The 2-cell block occurring during development of outbred mouse embryos is rescued by cytoplasmic factors present in inbred metaphase II oocytes.
ABSTRACT In mice, completion of preimplantation development in vitro is restricted to certain crosses between inbred strains. Most of the outbred and inbred strains cease development at the 2-cell stage, a phenomenon known as the "2-cell block". Reciprocal mating between blocking and non-blocking strains has shown that the 2-cell block is dependent upon female, but not male, developmental information. One question that still remains unanswered is whether the genome of the metaphase II (MII) oocyte is genetically programmed to express, during the very early stages of development, some factor(s) required to determine developmental competence beyond the 2-cell stage. In the present study, we have addressed this question by performing reciprocal MII-chromosome plate transfer between MII oocytes of a non-blocking inbred strain and MII oocytes of a blocking outbred strain. Here, we report that development beyond the 2-cell stage does not depend on the MII genome, but instead it relies on a cytoplasmic factor(s) already present in ovulated non-blocking oocytes, but absent, inactive or quantitatively insufficient in blocking oocytes. Further evidence of the ooplasmic origin of this component(s) was obtained by transferring a small quantity of ooplasm from non-blocking MII oocytes to blocking MII oocytes or 2-cell embryos. Following the transfer, a high percentage of blocking oocytes/embryos acquired developmental competence beyond the 2-cell stage and reached the blastocyst stage. This study shows that development beyond the 2-cell stage relies also on a factor(s) already present in the ovulated oocyte.
- SourceAvailable from: Paolo Giovanni Artini[Show abstract] [Hide abstract]
ABSTRACT: Cryopreservation of female reproductive cells allows preservation of fertility and provides materials for research. Although freezing protocols have been optimized, and there is a high survival rate after thawing, the in vitro fertilization (IVF) pregnancy rate is still lower in cycles with cryopreserved oocytes, thus highlighting the importance of identifying intrinsic limiting factors characterizing the cells at time of freezing. The aim of the present study is to investigate in the mouse model the impact of reproductive aging and postovulatory aging on oocyte biological competence after vitrification. Metaphase II oocytes were vitrified soon after retrieval from young and reproductively old mice. Part of the oocytes from young animals was vitrified after 6 h incubation (in vitro aged oocytes). All classes of oocytes showed similar survival rate after vitrification. Moreover, vitrification did not alter chromosomal organization in young cells, whereas in vitro aged and old oocytes presented an increase of slightly aberrant metaphase configurations. Compared to fresh young oocytes, in vitro aged and old oocytes showed increased ROS levels which remained unchanged after vitrification. By contrast, cryopreservation significantly increased ROS production in young oocytes. Both the aging processes negatively impacted oocyte ability to undergo pronucleus formation and first cleavage after vitrification by stimulating cellular fragmentation. These results could be helpful for establishing the correct time table for cryopreservation in the laboratory routine and improving its application in reproductively old females. Moreover, our observations highlight the importance of oxidative stress protection during vitrification procedures.Theriogenology 06/2011; 76(5):864-73. · 2.08 Impact Factor
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ABSTRACT: The hiatus between oocyte and embryonic gene transcription dictates a role for stored maternal factors in early mammalian development. Encoded by maternal-effect genes, these factors accumulate during oogenesis and enable the activation of the embryonic genome, the subsequent cleavage stages of embryogenesis and the initial establishment of embryonic cell lineages. Recent studies in mice have yielded new findings on the role of maternally provided proteins and multi-component complexes in preimplantation development. Nevertheless, significant gaps remain in our mechanistic understanding of the networks that regulate early mammalian embryogenesis, which provide an impetus and opportunities for future investigations.Development 03/2010; 137(6):859-70. · 6.60 Impact Factor
- Resuscitation 01/2010; 81(2). · 4.10 Impact Factor
The 2-cell block occurring during development of outbred
mouse embryos is rescued by cytoplasmic factors
present in inbred metaphase II oocytes
MARIO ZANONI1, SILVIA GARAGNA1, CARLO A. REDI2 and MAURIZIO ZUCCOTTI*,3
1Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia Animale and Centro di Ingegneria Tissutale,
Universita' degli Studi di Pavia, Pavia, Italy, 2Fondazione I.R.C.C.S. Policlinico San Matteo, Pavia, Italy and
3Sezione di Istologia ed Embriologia, Dipartimento di Medicina Sperimentale, Universita’ degli Studi di Parma, Parma, Italy.
