Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.
Agricola
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.
Biology of Reproduction 66, 1024-1032 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Chronic Cyclophosphamide Treatment Alters the Expression of Stress Response Genes in Rat Male Germ Cells1

Adriana Aguilar-Mahechaa, Barbara F. Halesa, and Bernard Robaire2,,a,b

a Departments of Pharmacology and Therapeutics b Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada H3G 1Y6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4–5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.

gene regulation, spermatid, spermatogenesis, stress, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer-related death rates for all cancers combined declined between 1990 and 1997 in the United States [1]. Earlier detection, due to advances in diagnostic technology, and more efficacious treatments have increased patient survival. However, these advances in treatment precipitate other concerns. One such concern is the effects of anticancer agents on the male reproductive system and the potential risks for progeny outcome [2]. Treatment of men with anticancer agents commonly results in male infertility and persistent gonadal damage [3]; the effect on the quality of spermatozoa remains unclear. Male sterility has led to the use of assisted reproduction techniques, such as intracytoplasmic sperm injection and in vitro fertilization; these approaches may solve infertility problems in cancer survivors but are of increasing concern with respect to their potential impact on progeny [4, 5].

There is evidence that exposure to drugs or toxicants results in damage to the male genome and induces heritable germ line mutations and malformations [6]. One such chemical is cyclophosphamide, a bifunctional alkylating drug commonly used in chemotherapeutic regimens [7]. The cytotoxicity of cyclophosphamide is mediated by alkylation of DNA at the N7 position of guanine and the formation of DNA-DNA cross-links, DNA-protein cross-links, and single-strand breaks [8, 9].

The male reproductive system is particularly sensitive to the damage induced by anticancer agents targeting rapidly dividing cells. The spermatogenic process is highly ordered and regulated; stem cells (spermatogonia) divide mitotically to form spermatocytes to initiate the process. Preleptotene spermatocytes go through two meiotic divisions to become round spermatids. During spermiogenesis, round spermatids differentiate. Changes such as chromatin remodeling, formation of the flagellum, and shedding of most of the cytoplasm take place to form mature spermatozoa [10].

Cyclophosphamide alters male fertility; treatment with 1–2 mg/kg for more than 4 mo increases the incidence of azoospermia and oligospermia in adult male patients [11]. Previous studies have shown that chronic paternal treatment of rats with cyclophosphamide damages sperm DNA [12], alters in vitro spermatozoal decondensation [13], and results in abnormal progeny outcome [14, 15]. The effects of cyclophosphamide on progeny outcome are time specific and dose dependent; maximum postimplantation loss occurs 4 wk after the initiation of treatment, whereas preimplantation loss is prominent at 5–6 wk after the initiation of treatment [16]. Chronic cyclophosphamide treatment alters the histology [17] and biochemistry [18] of the epididymal epithelium, but spermatogenesis is not arrested. Nevertheless, abnormal spermatozoa are found in the lumen of both the epididymis and the testis after chronic cyclophosphamide exposure [17]. The molecular mechanisms underlying the male-mediated developmental toxicity of cyclophosphamide remain to be elucidated.

Identifying the effects of cyclophosphamide treatment on gene expression in germ cells may provide insight into mechanisms underlying the male infertility and male-mediated developmental toxicity of this drug. DNA repair, heat shock proteins, and antioxidant defense mechanisms play an important role in the stress response to alkylating agents [19] and in male germ cell development and functionality. Recently, we reported the differential expression of stress response genes during spermatogenesis [20] and after acute exposure to cyclophosphamide [21]. Maximal adverse effects on progeny outcome were found after low-dose chronic cyclophosphamide exposure [14, 15], a common clinical regimen. Therefore, the goal of this study was to assess the effects of chronic cyclophosphamide treatment on the expression of stress response genes in male germ cells.


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

Adult male Sprague-Dawley rats (325–350 g) were obtained from Charles River Canada (St. Constant, QC, Canada), maintained on a 14L:10D light cycle, and provided with food and water ad libitum. Rats were randomly divided into groups of five animals and gavaged with saline or cyclophosphamide (6 mg/kg) daily, six times per week, for 5–6 wk. At the end of the treatment, rats were killed by decapitation. All animal handling and care were in accordance with the guidelines established by the Canadian Council on Animal Care.

Isolation of Spermatogenic Cell Types

Spermatogenic cells were isolated using the method described by Bellvé et al. [22], as adapted by Aguilar-Mahecha et al. [20]. Briefly, testes were removed, decapsulated, and incubated with collagenase (C9891; Sigma, Oakville, ON, Canada). After a washing, seminiferous tubules were incubated with trypsin (type 1, T8003; Sigma) and DNase I (type 1, DN-25, Sigma; 1 µg/ml), dissociated, and filtered through a nylon mesh (Sefar Canada, Montreal, QC, Canada; 70 µm) in the presence of DNase I. A total of 5.6 x 108 cells in 25 ml of 0.5% BSA (Sigma) in RPMI (RPMI medium 160; Gibco BRL, Burlington, ON, Canada) was loaded in a velocity sedimentation cell separator apparatus (STA-PUT; Proscience, Don Mills, ON, Canada) and separated by sedimentation velocity. With light microscopy, pachytene spermatocytes, round spermatids, and elongating spermatids were distinguished by their chromatin packaging and morphologic characteristics. Only fractions with high purity (>85%) were pooled. The average purities for each cell type were pachytene spermatocytes, 91%; round spermatids, 91%; and elongating spermatids, 97%. Five separate cell separations for each treatment were run, each with both testes from one animal.

