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Home / Equine Research Bank / Anim Reprod / Inhibition of Na+, K+ -ATPase with ouabain is detrimental to...
Animal reproduction2020; 17(1); e20190079; doi: 10.21451/1984-3143-AR2019-0079

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

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 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.
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Publication Date: 2020-01-22 PubMed ID: 32368275PubMed Central: PMC7189547DOI: 10.21451/1984-3143-AR2019-0079Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research examined if blocking Na, K-ATPase enzyme in equine blastocysts (early-stage embryos) using a compound called ouabain could reduce their size for more efficient cryopreservation. However, it was found that the process led to death of the blastocysts, making it an unsuitable method for size-reduction prior to freezing.

Study Design and Methodology

  • The study involved an experimental manipulation of 16 mares, each of which had been induced to ovulate using deslorelin acetate and inseminated.
  • Their embryos were harvested at two different developmental stages: days 7 (D7) and 9 (D9).
  • These embryos (blastocysts) were then exposed to 10 M ouabain in an attempt to inhibit the enzyme Na, K-ATPase. The exposure happened over two time intervals, 1 hour and 6 hours.
  • Some of the used ouabain was naturally occurring, while some was chemically conjugated to fluorescein (OuabainFL).

Measurements and Results

  • Evaluations were performed based on the embryos’ morphometric characteristics (analyzed through bright field microscopy) and their viability (evaluated using Hoescht 33342 and propidium iodide).
  • The results showed that the blastocysts underwent a significant reduction in size when incubated for 6 hours with ouabain (an average of 45.7% decrease). However, they displayed adverse morphologic changes, and did not re-expand after subsequent incubation in a neutral medium for 12 hours.
  • Further studies showed that even a shorter exposure to ouabain, of only 1 hour, caused a similar level of size reduction; 38.7 ± 6.7% and 33.6 ± 3.3% for D7 and D9 embryos, respectively.
  • All embryos lost their viability after OuabainFL exposure. The binding of ouabain to the embryos was confirmed through the fluorescence of OuabainFL.

Conclusion

  • The researchers concluded that the method of using ouabain to inhibit the Na, K-ATPase enzyme on equine blastocysts to reduce their size is not feasible for cryopreservation purposes, as it led to the death of the embryos.

Cite This Article

APA
do Nascimento AD, Marques JCC, Cezar ARR, Batista AM, Kastelic JP, Câmara DR. (2020). Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts. Anim Reprod, 17(1), e20190079. https://doi.org/10.21451/1984-3143-AR2019-0079

Publication

Animal reproduction
ISSN: 1984-3143
NlmUniqueID: 101272804
Country: Brazil
Language: English
Volume: 17
Issue: 1
PII: e20190079

Researcher Affiliations

do Nascimento, Agnelo Douglas
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Marques, Juliana Carla Cavalcanti
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Cezar, Allan Rodolf Ribeiro
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Batista, André Mariano
  • Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
Kastelic, John Patrick
  • Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Câmara, Diogo Ribeiro
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.

Conflict of Interest Statement

Conflict of interest: The authors have no conflict of interest to declare.

