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From the * Institute of Veterinary Anatomy,
Histology and Embryology, and Institute of
Veterinary Physiology, Department of Biomathematics, and
Department of Urology and Pediatric Urology,
University of Giessen, Germany.
| Correspondence to: PD Dr Klaus Steger, Klinich für Urologie und Kinderurologie, Rudolf-Buchheim, Straße 7, 35385 Giessen, Germany (e-mail: Klaus.Steger{at}chiru-med.uni-giessen.de). |
| Received for publication February 9, 2004; accepted for publication March 15, 2004. |
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Abstract |
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Key words: Histone, mouse, protamine, spermatogenesis
Global hyperacetylation of core histones, in addition, is known to play an important role during spermiogenesis; namely, the histone-to-protamine exchange in haploid spermatids (Gatewood et al, 1990; Meistrich et al, 1992; Hazzouri et al, 2000). Protamine-DNA interactions are essential for the DNA supercoiling process in haploid spermatids, representing a prerequisite for male fertility (Steger, 1999). Recently, it has been suggested that the production of fertile spermatozoa requires both hyperacetylation of core histones in elongating spermatids and maintenance of hypoacetylated core histones in precursor cells (Hazzouri et al, 2000). This conclusion was reached following the finding that treating mouse seminiferous epithelium with trichostatin-A (TSA) induced a pronounced increase of histone H4 acetylation solely in round spermatids, whereas the acetylation state of the other germ cells did not change.
This study investigates, for the first time, the in vivo effects of the HDAC inhibitor TSA on murine spermatogenesis. Daily treatment with TSA for the duration of one seminiferous epithelial cycle resulted in male infertility. This effect was completely reversible. Furthermore, no toxic effects on other organs could be observed. In vivo application of TSA predominantly resulted, interestingly, in an impairment of meiosis due to increased apoptosis.
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Materials and Methods |
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Experiment 1
Twenty-five 9-week-old male Balb-c mice (Harlan-Winkelmann, Borchen,
Germany) were divided into 5 groups, each containing 5 animals. Animals in
groups 2–5 were treated with TSA (Biomol, Hamburg, Germany) applied by
daily subcutaneous injection with concentrations of 0.8 mg/kg (group 2), 1.6
mg/kg (group 3), 2.4 mg/kg (group 4), and 3.2 mg/kg (group 5). TSA was
resolved in dimethylsulfoxide (DMSO) in order to obtain a stock solution with
a concentration of 20 mg/mL. Further dilutions of the stock solution in
phosphate-buffered saline (PBS) were created for each group of mice as
follows: 1 µL stock solution + 199 µL PBS (group 2), 2 µL stock
solution + 198 µL PBS (group 3), 3 µL stock solution + 197 µL PBS
(group 4), and 4 µL stock solution + 196 µL PBS (group 5). Animals in
group 1 received 4 µL DMSO + 196 µL PBS and served as the control. The
treatment lasted 35 days, which corresponded to the duration of one
seminiferous epithelial cycle (Russell et
al, 1990).
Experiment 2
Male Balb-c mice received 2.4 mg/kg (group 6, n = 5) and 3.2 mg/kg (group
7, n = 5) TSA under the same conditions as in experiment 1. They were allowed
to mate before TSA treatment (day 0, group A females, n = 10) and after TSA
treatment (days 35/36, group B females, n = 10; days 37/38, group A females;
days 73/74, group A females). Mating on days 35/36 was allowed to omit
false-positive results due to remaining sperm within the epididymis. The
mating conditions were designed according to the physiological mating behavior
of Balb-c mice (Harlan-Winkelmann).
Tissue and Histology
The relative testis weight was defined as the ratio between the weight of
both testes to the body weight. From each mouse, the right testis was fixed in
liquid nitrogen, and the left testis was fixed by perfusion in Bouins fixative
and embedded in paraffin using standard techniques. Five-micrometer sections
were stained with hematoxylin-eosin and evaluated according to the methods
described by Russell et al
(1990)
(Figure 2).
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Areas of the seminiferous tubules were calculated according to the formula:
/4 x long diameter x short diameter. The diameters were
measured with a micrometer ocular system, and the median tubule area was
assessed in at least 30 cross sectioned tubules per animal.
The following cell types were counted in at least 10 cross sectioned seminiferous tubules: spermatogonia, leptotene-zygotene spermatocytes, pachytene-diplotene spermatocytes, metaphase spermatocytes, and spermatids. For each cell type, the mean number per tubular cross section was assessed.
