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: firstname.lastname@example.org
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
134 M. Zanoni et al.
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Further Related Reading, published previously in the Int. J. Dev. Biol.
See our recent Special Issue Fertilization, in honor of David L. Garbers and edited by Paul M. Wassarman and Victor D. Vacquier at:
See our Special Issue Mammalian Reproduction and Development in honor of Anne McLaren and edited by Brigid Hogan at:
A reversible block at the G1/S border during cell cycle progression of mouse embryos.
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