Developmental Biology 243, 294 –300 (2002)

doi:10.1006/dbio.2001.0561, available online at http://www.idealibrary.com on

Mouse Parthenogenetic Embryos with Monoallelic

H19 Expression Can Develop
to Day 17.5 of Gestation

Tomohiro Kono,* ,1 Yusuke Sotomaru,* ,2 Yukiko Katsuzawa,*

and Luisa Dandolo†
*Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502,
Japan; and †Institute Cochin de Genetique Molecular, 24 Rue du Fbg. St. Jacques,
75014, Paris, France

In mammals, both maternal and paternal genomes are required for a fetus to develop normally to term. This requirement
is due to the epigenetic modification of genomes during gametogenesis, which leads to an unequivalent expression of
imprinted genes between parental alleles. Parthenogenetic mouse embryos that contain genomes from nongrowing (ng) and
fully grown (fg) oocytes can develop into 13.5-day-old fetuses, in which paternally and maternally expressed imprinted genes
are expressed and repressed, respectively, from the ng oocyte allele. The H19 gene, however, is biallelically expressed with
the silent status Igf2 in such parthenotes. In this study, we examined whether the regulation of H19 monoallelic expression
enhances the survival of parthenogenetic embryos. The results clearly show that the ng H19-KO/fg wt parthenogenetic embryos
carrying the ng-oocyte genome that had been deleted by the H19 transcription unit successfully developed as live fetuses
for 17.5 gestation days. Control experiments revealed that this unique phenomenon occurs irrespective of the genetic
background effect. Quantitative gene expression analysis showed that day 12.5 ng H19-KO/fg wt parthenogenetic fetuses
expressed Igf2 and H19 genes at <2 and 82% of the levels in the controls. Histological analysis demonstrated that the
placenta of ng H19-KO/fg wt parthenotes was afflicted with atrophia with severe necrosis and other anomalies. The present
results suggest that the cessation of H19 gene expression from the ng-allele causes extended development of the fetus and
that functional defects in the placenta could be fatal for the ontogeny. © 2002 Elsevier Science (USA)
Key Words: parthenogenesis; genomic imprinting; epigenetic modification; oocytes; H19 gene; nuclear transfer;
development; mouse.

INTRODUCTION
Mammalian parthenotes cannot be induced to develop
normally to term. Attempts to induce such development
have resulted in embryonic death before 10 days of gesta-
tion in mice (Surani and Barton, 1983; McGrath and Solter,
1984; Barton et al., 1984; Surani et al., 1986) and before 21
days of gestation in sheep (Hagemann et al., 1998) and pigs
(Kure-bayashi et al., 2000). Although the precise mecha-
nisms underlying the limited development of mammalian
parthenotes are still unclear, one persuasive explanation is
1
To whom correspondence should be addressed. Fax: 81-3-5477-
2543. E-mail: tomohiro@nodai.ac.jp.
2
Present address: Central Institute for Experimental Animals,
Kawasaki-shi, Kanagawa 216-0001, Japan.
that the functions of the male and female genomes are
definitively different as a result of epigenetic modifications
that occur during gametogenesis (Monk, 1988; Surani et al.,
1990; Sasaki et al., 1992; Brannan and Bartolomei, 1999).
This hypothesis is supported by our recent results, which
showed that the ability of maternal chromatin to support
full-term development is attained during the last half of
oocyte development (Bao et al., 2000). The epigenetic modi-
fications during gametogenesis lead to parental allele-
specific expression of the imprinted genes and result in the
normal development of fertilized biparental embryos to
term.
Previous studies (Kono et al., 1996; Obata et al., 1998)
have provided direct evidence of the epigenetic modifica-
tion that occurs in mammalian development as follows.
The parthenogenetic mouse embryos that carry two sets of

