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International journal of molecular sciences2023; 24(11); 9619; doi: 10.3390/ijms24119619

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

Abstract: In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts ( = 3) and blastocysts ( = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, = 5) or 9 (slow development, = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro ( = 5) or in vivo (after transfer into recipient mares, = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.
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Publication Date: 2023-06-01 PubMed ID: 37298570PubMed Central: PMC10253440DOI: 10.3390/ijms24119619Google Scholar: Lookup
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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 demonstrates that equine blastocysts created in laboratory settings, or in vitro, show greater dispersion and mixture of inner cell mass (ICM) cells compared to those developed naturally inside the horse’s body, or in vivo. The research also indicates that in vitro produced (IVP) embryos fare worse in terms of early embryonic loss and twin development compared to the in vivo derived (IVD) counterparts.

Key Objectives and Methodology of the Research

  • The objective of the study was to understand how embryo type (in vitro versus in vivo), developmental speed, and culture conditions affect the expression of certain cell lineage markers, CDX-2, SOX-2, and GATA-6, which identify different types of cells in early embryo development.
  • The study gathered data by analyzing two groups of blastocysts. The first group consisted of those which were IVD, and the second, those which were IVP. The cell expression of lineage markers in each group was then studied on day 7.
  • The researchers also observed what changes took place in the IVP embryos post an additional two days of in vitro culture, and what changes occurred post transferring the same into recipient mares.

Key Findings and Inferences

  • For the IVD group, on day 7, SOX-2 positive cells were seen enclosed by GATA-6 positive cells in the ICM. Some primitive endoderm (PE) cells also co-expressed SOX-2.
  • In the same group, on day 7, SOX-2 was uniquely expressed by compacted presumptive epiblast (EPI), while GATA-6 and CDX-2 expressions were associated with PE and trophectoderm (TE) specification, in that order.
  • For the IVP group, on day 7, SOX-2 and GATA-6 positive cells were intermixed and relatively scattered. There were also some TE cells that co-expressed either SOX-2 or GATA-6.
  • IVP embryos also showed lower TE and total cell numbers compared to IVD ones, and larger mean distances between EPI cells—factors that were even more significant in the slowly developing IVP embryos.
  • On the transfer of the IVP embryos into recipient mares, SOX-2 positive cells compacted into a presumptive EPI. However, extended in vitro culture did not yield the same results.

Conclusion

  • The study has concluded that in vitro produced (IVP) equine embryos display a poorly compacted ICM, with intermingled EPI and primitive endoderm (PE) cells.
  • This observation is even more prominent in slowly developing embryos. However, this issue can be corrected by transferring the IVP embryo to a recipient mare.

Cite This Article

APA
Umair M, Scheeren VFDC, Beitsma MM, Colleoni S, Galli C, Lazzari G, de Ruijter-Villani M, Stout TAE, Claes A. (2023). In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos. Int J Mol Sci, 24(11), 9619. https://doi.org/10.3390/ijms24119619

Publication

International journal of molecular sciences
ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 24
Issue: 11
PII: 9619

Researcher Affiliations

Umair, Muhammad
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Scheeren, Veronica Flores da Cunha
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Beitsma, Mabel M
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Colleoni, Silvia
  • Avantea srl, Via Porcellasco 7/F, 26100 Cremona, Italy.
Galli, Cesare
  • Avantea srl, Via Porcellasco 7/F, 26100 Cremona, Italy.
Lazzari, Giovanna
  • Avantea srl, Via Porcellasco 7/F, 26100 Cremona, Italy.
de Ruijter-Villani, Marta
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Stout, Tom A E
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.
Claes, Anthony
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands.

