P-169 Oocyte-to-embryo transition is regulated by availability of exogenous aminoacids in culture medium
Authors: Boiani M , Nolte T , Israel S , Drexler H , Makalowski W , Fuellen G 
Published: July 2024

Abstract

Study question
Is de novo protein synthesis during the conversion of oocytes into embryos coupled with aminoacid availability more than with gene transcription?

Summary answer
The oocyte-to-embryo transition is modulated post-transcriptionally by exogenous aminoacids, including translational reactivation of otherwise transcriptionally silent maternal genes.

What is known already
Countless studies documented how genes’ mRNAs are regulated up or down during the oocyte-to-embryo transition, with maternal genes being switched off and embryonic genes being switched on. This switching off and on has been poorly explored at the protein level, because it required to administer radioactive or non-canonical aminoacids to embryos in vitro or in the genital tract of the mother, in animal models. However, radioactive aminoacids could not be applied for too long as they harm cells, while non-canonical aminoacids are recognized by cells as unnatural and are metabolized differently. A promising solution is provided by non-radioactive isotopic aminoacids.

Study design, size, duration
In vivo-fertilized mouse oocytes (zygotes) were cultured to blastocysts in KSOM medium, which was adapted for live-cell proteomic labeling by replacing 2 of the 12 Eagle’s essential aminoacids (EAAs) with isotopic but non-radioactive counterparts (Arginine and Lysine, 13C 15N). The 7 non-essential Eagle’s aminoacids (NEAAs) were left untouched. Labeled blastocysts were sampled at 96 hours of culture in vitro. Controls were cultured in parallel under non-labeling conditions in conventional KSOM.

Participants/materials, setting, methods
B6C3F1 females were stimulated with eCG+hCG and mated to CD1 males. Zygotes were labeled in vitro by culture in KSOM containing polyvinylpyrrolidone in place of albumin, isotopic Arginine and Lysine in place of non-isotopic counterparts, with or without NEAAs and with or without the remaining 10 EAAs, at 37 °C under 5% CO2. In vitro-labeled blastocysts were transplanted to uterus to ascertain viability, or compared to non-labeled blastocysts by cell counting, mass spectrometry and RNA sequencing.

Main results and the role of chance
A total of 5426 mouse blastocysts were generated. Isotopically labeled embryos formed blastocysts with total cell numbers and full-term ability similar to unlabeled controls (blastocyst rate ≥68%, p ≥ 0.11; cell count ≥58, p ≥ 0.62; birth rate ≥25%, p ≥ 0.33; Wilcoxon test). When isotopic Arginine and Lysine were present as the sole aminoacids, the blastocyst proteome consisted of 54% unlabeled proteins, 30% semi-labeled proteins and 16% completely labeled proteins. The balance changed to 12%-51%-37% and 6%-56%-38% when NEAAs and NEAAs+EAAs, respectively, were added in culture on top of isotopic Arginine and Lysine (p = 1.9E-09; chi test). This rebalancing did not mirror in the transcriptomes, which were conserved in the three groups (p &gt; 0.08, Wilcoxon test). The proteins of 42 maternal-effect genes (PMID 35047854), which are downregulated during the oocyte-to-embryo transition, were unlabeled when isotopic Arginine and Lysine were present as the sole aminoacids, but became increasingly more labeled (49%, 66%) when NEAAs and NEAAs+EAAs were provided on top of isotopic Arginine and Lysine. Collectively, this information supports that the oocyte-to-embryo transition is not a cell-autonomous process, but features a dynamic post-translational response to the composition of culture medium, including an altered pace of degradation and de novo synthesis of maternal proteins.

Limitations, reasons for caution
This is an animal study. Mature and fertilized mouse oocytes were collected from oviduct, which is not how human embryos are produced in medically assisted reproduction. The role of autophagy as internal source of aminoacids was not investigated. The oocyte-to-embryo transition takes 2-3 days longer in humans than in mice.

Wider implications of the findings
As seen in mice, so also in humans the oocyte-to-embryo transition could be modulated by aminoacids, considering that human media are mouse embryo-tested and the requirement of aminoacids for development is conserved across species. To recognize that culture media actively change the embryos, also in medically assisted reproduction, is overdue.

Trial registration number
not applicable