Animal Reproduction (AR)
https://animal-reproduction.org/article/doi/10.21451/1984-3143-AR2019-0079
Animal Reproduction (AR)
Original Article

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts

Agnelo Douglas do Nascimento Júnior; Juliana Carla Cavalcanti Marques; Allan Rodolf Ribeiro Cezar; André Mariano Batista; John Patrick Kastelic; Diogo Ribeiro Câmara

http://dx.doi.org/10.21451/1984-3143-AR2019-0079 Anim Reprod, vol.17, n1, e20190079, 2020
PDF
Downloads: 1
Views: 1344

Abstract

Abstract: Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

Keywords

embryology, horse embryo, sodium pump

References

Aurich JE, Schonherr U, Hoppe H, Aurich C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology. 1997;48(2):185-92. http://dx.doi.org/10.1016/S0093-691X(97)84066-6. PMid:16728118.

Blanco G. Na, K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation. Semin Nephrol. 2005;25(5):292-303. http://dx.doi.org/10.1016/j.semnephrol.2005.03.004. PMid:16139684.

Betts DH, Macphee DJ, Kidder GM, Watson AJ. Ouabain sensitivity and expression of Na/K-ATPase α and β subunit isoform genes during bovine early development. Mol Reprod Dev. 1997;46(2):114-26. http://dx.doi.org/10.1002/(SICI)1098-2795(199702)46:2<114::AID-MRD2>3.0.CO;2-T. PMid:9021743.

Budik S, Walter I, Tschulenk W, Helmreich M, Deichsel K, Pittner F, Aurich C. Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus. Reproduction. 2008;135(4):497-508. http://dx.doi.org/10.1530/REP-07-0298. PMid:18367510.

Câmara DR, Kastelic JP, Thundathil JC. Role of the Na+, K+-ATPase ion pump on male reproduction and embryo development. Reprod Fertil Dev. 2017;29(8):1457-67. http://dx.doi.org/10.1071/RD16091. PMid:27456939.

Choi YH, Gustafson-Seabury A, Velez IC, Hartman DL, Bliss S, Riera FL, Roldán JE, Chowdhary B, Hinrichs K. Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction. 2010;140(6):893-902. http://dx.doi.org/10.1530/REP-10-0141. PMid:20843896.

Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS, Hinrichs K. Successful cryopreservation of expanded equine blastocysts. Theriogenology. 2011;76(1):143-52. http://dx.doi.org/10.1016/j.theriogenology.2011.01.028. PMid:21458049.

Clark KE, Squires EL, Mckinnon AO, Seidel GE Jr. Viability of stored equine embryos. J Anim Sci. 1987;65(2):534-42. http://dx.doi.org/10.2527/jas1987.652534x. PMid:3624099.

Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod. 2000;62(3):564-70. http://dx.doi.org/10.1095/biolreprod62.3.564. PMid:10684796.

Eckert JJ, Fleming TP. Tight junction biogenesis during early development. Biochim Biophys Acta. 2008;1778(3):717-28. http://dx.doi.org/10.1016/j.bbamem.2007.09.031. PMid:18339299.

Giannatselis H, Calder M, Watson AJ. Ouabain stimulates a Na+/K+-ATPase-mediated SFK-activated signaling pathway that regulates tight junction function in the mouse blastocyst. PLoS One. 2011;6(8):e23704. http://dx.doi.org/10.1371/journal.pone.0023704. PMid:21901128.

Hochi S, Maruyama K, Oguri N. Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose. Theriogenology. 1996;46(7):1217-24. http://dx.doi.org/10.1016/S0093-691X(96)00292-0. PMid:16727984.

Legrand E, Krawiecki JM, Tainturier PD, Cornière P, Delajarraud H, Bruyas JF. Does the embryonic capsule impede the freezing of equine embryos? In: Proceedings of the 5th International Symposium on Equine Embryo Transfer; 2000 Jul 6-9; Saari, Finland. Saari: Havemeyer Foundation Monograph Series; 2001. p. 62-65.

MacLellan LJ, Carnevale EM, Coutinho da Silva MA, McCue PM, Seidel GE Jr, Squires EL. 2002. Cryopreservation of small and large equine embryos pre-treated with cytochalasin-B and/or trypsin. Theriogenology. 2001;58:717-20.

Manejwala FM, Cragoe EJ Jr, Schultz RM. Blastocoel expansion in the preimplantation mouse embryo: role of extracellular sodium and chloride and possible apical routes of their entry. Dev Biol. 1989;133(1):210-20. http://dx.doi.org/10.1016/0012-1606(89)90312-6. PMid:2540052.

McKinnon AO, Squires EL. Morphologic assessment of the equine embryo. J Am Vet Med Assoc. 1988;192(3):401-6. PMid:3281922.

Rajasekaran SA, Palmer LG, Moon SY, Peralta Soler A, Apodaca GL, Harper JF, Zheng Y, Rajasekaran AK. Na, K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells. Mol Biol Cell. 2001;12(12):3717-32. http://dx.doi.org/10.1091/mbc.12.12.3717. PMid:11739775.

Samper JC. Ultrasonographic appearance and the use of uterine edema to time ovulation in mares. In: Proceedings of the 43rd Annual Convention of American Association of Equine Practicioners; 1997; Phoenix. Phoenix, AZ: AAEP; 1997. p. 189-191.

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-5. http://dx.doi.org/10.1038/nmeth.2089. PMid:22930834.

Stout TA. Cryopreservation of equine embryos: current state-of-the-art. Reprod Domest Anim. 2012;47(Suppl 3):84-9. http://dx.doi.org/10.1111/j.1439-0531.2012.02030.x. PMid:22681302.

Vajta G, Gjerris M. Science and technology of farm animal cloning: state of the art. Anim Reprod Sci. 2006;92(3-4):211-30. http://dx.doi.org/10.1016/j.anireprosci.2005.12.001. PMid:16406426.

Van Engeland M, Nieland LJW, Ramaekers FCS, Schutte B, Reutelingsperger CPM. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998;31(1):1-9. http://dx.doi.org/10.1002/(SICI)1097-0320(19980101)31:1<1::AID-CYTO1>3.0.CO;2-R. PMid:9450519.

Waelchli RO, Macphee DJ, Kidder GM, Betteridge KJ. Evidence for the presence of sodium and potassium dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus. Biol Reprod. 1997;57(3):630-40. http://dx.doi.org/10.1095/biolreprod57.3.630. PMid:9283001.

Watson AJ, Barcroft LC. Regulation of blastocyst formation. Front Biosci. 2001;6(3):708-30. http://dx.doi.org/10.2741/A636. PMid:11333210.

Willingham MC. Cytochemical methods for the detection of apoptosis. J Histochem Cytochem. 1999;47(9):1101-10. http://dx.doi.org/10.1177/002215549904700901. PMid:10449530.

Yamamoto Y, Oguri N, Tsutsumi Y, Hachinohe Y. Experiments in the freezing and storage of equine embryos. J Reprod Fertil Suppl. 1982;32:399-403. PMid:6962873.
 

5e31737f0e88250c0528e7b6 animreprod Articles
Links & Downloads
1984-3143 (Electronic) 1806-9614 (Printed)
Anim Reprod ©2024 All rights reserved.

Anim Reprod

Share this page
Page Sections