Inhibition of apical domain formation does not block blastocyst development in bovine embryos
S. A. A. dos Anjos A B , C. P. da Costa A , M. E. O. A. Assumpção A , J. A. Visintin A and M. D. Goissis
A C
+ Author Affiliations
- Author Affiliations
A Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, University of São Paulo, Av. Orlando Marques de Paiva, 87, Sao Paulo, SP 05508-270, Brazil.
B Institute of Biosciences, University of São Paulo, Av. Orlando Marques de Paiva, 87, Sao Paulo, SP 05508-270, Brazil.
C Corresponding author. Email: mdgoissis@usp.br
Reproduction, Fertility and Development 33(10) 665-673 https://doi.org/10.1071/RD20339
Submitted: 23 December 2020 Accepted: 4 May 2021 Published: 7 June 2021
Abstract
The first event of cellular differentiation consists of the segregation of the trophectoderm and the inner cell mass. Studies in mice suggest that cell contractility and the formation of an apical domain play important roles in this event; however, this remains unknown in the bovine. We tested the hypothesis that blocking apical domain formation would halt subsequent trophectoderm differentiation in bovine embryos. We first assessed the formation of an apical domain by the presence of Par-6 Family Cell Polarity Regulator Beta (PARD6B) and Ezrin (EZR), which appeared after the 8-cell stage. We inhibited apical domain formation by blocking cell contractility with 25 μM (–)-blebbistatin. Treatment from 90 to 186 h after insemination did not reduce blastocyst development compared with the untreated control group or the group treated with inactive (+)-blebbistatin. Immunofluorescence staining after blebbistatin treatment revealed the absence of EZR and the trophectoderm marker Caudal Type Homeobox 2 (CDX2). Following blebbistatin treatment, Yes1 Associated Transcriptional Regulator (YAP), which is involved in the Hippo signalling pathway, exhibited cytoplasmic staining instead of nuclear localisation. Despite changes in protein expression and localisation, no difference in trophectoderm or total cell numbers was observed. In conclusion, inhibition of cell contractility inhibited apical domain formation without impairing blastocyst formation, suggesting that a different biological mechanism is involved in trophectoderm and inner cell mass differentiation in bovine embryos.
Keywords: blastocyst, embryo differentiation, inner cell mass, trophectoderm.
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