RNA Extraction

Total RNA extraction was done with phenol-chloroform [23] followed by incubation with RNase-free DNase I (Sigma), for 1 h at 37°C, to avoid contamination by genomic DNA. The RNA quality was assessed by gel electrophoresis and spectrophotometric reading.

Probe Preparation and Hybridization to cDNA Atlas Arrays

RNA (5 µg) was reversed transcribed using Moloney murine leukemia virus reverse transcriptase and radiolabeled with [32P]dATP (10 µCi/µl; Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada). To purify labeled cDNAs from unincorporated 32P-labeled nucleotides, probes were filtered through CHROMA SPIN-200 DEPC-H2O columns (Clontech, Palo Alto, CA). The radioactive probes were added to nylon membranes (Rat Stress and Toxicology Atlas Arrays; Clontech) containing 216 cDNAs spotted in duplicate, and prehybridized with salmon testes DNA and ExpressHyb (Clontech) at 68°C; hybridization occurred overnight with continuous agitation at 68°C. After 18 h, the arrays were washed three times for 30 min with washing solution 1 (2x saline-sodium citrate [SSC], 1% SDS) and once for 30 min with washing solution 2 (0.1x SSC, 0.5% SDS), with continuous agitation at 68°C. After a final wash with 2x SSC at room temperature, the membranes were exposed to a phosphorimager plate for 24 h. Five separate replicates for each cell type and for each treatment were done.

Analysis of Gene Expression

Arrays were visualized after scanning with a phosphorimager (Storm, Molecular Dynamics, Sunnyvale, CA), and the images were imported to AtlasImage software (version 1.5; Clontech) for quantification. The data generated by AtlasImage software were imported into GeneSpring (version 3.2.8; Silicon Genetics, Redwood, CA) for further analysis. The raw signal intensity obtained was the result of the subtraction of the background level on each membrane from that of the intensity of each gene. The threshold for gene detection was set at a raw signal intensity for a given gene of two times the background intensity on that membrane. To minimize experimental variation and permit the comparison of different experiments to one another, an experiment-to-experiment normalization was done (GeneSpring), so that the raw value of a specific gene was divided by the median intensity of that single membrane. This calculation was done for all five replicates and averaged to generate what is referred to as the relative intensity of a specific gene. Changes in gene expression were considered only when the difference in expression level was at least 1.5-fold and consistent in at least three out of five replicate experiments; a 1.5-fold change is equivalent to an increase by 50% or to a decrease by 33%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Gene Expression

Large numbers of transcripts were detected in all three cell types after saline treatment (Fig. 1); 58% of the genes studied were detected in pachytene spermatocytes, 56% in round spermatids, and 38% in elongating spermatids. The lower expression in elongating spermatids was consistent with that found in previous studies [20]. In testicular cells obtained from male rats treated chronically with cyclophosphamide, the numbers of genes detected were reduced slightly; 52% were detected in pachytene spermatocytes, 50% in round spermatids, and 34% in elongating spermatids. Although most of the genes detected in each cell type were expressed in both saline and drug groups, some treatment-specific transcripts were observed. Thirteen genes that were expressed above threshold in pachytene spermatocytes in control animals became undetectable after treatment with cyclophosphamide; similar losses in gene expression were noted for round spermatids (14 genes) and elongating spermatids (13 genes) (Fig. 1, spotted areas). No genes were expressed selectively after cyclophosphamide treatment in pachytene spermatocytes or round spermatids, whereas four genes displayed intensity values above threshold level in elongating spermatids after cyclophosphamide treatment (Fig. 1, dashed areas).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 1. Numbers of genes expressed in pachytene spermatocytes (top), round spermatids (middle), and elongating spermatids (bottom) after chronic exposure to saline or cyclophosphamide. Spotted circles, number of genes detected exclusively after saline treatment; dashed circles, number of genes detected exclusively after chronic cyclophosphamide treatment. The overlap depicts the number of genes detected in both treatment groups. The boxes depict genes that were expressed exclusively in cells from one treatment group

Effects of Chronic Cyclophosphamide Treatment on Gene Expression

Overall changes in gene expression in the different spermatogenic cell types after chronic cyclophosphamide treatment were observed (Fig. 2). In pachytene spermatocytes, 91 of the 216 genes studied had intensity values below the threshold level (Fig. 2A). Transcript levels were not altered (decreased by less than 33%) after cyclophosphamide treatment for 51 of the genes. However, transcript levels for 50 of the genes studied decreased between 33% and 50%, while transcript levels for 24 decreased by 50% or more. Increases in the level of gene expression were not observed in this cell type after chronic cyclophosphamide treatment. Similar to the pattern noted in pachytene spermatocytes, 94 of the genes studied were not expressed in round spermatids (Fig. 2B). The expression of 59 of the genes remained unchanged after cyclophosphamide treatment. Transcript levels for 37 of the genes studied decreased by between 33% and 50%, while for 26 genes, transcript levels decreased by 50% or more. No increase in gene expression followed cyclophosphamide exposure in this cell type. The effect of cyclophosphamide treatment on elongating spermatids was distinct (Fig. 2C). Despite the fact that fewer genes were detected in this cell type (83 genes), the majority (58 genes) remained unaffected after drug exposure. A small number of genes had decreased expression levels: expression of six decreased between 33% and 50% and expression of only two decreased by 50% or more. Surprisingly, 17 genes had higher transcript levels after cyclophosphamide treatment, uniquely in elongating spermatids.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 2. Effects of chronic cyclophosphamide treatment on overall gene expression in male germ cells. The percent of genes affected from the total number of genes studied (216) in pachytene spermatocytes (A), round spermatids (B), and elongating spermatids (C). Light shading indicates not detected; heavy shading/lines to the right, unchanged; light shading/lines to the left, expression decreased 33%–50%; dark shading/crosshatched, expression decreased >50%; and intermediate shading/parallel lines, increased gene expression.

