BIOLOGY OF REPRODUCTION 53, 561-569 (1995)
Regulated Synthesis and Localization of DNA Methyltransferase
during Spermatogenesis'
Kathleen Jue, 3 Timothy H. Bestor,4 and Jacquetta M. Trasler 2' 3
The McGill University-MontrealChildren'sHospitalResearch Institute and Departmentsof Pediatrics,
of Pharmacology& Therapeutics, and of Human Genetics,3 McGill University, Montreal, Quebec, Canada
Departmentof Genetics and Development,4 Columbia University, New York, New York
ABSTRACT
Differences in the methylation patterns of male and female gamete DNA are likely to be involved in genomic imprinting. However,
little is known of the mechanisms that regulate de novo methylation and demethylation during gametogenesis. We report here that the
well-characterized Mr 190 000 form of DNA methyltransferase (the only known form) is present inisolated mitotic, meiotic, and postmeiotic
male germ cells, with the exception of meiotic pachytene spermatocytes, where the protein is undetectable by immunoblot analysis and
a novel 6.2-kb DNA methyltransferase transcript is present. Whereas replication and methylation are coupled insomatic cells, the presence
of DNA methyltransferase in postreplicative male germ cells is consistent with previously observed de novo methylation events in these
cells. Immunofluorescence experiments revealed that DNA methyltransferase is localized to the nuclei of male germ cells, with a subset
of spermatogonia and postreplicative leptotene/zygotene spermatocytes displaying prominent nuclear foci that are strongly enriched in
DNA methyltransferase. The data suggest that down-regulation of DNA methyltransferase expression during the pachytene stage of
meiosis utilizes an unusual mechanism that is associated with the production of alarger mRNA, and that de novo methylation inleptotene/
zygotene spermatocytes may take place in spatially restricted nuclear domains that are enriched in DNA methyltransferase.
INTRODUCTION
printing arose from experiments with transgenic mice [1014] and has been strengthened by several examples of allele-specific methylation at endogenous imprinted loci [1518]. Methylation is essential for normal development: embryos homozygous for a targeted partial loss of function
mutation in the DNA methyltransferase (DNA MTase) gene
are abnormal and die at mid-gestation [19]. Imprinting is
relaxed in the mutant embryos, and the H19, Igf2, and Igf2r
genes no longer show allele-specific expression [20].
To date, only one form of DNA (cytosine-5)-methyltransferase (EC 2.1.1.37) has been identified in mammals. The
enzyme has a relative mass (M) of 190 000 and is present
in all proliferating cells. DNA MTase has been purified to
homogeneity from cultured mouse cells [21], and the cDNA
has been cloned and sequenced [22]. DNA MTase consists
of a 1000-amino acid N-terminal domain linked by a series
of alternating glycyl and lysyl residues to a 500-amino acid
C-terminal domain. The C-terminal domain, whose sequence closely resembles those of bacterial DNA (cytosine5)-methyltransferases, contains the proposed S-adenosyl Lmethionine binding site and the catalytic center. The
N-terminal domain contains a Zn-binding site [231 and a targeting sequence that directs DNA MTase to sites of DNA
replication in S-phase nuclei [24]. DNA MTase is capable of
methylating both unmethylated DNA (de novo methylation)
and hemimethylated DNA (maintenance methylation);
however, the intact enzyme prefers hemimethylated DNA
as its substrate [21, 25, 26].
Although de novo methylation is rarely observed in somatic cells, dramatic changes in methylation patterns occur
during gametogenesis and early embryogenesis [27-33].
Mammalian development requires both maternal and paternal genomes, as evidenced by nuclear transplantation
studies [1, 2] and breeding experiments with mice carrying
Robertsonian translocations [31]. Studies of sex-dependent
differences between mammalian genomes have been extended to the level of individual genes such as Igf2 [4], Igf2r
[5], and H19 [6], which have been found to exhibit differential expression depending on whether the gene was contributed by the maternal or paternal gamete [7]. This phenomenon, genomic imprinting, allows a cell to distinguish
the parental origin of a gene and is believed to be initiated
during gametogenesis, a time when the male and female
genomes are physically separated.
The molecular nature of the imprint is unknown, although DNA methylation at the 5-position of cytosine is a
strong candidate, due to its heritability (through maintenance methylation), reversibility (through demethylation),
and ability to affect transcription [8]. Other unknown epigenetic modifications as well as the involvement of modifiers, differences in replication timing, and altered chromatin structure are also postulated to be involved in
genomic imprinting [9]. A link between methylation and imAccepted April 13, 1995.
Received January 4, 1995.
'This work was supported by grants from the Medical Research Council of Canada (to
J.M.T.) and by NIH grants GM43565, GM00616, and CA60610 (to T.H.B.). J.M.T. was the
recipient of a Queen Elizabeth II Research Fund/Medical Research Council of Canada Scientist Award.
2
Correspondence: Dr. Jacquetta M. Trasler, The McGill University-Montreal Children's
Hospital Research Institute, 2300 Tupper Street, Montreal, Quebec, Canada H3H 1P3. FAX:
(514) 934-4331.
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JUE ET AL.
