December 5 7, 1996, Hamburg, Germany. 231

6 K.Steger et al. References Arslan, M., Weinbauer, G.F., Schlatt, S. et al. (1993) FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta). J. Endocrinol., 136, Baker, H.W.G., Bremner, W.J., Burger, H.G. et al. (1976) Testicular control of follicle stimulating hormone secretion. Recent Progr. Horm. Res., 32, Becker, M.H.G., Key, G., Baron, B. et al. (1992) MIB 1 3 new monoclonal antibodies against the proliferation associated antigen previously defined by Ki-67. Histochem. J., 24, 610 (Abstract). Bergh, A. and Cajander, S. (1990) Immunohistochemical localization of inhibin alpha in the testes of normal men and in men with testicular disorders. Int. J. Androl., 13, Bergmann, M. and Kliesch, S. (1994) The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men. Anat. Embryol., 190, Bergmann, M., Behre, H.M. and Nieschlag, E. (1994) Serum FSH and testicular morphology in male infertility. Clin. Endocrinol., 40, Böckers, T.M., Nieschlag, E., Kreutz, M.R. and Bergmann, M. (1994) Localization of follicle-stimulating hormone (FSH) immunoreactivity and hormone receptor mrna in testicular tissue of infertile men. Cell Tissue Res., 278, Böswald, M., Harasim, I. and Maurer-Schultze, B. (1990) Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies. Cell Tissue Kinet., 23, Bravo, R. and MacDonald-Bravo, H. (1987) Existence of two populations of cyclin proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J. Cell Biol., 105, Bremner, W.J., Millar, M.R., Sharpe, R.M. and Saunders, P.T.K. (1994) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology, 135, Bruning, G., Dierichs, R., Stümpel, C. and Bergmann, M. (1993) Sertoli cell nuclear changes in human testicular biopsies as revealed by three dimensional reconstruction. Andrologia, 25, Bruno, S., Gorczyca, W. and Darzynkiewicz, Z. (1992) Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki-67 antibody. Cytometry, 13, Casasco, A., Callogaro, A., Casasco, E. et al. (1993) An immunocytochemical method for studying embryo cytokinetics. Basic Appl. Histochem., 32, Cattoretti, G., Dominoni, F., Fusilli, F. and Zanaboni, O. (1992) Microwave oven irradiation vs trypsin digestion for antigen unmasking in fixed, paraffin embedded material. Histochem. J., 24, 594 (Abstract). Chaturvedi, P.K. and Johnson, L. (1993) Architectural arrangement of stages of the spermatogenic cycle within human seminiferous tubules is related to efficiency of spermatogenesis. Cell Tissue Res., 273, Chowdhury, A.K. and Steinberger, E. (1977) In vitro 3 H-thymidine labeling pattern and topographic distribution of spermatogonia in human seminiferous tubules. In Troen, P. and Nankin, H.R. (eds), The Testis in Normal and Infertile Men. Raven Press, New York, pp Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, Clermont, Y. and Antar, M. (1973) Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arotoides. Am. J. Anat., 136, Cooper, T.G., Jockenhövel, J. and Nieschlag, E. (1991) Variations in semen parameters from fathers. Hum. Reprod., 6, Cordell, J.L., Falini, B., Erber, W.N. et al. (1984) Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J. Histochem. Cytochem., 32, De Kretser, D.M., Burger, H.G. and Hudson, B. (1974) The relationship between germinal cells and serum FSH levels in males with infertility. J. Clin. Endocrinol. Metab., 38, Duchrow, M., Gerdes, J. and Schlüter, C. (1994) The proliferation-associated Ki-67 protein: definition in molecular terms. Cell Prolif., 27, Du Manoir, S., Guillaud, P., Carius, E. et al. (1991) Ki-67 labeling in postmitotic cells defined different Ki-67 pathways within the 2c compartment. Cytometry, 12, Feig, L.A., Bellve, A.R., Erikson, N.H. and Klagsbrun, M. (1980) Sertoli cells contain a mitogenic polypeptide. Proc. Natl. Acad. Sci. USA, 77, Fouquet, J.P. and Dadoune, J.P. (1986) Renewal of spermatogonia in the monkey (Macaca fascicularis). Biol. Reprod., 35, Francavilla, S., Martini, M., Properzi, G. and Cordeschi, G. (1990) Quantitative parameters of seminiferous epithelium in secretory and excretory oligoazoospermia. Arch. Androl., 24, Gerdes, J., Lemke, H., Baisch, H. et al. (1984) Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki-67. J. Immunol., 133, Hall, P.A., Levison, D.A., Woods, A.L. et al. (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J. Pathol., 162, Hall, P.A., McKee, P.H., Menage, H.D.P. et al. (1993) High levels of p53 protein in UV irradiated normal human skin. Oncogene, 8, Haneji, T. and Nishimune, Y. (1982) Hormones and the differentiation of type A spermatogonia in mouse cryptorchid testes incubated in vitro. J. Endocrinol., 94, Holstein, A.F. and Schirren, C. (1983) Histological evaluation of testicular biopsies. Fortsch. Androl., 