Profiles of Gene Expression

Genes were classified according to their response to drug treatment in every cell type. Only expression of those genes that were detected throughout spermatogenesis was profiled; 12 different profiles of expression were obtained (Fig. 3). The most common response to chronic cyclophosphamide exposure was a decrease in expression in both pachytene spermatocytes and round spermatids with no effect in elongating spermatids (Fig. 3A). This profile was exhibited for 18 genes, which are involved in posttranslational modification (nine genes); DNA synthesis, recombination, and repair (three genes); DNA and chromatin binding (three genes); cell cycle (two genes); and antioxidant defense (one gene).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 3. Profiles of gene expression observed after chronic cyclophosphamide treatment in different spermatogenic cell types. The arrows represent the direction of the change in gene expression; only genes expressed in all cell types (81) were profiled

The expression of 15 genes was selectively affected in pachytene spermatocytes (Fig. 3). Of these, expression of extracellular signal-regulated kinase 1 (ERK1) decreased by 51%; while expression of the cell cycle genes cyclin-dependent kinase (CDK) 2-{alpha} and CDK2-ß (combined) and cyclin G-associated kinase (GAK) decreased by 41% and 33%, respectively. Round spermatids were also selectively targeted; expression of 11 genes decreased uniquely in round spermatids (Fig. 3); expression of apoptosis regulator bcl-x decreased by 63%, while expression of the DNA repair genes proliferating cell nuclear antigen (PCNA) and poly(ADP-ribose) polymerase (PARP) decreased by 37% and 35%, respectively. Other genes affected exclusively in round spermatids included genes that were involved in posttranslational modification (three genes), cell cycle regulation (two genes), and intracellular transduction (one gene). In elongating spermatids, there were fewer effects after drug treatment. Expression of the plectin, ornithine decarboxylase (ODC), and nucleoside diphosphate kinase A (NDKA) genes was unaffected up to midspermatogenesis but increased in elongating spermatids (Fig. 3); transcript levels increased by 106%, 98%, and 50%, respectively. Transcripts of the intracellular transducer extracellular signal-regulated kinase 2 (ERK2) and the cell cycle gene p55CDC were the only transcripts to decrease uniquely in mature spermatids after cyclophosphamide treatment (Fig. 3E); transcript levels for these genes decreased by 43% and 38%, respectively.

Interestingly, drug treatment had opposite effects on the expression of certain genes in different cell types. Nine genes that decreased in expression level in pachytene spermatocytes were unaffected in round spermatids and increased in expression in elongating spermatids after cyclophosphamide treatment (Fig. 3F). Genes within this pattern included cytoplasmic beta actin (ACTB) and tubulin alpha-1 (TUBA1); expression levels of these genes decreased by 40% and 57%, respectively, in pachytene spermatocytes and increased by 67% and 125%, respectively, in elongating spermatids. The same profile was found for the genes for ribosomal proteins S30 and S19, expression levels of which decreased by 36% and 39%, respectively, in pachytene spermatocytes and increased by 93% and 263%, respectively, in mature spermatids. Similarly, the expression of two genes, the 3-methyladenine DNA glycosylase (MPG) and dual-specific mitogen activated-protein kinase kinase 5 (MAPKK5) genes, was not affected in pachytene spermatocytes, was decreased in round spermatids, and was increased in elongating spermatids (Fig. 3G). The expression of genes for DNA excision repair cross-complementing in rodents 1 (ERCC1), xeroderma pigmentosum group D complementing protein (XPD), and DNA-binding protein inhibitor ID1 decreased up to midspermatogenesis and increased in mature spermatids (Fig. 3H).

Cyclophosphamide did not alter the expression of all genes during spermatogenesis; transcript levels for 12 genes remained unaffected by drug treatment (Fig. 3I). Interestingly, three genes involved in intracellular transduction (c-Jun NH[2]-terminal kinase 1 [JNK1], extracellular related kinase 3 [ERK3], and MAPKK2) and two involved in protein turnover (polyubiquitin and the ubiquitin-conjugating enzyme E2 [UBE2B]) did not respond to drug treatment. Other genes responded similarly in all cell types; p23 and heat shock 10-kDa protein (HSP10) responded to cyclophosphamide with decreased expression in all spermatogenic cell types (Fig. 3J). The expression of NADPH-cytochrome P450 reductase (CPR), high-mobility group protein 2 (HMG2), and HSP70/HSP90-organizing protein (HOP) was unaffected in pachytene spermatocytes but decreased in round and elongating spermatids (Fig. 3K). Transcript levels for multidrug resistance protein (MRP) decreased in both pachytene spermatocytes and elongating spermatids but were not affected in round spermatids (Fig. 3L).

Expression of Gene Families

The response to chronic cyclophosphamide exposure was analyzed in more detail by examining the effect of drug exposure on the expression of different gene families during spermatogenesis.

DNA repair The effects of chronic cyclophosphamide exposure on the expression of genes involved in different DNA repair pathways were analyzed. These pathways are 1) nucleotide excision repair or NER (replication protein A 32 kDa [RPA], replication protein A 70 kDa [RPA70], XPD, PCNA, ERCC1, and p44 subunit of the transcription factor IIH [TFIIHp44]); 2) base excision repair or BER (PARP, ribosomal protein S3 [RPS3], MPG, APEX); 3) homologous recombination repair or HRR (RAD51, breast cancer type 1 susceptibility protein [BRCA1]); and 4) mismatch repair or MMR (MutL homologue 1 [MLH1], MutS homologue 2 [MSH2], and MutS homologue 3 [MSH3]).

