Regulation of endocytosis in embryos: A mechanism to overcome poor maternal nutrition but with long-term health implications

Abstract

Our group have previously shown that the period around mammalian conception is sensitive to environmental factors that can affect the early embryo before implantation with enduring consequences over the lifetime. Our past work in mice has shown maternal low protein diet exclusively during preimplantation development with normal nutrition thereafter (Emb-LPD diet) leads to increased endocytosis in trophectoderm (TE) of blastocysts followed by growth, cardiovascular and metabolic dysfunctions. The maternal nutrient depletion associates with a reduction in mammalian target of rapamycin complex 1 (mTORC1) signalling (pathway regulating cellular growth) in the early embryos, which is responsive to insulin and BCAAs, both reduced in the Emb-LPD maternal serum and uterine luminal fluid, respectively. We reasoned that the reduced uterine metabolite composition is sensed by embryos through mTOR, activating endocytosis as an early biomarker of adversely programmed embryos, to increase nutrient uptake to counter the poor quality of maternal diet, yet associating with later health complications.

The current mouse study determined the role of individual BCAA and insulin depletion conditions on endocytosis in TE after establishing an improved in vitro embryo culture and endocytosis model. Thus, 2-cell embryos were cultured to the blastocyst stage in KSOM medium supplemented with BCAA and insulin at normal (N, NBCAA/N-INS; 100% of concentrations found in control-fed mothers) or with a combination or individual BCAA and/or insulin (INS) depleted by 50% (L-): low valine (L-VAL), low isoleucine (L-ISO), low leucine (L-LEU). After culture, blastocyst fluorescent markers for lysosomes (LysoTracker) and endocytosed and processed bovine serum albumin (BSA-BODIPY) were used with confocal microscopy and VOLOCITY imaging software for quantitative analysis. From many combinations of media with individually varying BCAAs and insulin, my 48 h cultures showed: (i) Culture with L-ISO was sufficient with control concentrations of other BCAAs and insulin to induce enhanced endocytosis in blastocysts; (ii) L-LEU alone had a weaker effect on endocytosis than L-ISO but L-VAL had no effect; (iii) L-INS with control BCAAs had no effect on endocytosis; (iv) Combinations of L-ISO and L-LEU enhanced endocytosis, also when combined with L-INS; (v) Control and experimental culture conditions had no effect on embryo development rate or blastocyst cell number. Additional studies on the enhanced endocytosis phenotype revealed that induction with L-ISO/N-INS also stimulated expression of the structural endocytosis vesicle protein, clathrin, and the main multi-ligand endocytic receptor, megalin, in blastocysts. Induction also increased active lysosomes containing the hydrolase cathepsin B. The timing of the change in endocytosis phenotype, assessed by screening earlier stage morulae, showed that the response only occurred from the early blastocyst stage onwards when the endocytosis system had fully matured.

I also explored whether deficiency in mTORC1 signalling in blastocysts could activate the enhanced endocytosis phenotype and, in particular, if the mTORC1-associated transcription factor for lysosome expression, TFEB, was involved. Experimental analysis showed that embryos cultured in control medium with NBCAA/N-INS with increasing concentrations of the mTORC1 inhibitor, rapamycin, led to a dramatic relocation of TFEB from mainly cytoplasmic (inactive) to nuclear (active) localisation, where it acts to promote transcription for lysosomal biosynthesis. Furthermore, treatment with the L-ISO/N-INS protocol in the absence of rapamycin also stimulated nuclear localisation of TFEB and subsequently, increased lysosomes.

Collectively, these mouse model experiments indicate L-ISO, and to lesser extents L-LEU and L-INS, act as environmental cues to stimulate compensatory blastocyst endocytosis in low nutrient conditions, mediated through altered TFEB activation shown to be responsive to reduced mTORC1 signalling.

In separate experiments, the same endocytosis assay markers were used (BSA-BODIPY and LysoTracker Red) on human embryos donated for research, and compared outcomes based upon maternal BMI and conventional IVF versus intracytoplasmic injection (ICSI). In the first experiment, patients were separated based on normal (18-23) or high (28-33) BMI as a measure of normal or dysfunctional physiological/nutritional states. Whilst no difference was found between blastocysts in terms of embryo survival and cell number, those from the high BMI group exhibited the enhanced endocytosis phenotype using LysoTracker Red. Moreover, the range of scores varied across the high BMI group showing individual embryo variation. Lastly, examination of human embryos for TFEB localisation revealed increased nuclear versus cytoplasmic location for the high BMI group, but not quite at significance level. ICSI procedure showed no effect on endocytosis phenotype, although ICSI embryo scores showed high variation in the high BMI group. Collectively, this preliminary data supports the concept that maternal nutrition/metabolism affects embryo endocytosis phenotype in the human as shown in the mouse model, with the potential to be a biomarker for embryo long-term health.

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