Male gametogenesis is a particularly advantageous system
for the study of de novo methylation and demethylation
because the process occurs in the adult, and large numbers
of purified germ cells at specific developmental stages can
be obtained. Previous studies have shown that methylation
patterns undergo changes both in replicating spermatogonia and also in postreplicative male germ cells
[27, 28, 31, 32]. It is shown here that the Mr 190 000 form of
DNA MTase is present in both replicative and postreplicative male germ cells, with a transient sharp down-regulation
in pachytene spermatocytes where crossing-over is occurring. At this time, the 5.2-kb mRNA that is present in nearly
all cells is replaced by a 6.2-kb mRNA that appears to be
translated only very inefficiently, if at all. Furthermore, the
subcellular localization of the enzyme was found to change
from a uniform nucleoplasmic distribution to concentration
at discrete nuclear foci or domains at specific times during
spermatogenesis. These DNA MTase-rich nuclear domains
may be the sites of active de novo methylation in postreplicative male germ cells.
MATERIALS AND METHODS
Isolation of Male Germ Cell Populations
Male CD-1 mice were obtained from Charles River Canada, Inc. (St. Constant, PQ). Purified populations of male
germ cells were obtained from the testes of 8-, 17-, and 70day-old mice by cellular sedimentation at unit gravity on 24% BSA gradients generated with the STA-PUT apparatus obtained from Johns Scientific (Toronto, Canada) [34-36].
Populations of type A spermatogonia (average purity = 86%,
n = 2 cell separations) and type B spermatogonia (purity =
85%, n = 2) were obtained from the testes of 8-day-old mice.
Preleptotene spermatocytes (average purity = 87%, n = 3),
leptotene/zygotene spermatocytes (average purity = 90%,
n = 3), and prepubertal pachytene spermatocytes (average
purity = 82%, n = 3) were obtained from the testes of 17day-old mice. Pachytene spermatocytes (average purity =
83%, n = 3), round spermatids (average purity = 94%, n =
3), and residual bodies (average purity = 80%, n = 3) were
obtained from 70-day-old mice.
RNA Extraction and Northern Analysis
Total RNA was extracted from isolated germ cell populations by the acid guanidium thiocyanate-phenol-chloroform method [371, electrophoresed on 1.5% agarose formaldehyde gels, and transferred to Zetabind nylon
membranes (CUNO, Meriden, CT). Blots were hybridized
[38] with a DNA MTase cDNA probe (pR5) [22] labeled to
specific activities of 5 x 108 to 8 x 108 cpm/gg of DNA by
the random priming method [39]. pR5 encodes nucleotides
1614-2055 of the cloned DNA MTase cDNA [22]. An endlabeled oligonucleotide probe for 18S rRNA was used to
correct for differences in RNA loading. Results were quantified by means of a phosphorimager (Fujix BAS 2000 BioImaging Analyzer; Fuji Medical Systems USA, Inc., Stamford,
CT). Quantification for each germ cell type was determined
in triplicate from three separate germ cell separations and
expressed as mean + SEM.
ProteinExtraction and Western Analysis
Lysates of purified germ cells were prepared by homogenization in 0.15 M NaC1, 0.05 M Tris Cl (pH 7.5), 2 gtg/ml
leupeptin, 2 tg/ml aprotinin, and 100 jtg/ml PMSF. Protein
concentrations were determined by the Bio-Rad protein assay (Bio-Rad Labs., Richmond, CA) according to the manufacturer's instructions. Aliquots (100 jg) of germ cell protein were heated at 65C for 10 min in reducing sample
buffer (2% SDS, 0.13 M Tris HCI [pH 6.8], 0.01% bromophenol blue, 10% glycerol, 0.005 M EDTA, and 5% 2-mercaptoethanol), electrophoresed on 10% SDS polyacrylamide gels, and transferred to nitrocellulose membranes.
Membranes were incubated for 4 h with the anti-pATH52
rabbit polyclonal antibody [23, 24] against DNA MTase
(1:10 000) in 5% borate Blotto buffer (0.1 M boric acid, 0.025
M sodium-borate, 0.075 M NaC1, pH 7.4, containing 5% Carnation [Los Angeles, CA] nonfat dried milk) followed by an
alkaline phosphatase-conjugated goat anti-rabbit IgG
(1:3333) from Promega (Madison, WI). Color was developed by use of nitro blue tetrazolium and 5-bromo-4 chloro3-indolyl phosphate as described for the Protoblot Western
Blot AP system from Promega.
Immunofluorescence
Freshly isolated germ cells were fixed in suspension for
10 min in 3.7% formaldehyde and rinsed in PBS with 1.5
mM MgCl 2 and 1 mM CaCl 2 for 5 min at room temperature.