MeSH Terms

  • Animals
  • Horses
  • Female
  • Blastocyst / metabolism
  • Embryo, Mammalian
  • Germ Layers
  • Cell Differentiation
  • Embryonic Development

Grant Funding

  • (PEEF/SSMS/18/222). / Punjab Educational Endowment Fund (PEEF), Punjab, Pakistan

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 50 references
  1. Cockburn K, Rossant J. Making the blastocyst: lessons from the mouse.. J Clin Invest 2010 Apr;120(4):995-1003.
    doi: 10.1172/JCI41229pmc: PMC2846056pubmed: 20364097google scholar: lookup
  2. White MD, Zenker J, Bissiere S, Plachta N. Instructions for Assembling the Early Mammalian Embryo.. Dev Cell 2018 Jun 18;45(6):667-679.
    doi: 10.1016/j.devcel.2018.05.013pubmed: 29920273google scholar: lookup
  3. Iqbal K, Chitwood JL, Meyers-Brown GA, Roser JF, Ross PJ. RNA-seq transcriptome profiling of equine inner cell mass and trophectoderm.. Biol Reprod 2014 Mar;90(3):61.
    doi: 10.1095/biolreprod.113.113928pmc: PMC4435230pubmed: 24478389google scholar: lookup
  4. Kuijk EW, Du Puy L, Van Tol HT, Oei CH, Haagsman HP, Colenbrander B, Roelen BA. Differences in early lineage segregation between mammals.. Dev Dyn 2008 Apr;237(4):918-27.
    doi: 10.1002/dvdy.21480pubmed: 18330925google scholar: lookup
  5. Plusa B, Piliszek A, Frankenberg S, Artus J, Hadjantonakis AK. Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst.. Development 2008 Sep;135(18):3081-91.
    doi: 10.1242/dev.021519pmc: PMC2768606pubmed: 18725515google scholar: lookup
  6. Niakan KK, Eggan K. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse.. Dev Biol 2013 Mar 1;375(1):54-64.
    doi: 10.1016/j.ydbio.2012.12.008pubmed: 23261930google scholar: lookup
  7. Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, Pfeffer PL. Trophectoderm lineage determination in cattle.. Dev Cell 2011 Feb 15;20(2):244-55.
    doi: 10.1016/j.devcel.2011.01.003pubmed: 21316591google scholar: lookup
  8. Zhi M, Zhang J, Tang Q, Yu D, Gao S, Gao D, Liu P, Guo J, Hai T, Gao J, Cao S, Zhao Z, Li C, Weng X, He M, Chen T, Wang Y, Long K, Jiao D, Li G, Zhang J, Liu Y, Lin Y, Pang D, Zhu Q, Chen N, Huang J, Chen X, Yao Y, Yang J, Xie Z, Huang X, Liu M, Zhang R, Li Q, Miao Y, Tian J, Huang X, Ouyang H, Liu B, Xie W, Zhou Q, Wei H, Liu Z, Zheng C, Li M, Han J. Generation and characterization of stable pig pregastrulation epiblast stem cell lines.. Cell Res 2022 Apr;32(4):383-400.
    doi: 10.1038/s41422-021-00592-9pmc: PMC8976023pubmed: 34848870google scholar: lookup
  9. Enders AC, Schlafke S, Lantz KC, Liu IKM. Endoderm Cells of the Equine Yolk Sac from Day 7 until Formation of the Definitive Yolk Sac Placenta.. Equine Vet. J. 2010;25:3–9.
    doi: 10.1111/j.2042-3306.1993.tb04814.xgoogle scholar: lookup
  10. Paris DB, Stout TA. Equine embryos and embryonic stem cells: defining reliable markers of pluripotency.. Theriogenology 2010 Sep 1;74(4):516-24.
    doi: 10.1016/j.theriogenology.2009.11.020pubmed: 20071015google scholar: lookup
  11. Desmarais JA, Demers SP, Suzuki J Jr, Laflamme S, Vincent P, Laverty S, Smith LC. Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos.. Reproduction 2011 Mar;141(3):321-32.
    doi: 10.1530/REP-09-0536pubmed: 21209071google scholar: lookup
