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Department of Experimental Radiation Oncology, University of Texas M. D. Anderson Cancer Center (G.A.S., G.S., G.W., M.L.M.), Houston, Texas 77030; the Department of Cell Biology, University of Utrecht (D.G.R.), 3584 CX Utrecht, The Netherlands; the Department of Physiology, University of Turku (I.H.), 20520 Turku, Finland; Central Research and Development ASTA Medica AG. (T.R.), D-60314 Frankfurt Aim Main, Germany; and the Department of Physiology, Southern Illinois University (L.D.R.), Carbondale, Illinois 62901
Address all correspondence and requests for reprints to: Dr. Gladis A. Shuttlesworth, Department of Experimental Radiation Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030. E-mail: shuttles{at}utmdacc.mdacc.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation; some were treated with the GnRH antagonist Cetrorelix
beginning 15 weeks after irradiation. Although the spermatogonia in the
irradiated rats without hormone treatment continue to proliferate
(labeling and mitotic indexes of 24% and 18%, respectively), they
underwent apoptosis (apoptotic indexes of 21% by the terminal
transferase-mediated end labeling assay and 9% by nuclear morphology),
resulting in a constant number of A spermatogonia. Whole mount analysis
of clones of A spermatogonia revealed that larger clones were more
likely to undergo apoptosis than mitosis. Hormone administration
decreased the intratesticular testosterone concentration to 6% of the
level in irradiated rats within 1 week. Concomitantly, there was a
decrease in spermatogonial apoptotic indexes to 43% of levels in
irradiated-only rats, leading to an increases in their numbers by
150%, their diameters by 11%, and their labeling indexes by 31%. The
sizes of the mitotic clones gradually increased, and clones of more
than eight cells appeared at week 3 of hormone treatment. A
spermatogonial differentiation began at week 4, and by week 6.6,
differentiation occurred in 30% of the tubules. Thus, suppression of
intratesticular testosterone by the GnRH antagonist may be responsible
for the immediate changes in spermatogonial numbers and kinetics, but
several additional steps are required before differentiation begins,
which did not occur until week 4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further studies in our laboratory demonstrated that the administration of hormones that suppress intratesticular testosterone, GnRH analogs, or testosterone itself after irradiation was effective in stimulating the differentiation of these spermatogonia and resulted in the recovery of spermatogenesis (3). When GnRH agonist was given to rats for 10 weeks immediately after 3.5 Gy irradiation, the percentage of tubules containing differentiating cells increased from 37% to 91%. Spermatogenic recovery was also stimulated when the GnRH agonist treatment was initiated 18 weeks after irradiation with 6 Gy, and the treatment was continued for 10 weeks, at which time the rats were killed.
Because in our previous studies of the stimulation of recovery of spermatogenesis by GnRH analogs, the earliest time point analyzed was 10 weeks after the initiation of hormone administration, in this study we addressed the question of when the initiation of spermatogenesis occurs and the sequence of events involved. We therefore examined the proliferation, clonal size, cell loss, and differentiation of A spermatogonia in irradiated rats within the first few weeks after hormone treatment to determine the time course and the degree of synchrony of initiation.
LBNF1 rats were irradiated with 6 Gy of
-irradiation, and GnRH analog treatment was initiated at 15 weeks
after irradiation, because at this time and dose, A spermatogonia are
the only germ cells remaining in the testis (2). A GnRH antagonist was
used rather than an agonist primarily because it produces a greater
suppression of testosterone and stimulation of spermatogenic recovery
(Meistrich, M.L., and G. Wilson, unpublished results).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Irradiation
Animals were anesthetized with 0.72 mg ketamine/kg and 0.022 mg
acepromazine/kg (im), and then a single 6-Gy dose was delivered to the
lower part of the abdomen by a 60Co -ray unit
(Eldorado 8, Atomic Energy of Canada Ltd., Ottawa, Canada). The
radiation field was 20 x 20 cm, and four rats were irradiated
simultaneously. Superflab material was placed on top of the rats to
ensure depth homogeneity of the delivered dose.
Hormone treatment
The GnRH antagonist, Cetrorelix,
(Ac-D-Nal(2)1-D-Phe(4Cl)2-D-Pal(3)3-D-Cit6-D-Ala10)LHRH
(SB-75), was used in two forms. Cetrorelix acetate has high initial
release and short duration due to its high solubility. Cetrorelix
pamoate has poor solubility and therefore is slowly released and
persists in the circulation for longer periods of time (4). Both
compounds were synthesized and provided by ASTA Medica, AG (Frankfurt,
Germany).
