Effect of Sphingosine-1-phosphate on trophectoderm differentiation in bovine embryos

Author(s)

Francieli Perroni Berling

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2024-06-07

Examining board members

Marcelo Demarchi Goissis; Flavia Regina Oliveira de Barros; Marcella Pécora Milazzotto

• Advisor: Marcelo Demarchi Goissis

Abstract

The early development of the embryo is marked by the first cellular differentiation that defines the inner cell mass (ICM) and the trophectoderm (TE). In mice, glucose (GLC) is essential for this process and oxygen tension (O2) also interferes with the formation of TE and there is evidence in embryos and in cultured cells that sphingosine-1-phosphate (S1P) can regulate the pathways involved in this event. We hypothesize that glucose is necessary and higher O2 tension is detrimental to TE differentiation, and that S1P induces TE differentiation in bovine embryos produced in vitro. This study had two objectives reported in two chapters: 1. To evaluate the effect of the presence (+GLC) or the absence (-GLC) of GLC at different O2 levels (5% or 20%) on the formation of the TE in bovine embryos; 2. to study the role of S1P stimulation in TE differentiation by adding S1P or the S1P receptor inhibitor, JTE-013. Initially, the embryos were randomly subjected to treatments on day 1, in a 2x2 factorial design, considering GLC (present/absent) and O2 (low/high voltage). In the second chapter, only the model without GLC was used in two different experiments. The first experiment consisted of treatment with S1P, while the second aimed to inhibit the pathway with JTE-013 treatment. Blastocyst formation rates and blastocysts were obtained 180 hours post-insemination. Total cell counts, TE (GATA3-positive) and MCI (GATA3-negative) were performed for all experiments, as well as qRT-PCR analysis for GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Rates and cell counts were analyzed by ANOVA, while for qRT-PCR a linear mixed model was considered. In the first study, there was an effect of O2 on cleavage rates, but not on blastocyst rates. There was an interaction between GLC and O2 on ICM cell count, CDX2 expression and SOX2 expression. In the second chapter, there was an increase in the rate of development for S1P compared to control and NaOH, although there were no changes to JTE- 013. Total cells and ICM cells were increased in the S1P group compared to control and NaOH. CDX2 expression increased in the S1P and NaOH groups, while TFAP2C decreased in the S1P group. The addition of JTE-013 did not interfere with gene expression or cell counts. In conclusion, TE differentiation occurred in the absence of glucose and, interestingly, this condition increased the expression of the TE-related gene CDX2 when exposed to greater O2 tension. Furthermore, S1P did not positively modulate TE differentiation, despite increasing CDX2, but induced an increase in the total number of cells and in the number of ICM cells, suggesting a different action of S1P in bovine embryos. (AU)

• FAPESP's process: 22/02748-1 - Effect of mTOR pathway stimulation during embryonic culture in the absence or presence of glucose on trophectoderm differentiation in embryos cattle.
• Grantee: Francieli Perroni Berling
• Support Opportunities: Scholarships in Brazil - Master