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-foetal 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 meagre. We recently identified the classically recognised 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 unrecognised 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.

p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation

“…On average, we observed significantly smaller cavities upon p38-MAPKi (3316µm 2 ) when compared to controls (6255µm 2 ). Taken together with our previous observations 25,26 these data indicate late blastocyst deficient PrE cell phenotypes caused by p38-MAPKi are coincident to significantly reduced cavity volumes.…”

“…In our previous studies, we also observed that p38-MAPKi throughout the entire blastocyst maturation period (E3.5 to E4.5) not only impaired PrE differentiation (recapitulated here, Fig. 1a (ii)) but appeared to cause reductions in blastocyst size and cavity volume, particularly in the absence of exogenous amino acid supplementation 26 . Therefore, as blastocyst cavity expansion and volume have recently been linked to EPI and PrE fate acquistion 12 , we decided to directly quantify late blastocyst cavity volumes following p38-MAPKi throughout the 24 hour maturation period and compare them to control treated embryos (Fig.…”

“…Collectively, these introductory data (together with our previous reports 25,26 ) confirm p38-MAPK (most probably the p38-α isoform) has a transient role during early blastocyst maturation, that feeds forward to ensure appropriate specification of PrE cells. Specifically, the developmental period of most significance is between third and tenth hours after E3.5 (and can be refined to a minimal 3-hour window within which p38-MAPKi is sufficient to significantly impair PrE differentiation).…”

“…However, notwithstanding such compensation, it is important to note p38-MAPKi throughout this same period, or simply in the identified minimal window of sensitivity, is still sufficient to impair PrE differentiation by E4.5 ( Fig. 1 and 25,26 ). Hence, the observed p38-MAPKi mediated transcript level changes may indeed be compensated by E3.5 +10h but the eventual PrE deficient phenotype persists, potentially due to impaired mRNA translation (see below).…”

“…Accordingly, we processed mRNA from 30 embryos per condition at three different time-points during early blastocyst maturation; informed by our initial analyses of the p38-MAPKi sensitive window ( Fig. 1) 25,26 . Consequently, the transcriptome was analysed after E3.5 +4h, +7h and +10h of p38-MAPKi and compared to that of control treatments (Fig.…”