At 15 weeks after irradiation, animals were given simultaneous (sc) injections of 1.5 mg Cetrorelix pamoate (suspended in carboxymethyl-cellulose and 1% Tween-80)/kg and 1.5 mg Cetrorelix acetate (dissolved in bacteriostatic water)/kg, each at a different site in the upper portion of the dorsal region. Both drugs were freshly prepared before they were used. Irradiated animals injected with diluents were used as controls. A second Cetrorelix pamoate injection was given 3.3 weeks after the first. The doses of the hormone were based on the results in Sprague Dawley rats, in which Cetrorelix pamoate at 1.5 mg/kg produced uniform suppression of testosterone levels for 27 days (4). Cetrorelix pamoate, however, did not appear to suppress testosterone levels rapidly. Hence, an initial bolus of Cetrorelix acetate at 1.5 mg/kg was also given to produce a high initial concentration of Cetrorelix in the serum and rapidly reduce testosterone levels. The serum level of Cetrorelix necessary to completely suppress testosterone in the rat is about 1 ng/ml (Reissmann, T., unpublished results).
Preparation of tissues
Two independent experiments were performed (Exp 1 and 2).
Animals (eight rats, Exp 1 and 2) were killed at each of the following
times except as noted: 1, 2, 3, 4, 5, and 6.6 (four rats, Exp 2) weeks
after hormone injection, which correspond to 16, 17, 18, 19, 20, and
21.6 weeks after irradiation, to measure spermatogonial changes.
Irradiated rats (8 rats, Exp 1 and 2) without hormone treatment, were
killed at 15 weeks and also at 20 weeks after irradiation. Intermediate
time points were not taken because previous studies demonstrated that
no changes in testicular histology, repopulation indexes, labeling
indexes, and serum hormone levels occurred between 10–30 weeks after
irradiation (2). All of these rats were injected ip with
bromodeoxyuridine (BrdU; Sigma, St Louis, MO) at a dose of
30 mg/kg 1 h before being killed.
Each testis was weighed with the tunica albuginea intact. They were carefully sliced into two pieces with a fresh, double edged razor blade, using a sawing motion. Half of the left testis was fixed in Bouin’s fluid for histological evaluation, counting of A spermatogonia, and determination of repopulation indexes. The tissue was embedded in plastic (JB4, Polysciences, Inc., Warrington, PA). The other half portion of the left testis was fixed in formalin and embedded in paraffin for terminal transferase-mediated end labeling (TUNEL) staining using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). One fourth of the right testis was fixed in 70% ethanol and embedded in plastic for BrdU immunohistochemistry. In all cases, sections were cut at 4-µm thickness and stained or counterstained with Harris hematoxylin. No compression or distortion of tubules or cells resulting from slicing the tissue was observed. Seminiferous tubules were collected from the remaining three fourths of the right testis; they were isolated under a dissecting microscope and immediately placed in Bouin’s fixative for whole mount preparations (5).
For electron microscopy (6), both testes from one normal and three irradiated (6 Gy, 27 weeks after irradiation) rats were prefixed by vascular perfusion using 5% electron microscopic grade glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in cacodylate buffer (0.05 M sodium cacodylate, pH 7.3–7.4). Then testes were postfixed with osmium tetroxide and embedded in araldite, and thin sections were cut between 50–100 nm and viewed under the electron microscope.
A Spermatogonial numbers and mitoses
A Spermatogonial cells and mitoses were counted in 200
nonrepopulating seminiferous tubular cross-sections of Bouin’s fixed
tissue. Sertoli cell nuclei, which were much more numerous, were
counted in every tenth seminiferous tubular cross-section, but only
when their nucleoli were visible. On the average, 220 A spermatogonia
and 330 Sertoli cells were counted per rat. For each animal, the
diameters of 10 A spermatogonia nuclei and 20 Sertoli cell nucleoli
were measured using a Magiscan Image Analysis System (Joyce-Loebl,
Ltd., Dukesway, UK) attached to a Nikon light microscope.
The average diameters of spermatogonial nuclei and Sertoli cell
nucleoli and the section thickness were used to calculate the
Abercrombie correction for bias in the estimation of nuclear numbers in
cross-sections (7). The corrected counts were used to calculate the
ratio of spermatogonia per 100 Sertoli cells, which is an accepted
method to account for tubular shrinkage in the comparison of germ cell
counts (8). Previous studies showed that the number of Sertoli cells
per tubule cross-section remained constant between 10–30 weeks after
irradiation (2) and was unaffected by GnRH antagonists (9).
The mitotic index was calculated by dividing the number of mitotic cells by the sum of A spermatogonia plus mitotic cells. The diameters of the A spermatogonial mitoses were calculated by averaging the width of the widest spread chromosomes and the distance covered by chromosomes in the perpendicular direction to the width. The Abercrombie correction was also used in the evaluation of mitotic index.
Apoptosis of spermatogonia.
