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Departments of Pediatrics, Pharmacology and Therapeutics, and Human Genetics, McGill University, and McGill University-Montreal Childrens Hospital Research Institute, Montréal, Québec, Canada H3H 1P3
Address all correspondence and requests for reprints to: Jacquetta M. Trasler, M.D., Ph.D., McGill University-Montreal Childrens Hospital Research Institute, 2300 Tupper Street, Montréal, Québec, Canada H3H 1P3. E-mail: mdja{at}musica.mcgill.ca
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Introduction |
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Genomic methylation patterns differ greatly in male and female germ cells (3, 7). In the male germ line, DNA methylation patterns undergo changes in both mitotic spermatogonia and postreplicative germ cells (8, 9, 10, 11). More recently, gamete-specific differences in methylation patterns have been found at imprinted loci, where they have important implications for allele-specific gene expression in the offspring (12, 13). The functional importance of gamete-specific differences at other loci is poorly understood, but has been proposed to play a role in normal germ cell development (6). Dnmt1 is highly regulated during both spermatogenesis (14, 15, 16, 17) and oogenesis (18), providing further evidence of a role for DNA methylation in gametogenesis. Dnmt1 messenger RNA and protein are expressed at high levels in mitotic and early meiotic male germ cells (14, 19, 20); the enzyme is translationally down-regulated in pachytene spermatocytes (14, 17, 18). The presence of Dnmt1 in mitotic spermatogonia is consistent with a role for the enzyme in maintaining DNA methylation patterns at the time of replication and possibly also of establishing new patterns. Dnmt1 protein is also abundantly expressed in postreplicative leptotene/zygotene spermatocytes, suggesting a role for the enzyme during meiotic prophase in male germ cell development (17, 18).
If DNA methylation plays an important role in normal germ cell development and the establishment of allele-specific imprints in the germ line, then altering DNA methylation levels during gametogenesis would be predicted to be deleterious to germ cells and their function in fertilization and embryo development. In previous work, in an approach designed to decrease DNA methylation levels in germ cells, we exposed male rats to low chronic doses of 5-azacytidine, a drug that inhibits Dnmt(s) and causes decreases in DNA methylation (21, 22, 23). After 4 weeks of treatment of male rats with 5-azacytidine, resulting in the exposure of spermatids and spermatozoa, epididymal sperm counts were unaffected in the males, and embryo development was normal when the males were mated with untreated females (24). In contrast, both abnormal germ cell and embryo preimplantation development were found when the males were treated for longer periods of time (11–16 weeks), a duration of treatment that resulted in exposure of mitotic, meiotic and postmeiotic germ cells. In studies by another group, 5-azacytidine and its analog 5-aza-2'-deoxycytidine were administered to 5-day-old neonatal mice and found to inhibit differentiation of spermatogonia to spermatocytes (25).
The present study was designed to determine whether both mitotic spermatogonia and meiotic spermatocytes are affected by 5-azacytidine, and the mechanisms that account for effects of the drug on sperm numbers and function. Testicular morphology, germ cell DNA methylation, and pregnancy outcome were compared in males treated with 5-azacytidine for 6 weeks (exposure of spermatocytes, spermatids, and sperm) with those in males treated for 11 weeks (exposure of all germ cell types including mitotic spermatogonia). Our results indicate that chronic treatment of male rats with 5-azacytidine results in the demethylation of sperm DNA and suggests that both spermatocytes and spermatogonia are affected by the drug exposure.
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Materials and Methods |
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Treatment groups and mating
Male rats were randomly assigned to one of three treatment
groups. Rats received ip injections of 1 ml/kg three times a week for 6
or 11 weeks, of saline (n = 8 rats/group), or of 5-azacytidine
(Sigma, St. Louis, MO) at doses of 2.5 or 4.0 mg/kg
(n = 6 rats/group). Males in all groups were weighed twice weekly.
