Extracellular vesicles improve embryo cryotolerance by maintaining the tight junction integrity during blastocoel re-expansion

Abstract

Cryopreservation is a process in which the intact living cells, tissues or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impact the survival and quality of the in vitro produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial-cells (BOECs) derived-EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between SOF and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryos ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4 %) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (Aquaporins, AQPs) and Na+/K+ ATPase (ATP1α1). The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.

GDF-8 improves in vitro implantation and cryo-tolerance by stimulating the ALK5-SMAD2/3 signaling in bovine IVF embryo development

“…Previous studies have discovered that TJ and AJ play a crucial role in maintaining the integrity of TJ during cryo-shrinkage ( Sidrat et al, 2022 ). TJ functions as a gate for the transport of ions and small molecules, whereas AJ provides structural support ( Eckert and Fleming, 2008 ; Marikawa and Alarcon, 2012 ).…”

“…TJ functions as a gate for the transport of ions and small molecules, whereas AJ provides structural support ( Eckert and Fleming, 2008 ; Marikawa and Alarcon, 2012 ). When these components are compromised during cryo-shrinkage, TJ integrity is not maintained, resulting in ineffective sealing of TJ post-thawing ( Sidrat et al, 2022 ). This difficulty in sealing prevents fluid accumulation within the blastocoel, making re-expansion difficult.…”

“…Tight junctions (TJ) and adherent junctions (AJ) determine the maintenance of TE cell integrity. Sustaining blastocoels is challenging when these structures undergo deformation or damage, and it becomes difficult to maintain the blastocoel ( Sidrat et al, 2022 ). Additionally, the Na + /K + -ATPase pump involving the ATPase Na + /K + Transporting Subunit Alpha 1 ( ATP1a1 ) located at the basal membrane of the TE facilitates the movement of Na + into the blastocoel through the TE, creating an osmotic pressure gradient that leads to fluid accumulation and blastocoel formation ( Kidder, 2002 ; Barcroft et al, 2003 ; Barcroft et al, 2004 ).…”

Messenger roles of extracellular vesicles during fertilization of gametes, development and implantation: Recent advances

“…Current research indicates that O-EVs can improve embryo blastocyst formation rate, embryo survival rate, embryo hatching rate, and post-embryo transfer birth rate ( Almiñana et al, 2017 ; Qu et al, 2019 ; Fang et al, 2022b ; de Alcântara-Neto et al, 2022 ). Furthermore, O-EVs can improve embryo development after cryopreservation and survival rate after vitrification ( Lopera-Vasquez et al, 2017 ; Leal et al, 2022 ; Sidrat et al, 2022 ). More than just O-EVs may influence embryo development in vivo .…”

“…EVs have been shown to improve the survival rate of frozen embryosLopera-Vasquez et al (2017) Bovine O-EVs can improve the re-expansion rate and hatching rate of frozen-thawed embryos, which may be related to O-EVs' ability to restore trophectoderm integrity and improve junctional cell contact and fluid fluxSidrat et al (2022) After vitrification, freezing, or heating, bovine O-EVs improved embryo survival, lipid content, and total cell numberLeal et al (2022) Pregnant mice's uterine cavity contains more I-EVs and higher levels of miRNA-21 than non-pregnant mice's uterine cavity. miRNA-21 may be involved in embryonic developmentI-EV Lv et al (2018)Cattle with endometritis have I-EVs that differ from normal I-EVs and are harmful to embryonic developmentWang et al (2019) EV secreted from the bovine blastocyst stage contains miRNA-378a-3p, which has been shown to increase the number of embryonic trophoblast cells and inner cells and improve hatching rate EO-EVPavani et al (2022) Mouse SP-EVs can improve blastocyst formation rate and ICM/TE index in sperm/oocyte mixed medium, as well as reduce embryonic cell apoptosis SP-EVMa et al (2022) Embryo implantation I-EVs are produced during pregnancy in pigs by both embryonic trophoblast cells and endometrial epithelial cells, but primarily by endometrial epithelial cells.…”