p38-Mitogen Activated Kinases Mediate a Developmental Regulatory Response to Amino Acid Depletion and Associated Oxidative Stress in Mouse Blastocyst Embryos

Abstract

Maternal starvation coincident with preimplantation development has profound consequences for placental-fetal development, with various identified pathologies persisting/manifest in adulthood; the ‘Developmental Origin of Health and Disease’ (DOHaD) hypothesis/model. Despite evidence describing DOHaD-related incidence, supporting mechanistic and molecular data relating to preimplantation embryos themselves are comparatively meager. We recently identified the classically recognized stress-related p38-mitogen activated kinases (p38-MAPK) as regulating formation of the extraembryonic primitive endoderm (PrE) lineage within mouse blastocyst inner cell mass (ICM). Thus, we wanted to assay if PrE differentiation is sensitive to amino acid availability, in a manner regulated by p38-MAPK. Although blastocysts appropriately mature, without developmental/morphological or cell fate defects, irrespective of amino acid supplementation status, we found the extent of p38-MAPK inhibition induced phenotypes was more severe in the absence of amino acid supplementation. Specifically, both PrE and epiblast (EPI) ICM progenitor populations remained unspecified and there were fewer cells and smaller blastocyst cavities. Such phenotypes could be ameliorated, to resemble those observed in groups supplemented with amino acids, by addition of the anti-oxidant NAC (N-acetyl-cysteine), although PrE differentiation deficits remained. Therefore, p38-MAPK performs a hitherto unrecognized homeostatic early developmental regulatory role (in addition to direct specification of PrE), by buffering blastocyst cell number and ICM cell lineage specification (relating to EPI) in response to amino acid availability, partly by counteracting induced oxidative stress; with clear implications for the DOHaD model.

DDX21 is a p38-MAPK sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

“…Our analysis of the contribution of Ddx21 downregulated cell clones to late blastocyst (E4.5) ICM lineages is ostensibly an extension of our previous observations obtained under p38-MAPKi culture conditions 11–13 , and is similarly characterised by a reduction in the number of GATA4 expressing PrE cells (Fig. 4).…”

“…Having previously confirmed a role for p38-MAPK during blastocyst maturation and PrE lineage differentiation 11–13 plus the effects of p38-MAPKi on DDX21 protein localisation (Fig. 2) and the remarkably reduced contribution of Ddx21 siRNA injected blastomeres towards inner cells (Fig.…”

“…Given the revealed p38-MAPKi sensitivity of blastocyst DDX21 nucleolar localisation, we speculated what the effect of targeted dysregulation of the Ddx21 gene expression would be on mouse preimplantation embryo development and, given our previously described reports 11–13 , on blastocyst ICM cell fate derivation and specifically PrE differentiation. Accordingly, we devised a siRNA microinjection mediated scheme to downregulate Ddx21 transcript levels (schematic described in Fig.…”

“…a) Scheme illustrating experimental protocol of p38-MAPK inhibition (p38-MAPKi) during blastocyst maturation (E3.5-4.5; previously reported by us to be a period during which p38- MAPK signalling is required for PrE specification and blastocyst maturation 11–13 ). b) d) Confocal microscopy z-projections of (b) control (DMSO treated; n=7) and (c, d) p38-MAPKi (SB220025 treated; n=5) embryos immuno-stained for DDX21 and NUCLEOSTEMIN (GNL3) (both displayed as inverted grayscale images).…”

“…In the earlier developmental stages, p38-MAPK activity is associated with formation of filamentous actin 17 and embryo compaction and subsequent appropriate functioning of the TE to enable expansion of the blastocoel cavity 15,16 . Our own p38-MAPK research has primarily focussed on the post E3.5 cavitated stages, thus specifically addressing blastocyst maturation and ICM specification towards EPI and PrE lineages 11–13 . In our recent work, we have identified a protein translation associated regulatory role for p38-MAPK that underpins PrE cell fate differentiation.…”