Fixed cells were dropped onto glass slides, air-dried, and
stored at 4°C. To stain for DNA MTase, cells were permeabilized for 12 min in 0.2% Triton X-100, blocked for 30 min
in blocking buffer (0.2% fish skin gelatin, 5% goat serum,
0.2% Tween 20), and incubated with anti-pATH52 for 2 h
or overnight (1:500). Incubations with preimmune sera
(1:500) or blocking buffer were also performed as negative
controls. Cells were incubated for 30 min with a biotinylated
goat anti-rabbit antibody (1:200) (Vector Laboratories, Burlingame, CA) followed by a 1-h incubation with 20 tg/ml
Texas Red Avidin D (Vector Laboratories). Hoechst 33258
(Polysciences, Warrington, PA) was applied at 0.02 mg/mL
for 5 min, and slides were subsequently mounted in mounting medium from Sigma Chemical Company (St. Louis,
MO). Microscopy was performed on a Zeiss Axiophot (Carl
Zeiss Canada, St-Laurent, PQ, Canada) photomicroscope
under oil immersion with use of a 100 x /1.30 Plan-NEOFLUAR (Zeiss) objective. Micrographs were obtained with
Kodak Ektachrome 400 ASA color slide film or Kodak Tmax
563
DNA METHYLTRANSFERASE DURING SPERMATOGENESIS
400 ASA black and white print film (Eastman Kodak, Rochester, NY).
RESULTS
DNA MTase mRNA Expression during Male Gametogenesis
Germ cells from dissociated testes were fractionated by
unit-gravity sedimentation in gradients of BSA as described
[34-36]. RNA blot hybridization (Fig. 1A) showed that the
ubiquitous 5.2-kb transcript [22, 40, 41] was present at very
high levels in mitotic type A spermatogonia and at somewhat
lower levels in type B spermatogonia, meiotic preleptotene
spermatocytes, and leptotene/zygotene spermatocytes. In
prepubertal and adult pachytene spermatocytes, the 5.2-kb
mRNA was barely detectable, and a more slowly migrating
transcript of 6.2 kb appeared. Expression of the 6.2-kb transcript was highest in pachytene spermatocytes, then fell to
low levels in haploid round spermatids coincident with the
reappearance of the 5.2-kb mRNA in these postmeiotic cells.
With exposure times that revealed DNA MTase mRNA in other
cell types (Fig. 1A), neither the 5.2-kb nor the 6.2-kb transcripts were detected in anuclear residual bodies.
Relative levels of DNA MTase mRNA in different germ
cell types were quantified after hybridization with an 18S
probe to normalize for RNA loading (Fig. 2). Meiotic preleptotene spermatocytes and leptotene/zygotene spermatocytes expressed similar amounts of the 5.2-kb transcript
that were 14- to 15-fold higher than the 5.2-kb transcript
levels observed in residual bodies (Fig. 2). In postmeiotic
round spermatids, 5.2-kb mRNA levels decreased to approximately 45% of that seen in leptotene/zygotene spermatocytes. The 6.2-kb DNA MTase mRNA is essentially specific to pachytene spermatocytes; none of the other 5 germ
cell fractions contained more than one-fourth to one-fifth
as much 6.2-kb mRNA.
Immunoblot Analysis of DNA MTase Protein Expression in
PurifiedMale Germ Cell Fractions
Having established the profile of steady-state DNA
MTase mRNA levels in male germ cells, we undertook a
similar analysis of protein expression. Lysates of purified
germ cell populations were examined by immunoblot
analysis (Fig. 3) with the anti-DNA MTase antibody antipATH52 [23, 24]. Levels of DNA MTase protein were proportional to levels of the 5.2-kb mRNA seen after RNA blot
hybridization. DNA MTase protein was absent or at very low
levels in prepubertal pachytene spermatocytes and adult
pachytene spermatocytes, where the 5.2-kb mRNA was
barely detectable and the 6.2-kb mRNA was prominent.
DNA MTase protein reappeared in round spermatids, where
the 5.2-kb transcript was also clearly observed by RNA blot
hybridization. Anuclear residual bodies did not express detectable DNA MTase protein. There was a consistent and
FIG. 1. Changes in DNA MTase transcript size and levels observed during male
germ cell development. A) Total RNA from purified male germ cells (10-15 ig)was
electrophoresed on 1.5% agarose-formaldehyde gels, transferred to nylon membranes, and probed with labeled DNA MTase cDNA. B) Filter shown in (A)was
reprobed for 18S rRNA, demonstrating similar RNA loading for all lanes. A, type A
spermatogonia; B,type Bspermatogonia; PL, preleptotene spermatocytes; L/Z, leptotene/zygotene spermatocytes; PP, prepubertal pachytene spermatocytes; P,pachytene spermatocytes; RS, round spermatids; RB, residual bodies; Tot, total mixed
germ cells from adult testes.
4
|2 5.2kb
*
6.2kb
3
Er
2
C)
U
PL
LZ
PP
P
RS
Germ Cell Type
RB
FIG. 2. Relative amounts of DNA MTase mRNA expressed during meiotic and postmeiotic periods of spermatogenesis. DNA MTase 5.2-kb and 6.2-kb transcript levels
were quantified with phosphorimager followed by 18S rRNA probing to correct for
differences inRNA loading. PL, preleptotene spermatocytes; LZ, leptotene/zygotene
spermatocytes; PP, prepubertal pachytene spermatocytes; P, pachytene spermatocytes; RS, postmeiotic round spermatids; RB, residual bodies (RB). DNA MTase
mRNA levels were determined for each germ cell type in triplicate and are expressed inarbitrary units as mean
SEM.
direct correlation between the levels of DNA MTase protein
and the 5.2-kb DNA MTase mRNA, and a reciprocal relationship between levels of the 6.2-kb mRNA and DNA
MTase protein.