0012-1606/02 $35.00
© 2002 Elsevier Science (USA)
294 All rights reserved.
Monoallelic H19 Expression
haploid genomes from nongrowing and fully grown oocytes
are able to develop to 13.5 days of gestation with a well-
developed placenta. In these embryos, the paternally ex-
pressed genes, Peg1/Mest, Peg3, and Snrpn, are activated,
while the maternally expressed genes, Igf2r and p57 Kip2, are
silent in the alleles derived from a nongrowing oocyte
genome. However, of the genes analyzed, it was determined
that the H19 gene was biallelically expressed while the Igf2
gene was repressed, as the enhancers that are shared by Igf2
and H19 act preferentially for the H19 gene (Bartolomei et
al., 1993; Hark et al., 2000). So far, studies have shown that
H19 is expressed in a broad range of tissues, although it is
not expressed in neural tissues (Ohlsson et al., 1994; Svens-
son et al., 1995). This high expression of H19 is also
detected in extraembryonic tissues. These findings imply
the possibility that the biallelic expression of H19 is in-
volved along with the silence of the Igf2 gene in the
developmental arrest of the ng/fg parthenotes.
To investigate this possibility, we constructed ng/fg par-
thenogenetic oocytes using nongrowing oocytes derived
from H19-null mutated mice (Ripoche et al., 1997) and
assessed whether the parthenogenetic embryos can develop
beyond 13.5 days of gestation.
MATERIALS AND METHODS
Oocyte Collection
MF1 mice, whose H19 transcription unit was targeted with a
Py-ty-neo cassette, and B6CBF1 (C57BL/6J ⫻ CBA) were used as
oocyte donors. Fully grown germinal vesicle (GV) oocytes were
collected from the ovarian follicles of adult females 44 – 48 h after
an eCG injection, and nongrowing stage oocytes, which are at the
diplotene stage of the first meiosis, were collected from the ovaries
of 1-day-old pups.
Embryo Production
Nuclear transfer was carried out as outlined previously (Kono et
al., 1996; Obata et al., 1998). Oocytes (ng H19-KO/fg wt) containing a
haploid set of genomes from H19-null mutant females and another
genome from ovulated MII oocytes of wild-type B6CBF1 mice were
constructed by nuclear transfer. Fusion of the diplotene oocytes
with enucleated GV oocytes was induced with an inactivated
Sendai virus (HVJ, 2700 hemagglutinating activity unit/ml). After
the fusion, the reconstituted oocytes were cultured for 14 h in
Waymouth medium (MB752/1; GIBCO). The GV oocytes were
manipulated in a medium containing 200 ␮M dbcAMP and 5% calf
serum throughout the experiment and were released from the
medium 1 h after fusion with a nongrowing stage oocyte. A set of
MII chromosomes from reconstituted oocytes was transferred into
an enucleated MII oocyte that had been collected from the oviducts
of superovulated B6CBF1 mice 15–16 h after hCG injection. ng wt/
fg wt parthenotes were also produced from wild-type MF1 outbreed
and B6CBF1 mice as described above. The reconstructed oocytes
were artificially activated with 10 mM SrCl 2 in Ca 2⫹-free M16
medium for 2 h (Bos-Mikich et al., 1995).