Two different measures of apoptosis were used. The numbers of
TUNEL-positive cells were scored in 200 round seminiferous tubular
cross-sections from formalin-fixed, paraffin-embedded material. A cell
was considered TUNEL positive when the nuclear staining was intense and
homogenous. Nuclear diameters of TUNEL-positive A spermatogonia were
measured, and the Abercrombie correction was applied. The apoptotic
index was calculated by dividing the numbers of TUNEL-positive
spermatogonia per Sertoli cell by the numbers of spermatogonia per
Sertoli cell obtained from Bouin’s-fixed tissue from the same testis.
This was performed because identification of spermatogonia in
formalin-fixed tissue was difficult.
In addition, cells that had apoptotic morphology were evaluated in 200 round seminiferous tubular cross-sections in Bouin’s-fixed plastic-embedded material. The criterion for apoptosis was the appearance of dense chromatin bodies distributed in the cell nucleus (10). The diameters of the apoptotic A spermatogonial nuclei were measured. The apoptotic index was calculated by dividing the number of apoptotic A spermatogonia by the total number of A spermatogonia scored in the same tubules after applying the Abercrombie correction.
BrdU labeling index
Ethanol-fixed, plastic-embedded, sectioned tissue was processed
for BrdU immunohistochemistry as previously described (2) with a slight
modification. Briefly, the section was digested with 0.1% protease
(protease, bacterial type XXIV, Sigma) for 15 min at room
temperature. Tissue was incubated with a monoclonal antibody against
BrdU (clone B44, BrdU Pure, catalogue no. 347580, Becton Dickinson and Co., San Jose, CA) diluted 1:20 in blocking
solution (PBS, 5% normal horse serum, 1% BSA, and 0.5% Tween 20).
The reaction was developed by the addition of antimouse IgG
biotinylated secondary antibody from the Vectastain kit
(catalogue no. PK-6102, Vector Laboratories, Inc.,
Burlingame, CA). After immunohistochemistry, slides were counterstained
with Harris hematoxylin. The labeling index was calculated by dividing
the number of A spermatogonial cell nuclei that were positive for BrdU
by the total number of A spermatogonia plus mitoses counted in 200
seminiferous tubular cross-sections. There were no differences between
the average diameters of BrdU-labeled and unlabeled A spermatogonia;
hence there was no need to apply the Abercrombie correction in this
case.
Assessment of A spermatogonial differentiation
Spermatogonial differentiation was assessed in Bouin’s-fixed,
plastic-embedded tissue by calculating the repopulation index, which is
the percentage of tubules showing repopulation, in 200 round
seminiferous tubular cross-sections. A tubule was scored as repopulated
if it contained 3 or more germ cells that had reached the B
spermatogonial stage or later.
Sizes of A spermatogonial clones
The clonal size of the A spermatogonia was determined in tubule
whole mounts. Tubules were dissected from irradiated rats (four rats,
Exp 1) at the following time points: 15 and 20 weeks after irradiation
and 1, 2, 3, 4, and 5 weeks after initiation of GnRH antagonist
treatment starting at 15 weeks after irradiation. Isolated tubules were
stained with hematoxylin and mounted on slides (5). Per animal, 15–25
randomly selected fragments, 1–3 cm in length, were studied. All
clones encountered along the length of the tubule were evaluated.
The spermatogonial clones were distributed over the basal membrane with a varying cell density. In general, the criterion used is that undifferentiated A spermatogonia lying within 20 µm of each other and showing the same morphology belong to the same clone (11, 12). However, because of the high density of A spermatogonia in the shrunken tubuli, it was often not possible to distinguish the individual clones reliably. The A spermatogonia that belong to the same clone are connected by intercellular bridges, and therefore, the cells comprising a clone will go through the cell cycle in a synchronous fashion. As mitosis is of short duration, neighboring cells simultaneously in mitosis will most likely belong to the same clone. Therefore, the determination of clonal size in the present study was carried out by only scoring clones in mitosis. Spermatogonia were considered to belong to the same clone when they were lying within 20 µm of each other and were synchronously in prophase, metaphase, or anaphase of mitosis.
Apoptotic spermatogonia were characterized by dense condensation of chromatin into peripheral masses or a marginated condensed chromatin. As apoptosis is also a process of short duration, apoptotic cells lying within 20 µm from each other were also considered to belong to the same clone.
Hormone measurements
In addition to the rats used for histological analysis,
irradiated animals (4 rats, Exp 1) were killed at 1, 2, 4, and 5 weeks
of hormone treatment in all cases, except at 3 weeks of hormone
treatment when more animals were used (7 rats, Exp 1 and 2), for
preparation of testicular homogenates for intratesticular testosterone
analysis. To measure immediate changes in hormone levels, animals (four
rats, Exp 2) were killed after 1 and 2 days of Cetrorelix treatment.
Blood was collected by cardiac puncture from all irradiated animals
used in the experiment (4–15/group) at the time the rats were killed.
