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BOR - Papers in Press, published online ahead of print January 26, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038620
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BIOLOGY OF REPRODUCTION 72, 1297–1304 (2005)
DOI: 10.1095/biolreprod.104.038620
© 2005 by the Society for the Study of Reproduction, Inc.

Effects of Acute and Chronic Cyclophosphamide Treatment on Meiotic Progression and the Induction of DNA Double-Strand Breaks in Rat Spermatocytes1

Adriana Aguilar-Mahecha 3, Barbara F. Hales 3, and Bernard Robaire 2 3,4

Departments of Pharmacology Therapeutics3 and of Obstetrics and Gynecology,4 McGill University, Montreal, Quebec, Canada H3G 1Y6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Male rats treated with cyclophosphamide, an alkylating agent commonly used clinically in both acute and chronic regimens, present with damaged male germ cells and abnormal progeny outcome. The extent and type of damage induced by cyclophosphamide largely depend on the germ cell type exposed to the drug and its ability to respond to insult. In the present study, the response of pachytene spermatocytes to damage was evaluated by assessing their ability to undergo meiotic G2/MI transition following exposure to acute or chronic cyclophosphamide. Male rats were given an acute high dose (70 mg/kg, once) or chronic low doses (6 mg/kg, daily for 5–6 wk) of cyclophosphamide. Pachytene spermatocytes were isolated, cultured, and induced to undergo G2/MI transition with okadaic acid. To determine the effect of DNA damage on meiotic progression, induction of DNA double-strand breaks was detected after each treatment regimen by the formation of foci of phosphorylated histone H2AX. The transition from G2 to MI was impaired after acute cyclophosphamide treatment; this impairment in the progression of pachytene spermatocytes was correlated with extensive DNA double-strand breaks. In contrast, despite the presence of significant levels of DNA damage, meiotic progression was not impaired in spermatocytes after chronic cyclophosphamide exposure. We suggest that the cell cycle impairment induced after acute cyclophosphamide treatment could be mediated by a G2/M checkpoint activated in response to DNA damage. The absence of impairment after chronic treatment raises concern about the functionality of defense mechanisms in male germ cells after repeated exposure to low doses of genotoxic agents.

meiosis, spermatogenesis, stress, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclophosphamide is an alkylating agent commonly used in acute regimens for the treatment of various neoplastic diseases and in chronic regimens for the treatment of autoimmune disorders [1]. Cyclophosphamide is cytotoxic to rapidly dividing cells, which makes the highly proliferative testis a good target for the damaging effects of this drug. Use of cyclophosphamide for the treatment of cancer in male patients increases the incidence of oligospermia and azoospermia, and results in male infertility [2, 3]. Cyclophosphamide binds covalently to DNA and induces DNA damage in the form of strand breaks, DNA-DNA cross-links, both interstrand and intrastrand, as well as DNA-protein cross-links [1].

The genotoxic effects of cyclophosphamide on male germ cells depend on the stage of germ cell development during which the cell is exposed to the drug. Previous studies from our laboratory demonstrate that it is postmeiotic germ cells that are most susceptible to the effects of cyclophosphamide [4]. However, mitotic and meiotic cells are also vulnerable to the damaging effects of this drug. Treatment of male rats with low doses of cyclophosphamide for 9 wk, a treatment that first exposes spermatogonia to the drug, resulted in an increased incidence of malformations and growth retardation in fetuses [5] as well as chromosomal aneuploidy [6]. Increased preimplantation loss was observed after a 4–6 wk treatment; this effect was mediated through the action of cyclophosphamide on germ cells first exposed as early spermatids and spermatocytes [4]. A single injection of high-dose cyclophosphamide given during the development of mouse spermatocytes resulted in heritable translocations [7] and increased the incidence of micronuclei [8–10]. In addition to the induction of gene conversions and frameshift mutations [11], exposure of spermatocytes to cyclophosphamide induced synaptonemal complex breakage and disrupted chromosome synapsis [12–14].

