Context Intracellular pH (pHi) regulation is critical for all cell types, including preimplantation embryos. Embryos maintain pHi homeostasis through ion exchangers that respond to both acidic and alkaline challenges. Aims While alkalosis defense mechanisms decline in later embryonic stages, this study investigated whether acidosis defense mechanisms – specifically Na+/H+ exchanger (NHE) activity alone and in conjunction with sodium-dependent chloride/bicarbonate exchanger (NDBCE) – remain active across developmental stages in in vivo- and in vitro-derived embryos. Methods In vivo-derived embryos (from 2-cell to blastocyst stage) were collected from BALB/c strain female mice. To obtain embryos at the same stages under in vitro conditions, in vivo-derived pronuclear (PN) zygotes were incubated in potassium simplex optimized medium (KSOM) medium. All embryos were loaded with the pH-sensitive fluorophore BCECF-AM (2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) and their recovery rates from induced acidosis were quantified microspectrofluorometrically in two different solutions (bicarbonate-free pHKFHM and pHKSOM). The contributions of NHE1 and NHE3 isoforms were assessed using specific inhibitors (cariporide and S3226). Additionally, indirect immunofluorescence was performed to visualize NHE and NDBCE proteins in a subset of embryos. Key results The exchanger activities were high at the 2-cell stage of the embryos and there was no significant decrease across in vivo or in vitro developmental stages. Both NHE1 and NHE3 isoforms contributed to acidosis recovery. Conclusions We found that both in vitro and in vivo embryos returned to resting pH at comparable rates in both solutions. Implications The absence of functional differences between in vivo- and in vitro-cultured embryos implies that advanced-stage in vitro-derived embryos retain physiological competence, supporting their potential use in IVF applications.
Gezgin et al. (2026) studied this question.