For the assessment of progeny outcome, six rats in each group were
mated overnight with two females in proestrous, as determined by
vaginal smears. After mating, males were killed, and tissues were
collected for testicular and epididymal histology studies and
epididymal sperm DNA methylation analysis. A separate group of males
(n = 4 rats/group) was treated for 6 weeks with saline or 2.5 or
4.0 mg/kg 5-azacytidine, and the testes were used for the preparation
of isolated germ cells for DNA methylation analysis.
Analysis of pregnancy outcome
Evidence of mating was obtained by examining vaginal smears for
the presence of spermatozoa. Females were killed on day 20 of gestation
(day 0 = morning when spermatozoa were found in the vagina).
Ovaries were removed, and corpora lutea were counted. The uteri were
opened, and the numbers of implantations, resorptions, and live fetuses
were determined. Preimplantation loss was determined by subtracting the
number of implantations from the number of corpora lutea to determine
the number of oocytes released that were not fertilized or were
fertilized but died before implantation. Postimplantation loss was
recorded as the difference between the number of implantation sites on
the uterus and the number of live fetuses. Fetuses were weighed, sexed,
and examined for gross malformations.
Histology
At the end of 6 or 11 weeks, males (n = 3/group) were
perfused through the abdominal aorta with Bouin’s fixative (BDH, Inc.,
Toronto, Canada) to prepare the right testes and epididymides for
histological examination. Before perfusion of each rat, the left testis
and epididymidis were ligated and removed. Both organs were weighed.
The testis was snap-frozen and stored at -80 C, and the cauda
epididymidis was used to isolate mature sperm. After perfusion, the
right testes were immersed in Bouin’s solution for 24 h, then
dehydrated and embedded in paraffin. Sections for histology (5 µm)
were cut, mounted on glass slides, deparaffinized with xylene, and
stained with hematoxylin and eosin. Slides were viewed on a Carl Zeiss Axiophot photomicroscope (New York, NY), and photographs
were taken using Kodak Tmax 400 ASA (Eastman Kodak Co., Rochester, NY) black and white print film. Analysis of
abnormal seminiferous tubule cross-sections was performed using a
series of photographs of testicular cross-sections from each animal.
The numbers of morphologically normal and abnormal tubule
cross-sections were counted; the number of abnormal tubule
cross-sections was expressed as a percentage of the total tubules
examined (81–172 tubules counted/animal).
Detection of apoptotic germ cells
Sections (6 µm) of Bouin’s-fixed testes embedded in paraffin
were used for terminal deoxynucleotidyl transferase-mediated deoxy-UTP
nick end-labeling (TUNEL) staining for in situ detection of
apoptosis using an Apoptag kit (Oncor, Gaithersburg, MD). Briefly,
after deparaffination and rehydration, sections were incubated with
proteinase K (20 µg/ml; ICN Biomedical Corp., Irving,
CA) in PBS, pH 7.4, for 15 min at room temperature, washed in distilled
water, and treated with 2% hydrogen peroxide in PBS for 30 min at room
temperature to quench endogenous peroxidase activity. Sections were
incubated with terminal deoxynucleotidyl transferase, supplied by the
Apoptag kit, at 37 C for 90 min in a humidified chamber.
Digoxigenin-UTP-labeled DNA was detected with
antidigoxigenin-peroxidase antibody, followed by peroxidase detection
with 0.05% diaminobenzidine (Sigma) and 0.02% hydrogen
peroxide in PBS for 6 min at room temperature. Slides were washed and
counterstained with Mayer’s hematoxylin for 40 sec; dehydrated in
70%, 95%, and 100% ethanol; cleared in xylene; and mounted with
Permount (Fisher Scientific, Fairlawn, NJ). Analysis of
TUNEL staining and photography was performed using a Carl Zeiss Axiophot photomicroscope. The seminiferous tubule
cross-sections were staged and divided into 4 groups (I–IV, V–VII,
VIII–X, and XI–XIV) (26), and TUNEL-stained cells were counted in
each staged tubule cross-section and identified as
spermatogonia/spermatocytes or spermatids by virtue of their location
in the seminiferous epithelium and by the features of surrounding
cells. At least 300 seminiferous tubule cross-sections from 3 different
regions of the testis, separated by 250 µm, from each of 3
males/treatment group were analyzed for apoptotic cells.