8, Jockenhövel, F., Khan, S.A. and Nieschlag, E. (1989) Diagnostic value of bioactive FSH in male infertility. Acta Endocrinol., 121, Johnson, L., Grumbles, J.S., Bagheri, A. and Petty, C.S. (1990) Increased germ cell degeneration during postprophase of meiosis is related to increased serum follicle-stimulating hormone concentrations and reduced daily sperm production in aged men. Biol. Reprod., 42, Johnson, L., Chaturvedi, P.K. and Williams, J.D. (1992) Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans. Biol. Reprod., 47, Johnson, L., McKenzie, K.S. and Snell, J.R. (1996) Partial wave in human seminiferous tubules appears to be a random occurrence. Tissue Cell, 28, Johnsen, S.C. (1970) Testicular biopsy score counts. A method for registration of spermatogenesis in human testes: Normal values and results in 335 hypogonadal males. Hormones, 1, Kancheva, L.S., Martinova, Y.S. and Georgiev, V.D. (1990) Prepubertal Sertoli cells secrete a mitogenic factor(s) that stimulates germ and somatic cell population. Mol. Cell Endocrinol., 69, Kliesch, S., Penttil, T.L., Gromoll, J. et al. (1992) FSH receptor mrna is expressed stage-dependently during rat spermatogenesis. Mol. Cell Endocrinol., 84, R45 R49. Kubbutat, M.H.G., Cattoretti, G., Gerdes, J. and Key, G. (1994) Comparison of monoclonal antibodies PC10 and MIN1 on microwave-processed paraffin sections. Cell Prolif., 27, Kujat, R., Bickel, D. and Wrobel, K.H. (1996) Optimal fixation allows reliable PCNA immunoreaction with highly diluted PC10 antibody. Ann. Anat. (Suppl. 178), 81. Kula, K. (1991) Hyperactivation of early steps of spermatogenesis comprises meiotic insufficiency in men with hypergonadotropism a possible quantitative assay for high FSH/low testosterone availabilities. Andrologia, 23, Lamont, M.A., Faed, M.J.W. and Baxby, K. (1981) Comparative studies of spermatogenesis in fertile and subfertile men. J. Clin. Pathol., 34, Maekawa, K., Ji, Z.S. and Abe, S.I. (1995) Proliferation of newt spermatogonia by mammalian FSH via Sertoli cells in vitro. J. Exp. Zool., 272, Martin-du-Pan, R.C. and Bischof, P. (1995) Increased follicle stimulating hormone in infertile men. Is increased plasma FSH always due to damaged germinal epithelium? Hum. Reprod., 10, Meistrich, M.L. and van Beek, M.E.A.B. (1993) Spermatogonial stem cells. In Desjardins, C. and Ewing, L.L. (eds), Cell and Molecular Biology of the Testis. Oxford University Press, Oxford, pp Miething, A. (1993) Proliferative activity of the developing seminiferous epithelium during prespermatogenesis in the golden hamster testis measured by bromodeoxyuridine labeling. Anat. Embryol., 187, Miyachi, K., Frizler, M.J. and Tan, E.M. (1978) Autoantibody to a nuclear antigen in proliferating cells. J. Immunol., 121, Oakberg, E.F. (1970) Spermatogonial stem-cell renewal in the mouse. Anat. Rec., 169, Paniagua, R., Codesal, J., Nistal, M. et al. (1987) Quantification of cell types through the cycle of the human seminiferous epithelium and their DNA content. A new approach to the spermatogonial stem cell in man. Anat. Embryol., 176, Sasaki, K., Murakami, R., Kawasaki, M. and Takahashi, M. (1987) The cell cycle associated change of Ki-67 reactive nuclear antigen expression. J. Cell Physiol., 133,

7 The proliferation of spermatogonia in the human testis Schlatt, S. and Weinbauer, G.F. (1994) Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testis. Int. J. Androl., 17, Sigg, C. (1979) Klassifizierung tubulärer Hodenatrophien der Sterilitätsabklärungen. Bedeutung der sogenannten bunten Atrophie. Schweiz. Med. Wochenschr., 109, Steger, K., Rey, R., Kliesch, S. et al. (1996) Immunohistochemical detection of immature Sertoli cell markers in testicular tissue of infertile adult men: a preliminary study. Int. J. Androl., 19, Terada, T. and Hatekayama, S. (1991) Morphological evidence of two types of idiopathic Sertoli cell only syndrome. Int. J. Androl., 14, Van Alphen, M.M.A., Van de Kant, H.J.G. and De Rooij, D.G. (1988) Folliclestimulating hormone stimulates spermatogenesis in the adult monkey. Endocrinology, 123, Weinbauer, W., Behre, H.M., Fingscheidt, U. and Nieschlag, E. (1991) Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist treated nonhuman primate (Macaca fascicularis). Endocrinology, 129, Werner, A., Meinhardt, A., Seitz, J. and Bergmann, M. (1997) Distribution pattern of heat shock protein 60 immunoreactivity in testes of infertile men. Cell Tissue Res., 288, World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction. Cambridge University Press, Cambridge. Wrobel, K.H., Bickel, D. and Kujat, R. (1996) Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen (PCNA). Cell Tissue Res., 283, Received on July 31, 1997; accepted on December 19,