The genes affected by drug treatment are depicted in Figure 4. In pachytene spermatocytes, the levels of expression of DNA repair genes decreased between 38% and 63% with the exception of expression of four genes, which remained unaffected after treatment. In round spermatids, fewer genes were detected, but expression of all decreased from 35% to 73% in response to cyclophosphamide. Surprisingly, in elongating spermatids, the few genes that responded to treatment did so by increasing transcript levels; MPG, XPD, and DNA excision repair protein ERCC1 increased in expression by 249%, 210%, and 96%, respectively.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. Effects of chronic cyclophosphamide treatment on the expression of DNA repair genes detected in male germ cells. The variation in expression level is expressed as the percent change from control saline values. Genes with intensity values below threshold level in saline ({delta}) or treated samples ({sigma}) were arbitrarily assigned to threshold level.

Heat shock proteins and cochaperones In pachytene spermatocytes, the expression level of genes for all HSPs detected, except HSP90-beta and HOP, decreased from 35% to 70% after cyclophosphamide treatment (Fig. 5A). Similar decreases were seen in round spermatids (34%–67%), although expression of HSP60 and HSP70 genes remained unaffected by treatment. In contrast, in elongating spermatids, most of the HSP genes expressed were not affected by drug treatment; decreased expression was seen for HSP10 (44%) and HOP (35%). Interestingly, the only member of this family showing increased expression was HSP70; transcript levels increased by 230% in elongating spermatids after drug treatment.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 5. Effects of chronic cyclophosphamide treatment on the expression of heat shock proteins (A) and cochaperones (B) in male germ cells. The variation in expression level is expressed as the percent change from control saline values. Genes with intensity values below threshold level in treated samples ({sigma}) were arbitrarily assigned the threshold value.

Expression of cochaperones decreased from 35% to 53% for all genes after cyclophosphamide treatment in pachytene spermatocytes, except for T-complex protein 1 {alpha} subunit (TCP1-{alpha}), calreticulin precursor (CALR), and probable protein disulfide isomerase ER60 precursor (Fig. 5B). In round spermatids, a greater range of decreases was seen; transcript levels were lowered from 35% to 68%, and only one gene, calcium-binding protein 2 (CABP2), did not respond to treatment. In elongating spermatids, expression of most cochaperones was unaffected by drug treatment; only p23 expression decreased, to undetectable levels in treated cells.

Antioxidant defense Genes involved in antioxidant defense behaved differently after drug treatment in the different cell types (Fig. 6). In pachytene spermatocytes, all of the genes detected decreased in expression level from 38% to 50%. In contrast, the transcript levels of genes expressed in round spermatids and elongating spermatids were relatively unchanged after treatment. Manganese-containing superoxide dismutase 2 precursor (Mn SOD2) and NAD(P)H:menadione oxidoreductase (NMOR1) were the only genes affected in round spermatids, decreasing by 35% and 46%, respectively. In elongating spermatids, heme oxygenase 1 (HO-1) was the only gene that responded to treatment; HO-1 levels increased from undetectable levels in control cells to reach a relative intensity of 0.30 in treated spermatids, an increase of at least 175%.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 6. Effects of chronic cyclophosphamide treatment on the expression of genes involved in antioxidant defense mechanisms. The variation in expression level is expressed as the percent change from control saline values. Genes with intensity values below threshold level in saline ({delta}) or treated samples ({sigma}) were arbitrarily assigned the threshold value.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chronic cyclophosphamide exposure resulted in a substantial decrease in gene expression, predominantly in pachytene spermatocytes and round spermatids. This finding contrasts sharply with the effects of acute cyclophosphamide exposure on germ cell gene expression [21]. Acute exposure to cyclophosphamide increases gene expression in all germ cell types, but most dramatically in round spermatids. Increased transcript levels are observed for 30 genes in round spermatids, 7 genes in pachytene spermatocytes, and 2 genes in elongating spermatids [21]. Previous studies have found that exposure to alkylating agents inhibits transcription and translation [24] as well as histone acetylation [25, 26]. An alteration in the transcriptional machinery, as a result of DNA damage, could explain the predominant decrease in gene expression in pachytene spermatocytes and round spermatids after chronic cyclophosphamide treatment. The large number of genes that were unaffected by drug treatment and the different levels of repression observed suggest that the reduction in transcript levels is not due to cytotoxicity but to a more specific response to cyclophosphamide. Alkylation of DNA and protein by cyclophosphamide induces the formation of cross-links. Cross-links in transcriptionally active regions of the chromosome can cause a pause or arrest of the RNA polymerases, adversely affecting transcription elongation and gene expression [27]. Treatment with nitrogen mustards inhibits the binding to DNA of specific transcription factors [28, 29], further supporting our observations that changes in the gene expression profile are specific. In elongating spermatids, chromatin is packed tightly after the replacement of histones by protamines, resulting in a transcriptionally inactive cell [30]. The condensed chromatin present at this stage of spermatogenesis may act to protect DNA against drug damage, thus decreasing susceptibility to cyclophosphamide and explaining why the expression of most of the genes detected in elongating spermatids was not altered by chronic drug treatment. Nevertheless, damage has been detected in sperm DNA after chronic cyclophosphamide treatment [12]; this damage may be induced earlier during spermatogenesis and carried on into mature spermatozoa.

