Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

Abstract

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Keywords: European roe deer (Capreolus capreolus); embryo development; embryonic diapause.

Fig. 1.

Developmental progression of preimplantation roe deer embryos from August to January with an example of an expanded hatched blastocyst in November. The embryonic number of cells were determined based on the embryonic genomic DNA content, and a doubling time of 2 to 3 wk in the number of cells was estimated prior to elongation. Dataset overlaps with previously published data (5).

Fig. 2.

(A and B) Principal component–based between-group analyses for the embryos (A) and luminal epithelium (B). Colors indicate the defined developmental stages. Early blastocysts form a separate cluster, embryos during diapause display a developmental-stage specific transcriptome profile, which, in parts, overlap, and the elongated embryos form a separate cluster. (C and D) The pseudotime backbone derived from the CellTree algorithm (68) of single embryos (C) and luminal epithelium (D). Each circle represents a sample and the colors indicate the assigned developmental stage based on the number of embryonic cells. Samples with largely similar transcriptome profiles are clustered together around one main sample (smaller circles attached to each big circle), while samples with less similar profiles are placed next to each other (big circles), connected by a backbone. (E and F) Transcriptome dynamics displayed by a self-organizing tree algorithm (SOTA) of the differentially abundant transcripts as calculated with the ImpulseDE2 algorithm (69) with mean centered log2 TPM+1 at predefined developmental stages including early blastocysts (EB), early (ED), mid (MD), and late diapause (LD), preelongated (PE), and elongated embryos (E) for the embryos (E) and for the LE (F). The embryonic and luminal epithelial transcriptome SOTA clusters are derived by unsupervised clustering, and derived clusters represent a group of transcripts with similar changes of the expressed transcripts.

Fig. 3.

(A) Uterine fluid AA abundance against developmental progression defined as number of embryonic cells, displayed with a locally weighted scatterplot smoothing (Loess) regression. (B) Developmental stage–specific uterine fluid AA profile, displayed with a principal component based between-group analyses. ED: early diapause, MD: mid-diapause, LD: late diapause, PE: preelongation, and E: elongated. (C) Correlation plot of the uterine fluid and plasma AA, indicating a lack of correlation between uterine fluid and plasma AA. Statistically significant correlations are displayed (P < 0.05). The sizes of the dots indicate the degree of the correlation. The red dots indicate a negative correlation, while the blue dots indicate a positive correlation.

Fig. 4.

(A) Uterine fluid serine abundance against developmental progression defined as number of embryonic cells, displayed with a locally weighted scatterplot smoothing (Loess) regression. (B) Abundance of uterine fluid mTORC1 priming and activating AA per developmental stage. Priming is achieved by a group of AAs including l-asparagine, l-glutamine, l-threonine, l-arginine, l-glycine, l-proline, l-serine, l-alanine, and l-glutamic acid. The group of activating AAs includes l-leucine, l-methionine, l-isoleucine, and l-valine (31). An increase in mTORC1-activating AAs was evident from preelongation onward. The letters indicate statistically significant differences at P < 0.05.

Fig. 5.

(A) Embryonic mTOR signaling during diapause, at preelongation, and during elongation. The pathway is derived from the Qiagen IPA software. All figures and data produced from IPA are available under an open-access CC-BY license for purposes of publication and are used with written permission. The mTORC1 is predicted to be inhibited during diapause and activated at preelongation. The red circles indicate transcriptional and predicted changes first observed at preelongation and elongation. (B) Embryonic expression of genes involved in the TCA cycle, fatty acid β-oxidation (FAO), glycolysis, oxidative phosphorylation (oxphos), and pentose phosphate pathway (PPP) displayed as z-scores of the fold changes. An increase in these metabolic pathways is observed from preelongation onwards. (C) Embryonic expression of genes involved in the one carbon metabolism. The embryonic gene expression and uterine fluid AA abundances are displayed in a heat map format in which each box represents one of the defined developmental stages, including the early blastocyst, early diapause, mid-diapause, late diapause, preelongation, and elongation stage.

Fig. 6.

Proposed mechanism of the regulation of embryonic diapause in the roe deer. Endometrial E2 signaling is inhibited during embryonic diapause. The increase in the rate of proliferation in the embryo coincides with a decrease in microRNA let-7–driven gene expression in the embryos and a simultaneous increase in mTORC1 signaling. The increase in mTORC1 signaling is related to an increased expression of genes involved in the glycolytic and pentose phosphate pathway, the TCA cycle, and one carbon metabolism.