The response of male germ cells to genotoxic damage relies in part on the presence and functionality of stress response mechanisms during germ cell development. Genes involved in the stress response, including DNA repair and apoptosis, are predominantly expressed at the pachytene stage [15]. Exposure to cyclophosphamide alters the expression of stress-response genes in male germ cells. The major effect of acute cyclophosphamide treatment was to increase transcript levels of stress-response genes in round spermatids [16]. In contrast, chronic treatment with cyclophosphamide resulted in a marked reduction in gene expression, predominantly in pachytene spermatocytes and round spermatids [17]. The decrease in expression of stress-response genes raises concern about the ability of male germ cells to respond to insult. Inability to repair damage or eliminate seriously damaged germ cells may provide an explanation for the persistence of genetic damage in spermatozoa [18, 19] and the deleterious consequences of paternal cyclophosphamide exposure on embryo development [20].

In the testis, spermatogonia divide mitotically to form preleptotene spermatocytes, the cell type that enters meiotic prophase. Most of the time in prophase is spent in the pachytene stage, during which chromosome synapsis is completed and genetic exchange or recombination takes place. From the pachytene stage, the cell enters the late substages of prophase, diplotene, and diakinesis, before reaching metaphase I [21]. Proper chromosome alignment and full-length synaptonemal complex formation, completion of recombination, and the accumulation of key cell division factors at the end of prophase are required to regulate the transition from G2 (prophase) to metaphase I (G2/ MI transition) [22, 23].

To maintain genomic stability, eukaryotic cells respond to genetic damage by arresting or delaying cell cycle progression. Such a delay, also known as a checkpoint, allows for the activation of DNA repair mechanisms, or when the damage is too overwhelming, of cell death pathways [24]. In mitotic cells, arrest at the G2 to M transition prevents cells from entering the division phase in the presence of genetic damage [25]. In male germ cells, arrest at different points of meiotic prophase has been reported for a number of null mutation mice [26–33]. Arrest and apoptosis of spermatocytes before the first division in such mutant mice have been associated with possible checkpoints monitoring chromosome structure and synapsis [26–32] as well as unrepaired DNA damage [33]. Transient meiotic delay has also been reported after in vivo exposure to colchicine, etoposide, irradiation, or 2,5-hexanedione [34–36]. Furthermore, the meiotic checkpoint/delay in spermatocytes exposed to irradiation-induced low levels of DNA damage is p53-dependent [37].

As assessed by light microscopy evaluation of the testis, spermatogenesis appears normal after exposure of male rats to chronic cyclophosphamide, yet progeny outcome is dramatically affected. The goal of this study was to elucidate the impact of acute or chronic cyclophosphamide exposure on meiosis, a critical event in spermatogenesis. To analyze the meiotic progression of pachytene spermatocytes to metaphase I, spermatocytes were cultured in vitro with okadaic acid to induce a premature G2/MI transition [38]. Okadaic acid releases pachytene spermatocytes from their inhibitory block, inducing chromosome condensation and progression into metaphase I characteristics of progression analogous to those in vivo [39]. To determine how the levels of DNA damage induced by acute and chronic cyclophosphamide exposure affect meiotic progression in spermatocytes, we analyzed the induction of DNA double-strand breaks by immunocytochemical detection of the phosphorylated form of histone H2AX ({gamma}H2AX) [40, 41]. Phosphorylation of H2AX is induced in response to double-strand breaks and plays a crucial role in the recruitment of DNA repair and signaling factors at the sites of damage [42]. The results from this study show that spermatocytes respond differentially to the damaging effects of acute and chronic cyclophosphamide exposure and provide further evidence in support of the existence of a G2/M DNA damage checkpoint in meiotic male germ cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Male Sprague-Dawley rats were obtained from Charles River (St-Constant, QC, Canada). Rats were housed in the McIntyre Animal Center, McGill University, maintained on a 14L:10D light cycle, and provided with water and food ad libitum. We randomly assigned rats to either acute or chronic treatment regimens. For the acute regimen, rats (425–450 g) were given an i.p. injection of either saline or a high dose (70 mg/kg) of cyclophosphamide (C0768; Sigma) and killed 16 h later. For the chronic regimen, rats (325–350 g) were gavaged with either saline or low doses of cyclophosphamide (6 mg/kg) daily, six times per week, for 5–6 wk; at the end of the chronic treatment regime rats weighed within the range of 425–450 g. All animal handling and care were performed in accordance with the guidelines established by the Canadian Council on Animal Care.