Interestingly, chronic cyclophosphamide exposure induced higher transcript levels uniquely in elongating spermatids. It is unlikely that these transcripts are newly synthesized in elongating spermatids after treatment, since there is normally a lack of transcription during the late phases of spermatogenesis. However, the presence of DNA strand breaks in elongating spermatids may promote an abnormal transcriptional response, increasing transcript steady-state concentrations after drug treatment. The expression of genes in mature spermatids for PCNA and for subunits of the transcription factor TFIIH (XPD and p44); genes involved in replication and transcription, respectively, may play a role in this response.

Stability of RNA may provide an alternate explanation for the elevated transcript levels observed in elongating spermatids. During spermatogenesis, transcriptional and translational control mechanisms allow haploid spermatids to acquire the factors necessary to complete their development [31, 32]. After genes are transcribed in early germ cell types, many mRNAs are bound to ribonucleoproteins and translationally repressed until the protein product is required. Cyclophosphamide may alter the interaction between mRNA and RNA-binding proteins, increasing the stability of the transcripts.

Whereas the predominant effect of acute cyclophosphamide exposure was the induction of gene expression in round spermatids [21], chronic cyclophosphamide exposure resulted mainly in decreased gene expression. These changes may impact spermatogenesis and sperm function. The decreased expression of cell cycle kinases in pachytene spermatocytes after chronic cyclophosphamide treatment may have important consequences for cell division because of the large number of cyclins with which they interact [33]. Altered meiosis during germ cell development may account for the damage to the synaptonemal complex [34] and the chromosome aberrations [35] observed after mouse spermatocytes are exposed to cyclophosphamide. Disruption of the meiotic process may lead to infertility in transgenic mice [36], emphasizing the importance of cell cycle regulation for proper germ cell development and fertility.

In round spermatids, PARP and PCNA, two key enzymes involved in DNA repair in response to alkylating agents, are specifically decreased. Inhibition of PARP potentiates the cytotoxicity induced by alkylating agents [37]. PCNA plays a crucial role in nucleotide excision repair, the main pathway of repair of alkylation damage [38]. The effect of cyclophosphamide exposure on progeny outcome reaches maximal plateau levels when germ cells are exposed at the round spermatid stage [14, 15]. The sensitivity of these cells to insult after chronic drug treatment may be enhanced by decreased expression of key DNA repair genes.

Expression of ODC was altered specifically in elongating spermatids after chronic cyclophosphamide exposure. ODC is involved in polyamine synthesis, catalyzing the formation of putrescine, from which spermidine and spermine are synthesized [39]. Polyamines are involved in cell growth and differentiation [40]; increasing concern about their role in spermatogenesis has emerged since overexpression of ODC has been shown to yield infertile mice [41, 42], and abnormal levels of polyamines have been found in semen from infertile men [43]. Spermine may be an inhibitory factor of sperm capacitation [44]. The increase in ODC transcript levels in elongating spermatids after chronic cyclophosphamide treatment may be one of the mechanisms by which this drug disturbs sperm function and fertility.

The persistence of genomic damage and DNA breaks in male gametes contributes to male infertility. Male germ cell functionality after cyclophosphamide treatment depends largely on the extent of damage and on the ability of the cell to undergo DNA repair. Unscheduled DNA synthesis, an indirect measure of DNA repair, has been detected after acute treatment with cyclophosphamide in male germ cells up to midspermatogenesis; no unscheduled DNA synthesis was detected in elongating spermatids or spermatozoa [45]. DNA repair genes are differentially expressed during spermatogenesis [20, 46], and different repair mechanisms can function in germ cells at specific stages of development [47, 48]. Clearly, the expression of DNA repair genes was affected by chronic cyclophosphamide treatment. The marked repression of genes involved in NER, BER, and MMR in pachytene spermatocytes and round spermatids may increase the susceptibility of these cell types to genotoxic damage. In addition, the role of MMR genes in meiotic recombination suggests that their decreased levels of expression after drug treatment, particularly in pachytene spermatocytes, may compromise proper germ cell development and male fertility [49]. Although no DNA repair has been detected in elongating spermatids, transcripts for DNA repair genes were present. The increased expression of DNA repair genes in elongating spermatids after chronic cyclophosphamide treatment may play a role in the repair of damage induced during spermiogenesis. Interestingly, transition protein 1 stimulates the repair of damage induced in spermatids during chromatin remodeling or after genotoxic damage [50].

To cope with aberrant peptides, the cell is equipped with a posttranslational modification system. HSPs and cochaperones are expressed ubiquitously and induced under a wide range of stressful stimuli, including exposure to cytotoxic antitumor agents [51]. In the testis, HSPs and cochaperones are differentially expressed during spermatogenesis and may have roles both in male germ cell development and in the response to stressful stimuli [20, 52]. Cyclophosphamide may alter protein conformation by binding to proteins and forming DNA-protein cross-links. How the cell responds to proteotoxic stress will depend largely on the expression of HSPs. The decreases in transcripts for HSPs and cochaperones noted after chronic cyclophosphamide treatment suggest that the ability of the cells to repair damaged peptides or properly synthesize proteins is hampered. The importance of an optimal response to proteotoxic stress in male germ cells is emphasized by the requirement for some of its members for fertility; infertility has been observed in HSP70-2 [53] and calmegin homozygous [54] null male mice. Interestingly, increased levels of HSP70 were detected in elongating spermatids, suggesting an important role of this protein in the response to cyclophosphamide.

