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Effect of medium conditioned with rat hepatoma BRL cells on ‘2-cell block’ of random-bred mouse embryos cultured in vitro

Published online by Cambridge University Press:  01 May 2009

Masayuki Kobayashi ,
Yoshinori Terawaki ,
Koichi Saito ,
Kano Kasuga  and
Ikuo Kojima
Masayuki Kobayashi*
Affiliation:
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Akita 010–0195, Japan. Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
Yoshinori Terawaki
Affiliation:
Dairy Science Institute, Rakuno Gakuen University, Ebetsu, Hokkaido 064–8501, Japan.
Koichi Saito
Affiliation:
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
Kano Kasuga
Affiliation:
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
Ikuo Kojima
Affiliation:
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
*
All correspondence to: Masayuki Kobayashi, Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Akita 010–0195, Japan. Fax: +81 18 872 1676. e-mail: makoba@akita-pu.ac.jp
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Summary

The phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the β-A or β-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

Keywords

ActivinDevelopmentEmbryoHepatomaIn vitro culture2-cell block
Type
Research Article
Information
Zygote , Volume 17 , Issue 2 , May 2009 , pp. 169 - 174
Copyright
Copyright © Cambridge University Press 2009

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