Germ cell isolation and sperm counts
Spermatozoa were obtained from the cauda epididymides using a
modification of previously described procedures (27). Briefly, the
cauda epididymides were slit open in PBS, pH 7.4, and agitated for 10
min at room temperature to allow release of spermatozoa. The
supernatant was removed, and cauda epididymides were again agitated in
PBS. After pooling, the supernatants from the two agitation steps were
spun at 1500x g for 15 min at 4 C. The sperm pellet was
washed five times in 0.45% NaCl to lyse somatic cells, and sperm were
centrifuged between washes at 4000 x g for 5 min at 4
C. The final pellet was snap-frozen at -80 C. Purified populations of
male germ cells were obtained from the testes by cellular sedimentation
at unit gravity on 2–4% BSA gradients as described previously (17, 28). Populations of pachytene spermatocytes (average cell purity:
control, 84.1 ± 5%; 2.5 mg/kg 5-azacytidine, 80.7 ± 3%;
4.0 mg/kg 5-azacytidine, 79.7 ± 3%), round spermatids (average
purity: control, 88.0 ± 1%; 2.5 mg/kg, 87.9 ± 1%; 4.0
mg/kg, 85.8 ± 2%), and residual bodies/cytoplasts (average
purity: control, 85.2 ± 2%; 2.5 mg/kg, 82.8 ± 2%; 4.0
mg/kg, 81.9 ± 3%) were isolated from both testes of each treated
rat (n = 4 rats/group) and snap-frozen at -80 C.
For assessment of sperm counts, the right testis and caput/corpus epididymidis were homogenized (Polytron, Brinkmann Instruments, Inc., Westbury, NY; setting 10) for three 15-sec periods, separated by 10-sec intervals, in 5 ml 0.9% NaCl, 0.1% thimersal, and 0.5% Triton X-100. Hemocytometric counts of condensed spermatids and/or spermatozoa were performed as described previously (29).
Preparation of DNA and quantification of methylated cytosine
in sperm and germ cell DNA
The methylation status of CCGG sites was determined using a
method previously described (4, 30). To isolate DNA, cauda sperm were
resuspended in 0.5–1.0 ml lysis buffer containing 20 mM
Tris (pH 8), 10 mM dithiothreitol, 150 mM NaCl,
10 mM EDTA (pH 8), and 1% SDS, then 12.5–25 µg
proteinase K (Roche Molecular Biochemicals, Mannheim,
Germany) were added to each sample. Samples were incubated at 37 C for
16–18 h, then DNA was extracted with phenol-chloroform and ethanol
precipitated. DNA was pulled out of the ethanol using flamed glass
Pasteur pipettes to prevent DNA breakage, then was resuspended in water
and incubated at 65 C for 5–10 min to aid in dissolution. DNA
isolation from germ cells was performed as described above, except the
lysis buffer consisted of 10 mM Tris (pH 8), 100
mM NaCl, 25 mM EDTA, and 0.5% SDS. Proteinase
K (0.1 mg/ml) was added to each sample, and samples were incubated at
50 C for 12–18 h. Digestion of DNA with the restriction enzyme
MspI cuts DNA at CCGG sequences regardless of the state of
methylation of the central cytosine and results in 5'-terminal
nucleotides that are mixtures of methylated and unmethylated cytosine
residues. By end labeling the restriction fragments and separating
nucleotides by TLC, it is possible to measure radioactivity in
unmethylated and methylated cytosine nucleotides. The
methylation-sensitive isoschizomer HpaII, which digests
CCGG sites only when unmethylated, allows labeling of only the
unmethylated cytosines and is used as a control for the integrity of
the isolated DNA; no signal should be seen in the methylated cytosine
position on TLC plates unless nonenzymatic cleavage of DNA has
occurred. Briefly, 1 µg purified germ cell DNA was treated with 2
µg ribonuclease A (Sigma) and 50 U ribonuclease T1
(Life Technologies, Inc., Burlington, Canada) for 1 h
at 37 C, then digested with either HpaII or MspI
(Life Technologies, Inc.) under conditions recommended by
the manufacturer. Digested DNA was treated with 1 U alkaline
phosphatase (Life Technologies, Inc.), then purified by