Induction of oxidative stress in male germ cells is associated with male infertility [55]. Sperm are highly susceptible to lipid peroxidation, and induction of reactive oxygen species (ROS) is correlated with decreased sperm motility [56, 57], decreased capacity to undergo the acrosome reaction [58], and DNA damage [59]. Aitken et al. [60] reported that induction of ROS was associated, in a dose-dependent manner, with increased DNA fragmentation in sperm nuclei; however, sperm-egg fusion was affected only in the presence of very high levels of ROS, thus raising concern about the quality of sperm at fertilization. Interestingly, male-mediated developmental toxicity was reported after oxidative stress; prooxidant treatment of male mice induced lethal mutations in offspring, resulting in increased postimplantation loss [61]. The presence of antioxidant defense mechanisms in the testis provides the male gamete with protection against oxidative stress-induced damage. An imbalance between the generation of ROS and the scavenging activities of antioxidants in the male gamete may lead to germ cell damage. Exposure to cyclophosphamide or acrolein, a metabolite of cyclophosphamide, induces the formation of ROS [62, 63] and lipid peroxidation [64]. Chronic cyclophosphamide treatment decreased the expression of all antioxidant genes detected in pachytene spermatocytes; this decrease may contribute to a redox imbalance, increasing the susceptibility of this cell type to oxidative stress.

In conclusion, chronic cyclophosphamide treatment alters the profile of gene expression in the male gamete. Reduced expression of important defense mechanisms after drug treatment may allow damage to accumulate in male germ cells and result in altered sperm function and infertility. The possibilities that damaged sperm can fertilize an egg and that mammalian development may be tolerant to epigenetic abnormalities [65] raise concerns about the male-mediated effects on progeny of drug and toxicant exposure. Whether the abnormal male germ cell gene expression induced by chronic cyclophosphamide exposure is causally related to fetal loss and abnormal development remains to be established.


   FOOTNOTES
 
First decision: 3 October 2001.

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, QC, Canada H3G 1Y6. FAX: 514 398 7120; brobaire{at}pharma.mcgill.ca Back

Accepted: November 5, 2001.