In addition, 4 unirradiated 13-week-old rats were used as controls. The
serum was collected by centrifugation. After removal of the tunica
albuginea, the right testis was homogenized in 5 ml deionized water
using a Polytron homogenizer (Brinkmann Instruments, Inc.,
Steinhofhalde, Switzerland).
Serum Cetrorelix concentrations were measured using a double antibody
RIA (13). Standards were prepared from a working stock of 4 µg/ml
Cetrorelix acetate in RIA buffer [sodium phosphate buffer (pH 7.2),
2% BSA, 1% EDTA, and 0.02% Triton X-100] by serial 1:2 dilution
steps. The RIA was set up by diluting the samples or standards,
iodinated Cetrorelix, and Cetrorelix antiserum in RIA buffer and
incubating at 4 C for 2 days. Antibody-bound and nonbound radiolabeled
Cetrorelix were separated by precipitation using goat antirabbit IgG,
rabbit IgG, and polyethylene glycol, and the pellet (antibody-bound
fraction) was counted in a -counter. The Cetrorelix concentration in
the samples was calculated; the lower limit of detection was 0.24
ng/ml.
Serum LH concentrations were measured by a supersensitive immunofluorometric assay for the rat (14) using NIDDK rLH RP-2 as the standard. FSH concentrations were measured by double antibody RIA using the NIDDK assay kits and rFSH RP-2 as standards (15). The lower limit of detection was 0.04 ng/ml for LH and 5 ng/ml for FSH.
Serum and intratesticular testosterone levels were measured using testosterone antiserum-coated tube RIA kits (catalogue no. DS 4000, Diagnostics Systems Laboratories, Inc., Webster, TX) with some modifications from the manufacturer’s instructions. The testosterone standards for serum testosterone measurements were prepared in rat serum stripped with dextran-coated charcoal (catalogue no. 6197, Sigma). Intratesticular testosterone standards were prepared in a solution containing 0.1% gelatin dissolved in PBS.
Statistical analysis
Results were presented as either the mean ±
SEM calculated from the untransformed data or as the mean
(+SEM, -SEM) calculated from log-transformed
data obtained from individual rats.
Data were tested for statistical significance using the SPSS, Inc. statistical computer program (SPSS, Inc., Chicago, IL). One-way ANOVA was used to test whether differences between groups were significant. If differences were significant (P < 0.05), further analysis were performed. The data for animals receiving irradiation alone and killed at 15 and 20 weeks were compared; their data were pooled if the values were not significantly different at P < 0.05. Dunnett’s pairwise multiple comparison test was then used for post-hoc evaluation of differences between different hormone-treated groups and the control irradiated-only groups. Tukey’s highest significant difference multiple comparison test was used for post-hoc evaluation of differences among GnRH antagonist-treated groups. The significance of the differences was P < 0.05, unless otherwise stated.
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Results |
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Cetrorelix and hormone analysis
Cetrorelix levels in serum at day 1 after hormone administration
were extremely high, but they continuously decreased through week 3
before the second injection (Fig. 1A
). At
week 3, Cetrorelix levels averaged 1.6 ng/ml. Five of 11 rats had
levels less than 1 ng/ml, and 4 of these had undetectable levels.
Hence, the sustained release of pamoate was sometimes, but not always,
sufficient to provide enough drug for 3–4 weeks. The second injection
(Cetrorelix pamoate alone) at 3.3 weeks (18.3 weeks after irradiation)
appeared to increase Cetrorelix levels, which then gradually
decreased.
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). GnRH antagonist administration significantly decreased serum FSH
levels to 74% of the levels in irradiated-only animals on day 3. A
slight progressive reduction to near the levels in unirradiated rats
appear to occur with longer treatment times.
Serum LH levels were increased about 2-fold by irradiation (Fig. 1C).
GnRH antagonist caused an immediate decrease in LH on day 1 of
treatment to about the levels observed in unirradiated rats. Levels
fell below the limit of detection at week 6.6 after hormone
administration.
An immediate reduction of serum testosterone to 20% of levels in
irradiated-only rats was observed on day 1 of Cetrorelix
administration (data not shown). Except for a slight increase at 3
weeks, levels progressively decreased to 5% at 5 weeks. In irradiated
rats, the intratesticular testosterone concentration (nanograms per g
testis) was approximately 3- to 4-fold higher in irradiated than in
unirradiated rats (Fig. 1D). At 1 day of GnRH antagonist
administration, levels of intratesticular testosterone were markedly
suppressed to 23% of the values in irradiated-only animals. In
general, there appeared to be a steady decline in intratesticular
testosterone levels between 1 day and 5 weeks. The transient
fluctuation in intratesticular testosterone levels observed at week 3
may have been due to the decline in serum levels of Cetrorelix in some
animals below the 1 ng/ml required for suppression of LH and
testosterone production. The second injection of Cetrorelix at 3.3
weeks produced an apparent further decline to less than 1% by week
5.
