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From the * Departments of Pediatrics, Human
Genetics, and Pharmacology & Therapeutics, McGill University, and McGill
University-Montreal Children's Hospital Research Institute, Montreal, Quebec,
Canada; and the Cardiovascular Research
Center, Massachusetts General Hospital, Department of Medicine, Harvard
Medical School, Charlestown, Massachusetts.
| Correspondence to: Dr Jacquetta M. Trasler, McGill University-Montreal Children's Hospital Research Institute, McGill University Health Centre, 2300 Tupper Street, Montreal, Quebec, Canada H3H 1P3. |
| Received for publication April 24, 2003; accepted for publication June 4, 2003. |
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Abstract |
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Key words: DNA methylation, 5-azacytidine, DNMT, DNMT1, mouse, fertility, DNA methyltransferase 1
Various members of the (cytosine-5)-DNA methyltransferase (DNMT) family are capable of catalyzing methylation in mammalian cells. Dnmt1 encodes the predominant mammalian maintenance DNMT, and several other enzymes, including Dnmt2, Dnmt3a, Dnmt3b, and Dnmt3l have recently been characterized (Okano et al, 1998; Aapola et al, 2001). Cytosine methylation is essential for normal development; mice homozygous for targeted partial (Dnmt1n/n or Dnmt1s/s) (Li et al, 1992) and complete loss of function (Dnmt1c/c) mutations in Dnmt1 have retarded growth and die by midgestation (Lei et al, 1996). Levels of methylation in Dnmt1c/c embryos are only 5% of those in wild-type mice (Lei et al, 1996). Dnmt1-deficient embryos have biallelic expression of imprinted genes (Li et al, 1993), ectopic X-chromosome inactivation (Panning and Jaenisch, 1996), hypomethylation and transcription of the normally silent endogenous retroviral IAP sequences (Walsh et al, 1998), and increased levels of apoptosis (Li et al, 1992). However, mice that are heterozygous for the null (Dnmt1c/+) mutation survive and are phenotypically normal, although they possess only half the wild-type DNMT1 levels (Lei et al, 1996).
Dnmt1 levels are highest in the testis and ovary, and their expression is highly regulated throughout spermatogenesis in both the rat and the mouse (Trasler et al, 1992; Benoit and Trasler, 1994; Jue et al, 1995). Both replicating and nonreplicating germ cells express DNMT1, although the enzyme is translationally down-regulated in pachytene spermatocytes (Trasler et al, 1992; Jue et al, 1995; Mertineit et al, 1998). The presence of DNMT1 in mitotically dividing spermatogonia suggests that it may function in maintaining methylation patterns after replication, and during meiotic prophase it may play a role in repair. For the more recently described DNMT enzymes, testicular expression has not yet been fully characterized.
Previous studies in rats exposed to cytidine analogues have underscored the importance of DNA methylation in normal male germ cell development. The long-term administration of 5-azacytidine to male Sprague-Dawley rats, exposing both mitotic and/or meiotic developing male germ cells, was associated with decreased germ cell DNA methylation, reduced sperm production, and aberrant embryo development (Doerksen and Trasler, 1996; Doerksen et al, 2000).
Cytidine analogues incorporate into replicating DNA, but, because of the presence of a nitrogen moiety at the fifth position of the pyrimidine ring, methylating DNMTs remain bound as covalent adducts (Gabbara and Bhagwat, 1995). DNMT adduct formation is thought to cause indirect genomic hypomethylation through the decreased activity of the DNMT enzymes (Gabbara and Bhagwat, 1995). 5-Azacytidine is a nonselective analogue that is incorporated into both RNA and DNA. In rat studies (Doerksen and Trasler, 1996; Doerksen et al, 2000), it was unclear whether 5-azacytidine's effects were due to cytotoxicity or to decreased DNA methylation. In the present study, we used the more selective and clinically useful cytidine analogue 5-aza-2'-deoxycytidine. This analogue is incorporated only into DNA and thus should affect DNA methylation without the toxicity from decreases in protein synthesis. Mice, rather than rats, were used, because the numerous gene-targeted mouse models may prove useful in unravelling the mechanisms underlying the effects of cytidine analogues on male germ cells. We used a combination of pharmacological (5-aza-2'-deoxycytidine) and genetic (Dnmt1 haploinsufficiency in Dnmt1c/+ mice) manipulation to determine how experimental approaches known to alter DNA methylation affect spermatogenesis in the mouse. We show that paternal administration of 5-aza-2'-deoxycytidine interferes with normal male germ cell development, without affecting the general health of the mice, and results in reduced fertility or function of treated sperm. Furthermore, we show that Dnmt1c/+ heterozygotes appear to be more resistant to 5-aza-2'-deoxycytidine-induced germ cell toxicity than their wild-type littermates.