References

This article includes 26 references
  1. Aurich JE, Schönherr U, Hoppe H, Aurich C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen.. Theriogenology 1997 Jul 15;48(2):185-92.
    doi: 10.1016/S0093-691X(97)84066-6pubmed: 16728118google scholar: lookup
  2. Blanco G. Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation.. Semin Nephrol 2005 Sep;25(5):292-303.
    doi: 10.1016/j.semnephrol.2005.03.004pubmed: 16139684google scholar: lookup
  3. Betts DH, MacPhee DJ, Kidder GM, Watson AJ. Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.. Mol Reprod Dev 1997 Feb;46(2):114-26.
    doi: 10.1002/(SICI)1098-2795(199702)46:2<114::AID-MRD2>3.0.CO;2-Tpubmed: 9021743google scholar: lookup
  4. 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 Apr;135(4):497-508.
    doi: 10.1530/REP-07-0298pubmed: 18367510google scholar: lookup
  5. Câmara DR, Kastelic JP, Thundathil JC. Role of the Na(+)/K(+)-ATPase ion pump in male reproduction and embryo development.. Reprod Fertil Dev 2017 Aug;29(8):1457-1467.
    doi: 10.1071/RD16091pubmed: 27456939google scholar: lookup
  6. 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 Dec;140(6):893-902.
    doi: 10.1530/REP-10-0141pubmed: 20843896google scholar: lookup
  7. 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 Jul 1;76(1):143-52.
    doi: 10.1016/j.theriogenology.2011.01.028pubmed: 21458049google scholar: lookup
  8. Clark KE, Squires EL, McKinnon AO, Seidel GE Jr. Viability of stored equine embryos.. J Anim Sci 1987 Aug;65(2):534-42.
    doi: 10.2527/jas1987.652534xpubmed: 3624099google scholar: lookup
  9. Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification.. Biol Reprod 2000 Mar;62(3):564-70.
    doi: 10.1095/biolreprod62.3.564pubmed: 10684796google scholar: lookup
  10. Eckert JJ, Fleming TP. Tight junction biogenesis during early development.. Biochim Biophys Acta 2008 Mar;1778(3):717-28.
    doi: 10.1016/j.bbamem.2007.09.031pubmed: 18339299google scholar: lookup
  11. Giannatselis H, Calder M, Watson AJ. Ouabain stimulates a Na+/K+-ATPase-mediated SFK-activated signalling pathway that regulates tight junction function in the mouse blastocyst.. PLoS One 2011;6(8):e23704.
    doi: 10.1371/journal.pone.0023704pmc: PMC3162003pubmed: 21901128google scholar: lookup
  12. Hochi S, Maruyama K, Oguri N. Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene glycol and sucrose.. Theriogenology 1996 Nov 1;46(7):1217-24.
    doi: 10.1016/S0093-691X(96)00292-0pubmed: 16727984google scholar: lookup
  13. Legrand E, Krawiecki JM, Tainturier PD, Cornière P, Delajarraud H, Bruyas JF. Does the embryonic capsule impede the freezing of equine embryos?. Havemeyer Foundation Monograph Series 2001. pp. 62–65.
  14. MacLellan LJ, Carnevale EM, Coutinho da Silva MA, McCue PM, Seidel GE Jr, Squires EL. Cryopreservation of small and large equine embryos pre-treated with cytochalasin-B and/or trypsin. Theriogenology 2001;58:717–720.
  15. 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 May;133(1):210-20.
    doi: 10.1016/0012-1606(89)90312-6pubmed: 2540052google scholar: lookup
  16. McKinnon AO, Squires EL. Morphologic assessment of the equine embryo.. J Am Vet Med Assoc 1988 Feb 1;192(3):401-6.
    pubmed: 3281922
  17. 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 Dec;12(12):3717-32.
    doi: 10.1091/mbc.12.12.3717pmc: PMC60750pubmed: 11739775google scholar: lookup
  18. Samper JC. Ultrasonographic appearance and the use of uterine edema to time ovulation in mares.. AAEP 1997. pp. 189–191.
  19. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis.. Nat Methods 2012 Jul;9(7):671-5.
    doi: 10.1038/nmeth.2089pmc: PMC5554542pubmed: 22930834google scholar: lookup
  20. Stout TA. Cryopreservation of equine embryos: current state-of-the-art.. Reprod Domest Anim 2012 Jun;47 Suppl 3:84-9.
    doi: 10.1111/j.1439-0531.2012.02030.xpubmed: 22681302google scholar: lookup
  21. Vajta G, Gjerris M. Science and technology of farm animal cloning: state of the art.. Anim Reprod Sci 2006 May;92(3-4):211-30.
    doi: 10.1016/j.anireprosci.2005.12.001pubmed: 16406426google scholar: lookup
  22. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure.. Cytometry 1998 Jan 1;31(1):1-9.
    doi: 10.1002/(SICI)1097-0320(19980101)31:1<1::AID-CYTO1>3.0.CO;2-Rpubmed: 9450519google scholar: lookup
  23. 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 Sep;57(3):630-40.
    doi: 10.1095/biolreprod57.3.630pubmed: 9283001google scholar: lookup
  24. Watson AJ, Barcroft LC. Regulation of blastocyst formation.. Front Biosci 2001 May 1;6:D708-30.
    doi: 10.2741/A636pubmed: 11333210google scholar: lookup
  25. Willingham MC. Cytochemical methods for the detection of apoptosis.. J Histochem Cytochem 1999 Sep;47(9):1101-10.
    doi: 10.1177/002215549904700901pubmed: 10449530google scholar: lookup
  26. 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.
    pubmed: 6962873

Citations

This article has been cited 2 times.
  1. Derisoud E, Jouneau L, Dubois C, Archilla C, Jaszczyszyn Y, Legendre R, Daniel N, Peynot N, Dahirel M, Auclair-Ronzaud J, Wimel L, Duranthon V, Chavatte-Palmer P. Maternal age affects equine day 8 embryo gene expression both in trophoblast and inner cell mass. BMC Genomics 2022 Jun 15;23(1):443.
    doi: 10.1186/s12864-022-08593-7pubmed: 35705916google scholar: lookup
  2. Pei J, Song R, Bao P, Yin M, Li J, Zhang G, Wu F, Luo Z, Wu X, Song W, Ba Y, Xiong L, Liang C, Guo X, Yan P. Differential proteomic analysis demonstrates follicle fluid participate immune reaction and protein translation in yak. BMC Vet Res 2022 Jan 14;18(1):34.
    doi: 10.1186/s12917-021-03097-0pubmed: 35031034google scholar: lookup
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