Received: September 13, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ries LA, Wingo PA, Miller DS, Howe HL, Weir HK, Rosenberg HM, Vernon SW, Cronin K, Edwards BK. The annual report to the nation on the status of cancer, 1973BB1997, with a special section on colorectal cancer. Cancer 2000; 88:2398-2424[CrossRef][Medline]
  2. Trasler JM, Doerksen T. Teratogen update: paternal exposures—reproductive risks. Teratology 1999; 60:161-172[CrossRef][Medline]
  3. Kenney LB, Laufer MR, Grant FD, Grier H, Diller L. High risk of infertility and long term gonadal damage in males treated with high dose cyclophosphamide for sarcoma during childhood. Cancer 2001; 91:613-621[CrossRef][Medline]
  4. Horne G, Atkinson A, Brison DR, Radford J, Yin JA, Edi-Osagie EC, Pease EH, Lieberman BA. Achieving pregnancy against the odds: successful implantation of frozen-thawed embryos generated by ICSI using spermatozoa banked prior to chemo/radiotherapy for Hodgkin's disease and acute leukaemia. Hum Reprod 2001; 16::107-109[Abstract/Free Full Text]
  5. Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998; 13:1864-1871[Abstract/Free Full Text]
  6. Brinkworth MH. Paternal transmission of genetic damage: findings in animals and humans. Int J Androl 2000; 23:123-135[CrossRef][Medline]
  7. Colvin OM. An overview of cyclophosphamide development and clinical applications. Curr Pharm Des 1999; 5:555-560[Medline]
  8. Hemminki K, Kallama S. Reactions of nitrogen mustards with DNA. IARC Sci Publ 1986; 78:55-70
  9. Crook TR, Souhami RL, McLean AE. Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res 1986; 46:5029-5034[Abstract/Free Full Text]
  10. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972; 52::198-236[Free Full Text]
  11. Qureshi MS, Pennington JH, Goldsmith HJ, Cox PE. Cyclophosphamide therapy and sterility. Lancet 1972; 16:1290-1291
  12. Qiu J, Hales BF, Robaire B. Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod 1995; 53:1465-1473[Abstract]
  13. Qiu J, Hales BF, Robaire B. Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol Reprod 1995; 52:33-40[Abstract]
  14. Trasler JM, Hales BF, Robaire B. Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod 1986; 34:275-283[Abstract]
  15. Trasler JM, Hales BF, Robaire B. Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature 1985; 316:144-146[CrossRef][Medline]
  16. Trasler JM, Hales BF, Robaire B. A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod 1987; 37:317-326[Abstract]
  17. Trasler JM, Hermo L, Robaire B. Morphological changes in the testis and epididymis of rats treated with cyclophosphamide: a quantitative approach. Biol Reprod 1988; 38:463-479[Abstract]
  18. Trasler JM, Robaire B. Effects of cyclophosphamide on selected cytosolic and mitochondrial enzymes in the epididymis of the rat. J Androl 1988; 9:142-152[Abstract/Free Full Text]
  19. Wassermann K. Intragenomic heterogeneity of DNA damage formation and repair: a review of cellular responses to covalent drug DNA interaction. Crit Rev Toxicol 1994; 24:281-322[Medline]
  20. Aguilar-Mahecha A, Hales BF, Robaire B. Expression of stress response genes in germ cells during spermatogenesis. Biol Reprod 2001; 65:119-127[Abstract/Free Full Text]
  21. Aguilar-Mahecha A, Hales BF, Robaire B. Acute cyclophosphamide exposure has germ cell specific effects on the expression of stress response genes during rat spermatogenesis. Mol Reprod Dev 2001; 60:302–311.
  22. Bellvé AR, Millette CF, Bhatnagar YM, O'Brien DA. Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem 1977; 25:480-494[Medline]
  23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159[Medline]
  24. Masta A, Gray PJ, Phillips DR. Nitrogen mustard inhibits transcription and translation in a cell free system. Nucleic Acids Res 1995; 23::3508-3515[Abstract/Free Full Text]
  25. Grunicke H. Csordas A. Helliger W, Hauptlorenz S, Loidl A, Multhaup I, Zwierzina H, Puschendorf B. Depression of histone acetylation by alkylating antitumor agents: significance for antitumor acitivity and possible biological consequences. Adv Enzyme Regul 1984; 22::433-446[CrossRef][Medline]
  26. Grunicke H, Helliger W, Hermann BJ, Hock W, Hofmann J, Puschendorf B. Alkylating antitumor agents reduce histone acetyl-transferase activity. Adv Enzyme Regul 1986; 25:87-97[CrossRef][Medline]
  27. Masta A, Gray PJ, Phillips DR. Molecular basis of nitrogen mustard effects on transcription processes: role of depurination. Nucleic Acids Res 1994; 22:3880-3886[Abstract/Free Full Text]
  28. Chen XM, Gray PJ, Cullinane C, Phillips DR. Differential sensitivity of transcription factors to mustard-damaged DNA. Chem Biol Interact 1999; 118:51-67[CrossRef][Medline]
  29. Chen X, Cullinane C, Gray PJ, Phillips DR. DNA damage by nitrogen mustard in a gene containing multiple Sp1-binding sites. Mutat Res 1999; 445:45-54[Medline]
  30. Monesi V, Geremia R, D'Agostino A, Boitani C. Biochemistry of male germ cell differentiation in mammals: RNA synthesis in meiotic and postmeiotic cells. Curr Top Dev Biol 1978; 12:11-36[Medline]
  31. Hecht NB. Molecular mechanisms of male germ cell differentiation. Bioessays 1998; 20:555-561[CrossRef][Medline]
  32. Steger K. Transcriptional and translational regulation of gene expression in haploid spermatids. Anat Embryol (Ber) 1999; 199:471-487[CrossRef][Medline]
  33. Ravnik SE, Wolgemuth DJ. Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage. Dev Biol 1999; 207:408-418[CrossRef][Medline]
  34. Backer LC, Gibson JB, Moses MJ, Allen JW. Synaptonemal complex damage in relation to meiotic chromosome aberrations after exposure of male mice to cyclophosphamide. Mutat Res 1988; 203:317-330[Medline]
  35. Pacchierotti F, Bellincampi D, Civitareale D. Cytogenetic observations, in mouse secondary spermatocytes, on numerical and structural chromosome aberrations induced by cyclophosphamide in various stages of spermatogenesis. Mutat Res 1983; 119:177-183[CrossRef][Medline]
  36. Venables JP, Cooke HJ. Lessons from knockout and transgenic mice for infertility in men. J Endocrinol Invest 2000; 23:584-591[Medline]
  37. Tatsumi-Miyajima J, Kupper JH, Takebe H, Burkle A. Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in Chinese hamster cells. Mol Cell Biochem 1999; 193:31-35[CrossRef][Medline]
  38. Shivji KK, Kenny MK, Wood RD. Proliferating cell nuclear antigen is required for DNA excision repair. Cell 1992; 69:367-374[CrossRef][Medline]
  39. Pegg AE, Shantz LM, Coleman CS. Ornithine decarboxylase: structure, function and translational regulation. Biochem Soc Trans 1994; 22:846-852[Medline]
  40. Thomas T, Thomas TJ. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 2001; 58:244-258[CrossRef][Medline]
  41. Kilpelainen PT, Saarimies J, Kontusaari SI, Jarvinen MJ, Soler AP, Kallioinen MJ, Hietala OA. Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility. Int J Biochem Cell Biol 2001; 33:507-520[CrossRef][Medline]
  42. Halmekyto M, Hyttinen JM, Sinervirta R, Utriainen M, Myohanen S, Voipio HM, Wahlfors J, Syrjanen S, Syrjanen K, Alhonen L, Jänne J. Transgenic mice aberrantly expressing human ornithine decarboxylase gene. J Biol Chem 1991; 266:19746-19751[Abstract/Free Full Text]
  43. Vanella A, Pinturo R, Vasta M, Piazza G, Rapisarda A, Savoca S, Nardo F, Bellia V, Panella M. Polyamine levels in human semen of unfertile patients: effect of S-adenosylmethionine. Acta Eur Fertil 1978; 9:99-103[Medline]
  44. Rubinstein S, Breitbart H. Role of spermine in mammalian sperm capacitation and acrosome reaction. Biochem J 1991; 278:25-28
  45. Sotomayor RE, Sega GA, Cumming RB. Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen. Mutat Res 1978; 50::229-240[Medline]
  46. Richardson LL, Pedigo C, Handel MA. Expression of deoxyribonucleic acid repair enzymes during spermatogenesis in mice. Biol Reprod 2000; 62:789-796[Abstract/Free Full Text]
  47. Jansen J, Olsen AK, Wiger R, Naegeli H, de Boer P, van Der Hoeven F, Holme JA, Brunborg G, Mullenders L. Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro. Nucleic Acids Res 2001; 29:1791-1800[Abstract/Free Full Text]
  48. Olsen AK, Bjortuft H, Wiger R, Holme J, Seeberg E, Bjoras M, Brunborg G. Highly efficient base excision repair (BER) in human and rat male germ cells. Nucleic Acids Res 2001; 29:1781-1790[Abstract/Free Full Text]
  49. Edelmann W, Cohen PE, Kane M, Lau K, Morrow B, Bennett S, Umar A, Kunkel T, Cattoretti G, Chaganti R, Pollard JW, Kolodner RD, Kucherlapati R. Meiotic pachytene arrest in MLH1-deficient mice. Cell 1996; 85:1125-1134[CrossRef][Medline]
  50. Caron N, Veilleux S, Boissonneault G. Stimulation of DNA repair by the spermatidal TP1 protein. Mol Reprod Dev 2001; 58:437-443[CrossRef][Medline]
  51. Smith DF, Whitesell L, Katsanis E. Molecular chaperones: biology and prospects for pharmacological intervention. Pharmacol Rev 1998; 50:493-514[Abstract/Free Full Text]
  52. Dix DJ, Hong RL. Protective mechanisms in germ cells: stress proteins in spermatogenesis. Adv Exp Med Biol 1998; 444:137-143[Medline]
  53. Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N, Poorman-Allen P, Goulding EH, Eddy EM. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci U S A 1996; 93:3264-3268[Abstract/Free Full Text]
  54. Ikawa M, Wada I, Kominami K, Watanabe D, Toshimori K, Nishimune Y, Okabe M. The putative chaperone calmegin is required for sperm fertility. Nature 1997; 387:607-611[CrossRef][Medline]
  55. Sikka SC. Relative impact of oxidative stress on male reproductive function. Curr Med Chem 2001; 8:851-862[Medline]
  56. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. J Androl 1992; 13:379-386[Abstract]
  57. de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl 1992; 13:368-378[Abstract/Free Full Text]
  58. Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J Reprod Fertil 1995; 103:17-26[Abstract/Free Full Text]
  59. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998; 13:1429-1436[Abstract/Free Full Text]
  60. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998; 59:1037-1046[Abstract/Free Full Text]
  61. Kumar TR, Muralidhara , Male-mediated dominant lethal mutations in mice following prooxidant treatment. Mutat Res 1999; 444:145-149[Medline]
  62. Sulkowska M, Sulkowski S, Skrzydlewska E, Farbiszewski R. Cyclophosphamide-induced generation of reactive oxygen species. Comparison with morphological changes in type II alveolar epithelial cells and lung capillaries. Exp Toxicol Pathol 1998; 50:209-220[Medline]
  63. Adams JD Jr, Klaidman LK. Acrolein-induced oxygen radical formation. Free Radic Biol Med 1993; 15:187-193[CrossRef][Medline]
  64. Berrigan MJ, Struck RF, Gurtoo HL. Lipid peroxidation induced by cyclophosphamide. Cancer Biochem Biophys 1987; 9:265-270[Medline]
  65. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout III WM, Biniszkiewicz D, Yanagimachi R, Jaenisch R. Epigenetic instability in ES cells and cloned mice. Science 2001; 293:95-97[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
G. Delbes, D. Chan, P. Pakarinen, J. M. Trasler, B. F. Hales, and B. Robaire
Impact of the Chemotherapy Cocktail Used to Treat Testicular Cancer on the Gene Expression Profile of Germ Cells from Male Brown-Norway Rats
Biol Reprod, February 1, 2009; 80(2): 320 - 327.
[Abstract] [Full Text] [PDF]