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Materials and Methods |
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Adult virgin female C57BL/6 and CD1 mice were obtained from Charles River Canada (St Constant, Canada). They were maintained on a 12: 12 hour light/dark cycle and were provided with food and water ad libitum. All animal studies were conducted in accordance with the principles and procedures outlined in the Guide to the Care and Use of Experimental Animals, by the Canadian Council on Animal Care.
Treatment and Mating
Male Dnmt1+/+ (age 7 weeks) mice were randomly
assigned to 1 of 4 treatment groups (n = 12/group) and, 3 times a week for 7
weeks, received intraperitoneal injections of saline or
5-aza-2'-deoxycytidine (5-azaCdR) (0.05, 0.1, and 0.15 mg/kg) (Sigma
Chemical Company, St Louis, Mo). Dnmt1c/+ males were
treated with either saline (n = 8) or 0.1 mg/kg 5-azaCdR (n = 9). Because
alterations induced by this drug can be repaired, a dose regimen of 3 times
per week was used to ensure continual incorporation of the drug into DNA while
limiting toxicity to the mice. A treatment regimen of 7 weeks was chosen to
target germ cells during their development; sperm collected after 7 weeks had
been treated throughout spermatogenesis. All mice were weighed twice weekly.
At the end of 7 weeks, each male was mated with 2 virgin CD1 females (age 8
weeks) that were checked daily for seminal plugs.
After treatment, male mice were killed, and blood was collected for white blood cell (WBC) counts and hemoglobin determination using a Coulter counter (Coulter Electronics, Hialeah, Fla). Testes, epididymides, and seminal vesicles were removed, weighed, snap frozen, and stored at -80°C for further analyses. Caudal sperm were isolated (Alcivar et al, 1989) and stored at -80°C for DNA methylation analysis.
Light Microscopy
For histological examination, the right testis was immersed in Bouin's
fixative (BDH Inc, Toronto, Canada) for 12 to 24 hours, dehydrated, and
embedded in paraffin. Sections (6 µm) were cut, mounted on glass slides,
deparaffinized with xylene, and stained with hematoxylin and eosin. A Zeiss
Axiophot photomicroscope was used to view the slides, and pictures were taken
using a SPOT RT Slider digital camera (Diagnostic Instruments Inc, Sterling
Heights, Mich). Tubules were staged according to the method of Oakberg
(1956), and abnormal
seminiferous tubules were quantified. The number of abnormal tubules was
expressed as a percentage of total tubules examined (90 tubules
counted/animal).
Sperm Counts
Hemocytometric counts of spermatozoa were done as described by Robb et al
(1978). To prepare for
counting, a weighed portion of the left testis was homogenized (Polytron,
setting 10; Brinkmann Instruments Inc, Westbury, NY) for 3 15-second periods,
separated by 10-second intervals, in 3 mL of 0.9% NaCl, 0.1% thimersal, and
0.5% Triton X-100.
Analysis of Pregnancy Outcome
To examine the effect of paternal treatment on progeny outcome, each male,
after 7 weeks of treatment, was mated with 2 untreated CD1 females over a
period of 6 days. The success of mating was determined each morning by the
presence of a vaginal plug; females were killed 19 days postcoitum (dpc).