Isolation of Pachytene Spermatocytes and Induction of G2/M Transition

Pachytene spermatocytes were obtained through cell separation by velocity sedimentation using the STA-PUT method as previously described by Bellvé et al. [43] and modified by Aguilar-Mahecha et al. [15]. In summary, both rat testes were decapsulated and digested by enzymatic treatment first with collagenase and then with trypsin and DNase I. After dissociation, tubules were filtered through a nylon mesh in the presence of DNase and washed with RPMI (RPMI medium 160; Gibco, Invitrogen, Burlington, ON, Canada) containing 0.5% BSA. Cells were centrifuged and filtered; a total of 5.6 x 108 cells in 25 ml of 0.5% BSA in RPMI were loaded in the velocity sedimentation apparatus (STA-PUT; Proscience, Don Mills, ON, Canada) followed by a 2%–4% BSA (Roche Diagnostics) gradient in RPMI for separation by sedimentation at unit gravity. Fractions of pachytene spermatocytes were identified by light microscopy and those fractions with greater than 80% purity were pooled.

We cultured pachytene spermatocytes using the method developed by Handel et al. [38]. Briefly, cells were washed and resuspended in a culture media composed of MEM{alpha} medium (M-0644; Sigma) supplemented with 25 mM Hepes (H-3375; Sigma), 5% fetal bovine serum (Gibco), 25 mM NaHCO3 (BP328-500; Fisher Scientific), 10 mM sodium lactate (L-1375; Sigma), 200 µg/ml streptomycin (S-9137; Sigma), 118 µg/ml penicillin G (PEN-K; Sigma), and 200 µl of 250 µg/ml fungizone (15290-018; Gibco). Pachytene spermatocytes were cultured at a density of 2.5 x 106 cells/ml, each milliliter of cell suspension was put into one of the four wells of Nunclon culture dishes (VWR, Montreal, QC, Canada) and cultured at 32°C under 5% CO2 in air. Two milliliters of cell suspension were cultured for each saline and cyclophosphamide experiment performed. Four replicate experiments per treatment group were carried out for the acute regimen, five saline and six cyclophosphamide replicates for the chronic regimen. To induce the G2/M transition, pachytene spermatocytes were cultured with the phosphatase inhibitor okadaic acid, as previously described [39]. Okadaic acid (459616; Calbiochem, Mississauga, ON, Canada) was dissolved in 100% ethanol to prepare a 244 µM stock solution. After overnight culture, okadaic acid was added to one of the wells at a concentration of 5 µM, the cells in the other well were left untreated. Cells were cultured for an additional 6 h and then collected for fixation and immunostaining.

Air-Dried Meiotic Spreads

At the end of the culture period, 400 µl of cells were collected from each culture well and processed for fixation using a modification of the protocol described by Evans et al. [44]. Briefly, cells were centrifuged at 1000 x g for 5 min and the supernatant was decanted. The pellet was resuspended and washed twice with 1 ml of sodium citrate (2.2%), added drop by drop to the suspension, followed by centrifugation for 5 min at 1000 x g. Subsequently, cells were left in 1 ml of hypotonic solution (1% sodium citrate) for 12 min. Cells were then fixed twice in 1 ml of 3:1 absolute ethanol and glacial acetic acid. Fixed cells were dropped on Superfrost microscope slides (Fisher Scientific) prewarmed at 45°C for 30 sec. Slides were air-dried and stained with Giemsa stain in phosphate buffer before analysis under light microscopy. Using cytogenetic criteria, 500 cells per experiment, for each treatment group, were scored to identify the different stages of meiotic progression from the pachytene stage to metaphase I. The same scoring criteria were repeated for both acute and chronic treatment regimens.

Immunocytochemistry

The immunofluorescence procedures used were adapted from methods previously described [45, 46]. Briefly, Superfrost microscope slides coated with 2% BSA were dipped in freshly made and filtered 2% paraformaldehyde (pH 8.5) containing 1% Triton X-100 (Sigma). A drop of cultured cells was placed on the left corner of the slide and distributed over the entire surface of the slide. Slides were briefly dried at room temperature and washed three times in 0.4% Kodak Photo-Flo pH 8.5. After air-drying, slides were stored at –80°C until further use or processed for immunostaining.