ABSTRACT In mice, completion of preimplantation development in vitro is restricted to certain
crosses between inbred strains. Most of the outbred and inbred strains cease development at the
2-cell stage, a phenomenon known as the "2-cell block". Reciprocal mating between blocking and
non-blocking strains has shown that the 2-cell block is dependent upon female, but not male,
developmental information. One question that still remains unanswered is whether the genome
of the metaphase II (MII) oocyte is genetically programmed to express, during the very early stages
of development, some factor(s) required to determine developmental competence beyond the 2-
cell stage. In the present study, we have addressed this question by performing reciprocal MII-
chromosome plate transfer between MII oocytes of a non-blocking inbred strain and MII oocytes
of a blocking outbred strain. Here, we report that development beyond the 2-cell stage does not
depend on the MII genome, but instead it relies on a cytoplasmic factor(s) already present in
ovulated non-blocking oocytes, but absent, inactive or quantitatively insufficient in blocking
oocytes. Further evidence of the ooplasmic origin of this component(s) was obtained by transfer-
ring a small quantity of ooplasm from non-blocking MII oocytes to blocking MII oocytes or 2-cell
embryos. Following the transfer, a high percentage of blocking oocytes/embryos acquired
developmental competence beyond the 2-cell stage and reached the blastocyst stage. This study
shows that development beyond the 2-cell stage relies also on a factor(s) already present in the
KEY WORDS: preimplantation, 2-cell block, nuclear transfer, cytoplasm transfer
In mice, completion of preimplantation development in vitro is
restricted to certain crosses between inbred strains. Most of the
outbred and inbred strains cease development at the 2-cell stage,
a phenomenon known as the ‘2-cell block’. Although it occurs in
particular in vitro culture conditions (for a review see Biggers,
1998), the 2-cell block may represent a model study that provides
important insights on a critical stage of development, when zygotic
genome activation (ZGA) occurs (Schultz, 2002). ZGA marks the
passage from maternal to embryonic control of development, and
represents the first crucial hurdle in the life of the new individual as
inhibiting the activation of embryonic genes arrests development
(Schultz and Worrad, 1995). Blocks to development at the time
Int. J. Dev. Biol. 53: 129-134 (2009)
THE INTERNATIONAL JOURNAL OF
*Address correspondence to: Maurizio Zuccotti. Sezione di Istologia ed Embriologia, Dipartimento di Medicina Sperimentale, Univesita’ degli Studi di Parma
43100, Parma, Italy. e-mail: email@example.com
Accepted: 6 June 2008. Published online: 18 December 2008.
ISSN: Online 1696-3547, Print 0214-6282
© 2008 UBC Press
Printed in Spain
Abbreviations used in this paper: MII, metaphase II
when ZGA occurs have been described in other mammals (Schultz,
2002), including humans at the 4-cell stage, when a high percent-
age of IVF embryos is lost.
The 2-cell block is rescued when cytoplasm is transferred from
2-cell non-blocking embryos to 2-cell blocking embryos, allowing
completion of preimplantation development (Muggleton-Harris et
al., 1982). Rescue does not occur when cytoplasm is injected from
a 1-cell G1 stage non-blocking embryo to a blocking 2-cell embryo
(Muggleton-Harris et al., 1982), suggesting that the component(s)
that allows to overcome the 2-cell block is yet not present, it has not
been translated or it is not functioning. Instead, rescue occurs
130 M. Zanoni et al.
consistently when injection is performed from a 2-cell G1 or G2
stage, but not S phase, non-blocking embryo to a 2-cell blocking
embryo at any phase of the cell cycle (Pratt and Muggleton-Harris,
1988). The blocking activity is not due to a blocking factor, since the
transfer of cytoplasm from blocking to non-blocking 2-cell embryos
did not impair their ability to complete preimplantation develop-
ment (Pratt and Muggleton-Harris, 1988).
The cyclic nature of these molecules indicates either a transcrip-
tional control of their expression by the embryonic genome or a
translational/post-translational regulation of transcripts/proteins.