Ovaries were removed, and the number of corpora lutea, which is representative
of the number of oocytes released, was counted. The uterus was then opened,
and numbers of implantations, resorptions, and live fetuses were determined.
The pregnancy rate is the number of plug-positive females that became
pregnant. Preimplantation loss was calculated as the difference between the
number of corpora lutea and implantations for each female. Thus,
preimplantation loss is the number of oocytes that were either unfertilized or
fertilized but were lost prior to implantation. The difference in the number
of live fetuses and uterine implantations is a measure of postimplantation
loss. Fetuses were weighed (as were resorption sites), sexed, and examined for
gross malformations. Placentas and liver segments from 2 female and 2 male
fetuses were snap frozen and stored at -80°C. All data concerning
pregnancy outcome were expressed on a per-male basis.
DNA Methylation
Thin-layer chromatography (TLC) was used to examine global methylation of
CCGG sites of genomic sperm DNA as described elsewhere
(Doerksen et al, 2000). Quantification was done by phosphorimager.
Statistical Analyses
Data were examined statistically using analysis of variance with Dunnett's
correction for pairwise comparisons or Student's 2-sided t test.
Fisher's exact test was used to analyze embryo data. The level of significance
used was P < .05 for all analyses (Sigma Stat; SPSS, Chicago,
Ill).
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Results |
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The effects of 5-azaCdR on the hematologic system are summarized in the Table. Hemoglobin, a measure of red blood cell numbers, and WBC counts were not significantly altered by treatment with 5-azaCdR.
Effects on the Male Reproductive System
As an accessory sex organ, the seminal vesicles are very sensitive to
changes in androgen status. The administration of 5-azaCdR did not appear to
alter hormone levels: seminal vesicle weights were unaffected after 7 weeks of
5-azaCdR treatment.
As illustrated in Figure 1A, testis weights declined by 35% and 55% after 7 weeks of treatment with 0.1 and 0.15 mg/kg 5-azaCdR, respectively. Although a significant decrease in testis weight (15%) was observed for Dnmt1c/+ animals treated with 0.1 mg/kg 5-azaCdR, this decline was significantly less (P < .05) than the decrease seen in Dnmt1+/+ mice treated with the same dose.
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Effects on Testicular Histology
To assess the histological consequences of 5-azaCdR administration in mouse
testis, detailed morphological examinations were conducted. A tubule was
considered abnormal if it possessed any of the following characteristics:
multinucleate/giant cells, degenerating germ cells, vacuole formation,
disorganization of germ cells, sloughing of immature germ cells, or a lack of
at least one germ cell population.
Considerable histological abnormalities were first noted in the testes of Dnmt1+/+ males treated with 0.1 mg/kg 5-azaCdR (Figures 1B and 2C); tubules in mice given this dose displayed disordered germ cell associations as well as vacuolization and multinucleate cells, which are indications of ongoing germ cell death. Seventy-five percent of tubules were abnormal after 7 weeks of treatment with 0.15 mg/kg 5-azaCdR (Figure 1B). Here, various germ cell populations were often absent, and sloughing of germ cells and vacuolization were also observed (Figure 2E). Of interest, saline-treated Dnmt1c/+ males had a higher baseline level of tubule abnormalities than their Dnmt1+/+ counterparts (Figure 1B); defects such as vacuolization and general germ cell disorganization were at an increased frequency compared with wild-type saline control mice. In direct contrast to the effect observed in Dnmt1+/+ males, there was no appreciable increase of histological abnormalities in Dnmt1c/+ males given 0.1 mg/kg 5-azaCdR compared with their saline controls (Figure 2B, D, and F).
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Sperm Counts
Reduced testicular weights were coupled with significant decreases of 33%
(0.1 mg/kg) and 66% (0.15 mg/kg) in sperm number, per gram testis weight, of
Dnmt1+/+ male mice
(Figure 1C). In keeping with
the histological results, sperm counts of
Dnmt1c/+ male mice treated with saline or 0.1
mg/kg 5-azaCdR were not significantly different.
