Molecular Human Reproduction vol.4 no.3 pp. 227–233, 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 Steger1, Ines Aleithe1, Hermann Behre2 and Martin Bergmann1,3
1Institute
of Anatomy and Cell Biology, University of Halle, Groβe Steinstraβe 52, D-06097 Halle (Saale) and 2Institute of
Reproductive Medicine, University of Münster, Germany
3To
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): 26.6 6 12.4%/46.3 6 9.5%. No stage-dependent
differences were found. Biopsies with mixed atrophy (score ø7) showed a significant (P , 0.05) decrease of
immunopositive spermatogonia to 19.9 6 3.0%/31.4 6 5.7% (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 (28.6 6 8.8%), sda (25.6
6 9.3%), sca (23.7 6 9.3%) and sga (16.2 6 6.0%) and of PCNA staining in sca (32.2 6 11.8%) and sga (20.0 6
9.5%), 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 Apale and type Adark 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 [3H]thymidine labelling
in humans (Chowdhury and Steinberger, 1977) and in nonhuman primates (Clermont and Antar, 1973), Adark 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, Apale and Adark spermatogonia were able to replicate
their DNA.
In testicular biopsies from oligozoospermic men, various
patterns of spermatogenic impairment are found in adjacent
© European Society for Human Reproduction and Embryology
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-
227
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 26.6
6 12.4%/46.3 6 9.5% 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: Apale, Adark and B
spermatogonia. Compared to type A spermatogonia (Ki-67positive: 22.1 6 8.9%; PCNA-positive: 45.3 6 9.5%) the type
B spermatogonia revealed a significantly higher immunostaining for both Ki-67 (43.8 6 15.7%) and PCNA (74.9 6
27.6%) (Table I).
According to the different stages of spermatogenesis (Clermont 1963), there were no significant differences between
stages I–III (Ki-67: 29.9 6 13.4%; PCNA: 45.2 6 10.1%)
and IV–VI (Ki-67: 23.5 6 10.4%; PCNA: 47.5 6 8.9%)
(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 19.9 6 3.0%
(Ki-67) and 31.4 6 5.7% (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 (32.2 6 11.8%) or
spermatogonial (20.0 6 9.5%) arrest compared to normal
biopsies. For the MIB-1 antibody this significant decrease (sca:
23.7 6 9.3%; sga: 16.2 6 6.0%) applied in addition to
spermatogenic arrest at the level of spermatids (25.6 6 9.3%)
and hypospermatogenesis (28.6 6 8.8%) (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 [3H]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
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 5 1 µm.
Table I. Percentage of type A and type B spermatogonia immunopositive for MIB-1 and PC10 in normal human seminiferous tubules. n 5 number of
patients
Type of spermatogonia
Spermatogonia immunopositive for the
anti-Ki-67 antibody (MIB-1)
A
B
anti-PCNA antibody (PC10)
n
Mean (%)
SD (%)
n
Mean (%)
SD (%)
4
4
22.1
43.8
8.9
15.7
5
5
45.3
74.9
9.5
27.6
Table II. Percentage of MIB-1 and PC10 immunopositive spermatogonia in normal human seminiferous tubules of different stages of the seminiferous
epithelial cycle. n 5 number of patients
Stages of the seminiferous
epithelium
Spermatogonia immunopositive for the
anti-Ki-67 antibody (MIB-1)
I–III
IV–VI
anti-PCNA antibody (PC10)
n
Mean (%)
SD (%)
n
Mean (%)
SD (%)
4
4
29.9
23.5
13.4
10.4
5
5
45.2
47.5
10.1
8.9
Table III. Percentage of MIB-1 and PC10 immunopositive spermatogonia in human testicular biopsies of different scores. n 5 number of biopsies
Score
Spermatogonia immunopositive for the
anti-Ki-67 antibody (MIB-1)
10, 9 (Normal)
8
7
6
5
4
3
2
1
anti-PCNA antibody (PC10)
n
Mean (%)
SD (%)
n
Mean (%)
SD (%)
15
3
4
7
5
10
5
5
6
30.2
28.6
20.9*
22.6
26.0
25.3
23.5*
23.3*
19.9*
6.6
1.9
1.9
9.0
4.9
5.8
3.0
4.7
3.0
16
2
4
9
5
6
7
5
16
46.5
44.4*
44.5
40.9
37.6*
37.1*
31.2*
33.2*
31.4*
4.5
0.8
4.8
7.9
4.5
3.3
4.2
11.7
5.7
*Indicates values which are significantly different (P , 0.05) from normal values (score 9, 10).
229
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 5 number of seminiferous
tubules
Spermatogenic defect
Spermatogonia immunopositive for the
anti-Ki-67 antibody (MIB-1)
Normal
hyp
sda
sca
sga
anti-PCNA antibody (PC10)
n
Mean (%)
SD (%)
n
Mean (%)
SD (%)
32
131
56
135
34
34.6
28.6*
25.6*
23.7*
16.2*
9.3
8.8
9.3
9.3
6.0
83
154
45
215
45
48.9
46.9
42.1
32.2*
20.0*
10.8
11.2
16.0
11.8
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
FSH (IU/l)
n
Mean (IU/l)
SD (IU/l)
Ki-67
n
Mean (%)
SD (%)
PCNA
n
Mean (%)
SD (%)
Normal
Without SCO
Unilateral SCO
Bilateral SCO
12
3.7
1.9
20
5.1
3.8
16
8.7*
5.3
12
16.1*
5.7
6
35.5
2.1
11
23.4*
5.1
11
24.3*
4.8
8
26.1*
3.9
9
44.5
5.0
16
36.9*
9.4
9
36.2*
6.6
10
33.6*
10.5
*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 G2-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 G1-, S-, G2- and Mphases of the cell cycle with a maximum in G2- and early Mphases (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 (G0-phase of the cell cycle) and in cells
during early G1-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 MIB1 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 [3H]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 26.6 6 12.4%/
46.3 6 9.5% 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 G1- and G0-phase of the cell cycle (Gerdes
et al., 1984).