Reciprocal mating between blocking and non-blocking strains
has shown that the 2-cell block is dependent upon a female, but not
male, developmental information (Goddard and Pratt, 1983). One
question that still remains unanswered is whether the genome of
the MII oocyte is genetically programmed to express, during the
very early stages of development, some factor(s) required to
determine the developmental competence beyond the 2-cell stage.
In the present study we have addressed this question by
performing reciprocal MII-chromosome plates (MII-plates) transfer
between MII oocytes of a non-blocking inbred strain and MII
oocytes of a blocking outbred strain. We report that development
beyond the 2-cell stage does not depend on the MII genome, but
instead it relies on a cytoplasmic factor(s) already present in
ovulated non-blocking oocytes, but absent, inactive or quantita-
tively insufficient in blocking oocytes. Further evidences of the
ooplasmic origin of this component(s) were obtained by transfer-
ring a small quantity of ooplasm from non-blocking MII oocytes to
blocking MII oocytes or 2-cell embryos. Following the transfer, a
high percentage of blocking oocytes/embryos acquired develop-
mental competence beyond the 2-cell stage and reached the
The presence in non-blocking embryos of a functional rescuing-
factor(s) beginning with the 2-cell stage (Pratt and Muggleton-
Harris, 1988) suggests either a developmentally regulated tran-
scription or a translational/post-translational control of transcripts/
proteins that could be present already in the ovulated MII oocyte.
Since the male genome is unimportant in determining/overcoming
the 2-cell block (Goddard and Pratt, 1983), to understand whether
the female genome is programmed to express the rescuing activity
or whether the rescuing factor(s) is already present in the MII
ooplasm, in the present study we have performed reciprocal MII-
transfer between MII oocytes of a non-blocking inbred strain
(B6C3F1, F1) and MII oocytes of a blocking outbred strain (CD1).
We argued that if the MII-plate of a non-blocking strain transferred
into an enucleated oocyte of a blocking strain was supporting
development beyond the 2-cell stage, whereas the reciprocal
transfer was not, then we could assume that the female F1 genome
was programmed to control the expression of this factor(s) in a
developmentally regulated fashion. On the contrary, we could infer
that the factor(s) was already present in the ooplasm of non-
blocking MII oocytes.
Reciprocal MII-plates transfer between MII oocytes of a non-
blocking inbred strain and MII oocytes of a blocking outbred
To this end, four different types of reconstructed MII oocytes
were produced: F1F1MII (F1MII-plates transferred into F1 enucle-
ated oocytes), F1CD1MII (CD1MII-plates transferred into F1 enucle-
ated oocytes), CD1CD1MII (CD1MII-plates transferred into CD1
enucleated oocytes) and CD1F1MII (F1MII-plates transferred into
CD1 enucleated oocytes). Following MII-transfer (Figure 1), oo-
cytes were inseminated in vitro with capacitated spermatozoa
isolated from the epidydimes of F1 males; 6 hr later, those oocytes
that presented two polar bodies (PBs) were transferred to M16
medium for further embryonic development. Control in vitro
fertilisation (IVF) experiments were conducted in parallel. For each
type of experiment, about 10 of the inseminated oocytes with two
PBs were fixed, stained with acetic orcein and observed under
phase microscopy for the presence of a male and a female
pronucleus. The great majority (98%) of all the reconstructed
oocytes presented two polar bodies and were correctly fertilised by
one single sperm (Fig. 2A). Then, 1-cell embryos were cultured for
up to 96 hrs to observe preimplantation development.
The MII-transfer procedure was tested, from fertilisation to birth,
on F1F1MII reconstructed oocytes. These experiments showed that
the whole procedure did not affect the preimplantation develop-
mental potential of the reconstructed oocytes. The frequency of
F1F1MII 2-cell embryos that developed to the blastocyst stage
(63.3%; Table 1) was not significantly different compared to that
obtained after IVF of normal F1 MII oocytes (71.4%; p= 0.218,
statistically not significant). When eight blastocysts obtained from
F1F1MII oocytes where transferred to a pseudopregnant female
recipient, three of them reached full-term development giving birth
to healthy mice (Fig. 2B). The majority of the zygotes obtained from
CD1CD1MII or CD1F1MII oocytes stopped development at the 2-cell
Fig. 1. An MII-plate was removed from an F1 MII oocyte (A,B) and
transferred to another, previously enucleated, F1 MII oocyte (C,D).
