Enhanced water and cryoprotectant permeability of porcine oocytes after artificial expression of human and zebrafish aquaporin‐3 channels

Abstract

One of the major obstacles for the vitrification of mature porcine oocytes with ethylene glycol is their low permeability to this cryoprotectant, which results in osmotic stress-induced cell damage and low survival. Pig blastocysts, on the other hand, show enhanced water and cryoprotectant permeability, which has been related to the transcriptional activation of aquaporin-3 (AQP3) channels at this stage of development. In this study, we asked if expression of cRNAs encoding two aquaglyceroporins, human AQP3 (hAQP3) or the zebrafish Aqp3b-T85A mutant, in porcine oocytes can increase their permeability. Microinjection of germinal-vesicle-stage oocytes with enhanced green fluorescent protein (EGFP) or AQP3 cRNAs resulted in the expression of the corresponding proteins in ∼26% of the metaphase-II stage oocytes at 40-44 hr of in vitro culture; co-injection of EGFP cRNA appeared to be a suitable marker for oocyte selection since all EGFP-positive oocytes also expressed the corresponding aquaporin. Using this method, we found that mature oocytes co-expressing EGFP and hAQP3 or EGFP and Aqp3b-T85A showed approximately a twofold increase of the hydraulic conductivity (Lp ) with respect non-injected or EGFP alone-injected oocytes in a 0.43 M sucrose or 1.3 M ethylene glycol solution, whereas the ethylene glycol permeability (PEG ) of EGFP + hAQP3 and EGFP + Aqp3b-T85A oocytes was 6.7- and 12-fold higher, respectively, than control oocytes. These data demonstrate that the artificial expression of aquaglyceroporins in porcine metaphase-II oocytes improves their permeability, and that the zebrafish Aqp3b-T85A mutant is more efficient than the human channel at increasing the oocyte permeability to ethylene glycol.

Expression and localization of aquaporins 3 and 7 in bull spermatozoa and their relevance to sperm motility after cryopreservation

“…In studies of oocytes and embryos, several researchers have demonstrated the function of AQP3 and AQP7 in the molecular pathways for transport of water and CPAs, and their crucial roles for cryotolerance in mouse [ 42 , 43 , 44 ], bovine [ 45 ], and porcine [ 46 , 47 ]. In order to elucidate the association of AQP3 and AQP7 with cryotolerance in bull spermatozoa in more detail, further investigations, such as studies using experimental systems permitting the specific inhibition of the channel function of AQP3 and AQP7 in bull spermatozoa, are necessary.…”

Aquaporins in the male reproductive tract and sperm: Functional implications and cryobiology

“…As far as oocytes are concerned, Edashige, Yamaji, Kleinhans, and Kasai () demonstrated that the injection of cRNA encoding for Aqp3 into mouse oocytes increased both glycerol permeability and cell survival after vitrification. Morató, Chauvigné, Novo, Bonet, and Cerdà () reported that the exogenous expression of AQP3 in in vitro‐matured porcine oocytes also increased the permeability to water and ethylene glycol. Moreover, AQP7 is involved in the tolerance to hyperosmotic stress and survival of human oocytes following cryopreservation (Tan et al., ), which agrees with the Aqp7 upregulation observed in mouse oocytes in response to the high hyperosmolality of vitrification media (Tan et al., ).…”

Activin‐A receptor expression patterns in prepubertal goat oocytes and derived embryos

“…Immunocytochemistry was performed according to the protocol described by Morató et al (). Oocytes and embryos were fixed in 4% paraformaldehyde for 30 min at 38.5°C, permeabilized in 0.25% Triton X‐100 in PBS for 10 min and incubated in blocking solution (10 µg/ml BSA in PBS) for 30 min at RT.…”

“…Protein extraction was conducted as described by Morató, Chauvigné, Novo, Bonet, and Cerdà (2014). Briefly, 10 oocytes or embryos from each group were solubilized in 5 µl of lysis buffer, frozen and stored at −80°C.…”