Tissue slides from other organs, routinely stained with hematoxylin-eosin (prostate, seminal vesicle, kidney, spleen, liver, intestine, salivary gland, and pancreas), were histologically assessed in each animal.
Antibodies and Immunohistochemistry
The polyclonal antibodies anti-penta-acetylated histone H4 (Biomol,
Hamburg, Germany) and anti-proliferating cell nuclear antigen (PCNA)
(Santa-Cruz, Heidelberg, Germany) were used. Immunohistochemistry was carried
out as previously described (Sonnack et
al, 2002). The primary antibodies were diluted 1:3000 (H4) and
1:100 (PCNA). The percentage of H4-positive spermatids was evaluated in stages
IX–XI of the seminiferous epithelial cycle.
Evaluation of Apoptosis
The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) method was used according to the manufacturer's instructions (ApopTag
7100 Kit; Chemicon, Hofheim, Germany). For each sample, foci of positive
signals were counted in the external cell layer of the seminiferous tubules
(corresponding to spermatogonia and leptotene-zygotene spermatocytes) and in
the internal cell layer (corresponding to pachytene-diplotene spermatocytes
and spermatids). The number was related to 1000 spermatogonia and
leptotene-zygotene spermatocytes (APOext) and 1000 pachytene-diplotene
spermatocytes and spermatids (APOint), respectively. APOtot comprises the
total number of apoptosis foci related to 1000 cells (spermatogonia,
leptotene-zygotene spermatocytes, pachytene-diplotene spermatocytes, and
spermatids).
Digoxigenin-Labeled Antiprotamine-1 and Antiprotamine-2 Complementary RNA Probes and In Situ Hybridization
The digoxigenin-labeled complementary RNA probes corresponding to
protamine-1 (GenBank accession number Y00443) and protamine-2 (GenBank
accession number X07862) genes were synthesized as previously reported
(Steger et al, 1998a), and in
situ hybridization was performed as previously reported
(Steger et al, 1998a). The
percentage of protamine-1 and protamine-2 positive spermatids was evaluated in
stages IX–XI of the seminiferous epithelial cycle.
Quantitative Evaluation and Statistical Analysis
Statistical evaluation was performed using the statistical software package
BMDP/Dynamic release 7.0 (Dixon,
1993) and TESTIMATE (Rahlfs,
2002). To describe the data, two-dimensional frequency tables
(BMDP4F) were formed. Associations between TSA concentration and body weight,
mean tubule area (MTA), number of different cell types per tubular cross
section, ratio between pachytene-diplotene and leptotene-zygotene
spermatocytes, ratio between metaphase and pachytene-diplotene spermatocytes,
APOext, and APOtot were assessed with linear regression analysis (BMDP6D). A
nonlinear but monotone relation was observed between TSA concentration and
each of the following variables: relative testis weight (RTW), occurrence of
the stages of the seminiferous epithelial cycle, occurrence of seminiferous
tubules revealing impaired spermatogenesis, ratio between spermatids and
pachytene-diplotene spermatocytes, APOint, H4 immunoreactivity, and
protamine-1 and protamine-2 in situ hybridization signals. The statistical
significance of these relations was assessed by Spearman rank correlation
coefficient (BMDP3D). Association between RTW and MTA was evaluated by partial
correlation coefficient (BMDP6R) considering RTW and MTA as dependent
variables and TSA concentration as an independent variable whose influence was
eliminated before calculating the correlation coefficient. The offspring
number before and after TSA treatment was compared with two factorial analysis
of variance with repeated measures in the factor mating number (BMDP2V). To
obtain nearly normal distribution data, a square root transformation was
performed before analysis.
The offspring number was compared in group 6 with regard to group 7 by the Wilcoxon-Mann-Whitney test (BMDP3D).