  12. Choi YH, Ross P, Velez IC, Macías-García B, Riera FL, Hinrichs K. Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations.. Reproduction 2015 Jul;150(1):31-41.
    doi: 10.1530/REP-14-0662pubmed: 25852156google scholar: lookup
  13. Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF. Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.. PLoS One 2020;15(9):e0238948.
    doi: 10.1371/journal.pone.0238948pmc: PMC7485800pubmed: 32915925google scholar: lookup
  14. Choi YH, Hinrichs K. Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program.. Theriogenology 2017 Jan 1;87:48-54.
    doi: 10.1016/j.theriogenology.2016.08.005pubmed: 27634397google scholar: lookup
  15. Claes A, Stout TAE. Success rate in a clinical equine in vitro embryo production program.. Theriogenology 2022 Jul 15;187:215-218.
    doi: 10.1016/j.theriogenology.2022.04.019pubmed: 35623226google scholar: lookup
  16. Cuervo-Arango J, Claes AN, Stout TAE. In vitro-produced horse embryos exhibit a very narrow window of acceptable recipient mare uterine synchrony compared with in vivo-derived embryos.. Reprod Fertil Dev 2019 Jan;31(12):1904-1911.
    doi: 10.1071/RD19294pubmed: 31587698google scholar: lookup
  17. Ducheyne KD, Rizzo M, Cuervo-Arango J, Claes A, Daels PF, Stout TAE, de Ruijter-Villani M. In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.. Reprod Fertil Dev 2019 Jan;31(12):1830-1839.
    doi: 10.1071/RD19227pubmed: 31771747google scholar: lookup
  18. Carnevale EM, Metcalf ES. Morphology, developmental stages and quality parameters of in vitro-produced equine embryos.. Reprod Fertil Dev 2019 Jan;31(12):1758-1770.
    doi: 10.1071/RD19257pubmed: 31718765google scholar: lookup
  19. Stout TAE. Clinical Application of in Vitro Embryo Production in the Horse.. J Equine Vet Sci 2020 Jun;89:103011.
    doi: 10.1016/j.jevs.2020.103011pubmed: 32563449google scholar: lookup
  20. Choi YH, Harding HD, Hartman DL, Obermiller AD, Kurosaka S, McLaughlin KJ, Hinrichs K. The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts.. Reproduction 2009 Sep;138(3):589-99.
    doi: 10.1530/REP-08-0394pubmed: 19525365google scholar: lookup
  21. Zhu M, Zernicka-Goetz M. Principles of Self-Organization of the Mammalian Embryo.. Cell 2020 Dec 10;183(6):1467-1478.
    doi: 10.1016/j.cell.2020.11.003pmc: PMC8212876pubmed: 33306953google scholar: lookup
  22. Mihajlović AI, Bruce AW. The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity.. Open Biol 2017 Nov;7(11).
    doi: 10.1098/rsob.170210pmc: PMC5717349pubmed: 29167310google scholar: lookup
  23. Keramari M, Razavi J, Ingman KA, Patsch C, Edenhofer F, Ward CM, Kimber SJ. Sox2 is essential for formation of trophectoderm in the preimplantation embryo.. PLoS One 2010 Nov 12;5(11):e13952.
    doi: 10.1371/journal.pone.0013952pmc: PMC2980489pubmed: 21103067google scholar: lookup
  24. Mistri TK, Arindrarto W, Ng WP, Wang C, Lim LH, Sun L, Chambers I, Wohland T, Robson P. Dynamic changes in Sox2 spatio-temporal expression promote the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos.. Biochem J 2018 Mar 20;475(6):1075-1089.
    doi: 10.1042/BCJ20170418pmc: PMC5896025pubmed: 29487166google scholar: lookup