All the other MII-plate transfers were obtained following the same
procedure. Arrows: an MII-plate (A); an MII-plate that is being removed
from the oocyte (B); an MII-plate enclosed into a small portion of
cytoplasm and oolemma, removed using a micropipette (C); an MII-plate
few minutes after its transfer into a previously enucleated oocyte (D).
Ooplasmic factors rescue the 2-cell block 131
stage (73.5% and 81.2%, respectively) (Table 1, Fig. 2C), a
number not significantly different (p= 0.351, statistically not signifi-
cant) to that obtained after IVF of CD1 oocytes with F1 sperm
(75.6%). As shown in Table 1, 5.9% of CD1CD1MII, none CD1F1MII
and 42.2% F1CD1MII 2-cell embryos developed to the blastocyst
stage (Fig. 2D).
As reported before (Whittingham, 1974; Pratt and Muggleton-
Harris, 1988), the 2-cell block is not an ‘all or none’ phenomenon,
but some of the embryos from blocking strains may develop
beyond the 2-cell stage and reach blastocyst. In the reconstructed
oocytes that we produced with a CD1 ooplasm, the number of
blastocysts varied from none (CD1F1MII) to 5.9% (CD1CD1MII). The
reconstructed embryos that best developed beyond the 2-cell
stage were those with an F1 ooplasm, namely F1F1MII and F1CD1MII.
The percentage of F1CD1MII 2-cell embryos that reached the 4-cell
stage (78.9%) was not significantly different (p= 0.085, statistically
not significant) to that of F1F1MII 2-cell embryos (89.4%) (Table 1,
Fig. 2E); however, although the majority of F1CD1MII embryos
developed beyond the 2-cell stage, further preimplantation devel-
opment was significantly different comparing the two types of
reconstructed embryos as 75.7% F1F1MII and 54.9% F1CD1MII 2-cell
embryos (p<0.05) reached the morula stage and 63.3% F1F1MII and
42.2% F1CD1MII embryos (p<0.05) reached the blastocyst stage
(Table 1). In those F1CD1MII embryos that reached the blastocyst
stage, the blastomere number was attested at 35+8 (Fig. 2F), a
number of cells not significantly different (p= 1, statistically not
significant) to that found in control IVF F1 embryos (35+5).
Cytoplasm transfer from non-blocking to blocking oocytes
Since this rescuing factor(s) is present in the ooplasm of
developmentally competent gametes, we then argued whether the
transfer of cytoplasm from F1 MII oocytes to CD1 MII oocytes could
result in the acquisition of developmental competence by this latter
blocking strain. When we injected approximately 8-10 pl of MII-F1
ooplasm into MII-CD1 oocytes, the majority of CD1 2-cell embryos
developed to the 4-cell stage (34 4-cell out of 49 2-cell embryos,
69.4%), and 38.7% of them developed to blastocyst (Table 2).
Cytoplasm transfer from MII-CD1 to MII-CD1 oocytes did not
rescue these oocytes from the 2-cell block (Table 2). This result
shows that a small quantity of F1 ooplasmic factor(s) is capable of
rescuing the majority of blocking CD1 oocytes to develop beyond
the 2-cell stage and also that the CD1 ooplasm does not represent
an inhibitory environment since it did not inactivate the functionality
of the transferred F1 factor(s). While the transfer of a small quantity
of ooplasm from F1 oocytes to CD1 oocytes rescues the latter from
the 2-cell block, the transfer of the whole F1 MII-plate to an
enucleated CD1 oocyte did not give any developmental improve-
ment, suggesting that the rescuing factor is not present on or
around the MII-plate.
Cytoplasm transfer from non-blocking MII oocytes to block-
ing 2-cell embryos
In a third type of experiment we addressed the question of
whether the ooplasm factor(s) activates a series of events, com-
prised between the oocyte and the 2-cell embryo, that lead to
completion of the second mitotic division or whether itself can
Fig. 2. Development of embryos obtained by reciprocal MII-plate
transfer. (A) About ten of the oocytes showing two polar bodies were
fixed and stained with acetic orcein and observed under phase micros-
copy. The micrograph shows the male (black harrowhead) and female
(white arrow) pronuclei. The male pronucleus is recognisable for the
presence of the sperm tail. (B) Three F2 mice born following IVF
fertilisation and embryo transfer of blastocysts obtained after insemina-
tion of F1F1MII reconstructed oocytes with F1 sperm. The mouse with the
white coat colour is the recipient female. (C) Most of the CD1F1MII
stopped development at the 2-cell stage. (D) The majority of F1CD1MII 2-
cell embryos developed to the blastocyst stage. (E) Summary of the
percentage of development of the 2-cell embryos obtained from the four
different types of reconstructed oocytes. (F) Blastomeres of a single
blastocyst developed from an F1CD1MII reconstructed oocyte.