SubcellularLocalization of DNA MTase
Previous immunofluorescence experiments in mouse
3T3 fibroblasts at S phase demonstrated that DNA MTase is
localized to discrete foci that are the sites of DNA replication
[24]. We were therefore interested in examining DNA MTase
staining patterns in germ cells, where de novo methylation
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JUE ET AL.
FIG. 3. Expression of DNA MTase protein in purified male germ cell populations.
Aliquots (80-100 pg) of isolated germ cell lysates were electrophoresed on 10% SDS
polyacrylamide gels and transferred onto nitrocellulose membranes, and DNA
MTase was detected with anti-pATH52 followed by goat anti-rabbit IgG conjugated
with alkaline phosphatase. Upper panel: Western blots of DNA MTase levels in each
cell type. Lower panel: Amounts of total protein loaded for each cell type in gels
electrophoresed under identical conditions but stained with Coomassie blue dye.
See Figure 1 legend for explanation of lane abbreviations.
is uncoupled from DNA replication [31]. Beginning with the
least developmentally mature germ cells isolated, type A
spermatogonia were stained with the anti-pATH52 antibody; many cells displayed DNA MTase concentrated to foci
or domains (Fig. 4A). Simultaneous staining of the cells with
Hoechst 33258 dye, which binds to the minor groove of
DNA, demonstrated the nuclear localization of these DNA
MTase foci (Fig. 4B). Because type A spermatogonia are
actively engaged in mitosis, the foci most likely represent
sites of DNA replication, similar to those observed in Sphase 3T3 cells. This staining pattern of DNA MTase was
not observed with preimmune serum (data not shown).
Levels of DNA MTase staining were lower in isolated type
B spermatogonia than in type A spermatogonia (Fig. 4C). In
contrast to type A spermatogonia, the majority of type B spermatogonia exhibited a homogeneous pattern of staining for
DNA MTase throughout the nucleus; only occasional type B
spermatogonia contained nuclear foci (arrow, Fig. 4C).
Preleptotene spermatocytes (which undergo the last
round of DNA replication prior to meiosis) were also stained
for DNA MTase with the anti-pATH52 antibody (data not
shown); Simultaneous staining with Hoechst dye (data not
shown) indicated that DNA MTase was localized to the nucleus in a uniform pattern, with some variation in intensity
of staining within the population.
There were two types of nuclear distributions for DNA
MTase observed in isolated fractions of postreplicative, meiotic leptotene/zygotene spermatocytes (Fig. 5, A-G). Stained
cells displayed either a uniform nucleoplasmic staining pattern, similar to that observed in preleptotene spermatocytes,
or the cells exhibited DNA MTase staining in nuclear foci that
seem to resemble replication foci in terms of size and number
per nucleus. The foci were distributed throughout the volume
of the nucleus and were not preferentially associated with the
FIG. 4. DNA MTase distribution and abundance in proliferating type A and type B spermatogonia. A)Immunofluorescent staining
of isolated type A spermatogonia fixed and incubated with anti-pATH52 antibody. Many cells exhibit DNA MTase in nuclear foci.
B) Same field as in (A)with Hoechst 33258 dye staining to indicate location of cell nuclei. C)Type B spermatogonia stained for
DNA MTase, showing lowered levels of enzyme compared to type A spermatogonia, and uniform, nucleoplasmic distribution. Bar
= 10pm.
DNA METHYLTRANSFERASE DURING SPERMATOGENESIS
565
FIG. 5. Changes in DNA MTase levels and localization among meiotic male germ cells. A) Leptotene/zygotene spermatocyte fraction showing DNA MTase staining uniformly
and also in foci within nuclei. B)Same field as in (A)showing nuclear Hoechst staining. C) Superimposed images generated by DNA MTase staining and Hoechst dye.
Localization of DNA MTase to nucleus is indicated by pink colour. D) Leptotene/zygotene spermatocytes incubated with preimmune serum. E)Same field as in ID)showing
superimposed view of staining with preimmune serum and Hoechst dye. F) Enlarged view of one cell displaying DNA MTase staining in nuclear foci. G)Same field as in
(F)showing superimposition of staining with DNA MTase and Hoechst. H) No DNA MTase staining was detected in pachytene spermatocytes. Nuclear staining intensity
within these germ cells was similar to levels observed with preimmune serum for all germ cell types (see [Dl for example in leptotene/zygotene spermatocytes). I) Same
field as HI), shown with phase contrast microscopy. Bar = 10 pm for A, B, C,D,E, H,and I.
566
JUE ET AL.
FIG. 6. Postmeiotic reappearance of DNA MTase protein among subpopulation of haploid round spermatids. A) Only in some round spermatids was DNA MTase present
and distributed in uniform nucleoplasmic pattern. Note elongating spermatid which also displays DNA MTase. B) Same field as in (A)with superimposed images of DNA
MTase staining and Hoechst staining. Bar = 10 Im.
nuclear envelope or with any other identifiable nuclear component (Fig. 5, F and G). The close similarity in size and the
lack of suitable immunological or cytochemical markers prevented separation and discrimination of leptotene and zygotene spermatocytes [35].
Pachytene spermatocytes appear to lack significant
amounts of DNA MTase protein (Fig. 5, H and I). The staining
intensity of prepubertal pachytene spermatocytes showed
only slight differences between specimens stained with antipATH52 and those incubated with preimmune serum from
the same rabbit (data not shown) while adult pachytene spermatocytes displayed no difference at all (Fig. 5H). This result
was consistent with immunoblot analysis, in which very low
levels and nondetectable levels were observed for prepubertal and adult pachytene spermatocytes, respectively.
After meiosis, DNA MTase protein was seen to reappear
in round spermatids (Fig. 6). Only a subpopulation of haploid round spermatids stained positively for DNA MTase.
The enzyme was again localized to nuclei in a uniform distribution as demonstrated by double staining (Fig. 6B). Morphological criteria indicate that DNA MTase is specifically
expressed in round spermatids at steps 7-8 [42, 43].
Interestingly, an elongating spermatid in the round spermatid fraction also appeared to stain positively for DNA
FIG. 7. DNA MTase distribution in elongating spermatids. A)Enlarged view of elongating spermatid where staining is seen at caudal end of head and along dorsal surface,
extending up to apex of head. B) Enlarged view of superimposed images arising from DNA MTase staining and Hoechst 33258 staining. C)Enlarged view of elongating
spermatid incubated with preimmune serum. Staining is absent in caudal and dorsal regions.
DNA METHYLTRANSFERASE DURING SPERMATOGENESIS
MTase (Fig. 6). A closer view of an elongating spermatid
reveals the presence of DNA MTase in the caudal region of
the spermatid head (Fig. 7, A and B). Further staining can
be observed in a crescent-shaped distribution along the dorsal aspect of the spermatid head. The DNA MTase staining
in both the caudal region and along the dorsal surface was
not observed with preimmune serum (Fig. 7C).
DNA MTase was not detected in residual bodies, staining
in these anuclear cells being similar with either anti-pATH52
or preimmune serum (data not shown). A number of elongating spermatids cofractionating with the residual bodies
displayed the dorsal and caudal pattern of DNA MTase expression, mentioned above.
DNA MTase is therefore concentrated into discrete nuclear foci in proliferating type A spermatogonia and in meiotic leptotene/zygotene spermatocytes, is absent from
pachytene spermatocytes, and has a uniform nuclear distribution in all other germ cells prior to the nuclear elongation
phase of spermiogenesis.
DISCUSSION
In this report we have demonstrated novel aspects of the
expression and localization of DNA MTase in male germ
cells at a time when changes in methylation patterns have
been observed. The possible significance of these findings
in terms of the establishment of methylation patterns during
gametogenesis is discussed below.
Levels of DNA MTase mRNA and Protein
during Spermatogenesis
The expression of a 5.2-kb DNA MTase mRNA was found
to be directly proportional to the amount of DNA MTase
protein in type A spermatogonia, type B spermatogonia,
preleptotene spermatocytes, leptotene/zygotene spermatocytes, and round spermatids. The low to undetectable levels of 5.2-kb mRNA in prepubertal pachytene spermatocytes, adult pachytene spermatocytes, and residual bodies
corresponded with the absence of detectable DNA MTase
protein on immunoblots of extracts from these cell types. A
novel 6.2-kb transcript was the predominant DNA MTase
mRNA expressed in pachytene spermatocytes. Furthermore, levels of the 6.2-kb mRNA in pachytene spermatocytes were higher than in any other germ cell type examined. Our localization of DNA MTase mRNA to
spermatogonia and spermatocytes is supported by recent in
situ hybridization studies [441. Using an oligonucleotide
probe that detects both the 5.2- and 6.2-kb transcripts, Numata et al. [44] localized DNA MTase predominantly in spermatogonia and spermatocytes.
To date, only one mouse DNA MTase gene has been
found [22]. Our previous results indicate that the 5.2- and
6.2-kb DNA MTase transcripts arise from the same gene [401.
In addition, the 6.2-kb mRNA has only a slight association
567
with polysomes [40] and does not appear to be efficiently
translated because of sequence differences at the 5' end
(data not shown). Crossing-over occurs at the pachytene
stage of meiosis, and the sharp down-regulation of DNA
MTase protein at this time might reflect an increased vulnerability of DNA to accidental de novo methylation due to
the exposure of DNA necessary for interstrand transfer and
formation of Holliday junctions at this time. It has been
pointed out [451 that the heteroduplex recombination intermediates formed between methylated and unmethylated
DNA present hemimethylated substrates to DNA MTase,
which strongly prefers such substrates. Cruciform DNA is
also a strongly preferred substrate of DNA MTase [46]. Since
DNA methylation represses transcription [9] and is heritable
[47], ectopic DNA methylation would be expected to interfere with gene expression during development. Why this
novel method of mRNA regulation arose for DNA MTase is
unknown at this time.
Changes in Localization of DNA MTase in
Isolated Germ Cells
Type A spermatogonia exhibited levels of DNA MTase
5.2-kb mRNA and protein expression that were similar to
those of proliferating cultured mouse cells. Because the
early mitotic phase of spermatogenesis is characterized by
frequent cell divisions of the spermatogonia, DNA MTase
would be required to maintain methylation patterns on each
daughter strand of DNA generated during DNA replication.
In addition, DNA MTase has been shown to localize to replication foci in all tested lines of cultured mouse cells [241.