  25. Schrode N, Saiz N, Di Talia S, Hadjantonakis AK. GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst.. Dev Cell 2014 May 27;29(4):454-67.
    doi: 10.1016/j.devcel.2014.04.011pmc: PMC4103658pubmed: 24835466google scholar: lookup
  26. Pérez-Gómez A, González-Brusi L, Bermejo-Álvarez P, Ramos-Ibeas P. Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates.. Front Vet Sci 2021;8:680539.
    doi: 10.3389/fvets.2021.680539pmc: PMC8239129pubmed: 34212020google scholar: lookup
  27. Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function.. Genes Dev 2003 Jan 1;17(1):126-40.
    doi: 10.1101/gad.224503pmc: PMC195970pubmed: 12514105google scholar: lookup
  28. Yu X, Zhao X, Wang H, Ma B. Expression Patterns of OCT4, NANOG, and SOX2 in Goat Preimplantation Embryos from in Vivo and in Vitro.. J. Integr. Agric. 2015;14:1398–1406.
    doi: 10.1016/S2095-3119(14)60923-0google scholar: lookup
  29. HosseinNia P, Hajian M, Jafarpour F, Hosseini SM, Tahmoorespur M, Nasr-Esfahani MH. Dynamics of The Expression of Pluripotency and Lineage Specific Genes in The Pre and Peri-Implantation Goat Embryo.. Cell J 2019 Jul;21(2):194-203.
    doi: 10.22074/cellj.2019.5732pmc: PMC6397601pubmed: 30825293google scholar: lookup
  30. Cuervo-Arango J, Claes AN, Stout TA. Effect of Embryo-Recipient Synchrony on Post-ET Survival of In Vivo and In Vitro-Produced Equine Embryos.. J. Equine Vet. Sci. 2018;66:163–164.
    doi: 10.1016/j.jevs.2018.05.058google scholar: lookup
  31. Santos JE, Thatcher WW, Chebel RC, Cerri RL, Galvão KN. The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs.. Anim Reprod Sci 2004 Jul;82-83:513-35.
    doi: 10.1016/j.anireprosci.2004.04.015pubmed: 15271477google scholar: lookup
  32. Lucy MC. Reproductive loss in high-producing dairy cattle: where will it end?. J Dairy Sci 2001 Jun;84(6):1277-93.
    doi: 10.3168/jds.S0022-0302(01)70158-0pubmed: 11417685google scholar: lookup
  33. Wiltbank MC, Baez GM, Garcia-Guerra A, Toledo MZ, Monteiro PL, Melo LF, Ochoa JC, Santos JE, Sartori R. Pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows.. Theriogenology 2016 Jul 1;86(1):239-53.
    doi: 10.1016/j.theriogenology.2016.04.037pubmed: 27238438google scholar: lookup
  34. Dijkstra A, Cuervo-Arango J, Stout TAE, Claes A. Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos.. Equine Vet J 2020 Mar;52(2):258-261.
    doi: 10.1111/evj.13146pmc: PMC7027474pubmed: 31232484google scholar: lookup
  35. McCue PM, Thayer J, Squires EL, Brinsko SP, Vanderwall DK. Twin Pregnancies Following Transfer of Single Embryos in Three Mares: A Case Report.. J. Equine Vet. Sci. 1998;18:832–834.
    doi: 10.1016/S0737-0806(98)80333-Xgoogle scholar: lookup
  36. Hansen PJ, Tríbulo P. Regulation of present and future development by maternal regulatory signals acting on the embryo during the morula to blastocyst transition - insights from the cow.. Biol Reprod 2019 Sep 1;101(3):526-537.
    doi: 10.1093/biolre/ioz030pmc: PMC8127039pubmed: 31220231google scholar: lookup
  37. Newcombe JR, Cuervo-Arango J. The Relationship Between the Positive Identification of the Embryo Proper in Equine Pregnancies Aged 18-28 Days and Its Future Viability: A Field Study.. J. Equine Vet. Sci. 2012;32:257–261.
    doi: 10.1016/j.jevs.2011.09.068google scholar: lookup