Home page
 Hum Exp ToxicolHome page
M. Rezvanfar, R. Sadrkhanlou, A Ahmadi, H Shojaei-Sadee, M. Rezvanfar, A Mohammadirad, A Salehnia, and M Abdollahi
Protection of cyclophosphamide-induced toxicity in reproductive tract histology, sperm characteristics, and DNA damage by an herbal source; evidence for role of free-radical toxic stress
Human and Experimental Toxicology, December 1, 2008; 27(12): 901 - 910.
[Abstract] [PDF]

Home page
 Biol. Reprod.Home page
A. M. Codrington, B. F. Hales, and B. Robaire
Chronic Cyclophosphamide Exposure Alters the Profile of Rat Sperm Nuclear Matrix Proteins
Biol Reprod, August 1, 2007; 77(2): 303 - 311.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
A.M. Codrington, B.F. Hales, and B. Robaire
Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa
Hum. Reprod., May 1, 2007; 22(5): 1431 - 1442.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
G. Delbes, B. F. Hales, and B. Robaire
Effects of the Chemotherapy Cocktail Used to Treat Testicular Cancer on Sperm Chromatin Integrity
J Androl, March 1, 2007; 28(2): 241 - 249.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
A. Aguilar-Mahecha, B. F. Hales, and B. Robaire
Effects of Acute and Chronic Cyclophosphamide Treatment on Meiotic Progression and the Induction of DNA Double-Strand Breaks in Rat Spermatocytes
Biol Reprod, June 1, 2005; 72(6): 1297 - 1304.
[Abstract] [Full Text] [PDF]

Home page
 J Natl Cancer Inst MonogrHome page
B. F. Hales, T. S. Barton, and B. Robaire
Impact of Paternal Exposure to Chemotherapy on Offspring in the Rat
J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 28 - 31.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
Y. Wang, R. Thuillier, and M. Culty
Prenatal Estrogen Exposure Differentially Affects Estrogen Receptor-Associated Proteins in Rat Testis Gonocytes
Biol Reprod, November 1, 2004; 71(5): 1652 - 1664.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
T. S. Barton, A. J. Wyrobek, F. S. Hill, B. Robaire, and B. F. Hales
Numerical Chromosomal Abnormalities in Rat Epididymal Spermatozoa Following Chronic Cyclophosphamide Exposure
Biol Reprod, October 1, 2003; 69(4): 1150 - 1157.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.
Agricola
Right arrow Articles by Aguilar-Mahecha, A.
Right arrow Articles by Robaire, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS