Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.
OBJECTIVE
We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis).


METHODS
ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining.


RESULTS
In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM.


CONCLUSIONS
GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.
Department of Obstetrics and Genecology, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita, 010-8543, Japan.
Kazue
K
Togashi
Jin
J
Kumagai
Emiko
E
Sato
Hiromitsu
H
Shirasawa
Yuki
Y
Shimoda
Kenichi
K
Makino
Wataru
W
Sato
Yukiyo
Y
Kumazawa
Yasufumi
Y
Omori
Yukihiro
Y
Terada
D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D003593:Cytoplasm / Q000648:ultrastructure; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D017629:Gap Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D019850:Heptanol / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D009829:Oleic Acids / Q000494:pharmacology; D059008:Time-Lapse Imaging
D009829:Oleic Acids; C029407:oleylamide; D019850:Heptanol