Effect of medium conditioned with rat hepatoma BRL cells on '2-cell block' of random-bred mouse embryos cultured in vitro.
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 beta-A or beta-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.
Kobayashi
Masayuki
M
Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Akita 010-0195, Japan. makoba@akita-pu.ac.jp
Terawaki
Yoshinori
Y
Saito
Koichi
K
Kasuga
Kano
K
Kojima
Ikuo
I
D028341:Activins / Q000378:metabolism*; D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000502:physiology; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D051379:Mice; D051381:Rats; D015053:Zygote / Q000502:physiology*
D017077:Culture Media, Conditioned; D028341:Activins