Testicular weight
The weight of this organ in the irradiated-only rats (0.53 g) was
35% of that in unirradiated rats (1.5 g) and did not change
significantly between 15 and 20 weeks after irradiation. Administration
of GnRH antagonist to the irradiated rats reduced testis weight from
0.51 g at 1 day of treatment to 0.28 g at 6.6 weeks.
Morphological analysis
In normal rats, Sertoli cell nuclei are ovoid, pyramidal, or
triangular (Fig. 2A). In irradiated rats,
Sertoli cell nuclei were more elongated and irregular, had more
invaginations, and occasionally appeared twisted or coiled (Fig. 2B).
These features were confirmed by electron microscopy (not shown). The
Sertoli cells had a similar irregular appearance in the irradiated rats
after GnRH antagonist administration (Fig. 2C). In the irradiated rats,
one to three Leydig cells per cross-section were undergoing mitosis
(not shown); dividing Leydig cells were not observed in adult
unirradiated rats. Administration of GnRH antagonist caused some
shrinkage in the nucleus and cytoplasm of the Leydig cells after
several weeks. No mitoses of Leydig cells were observed in the rats
that received GnRH antagonist.
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) with
fairly granular chromatin dispersed throughout the nucleus. There was
little heterochromatin along the nuclear membrane. Nucleoli (usually
one or two), which were not as round as those seen in Sertoli cells,
appeared just inside the nuclear membrane and/or near the center of the
nucleus. The spermatogonia from the irradiated rats were similar to
some of the undifferentiated A spermatogonia observed in stages II-III
of the cycle of the seminiferous epithelium of normal rats (Fig. 2A),
except that they were about 15% larger and were more uniformly round.
The morphology of the A spermatogonia in irradiated rats was not
changed by the hormone treatment (Fig. 2C). However, in ethanol-fixed
tissue the diameter of the A spermatogonia significantly increased from
7.8 ± 0.2 µm in the irradiated-only rats to 8.9 ± 0.1
µm in the hormone-treated rats (Fig. 3A).
This increase in spermatogonial diameter was significant already after
only 1 week of GnRH antagonist treatment. Similarly, spermatogonial
diameter in Bouin’s-fixed testicular cross-sections increased from
8.6 ± 0.1 µm in irradiated-only animals to 9.2 ± 0.2 µm
in the hormone-treated animals, but the differences at individual weeks
were significant only at weeks 2 and 5.
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) revealed no
alteration in cell morphology from control rats (not shown). The cell,
as in unirradiated rats, was in contact with the basement membrane and
contained a nucleus with dispersed chromatin and a nucleolus adjacent
to the nuclear membrane. The cytoplasm had a sparse content of cell
organelles, which is typical of these undifferentiated cells. In
addition, mitotic A spermatogonia (Fig. 4B) were observed in the
irradiated rats. The nuclear envelope was absent, and the chromatin was
compact and dense as it formed the chromosomes. The mitotic cells
remained attached to the basement membrane and showed no degenerative
changes.
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The numbers of A spermatogonia per Sertoli cell were determined in
Bouin’s-fixed tissue. The administration of GnRH antagonist
significantly increased A spermatogonial numbers (Fig. 3B). This
increase was readily evident at week 1 after the hormone
administration, and the level remained significantly above the values
for the irradiated-only animals throughout the duration of the
experiment.
Proliferation of A spermatogonia
To determine whether the increases in A spermatogonial numbers
were a result of increased cell proliferation, we measured their
labeling (Fig. 3C) and mitotic indexes (Fig. 3D). The BrdU labeling
index of A spermatogonia averaged 24% in irradiated-only animals. The
GnRH antagonist increased the labeling index to 35% within 1 week of
treatment and remained above the value for the irradiated-only animals
throughout the course of the GnRH antagonist administration. The
diameters of mitoses measured in Bouin’s-fixed material were 9.3
± 0.1 µm in the irradiated-only groups and 10.0 ± 0.3 µm in
the GnRH antagonist-treated groups. The mitotic index of A
spermatogonia in irradiated rats was 18%. Because this mitotic index
was unexpectedly high, we wondered whether the cells were arrested in
mitosis. However, no degenerating or TUNEL-positive mitotic cells were
apparent. Electron microscopy confirmed the normal morphology and the
absence of degenerative changes in the mitotic cells. In contrast to
the labeling index, the mitotic index decreased significantly at the
first week of GnRH antagonist treatment, to about 10%. Thereafter, the
mitotic index increased to values at or above those in irradiated-only
rats, reaching 23% at 6.6 weeks. This value was significantly
different from that obtained at week 1 of hormone administration.