For immunostaining, slides were washed in 1x PBS twice for 5 min each, followed by three 10-min washes with 10% ADB (10% goat serum [Vector Laboratories], 3% BSA [Fraction V; Sigma], and 0.05% Triton-X-100 in PBS). Slides were incubated overnight at 4°C with monoclonal anti-mouse primary antibody diluted in ADB. Primary antibody recognizing the phosphorylated form of H2AX at serine 139 ({gamma}H2AX) was used at a concentration of 1:1000 (clone JBW301; Upstate Biotech, Waltham, MA). After incubation, slides were washed twice for 5 min in 1x PBS and incubated in the dark with mouse immunoglobulin G secondary antibody (N1031; Amersham, Baie D'Urfe, QC, Canada) for 1 h at 37°C. Following incubation, slides were washed twice in PBS (5 min), once in double-distilled H2O (1 min), and mounted with Prolong Antifade (P-7481; Molecular Probes, Invitrogen, Burlington, ON, Canada) containing 2.5 µl/ml of DAPI to stain DNA. Slides were covered with coverslips. A total of four experiments for each saline and cyclophosphamide treatment group in the acute regimen and five in the chronic regimen were probed and analyzed.

Analysis of {gamma}H2AX Foci

The fluorescent signal was detected by using a fluorescence microscope (Leica AS LMD; Leica, Wetzlar, Germany) with single fluorochrome filters for DAPI (blue) and fluorescein isothiocyanate (FITC; green) (FITC, 41001 HQ:F; Chroma Technology Corp., Brattleboro, VT). Pictures of DAPI and {gamma}H2AX staining were captured using an RS Photometrics CoolSNAP fx camera (Roper Scientific, Tucson, AZ). A total of 50 pictures were taken for each experiment with a 100x objective and imported into Adobe PhotoShop 7.0. For quantitative analysis, the number of {gamma}H2AX foci present in each cell was counted by eye. Cells were classified according to the number of foci they presented: 0–4, 5–10, or >10 foci. At least 50 cells were scored for each experiment of cells cultured with or without okadaic acid; this was repeated for each treatment group (saline and cyclophosphamide) in each drug treatment regimen (acute and chronic).

Statistical Analysis

For metaphase spreads, the effect of cyclophosphamide on meiotic progression was assessed by chi-square analysis (P < 0.05). The proportion of cells at each prophase stage (pachytene, diplotene, diakinesis, and metaphase) from saline-treated rats was compared with the proportion of cells in each stage after cyclophosphamide treatment. A chi-square analysis (P < 0.05) was also performed to analyze the frequency of cells with different levels of double-strand breaks in saline and cyclophosphamide treatment groups. Statistical analyses were performed using the SigmaStat 3.0 software package (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The competence of spermatocytes to condense chromosomes and progress to metaphase I following acute and chronic cyclophosphamide treatment was analyzed using the in vitro G2/M transition system. We examined air-dried preparations of spermatocytes cultured with okadaic acid and identified the different prophase substages through analysis of their chromatin conformation and chromosome condensation [39]. Figure 1 illustrates the different substages of meiotic prophase observed: pachytene, diplotene, diakinesis, and metaphase I.



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  		FIG. 1. Different substages of late prophase observed in air-dried meiotic preparations of spermatocytes cultured with okadaic acid. Magnification x400

Acute cyclophosphamide treatment resulted in a significant reduction in the proportion of cells reaching metaphase I. While no apparent change was observed in the proportion of cells in the pachytene stage, the decrease in the number of cells with fully condensed bivalent chromosomes (metaphase I cells) resulted in a corresponding increase in the number of cells with intermediate levels of condensation: 23% in diakinesis and 19% in diplotene (Fig. 2, A and B). Thus, acute cyclophosphamide treatment impaired the meiotic transition of spermatocytes to metaphase I.