extraction with phenol-chloroform, and ethanol precipitated. The 5'-
termini were end labeled with [
-32P]ATP
(ICN Radiochemicals, Inc.) using T4 kinase (Life Technologies, Inc.) for 1 h at 37 C, and unincorporated isotope was
removed by centrifugation on Microspin S-300 HR columns
(Pharmacia Biotech Baie d’Urfe, Canada). The void volume
was diluted with 10 mM Tris (pH 7.4), 1 mM EDTA
(pH 8.0) buffer, and the DNA was ethanol precipitated and
dissolved in a solution containing 30 mM sodium
acetate (pH 5.3) and 0.1 mM
ZnCl2. Nuclease P1 (Amersham Pharmacia Biotech, Toronto, Canada) was added to a concentration of 100
µg/ml, and the samples were incubated at 65 C for 2 h. The DNA
was ethanol precipitated, resuspended in 10 µl distilled water, and
spotted on 20 x 20-cm Cel 300–25 cellulose TLC plates (Alltech
Associates, Inc., Deerfield, IL). The plates were developed in
isobutyric acid-water-ammonium hydroxide (66:33:1) and allowed to dry
overnight. Results were quantified using a phosphorimager (Fujix BAS
2000 Bioimaging analyzer, Fuji Medical Systems USA, Inc., Stamford,
CT).
Protein extraction and Western blot analysis
Lysates of purified germ cells were prepared by homogenization
in 0.15 M NaCl, 0.05 M Tris-HCl (pH 7.5), 2
µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml
phenylmethylsulfonylfluoride. Aliquots (100 µg) were denatured by
heating at 100 C in 2% SDS, electrophoresed on 10% acrylamide gels,
and transferred to nitrocellulose membranes. Membranes were incubated
for 4 h with the anti-pATH52 rabbit polyclonal antibody against
the DNA methyltransferase, Dnmt1 (1:10 000), as described previously
(4, 17), and developed using the Protoblot Western Blot AP System from
Promega Corp. (Madison, WI).
Statistical analysis
Data were examined using ANOVA with Dunnett’s correction for
pairwise comparison or by Kruskal-Wallis nonparametric test (31). Where
only two groups were compared, Student’s two-sided t test
was used. The level of significance was P 
0.05 for
all analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). After 6 weeks of 5-azacytidine
treatment, testis weight was not significantly affected; however,
epididymal weights were lower than control values for both drug
treatment groups. Testicular and epididymal sperm counts did not differ
between the control and treated rats in the 6-week treatment group
(Table 1). In contrast, treatment of rats for 11 weeks resulted in a
21% decrease in testis weight with the high dose of 5-azacytidine and
16% and 32% decreases in epididymal weight with the low and high
doses of 5-azacytidine, respectively, compared with control values.
Similar decreases in testis and epididymal weights were found in our
previous 11-week study with doses of 2.5–5.0 mg/kg (three times per
week) 5-azacytidine (24). In the earlier 11-week study, decreases
in epididymal and testicular weight were associated with decreases in
epididymal and testicular sperm numbers (24).
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). Preimplantation loss, representing
either death of embryos before implantation or failure of sperm to
fertilize the released ova, was 4.8-fold higher than control values
when males were treated with the high dose of 5-azacytidine (Fig. 1B).
Rates of postimplantation loss were similar in all groups (Fig. 1C).
The average incidence of gross morphological abnormalities in the pups,
such as increased or decreased number of digits, cleft palate, neural
tube defects, edema, as well as low or high fetal birth weights (<75%
or >125% of average weight for a litter), was very low, and did not
differ among treatment groups (data not shown). There were no
differences in fetal weights, average incidence of high or low
placental weights (>125% or <75% of average weight for the litter),
or altered male/female ratios among the treatment groups (data not
shown).
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, B and D–F). The morphological
appearance of spermatogonia, spermatocytes, spermatids, and spermatozoa
was similar in the testes of the saline- and 5-azacytidine-treated
rats; no abnormalities were detected in Sertoli or Leydig cells, and
the interstitium appeared normal. Eleven weeks of treatment with 2.5
mg/kg 5-azacytidine resulted in few visible abnormalities. All stages
of the seminiferous epithelium appeared to be present, with normal
cellular associations, and the most common abnormality observed was
vacuole formation in some of the seminiferous tubule cross-sections. In
contrast, a number of abnormalities in the seminiferous epithelium were
evident when males were treated with 4.0 mg/kg 5-azacytidine for
11 weeks (Fig. 2, C and G–I). After high dose exposure, a number of
defects were seen, including multinucleated giant cell formation (Fig. 2G), vacuole formation (Fig. 2H), disorganization of germ cell types,
sloughing of immature germ cells into the lumen, degeneration of germ
cells (Fig. 2I), and, sometimes, complete atrophy of tubules. Eleven
weeks of treatment with the high dose resulted in a statistically
significant increase in the average number of abnormal tubule
cross-sections as a percentage of total tubule cross-sections observed
(saline, 2.9 ± 0.9%; 2.5 mg/kg 5-azacytidine, 5.4 ±
2.4%; 4.0 mg/kg 5-azacytidine, 47.3 ± 14.3%; P
0.05). Tubules were considered abnormal if they contained vacuoles,
multinucleated giant cells, degenerating cells, sloughing of immature
germ cells into the lumen, disorganization of germ cells within the
seminiferous epithelium, or atrophy of the tubule.
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In situ detection of apoptosis in testicular germ cells of
5-azacytidine-treated rats
Although testicular histology and germ cell morphology in animals
treated with 5-azacytidine for 6 weeks appeared normal at the light
microscopic level, sperm from these animals did not function normally,
resulting in increases in preimplantation loss after mating with normal
females. Furthermore, 5 more weeks of treatment resulted in dramatic
changes in the seminiferous tubules, with multiple histological and
morphological abnormalities. Thus, we looked closer at the morphology
of testicular germ cells using the TUNEL method to detect apoptotic
cell death. This in situ detection method identifies
apoptotic cells by staining their nuclei dark brown and has been used
previously to detect apoptosis in the rat testes (32, 33, 34). Quantitative
analysis of TUNEL-stained testes resulted in a statistically
significant increase in the total number of apoptotic cells per 100
tubules in rats treated for 6 and 11 weeks with 4.0 mg/kg 5-azacytidine
over the saline-treated control value (Table 2). Furthermore, 6 weeks of treatment
resulted in a 2.3-fold increase in apoptotic premeiotic cells
(spermatogonia and spermatocytes) and a 3.3-fold increase in apoptotic
spermatids (Table 2). Compared with testes from saline-treated rats,
the increase in germ cell apoptosis after 6-week exposure was found
primarily in stages I–IV (Figs. 3 and 4, A and B). Stages V–XIV were not
significantly affected by drug treatment. The early (I–IV) and late
(XI–XIV) stages show the highest incidence of spontaneously occurring
apoptosis, as previously found (32, 33). Eleven weeks of treatment,
resulting in an 8.8-fold increase in germ cell apoptosis (Table 2), was
difficult to evaluate on a stage-specific and germ cell-specific basis
due to multiple abnormalities of the seminiferous tubules. Many
individual cells within tubules stained positively (Fig. 4C) as well as
some, but not all, multinucleated giant cells (Fig. 4D).
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). Sperm DNA methylation was
unaffected after 6 weeks of 5-azacytidine treatment, maintaining levels
similar to those in controls (Fig. 6). In
contrast, 11 weeks of treatment with the 2.5 and 4.0 mg/kg doses of
5-azacytidine resulted in 22% and 29% decreases, respectively, in
CCGG methylation of sperm DNA (Fig. 5). To confirm the findings and
determine whether DNA methylation was affected in earlier germ cells,
DNA methylation levels were also assessed in purified populations of
pachytene spermatocytes and haploid round spermatids from treated and
control rats. Rats treated with 5-azacytidine for 6 weeks were chosen
because germ cells appeared normal at this time. Overall methylation
levels of DNA from control pachytene spermatocytes and round spermatids
were similar to levels found in epididymal spermatozoa. Six weeks of
treatment resulted in 32% and 26% decreases in CCGG methylation of
pachytene spermatocytes exposed to the low and high doses of
5-azacytidine, respectively. There was no detectable change in round
spermatid or sperm DNA methylation after 6 weeks, suggesting that
5-azacytidine is incorporated into dividing spermatogonia to affect
overall levels of DNA methylation. This is the time during
spermatogenesis when Dnmt1 is most highly expressed (17, 18).
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Due to the tightly controlled kinetics of spermatogenesis in the rat, we were able to use specific treatment times to expose different populations of germ cells to 5-azacytidine (35). We used a similar approach to identify the effects of chronic cyclophosphamide exposure on defined germ cell types (36, 37). Six weeks of treatment exposes meiotic germ cells through to mature epididymal sperm. In the current study the 6-week treatment with the 4.0 mg/kg dose of 5-azacytidine resulted in increased preimplantation loss when rats were mated with normal females. In our previous study, with the 11-week treatment an increase in preimplantation loss was seen after administration of doses of 2.5, 4.0, and 5.0 mg/kg (24). Detailed follow-up studies of the mechanisms underlying the 5-azacytidine-induced preimplantation loss have indicated that both a lack of fertilization and early embryo death contribute to the preimplantation loss seen after 6 and 11 weeks of paternal treatment (our manuscript in preparation).
The exact cellular mechanism of action of 5-azacytidine is not yet established, although it is thought to involve covalent trapping of Dnmt(s) and subsequent decreases in DNA methylation (22, 23). In the present study protein levels, by Western analysis, of the predominant mammalian Dnmt, Dnmt1, were not affected in the germ cells of the 5-azacytidine-treated rats. The germ cell hypomethylation in the light of apparently normal Dnmt1 levels could be due to the fact that Dnmt1 that is bound to 5-azacytidine is inactive, but is detected by Western blot; Dnmt1 activity assays would be one approach to testing this possibility.
It is not surprising that exposure of germ cells to 5-azacytidine throughout spermatogenesis results in altered sperm function, because Dnmt is important to mitotic cells for maintaining DNA methylation levels at the time of replication, and it may have a role in de novo methylation as well (5, 38). The role of Dnmt1 in early meiotic germ cells is unknown. It may play a role in DNA repair, as Dnmt1 is closely linked to the repair marker proliferating cell nuclear antigen (39), or it may be involved in the establishment or maintenance of genomic imprinting (7). Thus, exposure of meiotic germ cells to 5-azacytidine may cause alterations in repair mechanisms or genomic imprinting events that later result in dysfunctional sperm.
This report shows quantitatively that in vivo germ cell exposure to 5-azacytidine decreases genomic methylation at CCGG sites. The germ cell separation technique allowed us to measure DNA methylation in specific cells after 6 weeks of treatment. The DNA methylation decreases in pachytene spermatocyte DNA, but not in round spermatid or spermatozoal DNA, indicated that incorporation of the drug into dividing mitotic spermatogonia is required for detectable hypomethylation. Administration of a similar analog, 5-aza-2'-deoxycytidine, to neonatal mice with only spermatogonial germ cells has been shown to interfere with the differentiation of spermatogonia to spermatocytes through loss of DNA methylation; however, only the whole testis was examined (25). In the current study the degree of hypomethylation of DNA did not always correlate with the physiology and function of germ cells. Six weeks of treatment resulted in preimplantation loss, although methylation in mature sperm was at control levels. Other sites not detectable by our assay may have been undermethylated. Small or single site decreases in methylation, as might occur if 5-azacytidine interferes with repair mechanisms, may affect single gene function without changing the overall level of DNA methylation. These effects will be studied in closer detail using techniques that detect site-specific changes in methylation, such as bisulfite sequencing (40).