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Results |
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Seminiferous tubules with impaired spermatogenesis were almost absent in the control group and did not exceed 2.5% in group 1, but reached 100% in group 5. This increase was correlated to TSA concentration (rs = .91, P < .001). The most prominent reduction was observed in spermatids (223.8 ± 32.0 per tubular cross section in group 1 and 12.6 ± 8.7 per tubular cross section in group 5), metaphase spermatocytes (18.8 ± 5.2 and 1.2 ± 0.9 in groups 1 and 5, respectively), and pachytene-diplotene spermatocytes (71.0 ± 1.8 and 6.2 ± 4.5 in groups 1 and 5, respectively). The regression lines and P values are y = 229.5–2.95 x TSA concentration, P < .001 for spermatids; and y = 18.2–0.22 x TSA concentration, P < .001 for metaphase spermatocytes; y = 73.3–0.81 x TSA concentration, P < .001 for pachytene-diplotene spermatocytes. The number of leptotene-zygotene spermatocytes and spermatogonia revealed a weaker relation to TSA concentration (y = 72.7–0.13 x TSA concentration, P = .03 and y = 41.2–0.09 x TSA concentration, P = .002, respectively). The ratio between spermatids and pachytene-diplotene spermatocytes negatively correlated with TSA concentration (rs = -.52, P = .009). The ratio between pachytene-diplotene and leptotene-zygotene spermatocytes significantly decreased with increasing TSA concentration (y = 1–0.01 x TSA concentration, P < .001). No sample displayed a complete absence of germ cells (Sertoli cell–only syndrome).
Immunohistochemistry with the polyclonal antibody against PCNA resulted in positive signals in spermatogonia and primary spermatocytes up to the pachytene stage (stage VII). No changes in the staining pattern of PCNA could be observed between untreated and treated mice (Figure 3g and h).
The TUNEL reaction revealed a decrease in APOext from 1.2 ± 0.8 (group 1) to 0.8 ± 0.3 (group 5); however, this did not correlate with TSA concentration (P = .23). In contrast, APOint increased from 0.3 ± 0.2 (group 1) to 17.0 ± 11.0 (group 5) and revealed a significant association with TSA concentration (rs = -.67, P < .001). As a consequence, APOtot increased significantly from 0.5 ± 0.4 (group 1) to 3.4 ± 1.5 (group 5) (y = 0.09 + 0.37 x TSA concentration, P < .001) (Figure 3i and j). These results are summarized in Table 1.
The polyclonal antibody against hyperacetylated histone H4 revealed a positive nuclear immunoreactivity in spermatogonia, leptotene spermatocytes, and elongating spermatids from stages IX to XII. Dividing spermatogonia, however, were immunonegative. Whereas the immunoreactivity of spermatogonia and leptotene spermatocytes remained constant, the percentage of immunopositive spermatids decreased from 97.5% ± 2.2% (group 1) to 64.8% ± 8.1% (group 5), exhibiting a significant relation to TSA concentrations (rs = -.78, P < .001) (Figure 3c and d; Table 1).
Transcripts for protamine-1 and protamine-2 were detected in spermatids from stages VIII–XII and I–II. The percentage of positive spermatids decreased from group 1 (P1, 96.6% ± 0.5%; P2, 97.5% ± 0.3%) to group 5 (P1, 73.2% ± 15.8%; P2, 85.6% ± 4.9%). For both protamines, there was a correlation with TSA concentration (P1, rs = -.82, P < .001; P2, rs = -.77, P < .001) (Figure 3e and f; Table 1).
The histology of other organs (prostate, seminal vesicles, kidney, spleen, liver, intestine, salivary glands, and pancreas) obtained from all mice revealed no significant modifications between treated and untreated animals. Although the mean body weight decreased from 26.6 g (group 1) to 24.7 g (group 5), this difference was not related to TSA concentrations (P = .13).
Experiment 2
On the basis of the data obtained in experiment 1, the fertility assay was
carried out with TSA concentrations of 2.4 and 3.2 mg/kg. Results are
summarized in Table 2. The
first mating (before TSA treatment) served as the control for the initial
fertility of each pair and was positive for all 10 pairs. The second mating
(after 35 days of TSA treatment) resulted in no offspring. The test to exclude
false-positive results due to remaining spermatozoa within the epididymis was
also negative. The third mating (after a 35-day recovery period without TSA
treatment) resulted in 7.5 ± 2.4 offspring in group 6 and 5.2 ±
3.7 offspring in group 7. Differences between the offspring number after the
first and the third matings in each group were not statistically significant
(P = .31). After the recovery period, histological evaluation of
testicular tissue revealed features similar to those in group 1.
