The 2-cell block occurring during development of outbred mouse embryos is rescued by cytoplasmic factors present in inbred metaphase II oocytes

Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia Animale and Centro di Ingegneria Tissutale, Universita' degli Studi di Pavia, Pavia, Italy.
The International journal of developmental biology (Impact Factor: 1.9). 02/2009; 53(1):129-34. DOI: 10.1387/ijdb.082617mz
Source: PubMed


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.

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    • "The reason to choose this specific developmental stage may rely on providing the more extended length of in vitro culture for positively fertilized embryos. However, this stage poses a risk in certain strains, since 2-cell block mostly appears linked to suboptimal culture conditions [7,27-29]. "
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    ABSTRACT: Background Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment. Methods Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR. Results In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C. Conclusions Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.
    Full-text · Article · Aug 2012 · Reproductive Biology and Endocrinology
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    • "In spite of enhanced reproductive success due to heterozygosity, [34], CD-1 mice have a low in vitro reproductive potential associated with a block at 2-cell stage of embryo development [35]. Thus additional studies may be warranted in other mouse strains and animal species in order to confirm the cruciality of aging effects observed in this work. "
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    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.
    Full-text · Article · Jun 2011 · Theriogenology
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    • "This block can be surmounted by injecting small amounts of cytoplasm from embryos that do not exhibit this block (Muggleton-Harris et al., 1982). This rescue occurs independently of the presence of maternal chromosomes, indicating the importance of stored, rather than newly synthesized, components (Zanoni et al., 2009). It has been suggested that in some types of clinical infertility, the transfer of donor ooplasm into human eggs might permit successful embryonic development (Cohen et al., 1997). "
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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.
    Full-text · Article · Mar 2010 · Development
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