DNA MTase also localized to foci within the nuclei of type
A spermatogonia. In contrast to cultured cells where a subset of mitotic cells demonstrate nuclear foci [24], nearly all
of the isolated type A spermatogonia exhibited foci. This
could be due to the long length of S phase in some type A
spermatogonia [48] and the selection of these cells during
the cell separation procedure.
The drop in DNA MTase mRNA and protein expression
in type B spermatogonia was unexpected since these cells
also undergo DNA replication and divide. A corresponding
drop in DNA MTase nuclear staining was also observed in
immunofluorescence studies. Furthermore, the enzyme was
distributed uniformly throughout the nucleus in most type
B spermatogonia and was present in foci in only a few cells.
The results indicate differences in expression and localization of DNA MTase between the two mitotic cell populations, type A and type B spermatogonia. Differences in
methylation levels between type A and B spermatogonia
have been observed for a number of testis-specific genes,
including mouse transition protein 1 and mouse protamine
2 [31]. Furthermore, genes such as c-myc, c-fos, and c-jun,
which might be involved in modulating the changes in gene
expression of germ cells as they develop, display differ-
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JUE ET AL.
ences in expression between type A and type B spermatogonia [49]. Thus the differential expression of DNA MTase
between type A and B spermatogonia is not inconsistent
with the large morphological and functional differences that
exist between these two mitotic germ cell types.
Preleptotene spermatocytes undergo the last round of
DNA synthesis prior to the start of meiosis. It was surprising
to observe by immunofluorescence that DNA MTase did not
localize to foci within the nuclei of preleptotene spermatocytes but instead exhibited a uniform nucleoplasmic staining pattern. The only other case, to our knowledge, in
which DNA MTase maintains a uniform nuclear distribution
during S phase is in the 8-cell mouse embryo [50]. However,
it should be noted that, in contrast to mitotic S phase, the
preleptotene S phase is unusually long and is incomplete in
that segments of DNA remain unreplicated until later in prophase [51]. In the present study, variations in the intensity
of DNA MTase staining were also noted within populations
of preleptotene spermatocytes, suggesting that levels of
DNA methyltransferase may vary in a temporal manner
within certain developmental phases.
After the last round of DNA synthesis, preleptotene spermatocytes develop into leptotene spermatocytes and subsequently zygotene spermatocytes. Chromosome condensation begins in leptotene spermatocytes, and pairing of the
condensed chromosomes begins in zygotene spermatocytes [521. Because of the close similarity in size between
leptotene and zygotene spermatocytes, both cell types tend
to cofractionate, as has been found by others [35]. Levels of
DNA MTase mRNA and protein expression in these germ
cells were similar to those of preleptotene spermatocytes.
The significance of high DNA MTase levels in postreplicative germ cells such as leptotene and zygotene spermatocytes is intriguing. It is interesting to note that high levels
of DNA MTase appear just prior to X inactivation, which
occurs during the pachytene phase of spermatogenesis [53].
X inactivation in male germ cells might involve de novo
methylation (as in somatic tissues of females), followed by
demethylation at the time of reactivation.
Immunofluorescence observations revealed that DNA
MTase could assume focal or uniform distributions within
nuclei of leptotene/zygotene spermatocytes. The focal pattern closely resembled that seen in S-phase 3T3 cells stained
for DNA MTase [24]. It was surprising to find a similar type
of DNA MTase distribution in meiotic leptotene/zygotene
spermatocytes, which do not undergo DNA replication.
Many nuclear activities (including DNA replication, RNA
splicing, and ribosome synthesis and assembly) take place
in micrometer-sized nuclear foci or domains that are
thought to represent biochemical machines [241. We suggest
that the foci that stain strongly for DNA methyltransferase
in leptotene/zygotene spermatocytes might represent sites
where specific DNA sequences are directed to undergo de
novo methylation.
Prepubertal pachytene spermatocytes represent germ
cells in the early stages of the prolonged pachytene phase
of spermatogenesis, whereas later stages of pachytene development are represented by adult pachytene spermatocytes. Immunofluorescence observations supported the immunoblot results in that adult pachytene spermatocytes did
not stain for DNA MTase in their nuclei, and prepubertal
pachytene spermatocytes stained only slightly. Thus DNA
MTase protein expression is down-regulated at a time when
events associated with chromosome pairing and recombination are occurring [54]. Changes in methylation levels
have been observed [31] in replicative and postreplicative
spermatocytes; these changes may be involved in the imprinting process thought to occur around this time. Evidence for a demethylase activity has recently been demonstrated in chicken tissues [55]. It would be interesting to
determine the levels of this activity in pachytene spermatocytes, which exhibit such low levels of DNA MTase. Imprinting might in fact involve an interplay between the processes of methylation and demethylation, with sequence
specificity exerted at either or both steps.
At the conclusion of meiosis, haploid germ cells proceed
through a lengthy period of cellular differentiation (spermiogenesis), which eventually yields the final product of
spermatogenesis: spermatozoa. Round spermatids represent the early steps of spermiogenesis. In these cells, the
5.2-kb mRNA and protein were once more expressed. Interestingly, immunofluorescence results revealed that only
some of the cells contained DNA MTase at very high levels.