% ± S.D. (N.) of preimplantation embryos
89.4 ± 10.1
26.5 ± 13.2
18.8 ± 6.3
78.9 ± 10.1
75.7 ± 9.1
20.6 ± 11.2
7.2 ± 3.6
54.9 ± 9.1
63.3 ± 13.8
5.9 ± 7.3
CD1 CD1 54 100 (34)
CD1 F1 87 100 (69)
F1 CD1 89 100 (71) 42.2 ± 10.1
RECIPROCAL MII-PLATE TRANSFER
Reciprocal MII-plate transfer between enucleated MII oocytes of a non-blocking inbred strain (F1)
and enucleated MII oocytes of a blocking outbred strain (CD1). Reconstructed oocytes were
inseminated with F1 spermatozoa and preimplantation development beyond the 2-cell stage was
*: the percentage was calculated considering the 2-cell stage as 100%.
132 M. Zanoni et al.
participate to unblock the second mitotic division. We have injected
approximately 10 pl of cytoplasm taken from F1 oocytes at the
opposite side of the MII-plate, into one single blastomere of early
(21 hr post insemination) 2-cell CD1 embryos obtained following
IVF with F1 spermatozoa. Out of a total of 58 CD1 2-cell embryos
injected, 36 (62%) developed to the 4-cell stage, 26 (44.8%) to the
morula and 23 (39.6%) to the blastocyst stage. Development
beyond the 2-cell stage in these experiments was significantly
(p<0.05) improved compared to that of embryos obtained after IVF
of CD1 MII oocytes. These results demonstrate that the same
factor(s) present in the F1 ooplasm is capable of rescuing CD1 2-
cell embryos and to induce further development, with a percentage
of embryos that reached the blastocyst stage similar (39.6%) to
that of reconstituted F1CD1MII embryos (42.2.%, p = 0.668, statisti-
cally not significant; Table 1). Also, the small quantity of cytoplasm
that is transferred in one blastomere is sufficient to induce the
second mitotic division in both blastomeres, as already shown
before (Pratt and Muggleton-Harris, 1988) when cytoplasm was
transferred between two 2-cell embryos. We think that a passage
of molecules from the injected blastomere to the other may occur
and induce cell division in the uninjected blastomere. This migra-
tion has been demonstrated to occur through gap junctions be-
tween blastomeres of cleavage embryos and fibroblasts, these
latter shown to express Oct-4, an embryonic-specific transcription
factor (Burnside and Collas, 2002).
The results of our study show that development beyond the 2-
cell stage does not depend on the MII genome, but instead it relies
on a cytoplasmic factor(s) already present in ovulated non-block-
ing oocytes, but absent, inactive or quantitatively insufficient in
blocking oocytes. These results also indicate that the CD1 MII-
plate, within an F1 cytoplasm, does not gain full developmental
competence, suggesting a strain-specificity of this genome. At
these very early stages of development, epigenetic modifications
from a gametic to an embryonic type of chromatin organisation
mold an expression profile required for development. Methylation
of cytosines in CpG sites is crucial for regulating the temporal,
spatial and parent-specific gene expression patterns (Haaf, 2006).
The paternal genome is actively demethylated within a few hours
after fertilisation, whereas the maternal genome is passively
demethylated after the 2-cell stage (Mayer et al., 2000; Barton et
al., 2001). Recent studies have demonstrated a strain-specific
difference in the efficiency of methylation reprogramming during
preimplantation development (Shi and Haaf, 2002; Haaf, 2006). In
vitro development of NMRI (an outbred strain that shows the 2-cell
block) 2-cell embryos show a higher frequency of abnormal methy-
lation patterns than B6C3F1, suggesting a functional link between
methylation reprogramming defects and lower developmental com-
petence (Shi and Haaf, 2002). The transfer of the CD1 genome
within the F1 ooplasm clearly improves its developmental compe-
tence, but the reprogramming activity is not completely efficient.