Evaluation of cell loss
As changes in numbers of A spermatogonia during the administration
of the GnRH antagonist were affected not only by changes in cell
proliferation but also by cell loss, we investigated A spermatogonial
apoptosis. The nuclear diameters of TUNEL-positive A spermatogonia were
6.8 ± 0.1 µm in the irradiated-only rats and 7.4 ± 0.3
µm in the irradiated GnRH-treated rats. The Sertoli cell nucleolar
diameter in this formalin-fixed tissue was 1.5 µm in all groups.
Likewise, the diameters of the apoptotic A spermatogonial nuclei were
7.8 ± 0.3 µm for the irradiated rats and 7.6 ± 0.2 µm
in the irradiated GnRH-antagonist-treated rats. The levels of apoptosis
in the irradiated-only rats as measured by the TUNEL assay (Fig. 2D)
and nuclear apoptotic morphology (Fig. 2E) averaged 31 ±
4% and 8.7 ± 0.3%, respectively (Figs. 5A and 5B). The quantitative difference
(P < 0.01) between these two values may be a result of
the different duration of times that an apoptotic cell spends in a
stage where it is TUNEL positive (probably an earlier stage of
apoptosis) and in a stage where chromatin fragmentation has occurred.
It was noted that some cells that had apoptotic nuclear morphology did
not show positive staining in the TUNEL reaction and vice
versa. All apoptotic cells had round nuclei and basal positions
within the seminiferous epithelium, indicating that they were
spermatogonia and not Sertoli cells.
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As GnRH antagonist treatment was continued, the apoptotic index as measured by both methods gradually increased, but generally remained below the values obtained for irradiated-only animals. The increased values obtained at 6.6 weeks by the TUNEL assay (51 ± 15%) and nuclear morphology (8.0 ± 0.7%) were significantly different compared with values at weeks 1 and 2. The increase in the apoptotic index by nuclear morphology from week 1 (3.0 ± 0.6%) to week 3 (6.6 ± 0.8%) was also significant. This increase in apoptosis at week 3 could have been a consequence of the failure to maintain suppression in some animals throughout the entire course of the study, but that does not explain the continued elevation at later times.
Clonal size of A spermatogonia
Whole mounts of seminiferous tubules, in which the topographical
arrangement of cells is visible, were used to determine the clonal size
of A spermatogonia. Except for rare differentiating clones of
intermediate and B spermatogonia and/or spermatocytes, the only germ
cells present at 15–20 weeks after irradiation were A spermatogonia.
Interphase clones were not counted because of uncertainty in
determining whether all cells belonged to a single clone (Fig. 6A). A striking feature of the clones of
undifferentiated spermatogonia in the irradiated tubules was the
occurrence of clumps of A spermatogonia, i.e. more than one
nucleus in one cytoplasm (Fig. 6C). Sometimes the morphological picture
suggested the breaking up of larger clones into smaller groups.
Interestingly, clumps containing mitotic nuclei were rare, suggesting
that the clumps could not develop further and were doomed to die.
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). Only about 5%
of mitotic clones were larger than 4 cells, and less than 1% were
larger than 8 cells (Fig. 7A). There were no
mitotic clones of 16 cells. These values are much lower than the
percentages of interphase clones containing 4, 8, and 16 cells found in
the normal rat (16).
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). The
morphology of the apoptotic cells within a clone varied considerably.
The apoptotic clones were primarily composed of 1, 2, 4, and 8 cells,
but 2.5% contained 16 cells (Fig. 7B). The percentages of apoptotic
clones relative to mitotic clones increased as the number of cells in
the clone increased; the ratios of the percentages at sizes 1, 2, 4,
and 8 were 0.7, 0.6, 4.0, and 14.4, respectively. These results
indicate that larger clones are more likely to undergo an apoptotic
rather than a mitotic process.
The administration of GnRH antagonist altered the clonal sizes of the
spermatogonia. The average size of the mitotic clones increased
progressively from a value of 2.1 in the irradiated-only rats to 2.9
after 5 weeks of treatment. The percentage of the mitotic clones
consisting of single cells decreased from 41% in the irradiated-only
rats to 29% after 5 weeks of hormone treatment (Fig. 8A). This trend appeared to begin at week 2.
The percentages of mitotic clones consisting of 8 cells in all groups
of GnRH antagonist-treated rats were above the values of the
irradiated-only animals (Fig. 8B). Although values at individual time
points were not significantly above control values, the difference
became significant when values of irradiated-only and irradiated,
hormone-treated animals at different time points were pooled and
compared. In addition, there appeared to be a gradual increase in the
percentage of mitotic clones containing more than 8 cells beginning at
week 3 after GnRH antagonist treatment and reaching 1% at week 5 (Fig. 8C). Furthermore, the size of the apoptotic clones was increased by
GnRH antagonist treatment from 3.4 cells in the irradiated rats to
about 5.4 after 4–5 weeks of treatment. Nevertheless, it was clear
that apoptotic clones of all sizes, from single cells up to more than
16 cells, were still present.
