Specific PKC isoforms regulate blastocoel formation during mouse preimplantation development

Abstract

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by Na+/K+ ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four (delta, theta, iota/lambda, zeta) PKC isoforms and PKCmicro/PKD1 showed partial colocalisation with the tight junction marker ZO-1alpha+ in TE and all four PKCs (delta, theta, iota/lambda, zeta) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKCdelta and zeta activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, Na+/K+ ATPase alpha1 subunit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including Na+/K+ ATPase.

Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

“…The activation of FGFR2 has been shown to regulate cell differentiation via PKC. 41,42 Interestingly, the activation of PKC has been shown to be involved in the regulation of blastocoel formation during mouse preimplantation development, 28,43 which is in accordance with our observation that the inhibition of PKC significantly suppressed the formation of expanded blastocysts. More importantly, p38 regulates cavitation and the function of tight junctions, which affects the expanded formation of the blastocyst.…”

“…The expression of certain protein markers in the development of expanded blastocysts has been shown to be mediated by a PKC-dependent pathway. 28 The addition of the PKC inhibitor G€ o6983 to the culture medium of embryos drastically inhibited the transformation of early blastocysts to expanded blastocysts compared with the DMSO-treated vehicle control (Fig. 5A).…”

Contribution of JAM-1 to epithelial differentiation and tight-junction biogenesis in the mouse preimplantation embryo

“…Apical surface PKCζ at compaction has also been reported by Pauken and Capco (Pauken and Capco, 2000). Subsequently, both atypical PKCs associate with the trophectoderm junctional complex in blastocysts (Eckert et al, 2004b), to which JAM-1 is mainly localized.…”

“…PKCζ was also present at cell contact and intracellular sites, including the nuclear region. During later cleavage, PKCζ is localized with the TJ marker ZO-1α+ at cell contact sites in the trophectoderm (Eckert et al, 2004b). By contrast, the atypical PKCι/λ displayed only intracellular cytoplasmic staining in compact eight-cell embryos (Fig.…”

“…By contrast, the atypical PKCι/λ displayed only intracellular cytoplasmic staining in compact eight-cell embryos (Fig. 5A), although it later localizes with the TJ in blastocyst trophectoderm (Eckert et al, 2004b). Compact eight-cell embryos were also screened for the localization of the novel PKC isoforms δ, µ and θ.…”