We have given a clear indication on the presence within the
mouse F1 ooplasm of a component(s) involved in conferring
developmental competence beyond the 2-cell stage. However, its
molecular feature remains unknown. The data of earlier studies
and a number of considerations that we may draw out from our
results may help its search.
One marked evidence is that this factor(s), or its functional form,
has a cyclic nature: it is present in MII oocytes (our data), it
disappears in the early 1-cell embryo, reappears in the early 2-cell
embryo, disappears in S-phase of the second cell cycle and
reappears again in the following G2-phase (Pratt and Muggleton-
Harris, 1988). This cyclic appearance speaks for a possible role of
this component(s) in the regulation of the embryo’s cell cycle and
since we have shown that its injection into a blastomere rescues 2-
cell blocking embryos from the block, the rescuing molecule is
probably the same during the first two cell cycles. At this regard, the
maturation promoting factor (MPF) has been considered a pos-
sible candidate. MPF is a cell cycle regulator in all eukaryotes and
failure of MPF activation leads to arrest of the cell cycle at the G2/
M phase (Norbury and Nurse, 1992). MPF is a complex of cyclin-
dependent kinase p34cdc2 and cyclin B; p34cdc2 kinase is
dephosphorilated and activated by cdc25 phosphatase (King et al.,
1994; Lew e Kornbluth, 1996) and cyclin B is phosphorylated by
Polo-like kinase I and translocated into the nucleus (Pines and
Hunter, 1991; Ookata et al., 1992). Microinjection of MPF purified
from Xenopus laevis eggs into 2-cell mouse embryos, rescued
these embryos from the block (Nakano et al., 2001). An earlier work
has proposed that 2-cell blocked embryos contain enough p34cdc2
to induce mitosis, but the mechanisms for its dephosphorilation
and activation is inefficient (Aoki et al., 1992). Absence of MPF
activation in G2/M-arrested 2-cell embryos occurs in the presence
of phosphate in the culture medium, it affects the dephosphorilation
of phosphorylated p34cdc2 (Aoki et al., 1992; Haraguchi et al.,
1996) and is correlated to a decrease in the levels of cyclin B and
cdc25B mRNAs (Haraguchi et al., 1999). When phosphate is
absent from the medium, the activation pattern of p34cdc2 kinase
during the second cell cycle is similar to that in the first cell cycle,
allowing completion of preimplantation development (Haraguchi et
Although a possible involvement of MPF as a crucial molecule
in rescuing embryos from the 2-cell block is intriguing and sup-
ported by a number of observations, our results suggest that it
might not be the sole factor. In mouse MII oocytes MPF is
compartmentalised, with the majority of this kinase around the MII-
plate (Fulka et al., 1995). The MII-transfer technique that we used
removes the whole MII-plate together with some cytoplasm around
the spindle (Figure 1), thus MPF from an F1 oocyte should be
transferred altogether into the recipient enucleated CD1 oocyte.
Since our results clearly show that the reconstituted CD1F1MII
oocytes maintained the 2-cell block (Table 1), MPF present in MII
oocytes is likely not enough to release 2-cell embryos from the
block. MPF is involved in the first mitotic division which occurs also
in blocking embryos, but some other molecule(s), perhaps other
cell cycle molecules, already present in the F1 ooplasm in an active
form (as shown by the transfer of small quantities of F1 cytoplasm
to MII oocytes or 2-cell CD1 embryos), is needed to complete the
N. (%) of preimplantation embryos
49 34 (69.4) 26 (53.1) 19 (38.7)
32 8 (25)
transferred N. of oocytes
82 5 (15.6) 1 (3.1)
TRANSFER OF OOPLASM FROM F1-MII TO CD1-MII RESCUES
THE DERIVED EMBRYOS FROM 2-CELL BLOCK
Ooplasmic factors rescue the 2-cell block 133
second cell cycle.