Effects on Progeny Outcome
Effects of 5-azaCdR treatment on pregnancy outcome were assessed by mating
2 CD1 female mice with each male. Mating behavior was similar for all
treatment groups; the number of sperm-positive females, per male, was not
affected at any dose (data not shown). However, although the pregnancy rate
remained the same as that in control mice in the 0.05 mg/kg group, it
decreased in female mice mated with Dnmt1+/+
males treated with 0.1 mg/kg (67%) and fell dramatically, to 0, with female
mice mated with males exposed to 0.15 mg/kg 5-azaCdR
(Figure 3A). In direct contrast
to the wild-type groups, the pregnancy rate did not differ between female mice
mated with Dnmt1c/+ males administered saline or
0.1 mg/kg 5-azaCdR (Fig. 3A);
indeed, the pregnancy rate for both groups was identical (100%). Mating of
female mice with Dnmt1+/+ males exposed to 0.1
mg/kg 5-azaCdR resulted in a considerably lower pregnancy rate than in those
females mated with Dnmt1c/+ males given the same
dose. In analyzing full-term pregnancies, a number of parameters were
ascertained, including the number of corpora lutea, implantations, resorptions
and live fetuses; litter size; sex ratios; fetal and placental weights (as
well as those <75% or >125% of mean weight); and preimplantation and
postimplantation loss. With the exception of preimplantation loss, none of
these factors was significantly different than those for control mice.
Regardless of genetic background, treatment with 0.1 mg/kg 5-azaCdR resulted
in significantly increased preimplantation loss (27%) compared with saline
controls (Dnmt1c/+, 6% and
Dnmt1+/+, 8%)
(Figure 3b).
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Methylation of Genomic Sperm DNA
The TLC/end-labeling assay is an established method to examine global
methylation within CCGG sites of genomic DNA and was used to determine changes
in overall methylation status caused by 7 weeks of treatment with 5-azaCdR
(Figure 4). Treatment with
5-azaCdR resulted in a dose-related decline in sperm DNA methylation; however,
only treatment with the highest dose (0.15 mg/ kg) elicited a significant
decrease (29%) in sperm DNA methylation (P < .01). Of interest,
treated Dnmt1+/+ and
Dnmt1c/+ males exhibited similar decreases in DNA
methylation of about 15% (Fig.
4).
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Discussion |
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Germ Cells Affected
Because of the precise timing of spermatogenesis
(Clermont, 1972), it is
possible to determine the germ cell types exposed from the onset of drug
treatment. After 7 weeks of treatment, mature spermatozoa had been exposed to
5-azaCdR throughout the course of spermatogenesis. It was the ability of these
sperm to fertilize that was tested when males were mated with untreated
females. In keeping with our results, rats exposed throughout spermatogenesis,
a regimen that targeted mitotic, meiotic, and postmeiotic germ cells
simultaneously, exhibited significant abnormal germ cell development
(Doerksen and Trasler, 1996;
Doerksen et al, 2000). Raman
and Narayan (1995)
demonstrated, with 5-day neonatal mice, that both 5-azacytidine and 5-azaCdR
inhibited the differentiation of spermatogonia to spermatocytes; experiments
in the rat have also shown that 5-azacytidine targets rapidly dividing cells
(Doerksen et al, 2000).
Effects of Cytosine Analogues on General Health
Similar to the results of rat studies with 5-azacytidine, there were
minimal effects of 5-azaCdR treatment on body weight in our study, which
suggests that the general health of the mice was unaffected. The absence of
treatment-induced reductions in seminal vesicle weights, together with
apparently normal mating behavior, as indicated by a similar number of
plug-positive females in the treated and control groups, suggests that
testosterone production was not significantly altered. Thus, it is likely that
the dose-dependent reduction in testis weight resulted from germ cell loss
rather than altered testosterone production, given that germ cells normally
account for 50% to 70% of testicular and epididymal weights. Unlike the rat
(Doerksen and Trasler, 1996), the treatment of male mice with 5-azacytidine (Kelly et al, unpublished data)
or 5-azaCdR did not affect hemoglobin levels or WBC numbers. Bone marrow, like
the testis, is a highly replicating tissue and is often affected by treatments
that alter germ cell numbers. Together, the lack of treatment effects on body
weight, weight gain, organ weights (other than the testis), and the
hematologic system indicates that, at the doses we chose, the effects of
cytosine analogues are highly selective for the mouse testis.