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  		FIG. 2. Percentage of cells at the different substages of late meiotic prophase. Meiotic figures were scored from air-dried meiotic spreads of spermatocytes cultured with okadaic acid; after acute (A, B) or chronic (C, D) exposure to saline (A, C) or cyclophosphamide (B, D). *Chi-square, P < 0.05

In contrast to the significant reduction in meiotic figures observed after acute cyclophosphamide exposure, chronic treatment with cyclophosphamide resulted in a slight increase in the number of cells reaching metaphase I (74% of the cells) (Fig. 2, C and D). An increase in the proportion of cells in the pachytene stage, but not in diplotene or diakinesis, was observed after chronic treatment with cyclophosphamide. Therefore, chromosome condensation associated with the progression of pachytene spermatocytes to the division phase was not impaired by chronic cyclophosphamide treatment.

Because acute exposure to a high dose of cyclophosphamide impaired meiotic progression, we investigated whether this difference in response between acute and chronic cyclophosphamide exposures was related to the extent of DNA damage induced in spermatocytes by each treatment regimen. To assess DNA damage, the incidence of DNA double-strand breaks after treatment with either saline or cyclophosphamide for both acute and chronic regimens was determined. Pachytene spermatocytes, cultured without okadaic acid, were analyzed to reflect the damage induced by the in vivo treatment; spermatocytes undergoing meiotic progression, cultured with okadaic acid, were assessed to establish whether the levels of damage changed after induction into metaphase.

At the sites of double-strand breaks, thousands of H2AX molecules become phosphorylated and form nuclear foci [40]. The direct correspondence between the number of {gamma}H2AX foci and the number of double-strand breaks allows a reliable quantification of this type of cellular damage in the cell [47]. Cells were classified according to the number of {gamma}H2AX foci (DNA double-strand breaks) detected in each cell: low (0–4 foci/cell), intermediate (5–10 foci/cell), or high (>10 foci/cell) levels, as shown in Figure 3.



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  		FIG. 3. DAPI and {gamma}H2AX staining of spermatocytes cultured without (A–C) and with (D–F) okadaic acid. Cells from rats exposed to acute saline (A, D) or cyclophosphamide (B–F) doses. Cells were classified according to the number of {gamma}H2AX foci: 0–4 (A, D), 5–10 (B, E), and >10 (C, F). {gamma}H2AX staining over the sex body is indicated by arrows and arrowheads. Magnification x1000

Following acute cyclophosphamide treatment, the percentage of cells with high levels of DNA damage (>10 foci/cell) increased by more than 3-fold (Fig. 3). At the same time, there was a dramatic, 80% decrease in the proportion of cells with low levels of strand breaks (0–4 foci/ cell) compared with saline-treated cells. No difference in the number of pachytene spermatocytes with intermediate levels of {gamma}H2AX foci (5–10) was observed between saline and cyclophosphamide treatment groups (Fig. 4A).



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  		FIG. 4. Frequency of cells presenting different levels of DNA damage as indicated by the numbers of {gamma}-H2AX foci. Data for cells cultured without (A, B) or with (C, D) okadaic acid after either acute (A, C) or chronic (B, D) treatment with saline or cyclophosphamide. Asterisks represent significant differences between treatment groups (chi-square analysis, P < 0.05)

After chronic exposure to cyclophosphamide the number of pachytene spermatocytes with high levels of DNA damage (>10 foci/cell) increased by 2.8-fold. A corresponding marked reduction in the number of pachytene spermatocytes (cultured without okadaic acid) presenting low numbers of {gamma}H2AX foci (0–4 foci/cell) was observed in the cyclophosphamide treated group. When we compared cells from saline- and cyclophosphamide-treated rats, we observed that drug treatment induced a 2-fold increase in the proportion of cells with 5 to 10 {gamma}H2AX foci per cell (Fig. 4B).

Of interest, cells treated with acute cyclophosphamide exhibited fewer overall DNA double-strand breaks following okadaic acid culture than preceding okadaic acid culture. Acute exposure to cyclophosphamide resulted in a less than 15% reduction in the percentage of cells presenting 0–4 foci/cell. This decrease in cells with low levels of DNA double-strand breaks was reflected in an analogous increase (1.5-fold increase) in the proportion of cells with intermediate levels of damage (5–10 foci/cell). Exposure to acute cyclophosphamide did not increase the proportion of cells presenting more than 10 immunofluorescent {gamma}H2AX foci per cell (Fig. 4C).