Testis histology after 11 weeks of 5-azacytidine exposure revealed numerous abnormalities in the seminiferous tubules, affecting all stages and germ cell types. Similar testicular pathology has been reported with numerous agents, including hormone disrupters, and certain DNA-damaging agents (41). This was expected after 11 weeks, assuming that 5-azacytidine is incorporated into mitotic germ cells and has its greatest effect on the maintenance methylation processes that take place early in spermatogenesis. Interestingly, 11 weeks of treatment with the same doses of 5-azacytidine used here resulted in alterations in fertility and early embryo development (24). After 6 weeks of 5-azacytidine exposure, we predicted that there would be abnormalities in spermatocytes and early spermatids, which would occur after incorporation of the drug into DNA during mitosis. The surprising lack of effect on testicular histology suggests that several rounds of division are necessary before abnormalities are seen, as would occur with stem cell exposure. Stem cell exposure appears to be necessary before seeing a decrease in germ cell DNA methylation in pachytene spermatocytes. We did not detect any decrease in round spermatid DNA methylation after 6 weeks of 5-azacytidine exposure, which would have exposed mitotic germ cells, but not stem cells. To confirm the effects of 5-azacytidine in altering DNA methylation in different types of spermatogonia, follow-up studies are required in which males are treated for enough time to expose the spermatogonia, the treatment is stopped, and DNA methylation is then examined once the treated cells reach the pachytene spermatocyte or round spermatid stage.
The effects of meiotic germ cell exposure to 5-azacytidine may be more complex than simply decreases in DNA methylation. Pachytene spermatocyte DNA methylation was decreased without detectable testicular pathology at the light microscopy level. Sperm from the males treated for 6 weeks had normal levels of genomic methylation; however, the sperm from these males did not appear to be completely normal functionally, as evidenced by the increases in preimplantation loss. We, thus, explored another possible mechanism of 5-azacytidine-induced disruption of sperm function using TUNEL detection in situ to detect apoptotic germ cells in sections of exposed testis. In situ 3'-end-labeling of internucleosomal DNA fragmentation is routinely used to detect apoptotic cells in various tissues, including testis. Spontaneous incidence of apoptosis is known to occur in the testis (32, 33) and is thought to be a way of eliminating cells with chromosomal abnormalities. Many agents induce apoptosis in male germ cells, including anticancer drugs (32, 42), GnRH antagonists (43), and events such as radiation (44), hypothermia (45), vasectomy (46), and cryptorchidism (47), although this list is certainly not exhaustive. 5-Azacytidine has been shown to induce apoptosis in various cell lines in culture (48), and when administered to pregnant mice, it induced fetal brain apoptosis (49). 5-Azacytidine could induce apoptosis either directly or indirectly through its ability to cause DNA demethylation. Findings of apoptosis in Dnmt1-deficient mice provide support for the indirect mechanism for 5-azacytidine. Mice deficient in the Dnmt1 enzyme, through targeted disruption of the Dnmt1 gene, have low levels of DNA methylation, die at midgestation, and show high levels of apoptosis (4) (Li, E., personal communication), suggesting that decreases in DNA methylation may lead to apoptosis. Both 6 and 11 weeks of treatment resulted in an increase in apoptotic germ cells, primarily in stages I–IV, and in both premeiotic cells and spermatids. We suggest that germ cell death may occur in animals treated with 5-azacytidine for 6 weeks, before the seminiferous tubule abnormalities that occur after 11 weeks of treatment. It has been postulated that apoptosis of germ cells plays a protective role, and that damaged cells are thus removed from spermatogenesis at specific checkpoints (50). Like many other DNA-damaging agents, 5-azacytidine induces apoptosis during those stages at which most of the spontaneous apoptosis occurs (32, 51). Germ cells exposed to 5-azacytidine may be recognized as damaged due to undermethylated DNA, which can lead to abnormalities in chromatin structure or gene expression and may persist if repair mechanisms have been impaired. As germ cell apoptosis occurs after 11 weeks of treatment at doses we have previously shown to result in abnormal preimplantation development (24), we speculate that some drug-damaged spermatozoa may have escaped cell death, resulting in abnormal progeny outcome.