MF1 and B6CBF1 embryos produced by in vitro fertilization were
used as controls.
© 2002 Elsevier Science (USA). All rights reserved.
295
In Vitro Culture and Embryo Transfer
These embryos were manipulated in M2 medium and cultured
in M16 medium (Whittingham, 1971) for 3 days in an atmosphere
of 5% CO 2, 5% O 2, and 90% N 2 at 37°C. Blastocysts obtained from
the constructed oocytes were transferred into the uterine horns of
CD-1 female mice at 2.5 days of pseudopregnancy. The postimplan-
tation development was assessed by an autopsy performed between
15.5 and 18.5 days of gestation.
Gene Expression Analysis
Total RNA was isolated from the control and parthenogenetic
embryos at 12.5 and 17.5 days of gestation by using the SV Total
RNA Isolation Kit (Promega). First-strand cDNA was synthesized
from 1 ␮g of total RNA from each embryo by Superscript reverse
transcriptase II (Gibco-BRL) according to the manufacturer’s in-
structions. The cDNA was subjected to PCR, which was carried
out in a 50-␮l reaction buffer containing 1.25 U of Taq DNA
polymerase (Takara), 1 pmol of each primer, 1.5 mM MgCl 2, and
250 ␮M dNTPs. Quantitative analysis of gene expression was
performed by means of Real-Time quantitative PCR (LightCycler
System; Roche Molecular Biochemicals, Germany) using a ready-
to-use reaction mixture kit (LightCycler FirstStart DNA Master
SYBR Green I; Roche Molecular Biochemicals). The primers used
for Igf2 were 5⬘-CTACTTCAGCAGGCCTTCAAG-3⬘ and 5⬘-GA-
TGGTTGCTGTACATCTCC-3⬘, and those used for H19 were
5⬘-CATGTCTGGGCCTTTGAA-3⬘ and 5⬘-TTGGCTCCAGGA-
TGATGT-3⬘. The quantity of PCR products was detected by
monitoring the luminous intensity. To provide a standard, day 12.5
fetuses from normal fertilized embryos were analyzed by the same
procedure.
Histological Analysis
The live fetuses and placentas at 17.5 days of gestation derived
from ng H19-KO/fg wt parthenogenetic embryos were fixed with 4%
paraformaldehyde and processed for wax embedding. Serial sec-
tions prepared in the cross planes were mounted on slides and
stained with hematoxylin and eosin.
RESULTS
Development of Parthenogenetic Embryos
For developmental assessment, we constructed 789 oo-
cytes (ng H19-KO/fg wt) using nongrowing stage oocytes from
H19-null mutant newborn mice. After maturation in vitro
and artificial activation, 87% of the oocytes resulted in
diploid one-cell parthenogenetic embryos that formed two
second-polar bodies and pronuclei, and 88% of them devel-
oped to the blastocyst stage in vitro (Table 1). In all, 505
blastocysts derived from ng H19-KO/fg wt oocytes were trans-
ferred to 43 recipients (Table 2). Since ng wt/fg wt parthenotes
can develop to 13.5 days of gestation (Kono et al., 1996), the
autopsy to assess postimplantation development was car-
ried out between 15.5 and 18.5 days of gestation. A total of
42 fetuses were estimated to have developed up to day 14.5
of gestation. At 17.5 days of gestation, 4 live fetuses were
recovered from 166 embryos transferred (Figs. 1a–1d). In
296 Kono et al.