The purpose of DNA MTase expression in haploid, postreplicative, and postmeiotic germ cells is unknown, although
it might be involved in further de novo methylation events
or in maintaining methylation patterns after DNA repair,
which occurs at this time [56].
DNA MTase protein was also observed at later steps of
spermiogenesis in elongating spermatids. Immunofluorescent staining of these cells revealed DNA MTase to be distributed in the caudal region and along the dorsal aspect of
the spermatid head, extending up to the apex. The finding
that DNA MTase is still present in the nuclei of germ cells
at the later steps of spermiogenesis complements recent indirect evidence for a methylating enzyme present in epididymal sperm. Pgk-2, ApoAl, and Oct-3/4 all became methylated at the particular sites monitored as they passed
through the corpus region of the epididymis [57]. This suggests that DNA MTase can be retained in an active form
within the highly compact nuclei of spermatozoa for a prolonged period of time that includes several steps of spermiogenesis and the time required for sperm maturation in
the epididymis.
ACKNOWLEDGMENTS
We thank Guylaine Benoit for technical assistance and Alan Forster for photography.
DNA METHYLTRANSFERASE DURING SPERMATOGENESIS
REFERENCES
1. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal
and paternal genomes. Cell 1984; 37:179-183.
2. Surani MAH, Barton SC, Norris ML. Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome. Cell 1986;
45:127-136.
3. Cattanach BM, Kirk M. Differential activity of maternally and paternally derived chromosome regions in mice. Nature 1985; 315:496-498.
4. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like
growth factor II gene. Cell 1991; 64:849-859.
5. Barlow DP, Stoger R, Herrmann BG, Saito K,Schweifer N.The mouse insulin-like growth
factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 1991;
349:84-87.
6. Bartolomei MS, Zemel S, Tilghman SM. Parental imprinting of the mouse H19 gene.
Nature 1991; 351:153-155.
7. Efstratiadis A. Parental imprinting of autosomal mammalian genes. Curr Opin Gen Dev
1994; 4:265-280.
8. Meehan R, Lewis J, Cross S, Nan X, Jeppesen P, Bird A. Transcriptional repression by
methylation of CpG. J Cell Sci Suppl 1992; 16:9-14.
9. Surani MA. Genomic imprinting: control of gene expression by epigenetic inheritance.
Curr Opin Cell Biol 1994; 6:390-395.
10. Hadchouel M, Farza H, Simon D, Tiollais P, Pourcel C. Maternal inhibition of hepatitis B
surface antigen gene expression in transgenic mice correlates with de novo methylation.
Nature 1987; 329:454-456.
11. Sapienza C, Peterson AC, Rossant J, Balling R. Degree of methylation of transgenes is
dependent on gamete of origin. Nature 1987; 328:251-254.
12. Swain JL, Stewart TA, Leder P. Parental legacy determines methylation and expression
of an autosomal transgene: a molecular mechanism for parental imprinting. Cell 1987;
50:719-727.
13. Chaillet JR, Vogt TF, Beier DR, Leder P. Parental-specific methylation of an imprinted
transgene is established during gametogenesis and progressively changes during embryogenesis. Cell 1991; 66:77-83.
14. Sasaki H, Hamada T, Ueda T, Seki R, Higashinakagawa T, Sakaki Y. Inherited type of
allelic methylation variations in a mouse chromosome region where an integrated transgene shows methylation imprinting. Development 1991; 111:573-581.
15. Sasaki H, Jones PA, Chaillet JR, Ferguson-Smith AC, Barton SC, Reik W, Surani MA.
Parental imprinting: potentially active chromatin of the repressed maternal allele of the
mouse insulin-like growth factor II (Igf2) gene. Genes &Dev 1992; 6:1843-1856.
16. Ferguson-Smith AC, Sasaki H, Cattanach BM, Surani MA. Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature 1993; 362:751-755.
17. Stoger R, Kubicka P, Liu CG, Kafri T, Razin A, Cedar H, Barlow DP. Maternal-specific
methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying
the imprinting signal. Cell 1993; 73:61-71.
18. Norris DP, Patel D, Kay GF, Penny GD, Brockdorff N, Sheardown SA, Rastan S. Evidence
that random and imprinted Xist expressionis controlled by preemptive methylation. Cell
1994; 77:41-51.
19. LiE, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results
in embryonic lethality. Cell 1992; 69:915-926.
20. LiE, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature 1993;
366:362-365.
21. Bestor TH, Ingram VM. Two DNA methyltransferases from murine erythroleukemia cells:
purification, sequence specificity, and mode of interaction with DNA. Proc Natl Acad Sci
USA 1983; 50:5559-5563.
22. Bestor T, Laudano A, Mattaliano R, Ingram V. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. J Mol Biol 1988; 203:971-983.
23. Bestor TH. Activation of mammalian DNA methyltransferase by cleavage of a Zn-binding
regulatory domain. EMBOJ 1992; 11:2611-2617.
24. Leonhardt HL, Page AW, Weier H-U, Bestor TH. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 1992; 71:865-873.
25. Gruenbaum Y,Cedar H, Razin A. Substrate and sequence specificity of a eukaryotic DNA
methylase. Nature 1982; 295:620-622.