  38. Claes A, Cuervo-Arango J, van den Broek J, Galli C, Colleoni S, Lazzari G, Deelen C, Beitsma M, Stout TA. Factors affecting the likelihood of pregnancy and embryonic loss after transfer of cryopreserved in vitro produced equine embryos.. Equine Vet J 2019 Jul;51(4):446-450.
    doi: 10.1111/evj.13028pubmed: 30269336google scholar: lookup
  39. Stout TA. Equine embryo transfer: review of developing potential.. Equine Vet J 2006 Sep;38(5):467-78.
    doi: 10.2746/042516406778400529pubmed: 16986609google scholar: lookup
  40. McCue PM. Embryo Evaluation.. .
  41. Lazzari G, Colleoni S, Crotti G, Turini P, Fiorini G, Barandalla M, Landriscina L, Dolci G, Benedetti M, Duchi R, Galli C. Laboratory Production of Equine Embryos.. J Equine Vet Sci 2020 Jun;89:103097.
    doi: 10.1016/j.jevs.2020.103097pubmed: 32563445google scholar: lookup
  42. Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova.. J Reprod Fertil 1972 Sep;30(3):493-7.
    doi: 10.1530/jrf.0.0300493pubmed: 4672493google scholar: lookup
  43. Lazzari G, Wrenzycki C, Herrmann D, Duchi R, Kruip T, Niemann H, Galli C. Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome.. Biol Reprod 2002 Sep;67(3):767-75.
    doi: 10.1095/biolreprod.102.004481pubmed: 12193383google scholar: lookup
  44. Goszczynski DE, Tinetti PS, Choi YH, Hinrichs K, Ross PJ. Genome activation in equine in vitro-produced embryos.. Biol Reprod 2022 Jan 13;106(1):66-82.
    doi: 10.1093/biolre/ioab173pubmed: 34515744google scholar: lookup
  45. Frank BL, Doddman CD, Stokes JE, Carnevale EM. Association of equine oocyte and cleavage stage embryo morphology with maternal age and pregnancy after intracytoplasmic sperm injection.. Reprod Fertil Dev 2019 Jan;31(12):1812-1822.
    doi: 10.1071/RD19250pubmed: 31630724google scholar: lookup
  46. Salamone DF, Canel NG, Rodríguez MB. Intracytoplasmic sperm injection in domestic and wild mammals.. Reproduction 2017 Dec;154(6):F111-F124.
    doi: 10.1530/REP-17-0357pubmed: 29196493google scholar: lookup
  47. Yao Y, Yang A, Li G, Wu H, Deng S, Yang H, Ma W, Lv D, Fu Y, Ji P, Tan X, Zhao W, Lian Z, Zhang L, Liu G. Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells.. Cell Cycle 2022 Jul;21(13):1360-1375.
    doi: 10.1080/15384101.2022.2051122pmc: PMC9345622pubmed: 35311450google scholar: lookup
  48. Olivera R, Moro LN, Jordan R, Luzzani C, Miriuka S, Radrizzani M, Donadeu FX, Vichera G. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.. PLoS One 2016;11(10):e0164049.
    doi: 10.1371/journal.pone.0164049pmc: PMC5061425pubmed: 27732616google scholar: lookup
  49. Wiater J, Samiec M, Wartalski K, Smorąg Z, Jura J, Słomski R, Skrzyszowska M, Romek M. Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes-In Vitro Studies.. Int J Mol Sci 2021 Sep 7;22(18).
    doi: 10.3390/ijms22189683pmc: PMC8469251pubmed: 34575846google scholar: lookup
  50. Samiec M, Skrzyszowska M. Biological transcomplementary activation as a novel and effective strategy applied to the generation of porcine somatic cell cloned embryos.. Reprod Biol 2014 Apr;14(2):128-39.
    doi: 10.1016/j.repbio.2013.12.006pubmed: 24856472google scholar: lookup

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