TABLE 1 MF1 outbreed and B6CBF1 mice, respectively, were trans-

Development of Constructed Parthenogenetic Mouse Embryos ferred to the recipient females as before. The results from
in Vitro the autopsy at 14.5 days of gestation confirmed that ng wt/
fg wt parthenotes cannot develop beyond 13.5 days of gesta-
No. of No. of No. of embryos
tion. Further, we have confirmed that fg H19-KO/fg wt parthe-
oocytes oocytes developed to
Genotypes constructed activated (%) blastocysts (%)
nogenetic embryos die before day 10 of gestation (Tables 1
and 2). These results suggest that the extended develop-
ng H19-KO
/fg wt 789 698 (88) 613 (88) ment of the parthenogenetic embryos to 17.5 days of gesta-
wt wt
ng /fg (MF1) 282 254 (90) 201 (79) tion was caused by the monoallelic expression of the H19
ng wt/fg wt (B6CBF1) 458 424 (93) 326 (77) gene and by a unique phenomenon seen in the ng H19-KO/fg wt
fg H19-KO/fg wt 133 123 (92) 113 (92) parthenogenetic mouse embryos.

Expression of Igf2 and H19

these fetuses, the skin was wrinkled and thickened, and the
subcutaneous veins were no longer distinctly visible. The
eyelids were fused and thickened, and the fingers and toes
were parallel. The umbilical hernia had already reposited.
However, the weight of the fetuses (503 mg, n ⫽ 4) was
significantly (P ⬍ 0.01) less than that of the controls, which
was 708 mg (n ⫽ 12) at stage 25–26. When the remaining
fetuses were autopsied at 18.5 days of gestation, only two
dead fetuses, which were estimated as day 17.5 fetuses
(Figs. 1e and 1f), were recovered, suggesting that term
development was difficult for them.
To demonstrate that the present results were due to the
monoallelic expression of the H19 gene irrespective of the
genetic background effect and of the particular case, we
constructed an adequate number of ng wt/fg wt parthenotes
using both MF1 outbreed and B6CBF1 mice (Tables 1 and 2).
The 185 and 239 blastocysts that were obtained from the
To obtain further insight into the mechanism underlying
the extended development of the parthenogenetic embryos,
quantitative expression analysis was performed in indi-
vidual ng H19-KO/fg wt parthenotes (n ⫽ 10) by using quantita-
tive Real-Time PCR (Fig. 2). The level of Igf2 in these
parthenotes at day 12.5 of gestation was found to be ⬍2%
that of the level in the control fetuses at day 12.5 of
gestation. The expression level is not significantly different
from that of the level in day 12.5 of ng wt/fg wt parthenotes
(n ⫽ 7), suggesting that this leaking expression did not
affect the extended development in ng H19-KO/fg wt parthe-
notes. The low expression of Igf2 may correlate with the
significantly lighter weight of the parthenotes. The day 17.5
parthenotes (n ⫽ 3) expressed Igf2 at a level equal to that of
the day 17.5 controls, which was about 20% in the level in
the control fetuses at day 12.5 of gestation.
In contrast, H19 gene expression in ng H19-KO/fg wt parthe-
notes, which have a single expression allele, was only

TABLE 2
Postimplantation Development of Constructed Parthenogenetic Mouse Embryos

Day of No. of No. No. of fetuses developed to

biopsy embryos pregnants/ No. of
Genotypes (dpc) transferred recipients implantation (%) 12.5 dpc ⬍ 14.5 dpc ⬍ 16.5 dpc ⬍ 17.5 dpc Notes
H19-KO wt
ng /fg 15.5 104 7/8 60 (58) D6 D7, L2
16.5 79 6/7 35 (44) D9 D2 L1 fetus, 13.7 mm, 360 mg;
placenta, 117 mg
17.5 166 13/14 117 (70) D14 D12 D2 L4 fetus (a), 17.7 mm, 532 mg;
placenta, 78 mg
fetus (b), 18.3 mm, 503 mg;
placenta, 60 mg
fetus (c), 17.5 mm, 491 mg;
placenta, 77 mg
fetus (d), 16.8 mm, 486 mg;
placenta, 103 mg
18.5 156 12/14 96 (62) D18 D8 D2 D2 fetus (e), 17.8 mm, 542 mg;
placenta, 75 mg
fetus (f), 17.2 mm, 520 mg;
placenta, 51 mg
ng wt /fg wt (MF1) 14.5 185 13/15 127 (69) D59 0
ng wt /fg wt (B6CBF1) 14.5 239 16/18 155 (65) D70 0
fg H19-KO /fg wt 12.5 113 8/10 74 (65) 0

Note. dpc, day post coitum; D, dead fetuses; L, live fetuses.

© 2002 Elsevier Science (USA). All rights reserved.