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). The most
advanced cell populations observed in the repopulated tubules at 5
weeks of GnRH antagonist treatment were generally primary spermatocytes
(Fig. 2F). The initiation of differentiation of spermatogonia did not
occur synchronously in all tubules, as some tubules showed initiation
at 4 weeks, others not until 6.6 weeks, and 70% of the tubules did not
show any differentiation at all in this experiment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), isolated A spermatogonia
(As) divide to produce either new
As spermatogonia or a pair of A spermatogonia
(Apr) joined by an intercellular bridge. Most of
these Apr then undergo further divisions to
produce aligned spermatogonia (Aal) in chains of
4, 8, 16, and even occasionally 32. These chains enter a long
G1 phase of their cell cycle, and at about stage
VII of the cycle of the seminiferous epithelium morphologically
differentiate into A1 spermatogonia. No apoptosis
of the undifferentiated spermatogonia has been reported during normal
spermatogenesis.
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These cells were actively proliferating, as shown by their high
labeling index and mitotic index. Thus, the model of stem cell renewal,
in which the undifferentiated A spermatogonia proliferate (16), also
applies to the irradiated rat (Fig. 9B). The 18% mitotic index is
unusually high, even for cells in culture, of which 100% are
proliferating. For example, the mitotic index of HeLa cells in
exponential growth is 2% (19). The high mitotic index in the
irradiated-only animals could be due to either a long mitotic phase
(18% of the cycle) or to a block at mitosis, although the latter is
unlikely based on the failure to observe degenerating mitotic
cells.
Despite this proliferation, the number of A spermatogonia remains
constant. This was due to extensive apoptosis of the undifferentiated
spermatogonia, which appears to be the major mechanism by which these
cells are lost. Hence, in the model proposed in Fig. 9B, a probability
of apoptosis of the undifferentiated spermatogonia is included.
Although we do not know in which phase of the cell cycle this apoptosis
occurs, the assumption that it occurs at several phases of the cell
cycle, including G1 and G2,
is most consistent with the responses seen after GnRH antagonist
administration. The probabilities of apoptosis at the various stages
were estimated from the ratios of apoptotic index to mitotic index for
different clonal sizes and the ratios of numbers of mitotic clones of
different sizes. The increase in the probability of apoptosis with
increasing clonal size supports the suggestion that these cells are
more likely to die as the numbers of cells in the clone increase, and
most of the clones of undifferentiated spermatogonia are lost before
the mitotic divisions of the Aal (4). The
apoptosis observed in the undifferentiated Aal
spermatogonia of irradiated rats is in contrast to the situation in
normal rats, in which apoptosis occurs primarily in the
A2 and A3 spermatogonia
(20). Interestingly, apoptosis in A3
spermatogonia was described in rats at long times after exposure to
2,5-hexanedione (21). This toxicant’s effects in testicular tissue are
similar to those of irradiation. After exposure to either agent, no
cells beyond A spermatogonia are produced, and cell loss occurs
primarily by apoptosis.
In these models for both normal and irradiated rats, only clones of size 2n are predicted. In the present study, clones consisting of odd numbers of cells (i.e. not 2n) constituted 11% of the mitotic clones in the irradiated rats. It is not clear whether this is abnormal, as disparate results have been reported for the percentages of odd-numbered clones in normal mice, ranging from less than 1% (22) to 12% (23). On the other hand, Huckins (16) did not report the incidence of odd-numbered clones in normal rats, but stated that clones invariably followed a 2n distribution. The fact that we found odd-numbered clones may have been due to a failure to count all cells in the clone due to their position in the tubule or to some asynchrony of the cells. Alternatively, clonal fragmentation (24) could have occurred. If the odd-numbered apoptotic clones are indeed real, then this could indicate that only part of a clone could undergo apoptosis, leaving behind an odd-numbered clone to undergo the next mitosis.
Hormonal effects of GnRH antagonist treatment
The effects of Cetrorelix were quite rapid, resulting in declines
in FSH and LH within 1 day, followed by a steady decrease over the
6.6-week treatment period. The higher values of intratesticular
testosterone at 3 weeks were a result of the low serum Cetrorelix
concentration in some animals of this group.
However, despite the failure to maintain serum levels of the antagonist throughout the period, its effectiveness in stimulating the recovery of spermatogenesis surpassed that achieved by a GnRH agonist. In this study the repopulation index was 28% after 6.6 weeks of antagonist administration, exceeding the value of 14.5% obtained after 10 weeks of GnRH agonist administration, which was also given in a depot form beginning at 18 weeks after irradiation with 6 Gy (3).
Spermatogonial kinetics after GnRH antagonist treatment
The responses to GnRH antagonist treatment will be discussed in
terms of the model for spermatogonial kinetics in the irradiated rat.