26. Bestor TH, Verdine GL. DNA methyltransferases. Curr Opin Cell Biol 1994; 6:380-389.
27. Rahe B, Erickson RP, Quinto M. Methylation of unique sequence DNA during spermatogenesis in mice. Nucleic Acids Res 1983; 11:7947-7958.
28. Groudine M,Conklin KF. Chromatin structure and de novo methylation of sperm DNA:
implications for activation of the paternal genome. Science 1985; 228:1061-1068.
29. Monk M, Boubelik M,Lehnert S. Temporal and regional changes in DNA methylation in
the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 1987; 99:371-382.
569
30. Sanford JP, Clark HJ, Chapman VM, Rossant J. Differences in DNA methylation during
oogenesis and spermatogenesis and their persistence during early embryogenesis in the
mouse. Genes &Dev 1987; 1:1039-1046.
31. Trasler JM, Hake LE, Johnson PA, Alcivar AA, Millette CF, Hecht NB. DNA methylation
and demethylation events during meiotic prophase in the mouse testis. Mol Cell Biol
1990; 10:1828-1834.
32. Ariel M, McCarrey J, Cedar H. Methylation patterns of testis-specific genes. Proc Natl
Acad Sci USA 1991; 88:2317-2321.
33. Kafri T, Ariel M, Brandeis M, Shemer R, Urven L, McCarreyJ, Cedar H, Razin A. Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line.
Genes &Dev 1992; 6:705-713.
34. Romrell LJ, Bellve AR, Fawcett DW. Separation of mouse spermatogenic cells by sedimentation velocity. Dev Biol 1976; 49:119-131.
35. Bellve AR, CavicchiaJC, Millette CF, O'Brien DA, Bhatnagar YM, Dym M. Spermatogenic
cells of the prepubertal mouse. J Cell Biol 1977; 74:68-85.
36. Bellve AR, Millette CF, Bhatnagar YM, O'Brien DA. Dissociation of the mouse testis and
characterization of isolated spermatogenic cells. J Histochem Cytochem 1977; 25:80-494.
37. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159.
38. Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 1984; 81:19911995.
39. Feinberg AP, Vogelstein B. A technique for radiolabelling DNA restriction endonuclease
fragments to high specific activity. Anal Biochem 1984; 137:266-267.
40. TraslerJM, Alcivar AA, Hake LE, Bestor T, Hecht NB. DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells. Nucleic Acids
Res 1992; 20:2541-2545.
41. Benoit G, Trasler JM. Developmental expression of DNA methyltransferase messenger
ribonucleic acid, protein, and enzyme activity in the mouse testis. Biol Reprod 1994;
50:1312-1319.
42. Leblond CP, Clermont Y. Spermiogeneses of rat, mouse, hamster and guinea-pig as revealed by the "periodic acid-fuchsin sulfurous acid" technique. AmJ Anat 1952; 90:167215.
43. Oakberg EF A description of spermiogenesis in the mouse and its use in analysis of the
cycle of the seminiferous epithelium and germ cell renewal. Am J Anat 1956; 99:391413.
44. Numata M,Ono T, Iseki S. Expression and localization of the mRNA for DNA (cytosine5)-methyltransferase in mouse seminiferous tubules. J Histochem Cytochem 1994;
42:1271-1276.
45. Matzke MA, Matzke AJM. Homology-dependent gene silencing in transgenic plants: what
does it really tell us? TIG 1995; 11:1-3.
46. Bestor TH. Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase. Nucleic Acids Res 1987; 15:3835-3843.
47. Wigler M, Levy D, Perucho M. The somatic replication of DNA methylation. Cell 1981;
24:33-40.
48. Monesi V.Autoradiographic study of DNA synthesis and the cell cycle in spermatogonia
and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 1962; 14:1-18.
49. Wolfes H, Kogawa K, Millette CF, Cooper GM. Specific expression of nuclear protooncogenes before entry into meiotic prophase of spermatogenesis. Science 1989;
245:740-743.
50. Carlson LL,Page AW, Bestor TH. Localization and properties of DNA methyltransferase
in preimplantation mouse embryos: implications for genomic imprinting. Genes &Dev
1992; 6:2536-2541.
51. Handel MA. Genetic control of spermatogenesis in mice. Results Probl Cell Differ 1987;
15:1-62.
52. de Kretser DM, Kerr JB. The cytology of the testis. In: Knobil E, Neill J (eds.), The
Physiology of Reproduction. New York: Raven Press Ltd.; 1988: 837-932.
53. Handel MA, Hunt PA, Kot MC, Park C, Shannon M. Role of sex chromosomes in the
control of male germ-cell differentiation. In: Robaire B (ed.), The Male Germ Cell: Spermatogonium to Fertilization. New York: New York Academy of Sciences; 1991: 64-73.
54. Hawley RS, Arbel T. Yeast genetics and the fall of the classical view of meiosis. Cell
1993; 72:301-303.
55. Jost J-P Nuclear extracts of chicken embryos promote an active demethylation of DNA
by excision repair of 5-methyldeoxycytidine. Proc Natl Acad Sci USA 1993; 90:46844688.
56. 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.
57. Ariel M,Cedar H, McCarrey J. Developmental changes in methylation of spermatogenesisspecific genes include reprogramming in the epididymis. Nature Genet 1994; 7:59-63.