Monoallelic H19 Expression
FIG. 1. Postimplantation development of parthenogenetic
ng H19-KO/fg wt embryos. Four individual live fetuses were recovered at
17.5 days of gestation. (a) The fetus inside of the yolk sac with its
placenta. (b) The fetus after being released from the yolk sac. (c, d)
The fetuses after being removed from the extraembryonic tissues.
(e, f) Two dead fetuses recovered at 18.5 days of gestation.
slightly lower (82%) than that of day 12.5 controls (Fig. 2).
This level of H19 gene expression was almost half the level
of ng wt/fg wt parthenotes, both of whose maternal alleles
were active. The expression of H19 in day 17.5 ng H19-KO/fg wt
parthenotes was 88, 153, and 258% of the level in the day
17.5 and 12.5 controls and day 12.5 ng wt/fg wt parthenotes,
respectively.
© 2002 Elsevier Science (USA). All rights reserved.
297
Histological Analysis
The placenta of ng H19-KO/fg wt parthenotes was afflicted
with atrophia with severe anomalies (Fig. 3). Histological
analysis showed that severe necrosis had occurred through-
out the tissue, especially in the labyrinthine layer. Throm-
bus and agglutination of red blood cells were also seen in
some blood vessels. A number of endometrial glycogen
cells, which were vacuolated, were also distributed in the
labyrinthine layer. The border between the spongiotropho-
blast and labyrinth layers was disrupted. The necrosis and
vacuolation led to severe defects in the distribution of the
maternal and fetal blood vessels in the spongiotrophoblast
and labyrinthine layers, respectively. In contrast, no cardiac
defect, which are the most likely reason for fetal death at
17.5 days of gestation, was detected, and neither were any
histological anomalies (Fig. 4). This result suggests that
defects in the placenta, rather than in the fetus itself,
caused the ng H19-KO/fg wt parthenotes to fail to develop to
term.
DISCUSSION
The present experiment provided the parthenotes an
opportunity to develop to 17.5 days of gestation. The
phenotypes were quite similar to those of the control
FIG. 2. Expression of Igf2 and H19 in individual ng H19-KO/fg wt
parthenotes. The expression levels were determined by quantita-
tive Real-Time PCR and are indicated as ratios to the standard level
obtained from day 12.5 control fetuses. Individual expression of the
Igf2 and H19 genes by day 12.5 (circles) and day 17.5 (squares)
fetuses is represented by closed and open symbols for ng H19-KO/fg wt
parthenotes and controls, respectively. Oblique lined symbols
represent individual expression by ng wt/fg wt parthenotes.
298
FIG. 3. Histological sections of the placenta from control (a, ⫻40; b, ⫻200) and ng H19-KO/fg wt parthenogenetic (c, ⫻40; d, ⫻200) live fetuses
at day 17.5. SZ, spongiotrophoblastic zone; LZ, labyrinthine zone; CP, chorionic plate; GC, glycogen cells; NE, necrotic cells; TB, thrombus.
fetuses at the corresponding stage and were estimated to be
at stage 25 (Theiler, 1989). This extended development of
mouse parthenotes is 4 days longer than that observed
previously with nongrowing and fully grown oocyte ge-
nomes. The deletion of the transcription unit in the H19
gene and the corresponding monoallelic expression of H19
gene may be responsible for this extended development of
the parthenotes.
FIG. 4. Sagittal sections of a ng H19-KO/fg wt parthenogenetic live
fetus (a, ⫻10), and its heart (b, ⫻100). TH, thymus; HA, heart; LI,
liver; AO, aorta; AS, auricula sinistra; AD, auricula dextra; VD,
ventriculus dextra; LU, lung.
© 2002 Elsevier Science (USA). All rights reserved.
Kono et al.
Assuming that silencing the H19 gene in the ng allele
results in the extended development of ng H19-KO/fg wt parthe-
notes, the question remained as to what the mechanism of
this extension of development might be. It is known that
the H19 gene is expressed abundantly in a broad range of
tissues of both endodermal and mesodermal origin in de-
veloping mouse embryos (Ohlsson et al., 1994; Svensson et
al., 1995), but the 2.5-kb H19 spliced and polyadenylated
RNA does not encode a protein. Earlier studies have been
unable to identify any function for the H19 transcript,
except one report that found that growth retardation was
induced when an H19 expression construct was transfected
into human embryo tumor cells (Hao et al., 1993). It has
recently been shown that the H19 transcript is associated
with polysomes in a variety of cells and that there is a
reciprocal correlation in trans between cytoplasmic H19
and Igf2 mRNA levels, suggesting that the H19 transcript
represses Igf2 expression in trans (Li et al., 1998). In
contrast, the replacement of the H19 gene with a protein-
coding gene does not affect the expression of Igf2 (Jones et
al., 1998). The deletion of a silencer element located be-
tween 11.7 and ⫺2.9 kb from the transcription start site of