At week 1 of hormone treatment the reduction of the apoptotic and
mitotic indexes as well as the increases in nuclear diameter and the
labeling index clearly indicate the immediate effect of hormonal
administration on these cells.
We believe that the decrease in the apoptotic index to about 40% of
the level in irradiated-only rats within 1 week of GnRH antagonist
administration is responsible for subsequent changes. The decline in
apoptosis affects all clonal sizes, so we will assume that the values
of P for As to
Aal (4) spermatogonia are reduced to 40% of
those in the irradiated-only rats shown in Fig. 9C. This decrease in
apoptosis results in an increase in the number of cells, as those cells
that would have died survive and accumulate. Three observations suggest
that apoptosis was reduced by GnRH antagonist preferentially in the
G1 phase of the cell cycle. These are the
increase in labeling index when apoptosis is reduced due to a greater
proportion of cells entering S phase, the increase in spermatogonial
nuclear diameter resulting from further progression through the cell
cycle before undergoing apoptosis, and the initial suppression of the
mitotic index due to the increase in the number of cells that do not
reach mitosis because apoptosis still occurs in
G2.
Another change, which was observed beginning at weeks 2 and 3 of GnRH administration, was a decrease in the percentages of mitotic clones that consisted of one cell and an increase in the percentages of clones with eight or more cells. This increase was expected, because the reduction of apoptosis allows the smaller clones to become larger by mitotic division. However, after several weeks of GnRH antagonist administration, the mitotic index and apoptotic index again rise. The rise in the apoptotic index could have been a result of the increase in clonal size, as larger clones are more susceptible to apoptosis in the irradiated testis. If G1 apoptosis is still active in Aal (8) and Aal (16), there will be a restoration of the mitotic index toward the level in the irradiated rat. However, this does not explain why the labeling index and nuclear size do not decrease.
Finally, between weeks 3–4 of GnRH antagonist treatment, the differentiation of spermatogonia was initiated, although in only a small percentage of tubules. It is not known whether the pathway of differentiation completely replaces apoptosis in those tubules. However, if we assume that efficient recruitment to A1 only occurs in the Aal (16) and is competing with G1 apoptosis, this explains why the repopulation index follows the incidence of clones of more than eight cells.
Implications regarding mechanisms of recovery
Although testosterone and, to a lesser degree, FSH are required
for survival of the later differentiated germ cells in normal adult
rats, spermatogonial numbers are unaffected by GnRH antagonist
treatment (9), and the undifferentiated A spermatogonia are only
marginally reduced in number by hypophysectomy (25). Thus, these cells
do not have a requirement for high levels of FSH and testosterone for
survival. However, what is unique about this study is that GnRH
antagonist treatment actually stimulates spermatogonial survival and
differentiation in the irradiated rat.
Among the various hormonal changes that ensue from GnRH antagonist treatment, we have evidence that it is only the reduction in levels of testosterone (intratesticular testosterone and serum) that is responsible for the recovery of spermatogenesis (Shetty, G., unpublished results). It is interesting to compare the effect of testosterone on the reinitiation of spermatogenesis in the irradiated rat with the initiation of spermatogenesis in normal immature rats, which occurs when testosterone levels are very low. Whereas exogenous testosterone inhibits initiation of spermatogenesis in the irradiated and GnRH analog-treated rat (Shetty, G., unpublished results), it failed to inhibit initiation of spermatogonial development and differentiation in immature rats (26, 27).
In this study we have demonstrated that the administration of Cetrorelix, a GnRH antagonist, to rats beginning 15 weeks after irradiation stimulates the proliferation and differentiation of A spermatogonia. As A spermatogonia do not possess androgen receptors (28), it is assumed that alterations in factors secreted by surrounding androgen-responsive cells as a consequence of the decrease in intratesticular testosterone are responsible for the changes observed in spermatogonia. The Sertoli cell is the most likely candidate because of its proximity to the germ cells and its known production of paracrine factors (29). Changes in the paracrine environment must occur within 1 week (concomitant with the fall in intratesticular testosterone concentration), as changes in spermatogonia were observed at this time. The 4-week time interval before the start of spermatogonial differentiation indicates that slow acting intermediate steps might be required to establish appropriate paracrine environment for this event.
These results demonstrate that even as late as 15 weeks after a dose of radiation (6 Gy) that would produce prolonged azoospermia in humans (30), recovery of spermatogenesis could be stimulated by suppression of testosterone. However, the delay in initiation and the incomplete nature of recovery indicate that treatment times with GnRH analogs longer than 6.6 weeks are necessary in rodents and in potential clinical trials of recovery of spermatogenesis in cancer patients.
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Acknowledgments |
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Footnotes |
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Received May 25, 1999.
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References |
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radiation.
Radiat Res 86:34–51[CrossRef][Medline]
-irradiated
mouse and rat. Mutat Res 108:317–335[Medline]
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