Progeny Outcome
To investigate the consequences of paternal treatment on progeny outcome,
males treated for 7 weeks were mated, and females were killed at 19 dpc.
Similar to rat studies, we observed a significant increase in preimplantation
loss but not in postimplantation loss. Moreover, there was a sharp
dose-related decline in the percentage of plug-positive females that became
pregnant after mating with Dnmt1+/+ males.
Treatment with 0.1 mg/kg 5-azaCdR resulted in a threefold increase in preimplantation loss in both the Dnmt1+/+ and Dnmt1c/+ groups. The etiology of pregnancy failure could have been due to the inability of treated sperm to fertilize or to the failure of normal embryo development after fertilization. Transcription in mouse embryos is believed to initiate in the paternal pronucleus as early as the late 1-cell stage (Bouniol et al, 1995); it follows that any defect in the paternal genome induced by 5-azaCdR could have deleterious consequences on embryo viability and normal development.
DNA methylation is intimately linked to gene expression. Alterations in methylation, such as those seen after the exposure of mouse male germ cells to 5-azaCdR, could affect gene expression in developing sperm and the resultant embryos. Although not statistically significant, perhaps the hypomethylation at the 0.1 mg/kg 5-azaCdR dose is adequate to compromise the ability of some sperm to participate in fertilization or early embryo development. Imprinted genes have been implicated in embryogenesis, and it is likely that DNA methylation plays a role in the establishment or maintenance of genomic imprinting (Reik et al, 2001). Thus, any disruption of the methylation patterns of imprinted genes could alter the expression of paternally imprinted genes and interfere with early embryo development. Current investigations examining the effect of paternal treatment with 5-azaCdR on preimplantation embryo development should shed some light on this issue.
Combination of Cytosine Analogues and Dnmt1 Deficiency
We proposed that 5-azaCdR and Dnmt1 deficiency might act
synergistically to lower genomic methylation levels in the male germ line.
Mice homozygous for deficiencies in one or more of the known DNA
methyltransferases exist; however, these animals are inappropriate for studies
of male germ line methylation, because most of them die before midgestation,
prior to the onset of spermatogenesis (Li
et al, 1992; Okano et al,
1999). Moreover, the existing Dnmt-deficient mouse models
may not reflect the full extent of the Dnmt family; it has been
suggested that additional enzymes may exist. Here, use of the hypomethylating
drug 5-azaCdR offered an advantage. This agent is incorporated into
replicating DNA and can then inhibit cytosine methylation by binding both
known, and presumably unknown, methyltransferases. The combination of
hypomethylating drugs with mice deficient in DNMT1, the principal mammalian
methyltransferase, provides an interesting approach by which to reduce
methylation levels and avoid toxicity of high-dose drug treatment.
Despite differing DNMT1 levels, both Dnmt1+/+ and Dnmt1c/+ mice treated with 0.1 mg/kg 5-azaCdR responded similarly with respect to sperm DNA methylation and preimplantation loss. Yet treated Dnmt1c/+ mice appeared to be resistant to other deleterious effects of 5-azaCdR. Dnmt1c/+ males displayed a smaller reduction in testis weight and considerably less histological abnormalities; furthermore, the pregnancy rate was not affected. These results suggest less germ cell toxicity in the treated Dnmt1c/+ male mice. Our results are consistent with the findings of Jutterman et al (1994), who demonstrated that 5-azacytidine's primary toxicity may be mediated via the formation of "toxic" covalent adducts with DNMT1. Dnmt1c/+ males may be more resistant to the cytotoxic effects of these drugs, because they possess only half the wild-type level of DNMT1, and the chances of adduct formation are thus reduced. Our findings here suggest that Dnmt1c/+ mouse testes are more resistant to the toxic effects of cytosine analogues, possibly because of the drug's mechanism of toxicity. We propose that the adverse consequences of 5-azaCdR in Dnmt1+/+ mice may be due to a combination of drug toxicity (through the formation of toxic adducts) and hypomethylation (secondary to the inhibition of DNMTs bound to 5-azaCdR incorporated into DNA), whereas decreased methylation itself may explain the majority of the effects observed in Dnmt1c/+ mice.
