Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos
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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).
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.
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.
GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.
KeywordsGap junction inhibitor Time-lapse movie observation Delayed blastocyst formation Blastocoel collapse ICM division
We would like to thank Yasuyuki Mio and his colleagues for their constructive comments, face-to-face discussion, and helpful advice on analyzing the TLM data regarding the aberrant ICM behavior, and Masahito Tachibana for practical suggestions on the IF protocol, especially Nanog protein. We appreciate Mr. Jeffrey G. Stocker's contributions to proofreading.
All staff in the Department of Obstetrics and Genecology, Akita University Graduate School of Medicine provided considerable long-term encouragement. In particular, Hideya Kodama and Katsuya Kabashima provided valuable advice. Hisataka Hasegawa contributed constructive discussion to this work. We greatly appreciate the protocols provided by “Cell Community in early mammalian development” in Grant-in-Aid for Scientific Research on Innovative Areas.
This work was supported by JSPS KAKENHI, Grant-in-Aid for Scientific Research (C) [Grant Number 25462549]; and a Grant of National Centre for Child Health and Development (24–6).
Conflict of interest
TLM observation of mouse embryos. 1A: TLM observation of mouse embryos co-cultured with 0.1 % DMSO. They developed smoothly and finished hatching. The recording interval was 3 min at 15 frames per second. Scale bar: 100 μm (MPG 48940 kb)
1B: TLM observation of mouse embryos co-cultured with oleamide 50 μM. Aberrant ICM behavior and frequent collapses were recorded. The recording interval was 3 min at 15 frames per second. Scale bar: 100 μm (MPG 43864 kb)
TLM observation of human freeze/thaw embryos. Two embryos were obtained from a patient. They were conventionally fertilized and frozen on day 5. Both of them were frozen at 3AA (blastocyst grading system introduced by Gardner and Schoolcraft in 1999). The aberrant ICM divisions were traced in both embryos. The embryo on the left has finished hatching. The embryo on the right contained fragment blastomeres, exhibited delayed expansion, and failed to finish hatching. The recording interval was 3 min at 15 frames per second. Scale bar: 100 μm (MPG 29904 kb)
Immunofluorescent staining and confocal imaging of divided ICMs. These are confocal images taken using the Zeiss LSM 780 confocal microscope. 3A: Mouse embryo cultured with oleamide 50 μM. The same embryo as that in Fig. 4B (MPG 16222 kb)
3B: Human freeze/thawed blastocyst. Same embryo as that in Fig. 4D green, Cx43; red, Nanog; blue, DNA (MPG 26928 kb)
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