After chronic cyclophosphamide exposure the distribution of cells with different levels of DNA double-strand breaks was similar in spermatocytes cultured without or with okadaic acid; however, the magnitude of the changes between saline and drug groups was less prominent. The percentage of cells with low levels of DNA double-strand breaks decreased by 26%, while the percentage of cells with more than 10 foci per cell increased by 1.4-fold in the cyclophosphamide treatment group. Close to a 2-fold increase in the proportion of cells with intermediate levels of damage (5–10 foci/cell) was observed in cells exposed chronically to cyclophosphamide (Fig. 4D).

In pachytene spermatocytes {gamma}H2AX presents an interesting profile of expression, localizing over the condensed XY chromosomes [48, 49]. In our study we found that the majority of pachytene spermatocytes (cells cultured without okadaic acid) presented H2AX phosphorylation on the sex chromosomes independent of the presence of any other {gamma}H2AX foci (arrows in Fig. 3). After either acute or chronic saline treatment, 90% of pachytene spermatocytes had clearly defined sex bodies stained with {gamma}H2AX. This percentage was, interestingly, significantly reduced (chi-square, P < 0.05) after acute (80%) and chronic (83%) cyclophosphamide treatment. In cells induced to undergo meiotic transition with okadaic acid, the sex body remained condensed for a significant number of metaphase figures [39]. As expected, {gamma}H2AX also localized on the sex body of metaphase cells and cells at intermediate stages of prophase (arrowheads in Fig. 3). On average, 70% of the cells cultured with okadaic acid presented sex bodies with positive {gamma}H2AX staining; this number was not affected by either acute or chronic cyclophosphamide treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The progression of spermatocytes through meiotic prophase was analyzed using the in vitro G2/M transition system to determine the effects of in vivo cyclophosphamide exposure. This is the first study to report the damaging effects of in vivo treatment with a genotoxic agent on spermatocytes using this in vitro assay.

Competence to condense and individualize chromosomes is tested by treating pachytene spermatocytes with okadaic acid. The chromosome condensation induced upon treatment with okadaic acid is associated with progression to meiotic metaphase I; this progression has been shown to be similar in many aspects to the meiotic progression observed in vivo [39, 45]. In the present study we found that acute exposure to a high dose of cyclophosphamide impaired meiotic progression of spermatocytes from G2 to metaphase I. The numbers of cells at intermediate substages of late prophase, diplotene, and diakinesis increased after acute drug treatment, thus indicating that spermatocytes were impaired in their progression to the division phase. Clearly, spermatocytes maintain their ability to condense and individualize chromosomes as seen by the presence of metaphase figures in cells cultured with okadaic acid. However, okadaic acid induces the entry of pachytene spermatocytes into the diplotene stage during the first hour of treatment, and by 6 h the majority of cells reach metaphase [39, 45]; this suggests that completion of full chromosome condensation is slowed down or arrested in a number of cells after acute cyclophosphamide exposure.

Delay in cell cycle progression is often triggered in somatic cells after exposure to alkylating agents and radiation [50, 51]. In male germ cells, however, the kinetics of spermatogenesis are so tightly regulated that a delay in the progression of male germ cells throughout the spermatogenic cycle is rather unusual. To date, few in vivo exposures have been reported to result in meiotic delay in spermatocytes [34–36]. In the present study we report that a clinically-relevant dose of cyclophosphamide given in vivo can affect the transition of spermatocytes to metaphase.

The cytotoxic effects of cyclophosphamide in male germ cells are mediated through the induction of DNA adducts, DNA strand breaks, and cross-links [52]. Repair of such damage, particularly of cross-links, results in the indirect induction of DNA double-strand breaks by cyclophosphamide [53, 54]. A remarkable increase in the number of pachytene spermatocytes (cultured without okadaic acid) with high levels of DNA double-strand breaks ({gamma}H2AX foci) was observed after acute cyclophosphamide exposure, further demonstrating the genotoxic effects of this alkylating agent on male germ cells. Cyclophosphamide may induce DNA double-strand breaks directly or indirectly as a consequence of an attempt to repair damage.

