Development of 1-Cell Embryos from Different Strains of Mice in CZB Medium1

Chatot, Clare L.; Lewis, J. L.; Torres, Ioannis; Ziomek, Carol A.

doi:10.1095/biolreprod42.3.432

Cited by 258 publications

(153 citation statements)

References 20 publications

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“…Female B6D2F1 and C57BL͞6 (B6) mice (7-10 weeks old) were each injected with 7.5 units of equine chorionic gonadotropin followed by injection of 7.5 units of human chorionic gonadotropin (hCG) 48 h later. Mature oocytes were collected from oviducts 15-17 h after hCG injection and were freed from cumulus cells by a 3-min treatment with 0.1% hyaluronidase in Chatot, Ziomet, and Bavister (CZB) medium (22). The oocytes were transferred to fresh CZB medium and incubated in it at 37°C in an atmosphere of 5% CO 2 in air for up to 90 min before micromanipulation.…”

Section: Methodsmentioning

confidence: 99%

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Ogonuki

Mochida

Miki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. We tested whether mouse male germ cells could be cryopreserved without cryoprotection by simply freezing epididymides, testes, or whole bodies. The reproductive organs were isolated from killed mice and frozen for 1 week to 1 year at ؊80°C before spermatozoa and spermatids were collected and injected into mature oocytes. Normal pups were born irrespective of strains tested (ICR and C57BL͞6). Epididymides and testes frozen and transported internationally to another laboratory by air could produce pups of inbred C57BL͞6 mice. Testicular spermatozoa retrieved from the bodies of male mice (BALB͞c nude and C3H͞He strains) that had been kept frozen (؊20°C) for 15 years could also produce normal offspring by microinsemination. Thus, freezing of either male reproductive organs or whole bodies is the simplest way to preserve male germ cells. Restoration of extinct species could be possible if male individuals are found in permafrost.cryopreservation ͉ gametes ͉ intracytoplasmic sperm injection ͉ microinsemination ͉ mouse S perm cryopreservation has been widely used for both human reproduction and animal breeding. Because much of basic research of mammalian genetics and early development has been done by using laboratory mice, and the number of genetically engineered mice (transgenesis, gene targeting, and mutagenesis) is increasing exponentially, development of simple, cost-saving, and space-effective means of mouse sperm preservation is much needed (1). Mouse sperm cryopreservation using raffinose and skim milk as cryoprotectants has been successful, but defrosted spermatozoa of some strains of mice do not fertilize eggs well (2). This is especially true for C57BL͞6 (B6) mice, most frequently used for generation of genetically engineered mice. This problem has been overcome by partial zona dissection before in vitro fertilization (3) or intracytoplasmic sperm injection of frozenthawed or freeze-dried spermatozoa (4-7).Immature male germ cells such as spermatids and spermatocytes are currently used to produce offspring (8-11) when spermatozoa cannot be obtained due to spermatogenic arrest because of genetic mutations or as the result of in vitro manipulation of germ cells (7, 10, 11). Therefore, cryopreservation of these immature sperm cells is necessary, but they suffer more cryodamage than mature epididymal spermatozoa. A cryopreservation protocol we developed for mouse spermatogenic cells in the past is rather complicated, and therefore the development of simpler techniques is required (12).Thus far, the simplest method of cryopreserving mouse spermatozoa is to freeze spermatozoa in simple salt solutions without any cryoprotectants (6). Although defrosted spermatozoa are not alive in the conventional sense, they apparently maintain their genetic integrity, because they are able to produce live offspring by microinsemination. Here we report that spermatozoa or spermatids, retrieved from f...

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Section: Methodsmentioning

confidence: 99%

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Ogonuki

Mochida

Miki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…DMEM (GIBCO͞BRL) supplemented with 10-20% (vol͞vol) FBS (HyClone) was used for dissecting embryos and isolating PGCs. Oocytes and preimplantation embryos were cultured in bicarbonate-buffered CZB medium (12) at 37°C under 5% CO 2 in air. Oocyte manipulation was carried out in Hepes-buffered CZB (Hepes-CZB) (13) at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning

Yamazaki

Mann

Lee

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

161

138

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Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19-and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.S omatic cell cloning has been successfully used to produce live cloned offspring in a variety of mammals (1-3). Although the rate of obtaining healthy offspring is very low, such successes illustrate the capacity of the egg cytoplasm and the donor somatic cell nucleus to support embryonic development.As part of the normal course of development, embryonic germ cells are dynamically reprogrammed during germ cell migration and differentiation. In this context, reprogramming is defined as a stepwise process whereby the somatic-like epigenetic pattern, hypothesized to be characteristic of the earliest detectable primordial germ cells (PGCs), undergoes erasure and is transformed into the sex-specific pattern of mature germ cells (4, 5). This process is exemplified by reprogramming of methylation associated with imprinted genes during germ-cell development; the somatic methylation imprints associated with early PGCs are erased around 11.5 days postcoitum (dpc) in both male and female PGCs and are reestablished in a sex-specific manner with paternal methylation imprints acquired early and maternalspecific methylation obtained late in the gametogenic process (6-10).Because embryonic germ cells by their very nature are in the process of reprogramming their genome, cells with different epigenetic patterns can be obtained and, in this study, were isolated by using a germ cell marker, EMA-1. Before migration into the genital ridges (10.5 dpc), PGCs are thought to possess a somatic-like epigenetic pattern, whereas, after migration (11.5 dpc and onward), germ cells are in various stages of r...

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“…Oocytes that exited MII arrest and resumed meiosis were considered to have been activated. At 48 h post-activation, glutamine and glucose were directly injected into one drop of CZB to give a final concentration of 1 and 5.56 mM, respectively, which supported embryo development to the morula and blastocyst stages (25).…”

Section: Parthenogenetic Activationmentioning

confidence: 99%

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Feng

Zhou

Ling

et al. 2009

Braz J Med Biol Res

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Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca 2+ ] i ), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca 2+ ] i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 μg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca 2+ ] i oscillations, completion of meiosis and parthenogenetic development.

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Development of 1-Cell Embryos from Different Strains of Mice in CZB Medium1

Cited by 258 publications

References 20 publications

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

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