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Discussion |
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In somatic immortalized cells, TSA and other HDAC inhibitors are able to induce cell cycle arrest followed by differentiation or apoptosis (Marks et al, 2000) as a result of a selective transcriptional stimulation of genes that negatively control the cell cycle (Johnstone, 2002). This effect is due to either acetylation of core histones and consequent generation of "open" chromatin areas, which favor transcription (Spencer and Davie, 1999; Jenuwein and Allis, 2001), or to a direct acetylation and activation of certain transcription factors such as p53, GATA, TFIIE, and TFIIF (Imhof et al, 1997; Vigushin and Coombes, 2002). Therefore, in recent years, HDAC inhibitors have emerged as promising anticancer drugs (Marks et al, 2000; Johnstone, 2002; Vigushin and Coombes, 2002). Spermatogonia are known to represent the mitotic active germ cells within the seminiferous epithelium (Steger et al, 1998b). It is interesting that in the present study, this cell type revealed neither a decrease in the proliferation activity, nor an increase in apoptosis in TSA-treated mice compared to control animals. Moreover, a complete regeneration of the seminiferous epithelium after TSA withdrawal was observed in the fertility assay, suggesting that the reserve cells of the testis (spermatogonia) remained intact (de Rooij, 2001). These results suggest that TSA administered in vivo under the conditions used in this work exert no significant effect on the mitotic cell cycle of the male germ cells.
Contrary to mitosis, information about TSA effects on meiosis are scarce. In addition, the role that core histone acetylation plays in the meiotic cell cycle is yet unclear. In situ studies on the expression of H4 demonstrated that highly acetylated isoforms of this core histone are absent in pachytene spermatocytes (Grimes and Henderson, 1984; Meistrich et al, 1992; Hazzouri et al, 2000). In the present study, we could not detect hyperacetylated H4 in murine spermatocytes before and after TSA treatment. However, as reflected by the pachytene-diplotene to leptotene-zygotene ratio, we found a strong impairment of meiosis under TSA treatment. Moreover, apoptosis of spermatocytes and spermatids significantly increased with increasing TSA doses. Like the mitotic cell cycle, the meiotic cell cycle harbors regulators and probably restriction points that monitor genomic integrity and successful completion of upstream events (Wolgemuth et al, 2002). Spermatocytes that are unable to complete meiosis are eliminated by apoptosis (Odorisio et al, 1998; Print and Loveland, 2000; Wolgemuth, 2002). Combining these data with our results, one can presume that TSA has induced defects in the meiotic cell cycle followed by apoptosis of the affected cells. Because the ratio of metaphase spermatocytes to pachytene-diplotene spermatocytes remained nearly constant, it is likely that TSA did not disturb the progression of metaphases I and II in meiosis; but rather, it acted during prophase. The mechanisms involved in this process remain unclear. Hyperacetylation of core histone H4 appears not to be implicated. However, the possibility exists that hyperacetylation occurred only at certain gene promoters and, therefore, was undetectable with the immunohistochemical method used.
The incomplete differentiation of spermatocytes induced by TSA treatment was obviously an important cause of reduction of the number of spermatids observed in TSA-treated testes. However, the ratio of spermatids to pachytene-diplotene spermatocytes significantly decreased with increasing TSA concentration, suggesting that an independent mechanism also might act beyond meiosis. The expected hyperacetylation of core histone H4 in round spermatids (Hazzouri et al, 2000) could not be detected, indicating that TSA did not act through changing the acetylation status of these cells. Unexpectedly, the percentage of elongating spermatids positive for hyperacetylated H4 decreased as a result of TSA treatment, suggesting a hypoacetylation. This was accompanied by a decrease of protamine-1 and protamine-2 messenger RNA (mRNA) expression in the same cells. The mechanism implicated in this phenomenon remains unknown. The correlation between hyperacetylated H4 immunoreactivity and mRNA in situ hybridization signals for protamines in the seminiferous epithelium is in line with our previous findings linking the reduction of protamine-1 and protamine-2 transcripts to round spermatid maturation arrest and to defects in histone H4 acetylation (Steger et al, 2001; Sonnack et al, 2002). It appears paradoxical that TSA, a hyperacetylating agent, triggers the occurrence of hypoacetylated elongating spermatids. However, round spermatid maturation arrest may represent only the result of a disturbed meiosis.
In conclusion, our results recommend TSA as a potent, reversible inhibitor of male fertility in mice with no obvious toxic effects on other organs. Although the one or more mechanisms of TSA action on spermatogenesis are still not known in detail, we have demonstrated that TSA predominantly affects meiosis. Other male reproductive organs displayed no morphological changes in TSA-treated mice, suggesting that TSA action on testis was not mediated by sex hormones.
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Acknowledgments |
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Footnotes |
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? I.F. and V.S. contributed equally to this work.
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