H19 has been found to cause a loss of imprinting after
paternal inheritance, but the expression of the neighboring
imprinted Igf2 gene is not affected (Drewell et al., 2000).
Thus, the function of H19 is still not clear and appears to be
quite complicated.
For the H19-deletion mice used here as ng oocyte donors,
it has been reported that homozygous mutants are viable
and exhibited an 8% overgrowth (Ripoche et al., 1997). To
Monoallelic H19 Expression
correlate the extended development of parthenotes with the
expressions of Igf2 and H19 genes, we detected the levels of
the imprinted genes in ng H19-KO/fg wt and ng wt/fg wt parthe-
notes and in the control fetuses. The expression of Igf2 gene
in both ng H19-KO/fg wt and ng wt/fg wt parthenotes was a very
small percentage (⬍2%) of that of the controls. The normal
level of Igf2 expression detected in day 17.5 ng H19-KO/fgwt
parthenotes is not likely to be the primary reason for the
extended development, as mice carrying Igf2 deletions are
viable and fertile (Baker et al., 1993), albeit with a reduction
in body weight. Similar expression is seen in H19 deletion
mice. The maternal Igf2 allele, which is normally silent, is
altered to have an active status in cis (Ripoche et al., 1997).
Thus, the Igf2 expression, although at a low level, may
provide feasible circumstances under which the embryo can
develop to day 17.5 of gestation.
It is expected that maternally expressed imprinted genes
could be expressed in parthenogenetic embryos at twice the
level of the controls. As expected, ng wt/fg wt parthenotes
expressed H19 gene at 240% of the levels found in the
controls, while ng H19-KO/fg wt, which has a single H19 gene
allele, expressed it at a level almost equal to the level found
in the controls, suggesting that the expression of H19 gene
at an appropriate dose from a single allele is involved in the
extended development beyond day 13.5 of gestation.
So far, studies have shown that overexpression of the H19
gene itself does not have deleterious effects. No obvious
phenotype has been detected with an excess gene dosage of
H19 in transgenic experiments using a 130-kb YAC clone
(Ainscough et al., 1997). An earlier report based on results
in transgenic mice suggested that ectopic expression of the
H19 gene can cause prenatal lethality (Brunkow and Tilgh-
man, 1991), but this suggestion was later retracted by the
same group (Pfeifer et al., 1996). However, we cannot
remove the possibility that it is H19 itself that appears to be
responsible for the extended development of ng H19-KO/fg wt
parthenotes. It is possible that the biallelic expression of the
gene itself is responsible for the developmental arrest at day
13.5 of gestation in ng wt/fg wt parthenotes. Although the
mechanism is not clear, it is supposed that the transcripts
facilitate or block the binding of transcription factors and
thus alter the DNA conformation. Therefore, the biallelic
expression may have deleterious effects at the cis and/or
trans positions on a certain gene that regulates embryo
development (Forne et al., 1997; Duvillie et al., 1998).
On the other hand, although the reason for death at 17.5
days of gestation is not clear, histological analysis suggests
that a defect of the placenta led to death. Severe ischemic
necrosis was observed throughout the placental tissues,
suggesting that this placental defect causes hypoxidosis and
metabolic defects in the parthenotes. In contrast, no
anomaly was detected in the cardiac tissue. These observa-
tions suggest that defects in the placenta, rather than in the
fetus itself, are the main reason that the ng H19-KO/fg wt par-
thenotes failed to develop to term.
Thus, together with our previous work, the present study
clearly demonstrates that epigenetic modifications that
© 2002 Elsevier Science (USA). All rights reserved.
299
occur during oocyte growth, namely maternal primary
imprinting, have a critical effect on the development of
parthenogenetic mouse embryos, and that an alteration of
imprinting status leads to extended parthenogenetic devel-
opment. However, it remains unclear whether further al-
terations of the imprint status of the ng allele can bring
about term development of parthenogenetic mouse em-
bryos.
ACKNOWLEDGMENTS
We would like to thank Dr. J. Carroll (UCL, UK), Dr. Y. Obata
(Gunma University, JP), and Dr. S. Kubo (National Institute of
Animal Health, JP) for their helpful discussion. This work was
supported by grants from the Ministry of Education, Science,
Culture and Sports of Japan, the Ministry of Agriculture of Japan,
and the Japanese Society for the Promotion of Science.
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Received for publication July 9, 2001
Revised November 26, 2001
Accepted December 4, 2001
Published online February 11, 2002