In this study we have shown that there is an increase in preimplantation loss after meiotic germ cell exposure (6-week treatment) to 5-azacytidine, suggesting that the sperm may be functionally abnormal and unable to either fertilize eggs or produce normal preimplantation embryos. After 6 weeks of treatment, testes appear histologically normal, compared with after 11 weeks of treatment, when testes show multiple abnormalities; however, TUNEL staining of the testis of animals treated for 6 and 11 weeks suggests that germ cells are undergoing apoptosis. Germ cell separation techniques have allowed us to study DNA methylation levels in different populations of germ cells, and we have shown that after 11 weeks of treatment, mature epididymal sperm DNA is undermethylated, whereas after 6 weeks of treatment, only pachytene spermatocytes have decreased DNA methylation. We suggest that the events underlying the testicular toxicity of 5-azacytidine are decreased DNA methylation and germ cell apoptosis, and that these events precede alterations in testicular histology.
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Acknowledgments |
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Footnotes |
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2 Scientist of the Medical Research Council of Canada and Scholar of
the Fonds de la Recherche en Santé du Québec.
Received November 15, 1999.
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 T. Forges, P. Monnier-Barbarino, J.M. Alberto, R.M. Gueant-Rodriguez, J.L. Daval, and J.L. Gueant Impact of folate and homocysteine metabolism on human reproductive health Hum. Reprod. Update, May 1, 2007; 13(3): 225 - 238. [Abstract] [Full Text] [PDF]
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H.-C. Lee, Y.-M. Jeong, S. H. Lee, K. Y. Cha, S.-H. Song, N. K. Kim, K. W. Lee, and S. Lee Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility Hum. Reprod., December 1, 2006; 21(12): 3162 - 3170. [Abstract] [Full Text] [PDF]
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E. Seli and D. Sakkas Spermatozoal nuclear determinants of reproductive outcome: implications for ART Hum. Reprod. Update, July 1, 2005; 11(4): 337 - 349. [Abstract] [Full Text] [PDF]
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 M. Imamura, Y. Mochizuki, E. Engvall, and S. Takeda {varepsilon}-Sarcoglycan compensates for lack of {alpha}-sarcoglycan in a mouse model of limb-girdle muscular dystrophy Hum. Mol. Genet., March 15, 2005; 14(6): 775 - 783. [Abstract] [Full Text] [PDF]
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T. L.J. Kelly, O. R. Neaga, B. C. Schwahn, R. Rozen, and J. M. Trasler Infertility in 5,10-Methylenetetrahydrofolate Reductase (MTHFR)-Deficient Male Mice Is Partially Alleviated by Lifetime Dietary Betaine Supplementation Biol Reprod, March 1, 2005; 72(3): 667 - 677. [Abstract] [Full Text] [PDF]
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 T. L. J. Kelly, E. Li, and J. M. Trasler 5-Aza-2'-Deoxycytidine Induces Alterations in Murine Spermatogenesis and Pregnancy Outcome J Androl, November 1, 2003; 24(6): 822 - 830. [Abstract] [Full Text] [PDF]
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 C. C. Oakes, D. J. Smiraglia, C. Plass, J. M. Trasler, and B. Robaire Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats PNAS, February 18, 2003; 100(4): 1775 - 1780. [Abstract] [Full Text] [PDF]
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 J. Trasler, L. Deng, S. Melnyk, I. Pogribny, F. Hiou-Tim, S. Sibani, C. Oakes, E. Li, S. J. James, and R. Rozen Impact of Dnmt1 deficiency, with and without low folate diets, on tumor numbers and DNA methylation in Min mice Carcinogenesis, January 1, 2003; 24(1): 39 - 45. [Abstract] [Full Text] [PDF]
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