83 CHANGES IN BLASTOCYST LINEAGE DIFFERENTIATION AND NUTRIENT SENSING ARE MEMORIZED IN RESPONSE TO MATERNAL LOW-PROTEIN DIET

Abstract

Studies have consistently shown that conditions experienced during the periconceptional period including culture in vitro and maternal diet composition can profoundly alter the development of the embryo/fetus and can result in adult diseases such as obesity, hypertension, and cardiovascular disease. This programming occurs during the pre-implantation period. Here, we have examined blastocyst phenotype changes in response to maternal protein undernutrition during the pre-implantation period and determined whether such adaptations are stable through a change in environment. Embryos were flushed at Day 3.25 to 3.75 from naturally mated MF1 mice fed either a control (NPD) or low-protein diet (LPD) from day of plug. Blastocysts were differentially labeled using the TNBS-anti-DNP-complement method with propidium iodide and Hoechst, and cells were counted on z-series with overlays using Metamorph software (Molecular Devices, Sunnyvale, CA). For outgrowths, blastocysts flushed from LPD or NPD mothers were placed into KSOM supplemented with 10% FCS and amino acids at uterine fluid concentrations (Porter et al. 2003 Pediatr. Res. 53, 46A abst). Outgrowths were cultured for an additional 96 h and scored daily with final 4′,6-diamidino-2-phenylindole (DAPI) nuclei counts. Neither total cell numbers nor lineage divergence was affected by maternal diet until just before implantation (Day 3.75) when blastocysts from LPD mothers had significantly more total cells due to an increase in trophectoderm (TE) compared with NPD blastocysts (ANOVA; Table 1). Upon outgrowth, LPD embryos spread over a greater area although with similar nuclei numbers compared with NPD blastocysts. Moreover, inhibition of the mTOR signaling pathway with rapamycin (rapa) showed the expected dose-responsive decrease of spreading in NPD but not in LPD outgrowths (Table 1). Our data suggest a compensatory upregulation of the TE lineage after mild maternal protein undernutrition. This response becomes evident just before implantation and is, together with altered nutrient sensing and signaling sensitivity, sustained throughout peri-implantation development in vitro regardless of culture environment. Thus, we show that adaptations evident by the blastocyst stage and induced by maternal environment can subsequently be memorized and are likely to contribute to long-term programming of phenotype independent of that environment.

Table 1.  Blastocyst and peri-implantation development respond to maternal diet

Funding by DOHaD, Gerald Kerkut Trust, and NICHD are gratefully acknowledged.