Other candidates might be found focusing on the events that
most characterise these early developmental stages. For example,
the female gamete accumulates during growth a group of gene
transcripts and proteins that become necessary, after fertilisation,
for successful embryogenesis. These maternal-effect genes are
crucial during the oocyte-to-embryo transition (Zheng and Dean,
2007), since their misregulation determines a developmental block,
mainly at the 2-cell stage. Our next experiments will try to compare
the pattern of expression of these genes and of their proteins in
preimplantation embryos derived from oocytes of blocking and
Materials and Methods
Mice of the strains B6C3F1 (3-4 month-old males and 4-5 week-old
females) and CD1 (4-5 week-old females) were purchased from Charles
River (Como, Italy). Animals were maintained under controlled room
conditions (22°C, with 60% air moisture and 14L:10D photoperiod) and
investigations were conducted in accordance with the guiding principles of
European (n. 86/609/CEE) and Italian (n. 116/92, 8/94) laws protecting
animals used for scientific research.
All reagents were purchased from Sigma Chemical Co. (Milano, Italy),
unless otherwise stated.
Micromanipulation of metaphase II oocytes
Ovulated MII oocytes were collected from the oviducts of females
injected with 3.5 I.U. Folligon (Intervet International, Netherlands), followed
48 hr later by an injection of 3.5 I.U. Corulon (Intervet International,
Netherlands). MII oocytes were placed in M2 medium containing hyalu-
ronidase (Type II, 500 IU/ml in BSA-free M2—medium) for 4-5 min., then
cumulus-free oocytes were thoroughly washed in Whittingham medium
(Whittingham, 1971) and transferred to a drop of fresh Whittingham
medium in a 35x10 mm plastic petri dish (Corning, Bibby Sterilin, UK) under
mineral oil and placed in incubator at 37°C and 5% CO2, before further
treatment. Groups of 20-30 MII oocytes were then transferred to HEPES-
buffered CZB (H-CZB) medium (Chatot et al., 1989) containing 0.1% PVP
(Polyvinylpyrrolidone), 5 µg/ml cytochalasin B and 0.1% DMSO, for enucle-
ation. Enucleation and MII transplantation were performed using piezo-
driven (PMM 150 FU, Prime Tech, Ibaraki, Japan) borosilicate micropi-
pettes (Clark Instruments, Edenbridge, UK) under an inverted microscope
equipped with DIC optics (Olympus, Ibaraki, Japan). Following enucle-
ation, oocytes were transferred in M16 medium (Biggers, 1998) and
incubated at 37° C in 5% CO2 in air for 1 1/2 hr. Then they were transferred
in H-CZB containing 1% PVP and 5 µg/ml cytochalasin B and following MII
transplantation, reconstructed oocytes were incubated in M16 for 1 1/2 hr,
washed in Whittingham medium and finally transferred in fresh Whittingham
medium for insemination with capacitated spermatozoa.
In vitro fertilisation and embryo culture
Sperm were isolated as previously described (Zuccotti et al., 1998) and
incubated for 60 min in 100 µl drops of Whittingham medium at a final
concentration of 1.8x106 sperm/ml. MII oocytes were transferred into the
insemination drop and incubated at 37°C under a 5% CO2 in air for 2 hr.
Based on the presence of a second polar body, as a sign that fertilisation
had occurred, presumptive 1-cell stage embryos were pooled, transferred
from Whittingham medium to a 30 µl drop of M16 medium (Biggers, 1998),
for further development.
For each experiment type, about 10 oocytes with two polar bodies were
used to check that fertilisation was occurred. Oocytes were placed under
a coverslip suspended with drops of beeswax at each corner; the coverslip
was slightly pushed down until the oocytes were held in place. Oocytes
were then treated with a series of fixatives and washing. Glutaraldehyde
(2.5%) was drawn under the coverslip using a filter paper placed at one side
of the coverslip; this fixative was followed by 10% formalin, a wash with
distilled water, 95% ethanol and finally 1% acetic orcein.
Statistical analysis was done with the SigmaStat 3.0 software, introduc-
ing the raw data of each single experiment performed and using the
Student’s t test. A value of p<0.05 was considered statistically significant.
This work was supported by grants from: FIRB 2005 (Project N.
RBIP06FH7J), Regione Lombardia (Project REGLOM06), Millipore, Olym-
pus Foundation Science for Life, Fondazione CARIPLO.
AOKI, F., CHOI, T., MORI M., YAMASHITA, M., NAGAHAMA, Y. and KOHMOTO, K.
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