Furthermore, the dose-related effects of 5-azaCdR on mouse sperm DNA methylation were reminiscent of results seen in previous rat studies with 5-azacytidine. CpG methylation occurs at 3 x 107 sites throughout the mammalian genome, and the TLC assay assesses only those CpGs within CCGG sites. CCGG sites represent only about 5% of the total number of CpGs that become methylated. To detect gene-specific alterations in methylation and effects at low doses, more sensitive techniques such as restriction landmark genome scanning (RLGS) may be needed. In a recent study that compared old and young rats, RLGS revealed alterations in DNA methylation that were not detected by the TLC assay (Oakes et al, 2003).
Mechanisms of Germ Cell Damage by Cytosine Analogues
The mechanism of action of this cytosine analogue is complex and may
involve many events associated with a reduction in DNA methylation, including
altered gene expression, alterations in chromatin structure, chromosome
rearrangements, the induction of apoptosis, and abnormal genomic imprinting.
The treatment of cell cultures with 5-azacytidine results in the expression of
normally silent genes (Jones et al,
1982; Eversole-Cire et al,
1993; Jones,
1995), chromatin decondensation, and micronuclei formation
(Schmid et al, 1984;
Davidson et al, 1992;
Stopper et al, 1995). The
reactivation of genes may involve the formation of a more open chromatin
structure, as has been shown by increased sensitivity to nucleases in
5-azacytidine-treated cultures (Litt et
al, 1997). Similarly, the treatment of cells with the histone
deacetylase inhibitor trichostatin-A has been shown to increase the expression
of imprinted genes (Cameron et al,
1999). DNA methylation and histone deacetylation are intrinsically
linked and together help form the closed chromosomal conformation
characteristic of silenced gene areas (heterochromatin)
(Jones et al, 1998; Nan et al, 1998;
Cameron et al, 1999); if one of
these elements is disrupted, so in turn may be the other. Understanding the
mechanisms underlying the effects of cytosine analogues on male germ cells,
with or without DNMT deficiency, will clearly require an examination of their
effects on end points such as gene expression, chromatin structure, and
chromosomal instability. The fact that the results of both rat and mouse
studies have suggested that mitotic and meiotic male germ cells are affected
by cytosine analogues is consistent with drug effects on any one of these end
points. To date, most studies on the mechanisms of action of cytosine
analogues have been done in cultured cells, and none have been done in germ
cells. Our studies provide an in vivo model to study cellular effects of
cytosine analogues in male germ cells.
Implications
Recently, there has been a resurgence of interest, and encouraging results,
in the use of cytosine analogues to treat various diseases, including sickle
cell anemia and malignant hematologic disease, such as acute myelogenous
leukemia (Koshy et al, 2000;
Wijermans et al, 2000;
Silverman et al, 2002). The
results of the experiments described here have implications for the clinical
use of such drugs. The treatment of these diseases involves chronic drug
courses as either the primary therapy, or in conjunction with other drugs, at
doses equal to or greater than those used in our study
(Koshy et al, 2000;
Wijermans et al, 2000). The
fact that effects on spermatogenesis are similar in rats and mice suggests
that adverse effects on male germ cells will also be seen in humans. With
increasing implications of the role of epigenetic factors such as DNA
methylation and chromatin structure in germ cell and embryo development, we
suggest that a thorough study of the mechanisms underlying the germ cell
effects of cytosine analogues is warranted.
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Footnotes |
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We are grateful to Tonia Doerksen for her help in the conception of this project and to Daniel Leclerc and Liyuan Deng for establishing the assay to genotype the Dnmt1c/+ mice. We thank Eric Simard and Xinying He for their superb technical assistance.
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