Mouse early extra-embryonic lineages activate compensatory endocytosis in response to poor maternal nutrition

Abstract

Mammalian extra-embryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth. These lineages derive from outer trophectoderm (TE) and internal primitive endoderm (PE) in the blastocyst and subsequently give rise to chorio-allantoic and visceral yolk sac placentae, respectively. We have shown maternal low protein diet exclusively during mouse preimplantation development (Emb-LPD) is sufficient to cause a compensatory increase in fetal and perinatal growth that correlates positively with increased adult-onset cardiovascular, metabolic and behavioural disease. Here, to investigate early mechanisms of compensatory nutrient provision, we assessed the influence of maternal Emb-LPD on endocytosis within extraembryonic lineages using quantitative imaging and expression of markers and proteins involved. Blastocysts collected from Emb-LPD mothers within standard culture medium displayed enhanced TE endocytosis compared with embryos from control mothers with respect to the number and collective volume per cell of vesicles with endocytosed ligand and fluid and lysosomes, plus protein expression of megalin (Lrp2) LDL-family receptor. Endocytosis was also stimulated using similar criteria in the outer PE-like lineage of embryoid bodies formed from embryonic stem cell lines generated from Emb-LPD blastocysts. Using an in vitro model replicating the depleted amino acid (AA) composition found within the Emb-LPD uterine luminal fluid, we show TE endocytosis response is activated through reduced branched-chain AAs (leucine, isoleucine, valine). Moreover, activation appears mediated through RhoA GTPase signalling. Our data indicate early embryos regulate and stabilise endocytosis as a mechanism to compensate for poor maternal nutrient provision.

Early sex-dependent differences in response to environmental stress

“…The effect of this low-protein diet was mediated through changes in branched-chain amino acids and insulin levels in the uterine fluid, which were detected by embryos via the mTOR signaling pathway. These embryos were able to activate compensatory mechanisms in order to enhance maternal nutrient retrieval, by stimulating trophectoderm and primitive endoderm proliferation, endocytosis and cellular motility (Eckert et al 2012, Sun et al 2014. Although these responses protected foetal growth, at the same time, they led to abnormal growth and increased adult adiposity, resulting in adverse long-term effects, with female offspring more severely affected.…”

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage

“…mTSC show optimal growth, potency, lowest stress (defined by lowest stress-activated protein kinase activity) and differentiation at 2% O 2 level, while hypoxic stress below this level leads to decreased growth and potency and higher differentiation despite culture conditions that otherwise favor growth and potency [44]. Pre-implantation isocaloric protein deficient malnutrition diminishes the numbers of ESCs and TSCs in blastocysts [51], and this type of malnutrition also increases LRP2 expression in extraembryonic endoderm and increases endocytosis [52] (Fig. 2).…”

“…However, animal studies suggest that stress limited only to preimplantation does have long-term consequences. Low protein diet (LPD) only during preimplantation period restricted rat ICM/ESC and trophectoderm/TSC proliferation, led to increases in extraembryonic endoderm markers, decreased birth weight and caused hypertension in offspring [51,52]. Exposure to maternal diabetes during mouse oogenesis or embryogenesis is sufficient to cause increased rate of fetus malformation and growth retardation, and developmental defects can be partially explained by a high glucose environment [65].…”

“…Maternal LPD leads to up-regulation of ectoplacental outgrowth, which is an adaptive mechanism to enhance embryonic-maternal connection and maintain fetal nutrient supply. Moreover, enhanced endocytic activity in trophectoderm and 1st lineage primitive endoderm was reported to compensate for maternal pre-implantation malnutrition in rodents [52]. It is essential that mammalian extraembryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth.…”

Cell Signalling During Blastocyst Morphogenesis

“…Experimental evidence suggests that signalling through the mTORC1 pathway plays an important role in trophoblast motility (Martin and Sutherland 2001;Martin et al 2003;Gonzalez et al 2012), proliferation ) and implantation (Zeng et al 2013) since embryos null for the mTOR gene arrest at E5.5 with implantation failure (Gangloff et al 2004). In addition, the reduced availability of branched chain AAs following LPD acts to stimulate TE endocytosis through Rho A signalling, acting as a further mechanism of dietary compensation (Sun et al 2014). Indeed, compensatory morphological organisation and transport activity in response to LPD in placentas in later gestation has been reported (Coan et al 2011).…”