Thus, the change of spermatogonial immunoreactive pattern
obtained with both antibodies reflects the differences associated
The proliferation of spermatogonia in the human testis
Table VI. Comparison of Ki-67 antigen and proliferating cell nuclear antigen (PCNA)
Antigen
Monoclonal antibody
Cellular localization
Biological half-life
Expression during cell cycle
Maximum
Absent
Additional characteristics
Ki-67
PCNA
MIB-1 (Dianova)
Nuclear matrix
~1 h
Late G1-, S-, G2-, M-phase
G2-, early M-phase
G0-, early G1-phase
PC10 (Dakopatts)
Nuclear matrix
~20 h
G1-, S-, G2-, M-phase
S-, G2-phase
G0-phase
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 antiMuellerian 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
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, 235–243.
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,
429–476.
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, 463–469.
Bergmann, M. and Kliesch, S. (1994) The distribution pattern of cytokeratin
and vimentin immunoreactivity in testicular biopsies of infertile men. Anat.
Embryol., 190, 515–520.
Bergmann, M., Behre, H.M. and Nieschlag, E. (1994) Serum FSH and
testicular morphology in male infertility. Clin. Endocrinol., 40, 133–136.
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, 595–600.
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, 169–181.
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, 1549–1554.
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, 1227–1234.
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, 311–316.
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, 496–501.
Casasco, A., Callogaro, A., Casasco, E. et al. (1993) An immunocytochemical
method for studying embryo cytokinetics. Basic Appl. Histochem., 32,
293–296.
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, 65–70.
Chowdhury, A.K. and Steinberger, E. (1977) ‘In vitro’ 3H-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. 69–83.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J.
Anat., 112, 35–51.
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, 153–166.
Cooper, T.G., Jockenhövel, J. and Nieschlag, E. (1991) Variations in semen
parameters from fathers. Hum. Reprod., 6, 859–866.
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, 219–229.
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, 787–793.
Duchrow, M., Gerdes, J. and Schlüter, C. (1994) The proliferation-associated
Ki-67 protein: definition in molecular terms. Cell Prolif., 27, 235–242.
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, 455–463.
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, 4774.
232
Fouquet, J.P. and Dadoune, J.P. (1986) Renewal of spermatogonia in the
monkey (Macaca fascicularis). Biol. Reprod., 35, 199–207.
Francavilla, S., Martini, M., Properzi, G. and Cordeschi, G. (1990) Quantitative
parameters of seminiferous epithelium in secretory and excretory
oligoazoospermia. Arch. Androl., 24, 277–285.
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, 1710–1715.
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, 285–294.
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, 203–207.
Haneji, T. and Nishimune, Y. (1982) Hormones and the differentiation of
type A spermatogonia in mouse cryptorchid testes incubated in vitro.
J. Endocrinol., 94, 43–50.
Holstein, A.F. and Schirren, C. (1983) Histological evaluation of testicular
biopsies. Fortsch. Androl., 8, 108–117.
Jockenhövel, F., Khan, S.A. and Nieschlag, E. (1989) Diagnostic value of
bioactive FSH in male infertility. Acta Endocrinol., 121, 802–810.
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, 281–287.
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, 1091–1098.
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,
127–136.
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, 2–25.
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, 121–128.
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, 553–559.
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, 127–134.
Lamont, M.A., Faed, M.J.W. and Baxby, K. (1981) Comparative studies of
spermatogenesis in fertile and subfertile men. J. Clin. Pathol., 34, 145–150.
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, 363–373.
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, 1940–1945.
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. 266–295.
Miething, A. (1993) Proliferative activity of the developing seminiferous
epithelium during prespermatogenesis in the golden hamster testis measured
by bromodeoxyuridine labeling. Anat. Embryol., 187, 249–258.
Miyachi, K., Frizler, M.J. and Tan, E.M. (1978) Autoantibody to a nuclear
antigen in proliferating cells. J. Immunol., 121, 2228–2234.
Oakberg, E.F. (1970) Spermatogonial stem-cell renewal in the mouse. Anat.
Rec., 169, 515–532.
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, 225–230.
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, 579–584.
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, 214–222.
Sigg, C. (1979) Klassifizierung tubulärer Hodenatrophien der
Sterilitätsabklärungen. Bedeutung der sogenannten bunten Atrophie.
Schweiz. Med. Wochenschr., 109, 1284–1293.
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, 122–128.
Terada, T. and Hatekayama, S. (1991) Morphological evidence of two types
of idiopathic Sertoli cell only syndrome. Int. J. Androl., 14, 108–116.
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, 1449–1455.
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, 1831–1839.
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, 539–544.
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, 191–201.
Received on July 31, 1997; accepted on December 19, 1997
233