Meiotic disruption during pachytene in several DNA repair gene null mutation mice [31–33] and apoptosis of pachytene spermatocytes after exposure to genotoxic agents [55, 56] suggest that the pachytene stage may act as a checkpoint to monitor DNA damage and meiotic events during germ cell development. In addition to the DNA double-strand breaks detected in the present study, synaptonemal complex damage, chromosome breaks, and asynapsis were observed after exposure of spermatocytes to a single, high dose of cyclophosphamide [12, 14, 57]. Despite the induction of such damage, okadaic acid triggered the exit of >99% of spermatocytes from the pachytene substage to later prophase substages. Our results suggest that okadaic acid inhibits the checkpoint that maintains cells at the pachytene stage regardless of the presence of abnormal meiotic events and DNA damage. These results contrast with the severe inhibition in G2/M chromosome condensation observed after treatment of spermatocytes with teniposide. In vitro culture of spermatocytes with this topoisomerase II inhibitor resulted in the majority of cells remaining in a pachytene-like stage upon okadaic acid treatment; this effect seemed to be associated with the induction of DNA double-strand breaks by teniposide and the potential presence of a DNA damage checkpoint [58].

In the present study, the progression of cells past the pachytene substage was impaired after acute cyclophosphamide treatment. Genetic damage and changes in chromatin conformation associated with the effects of cyclophosphamide could perhaps slow down or arrest chromosome condensation and result in the accumulation of cells at the diplotene and diakinesis substages. It is also possible that the cyclophosphamide-induced impairment in the G2 to metaphase transition is checkpoint-mediated. Handel et al. [23] have suggested that delayed or inhibited response to okadaic acid in the presence of DNA damage constitutes evidence for a spermatocyte DNA damage checkpoint. We propose that the transient arrest at the diplotene and diakinesis substages of prophase induced by acute cyclophosphamide exposure are due to a late prophase G2/M checkpoint activated in response to the damaging effects of this alkylating agent. Arrest during the cell cycle before metaphase may provide the cell with an opportunity to repair DNA damage or to induce cell death in severely damaged cells. In support of this suggestion, increased apoptosis and evidence of DNA repair have been reported in spermatocytes following acute exposure to cyclophosphamide [59, 60].

Fewer {gamma}H2AX foci were detected in spermatocytes cultured with okadaic acid. This decrease in the detection of double-strand breaks appears to be related more to the effect of the phosphatase inhibitor than to the effect of acute or chronic cyclophosphamide exposure. Chromosome condensation induced by okadaic acid may modify the process by which {gamma}H2AX is detected. We consider it unlikely that chromosome condensation affects the exposure of H2AX antigens and impedes the access of antibodies because we still see metaphase cells with high numbers of foci. Alternatively, the attenuation in the levels of DNA double-strand breaks seen in those cells induced to undergo premature G2/M transition may be due to the activation of DNA repair mechanisms during the progression of the cell through late prophase. Dephosphorylation of H2AX is associated with the repair of double-strand breaks [61]. Those cells delayed in their progression to metaphase after acute drug treatment may have undergone or be in the process of undergoing repair, further supporting the G2/M checkpoint function. It would be interesting to determine whether H2AX colocalizes with other repair factors at the sites of damage in cells arrested at diplotene and diakinesis. However, because H2AX phosphorylation also occurs during apoptosis [62], we cannot rule out the possibility that some of the {gamma}H2AX-positive cells in our study may be apoptotic and thus be eliminated after okadaic acid treatment.

Spermatocytes from rats chronically exposed to low doses of cyclophosphamide were not impaired in their progression to metaphase I after induction with okadaic acid. This observation correlates with the absence of spermatogenic arrest or alteration in the kinetics of rat spermatogenesis observed after histological analysis of rat testes exposed to cyclophosphamide for 9 wk [5, 63]. However, a transient delay of a few hours in meiotic progression would be difficult to detect through histological analysis and therefore cannot be ruled out. The results from the present study confirm that, under in vitro induction, chronic exposure to cyclophosphamide does not affect the competence of spermatocytes to condense chromosomes and reach metaphase.

Chronic treatment for 5–6 wk with cyclophosphamide before the pachytene stage corresponds to germ cells that are exposed to this drug from their mitotic division phase (spermatogonia) onward. The amount of DNA damage induced by chronic, low-dose cyclophosphamide treatment up to the pachytene stage had not been assessed previously. Our data show that repeated exposure to low doses of cyclophosphamide clearly induces significant DNA damage in spermatocytes. It is striking that despite the damage detected in chronically treated spermatocytes, chromosome condensation is not altered. The contrasting results between acute and chronic cyclophosphamide treatments raise further questions about the nature of the damage induced by each treatment regimen and the effects on factors regulating chromosomal changes and meiotic progression.

If the impairment in meiotic progression observed after acute cyclophosphamide treatment is in fact checkpoint-mediated, a lack of meiotic delay in the presence of substantial DNA strand breaks after chronic drug treatment may imply that spermatocyte quality is affected. Of interest, we reported previously that chronic cyclophosphamide exposure for 5–6 wk decreased the expression of stress-response genes in pachytene spermatocytes [17]. A reduction in transcript levels for genes involved in regulation of the checkpoint response (DNA repair, apoptosis, cell cycle) could prevent the activation of the G2/M checkpoint and of DNA repair and apoptosis mechanisms. Inability to repair DNA damage or undergo apoptosis after chronic exposure to cyclophosphamide may explain our observation that when compared to the acute regimen, chronic exposure to cyclophosphamide results in a greater proportion of cells with high numbers of {gamma}H2AX foci upon culture with okadaic acid. Faulty activation of surveillance mechanisms may result in accumulation of unrepaired genetic damage and consequent genomic instability, affecting both germ cell quality and embryo development [64]. Genomic instability is induced in male germ cells by cyclophosphamide as demonstrated by the increased incidence in numerical chromosomal abnormalities found in spermatozoa after chronic cyclophosphamide treatment; the aneuploidy of autosomal chromosomes reported is, interestingly, suggestive of defects in meiosis I [6].

Independent of its role in double-strand break signaling and repair, H2AX plays an important role in sex chromosome condensation, synapsis, and transcriptional inactivation [49]. We found it interesting that the majority of pachytene cells that were found to lack sex bodies after either acute or chronic cyclophosphamide treatment were those cells presenting >10 {gamma}H2AX foci. The decrease in the number of pachytene spermatocytes with sex bodies presenting {gamma}H2AX localization after drug treatment is likely to be due to the inability of {gamma}H2AX to localize on the sex body when a high number of DNA double-strand breaks are present in the cell. A similar observation has been reported after in vitro exposure of spermatocytes to gamma-irradiation [65]. The fact that after culture with okadaic acid fewer cyclophosphamide-treated cells presented high numbers of {gamma}H2AX foci (>10) compared with those of pachytene spermatocytes could explain why treatment with cyclophosphamide does not affect the number of cells with {gamma}H2AX-positive sex bodies upon culture with okadaic acid.

In conclusion, we demonstrate that DNA double-strand breaks are detected in pachytene spermatocytes and cells induced to undergo meiotic progression after acute or chronic cyclophosphamide treatments. The impairment in meiotic progression observed after acute cyclophosphamide treatment is an indication of the responsiveness of the cell to damage. In contrast, the absence of meiotic delay after chronic drug exposure raises concern about the consequences of repetitive exposure to low doses of genotoxic agents on cell functionality. Whether the effects of cyclophosphamide on chromosome condensation and meiotic progression affect germ cell quality and embryo development needs to be further assessed.


   ACKNOWLEDGMENTS
 
We thank Drs. Amy Inselman and Mary Ann Handel, University of Tennessee, Knoxville, for their assistance in setting up the in vitro G2/M transition system.


   FOOTNOTES
 
1 Supported by the Canadian Institutes of Health Research (CIHR). Back

2 Correspondence: B. Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montréal, Québec, Canada H3G 1Y6. FAX: 514 398 7120; Bernard.robaire{at}mcgill.ca Back

Received: 29 November 2004.

First decision: 3 January 2005.

Accepted: 24 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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