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Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada2017; 23(6); 1197-1206; doi: 10.1017/S1431927617012740

Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro.

Abstract: Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ 2 and Fisher's exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.
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Publication Date: 2017-12-06 PubMed ID: 29208065PubMed Central: PMC5976488DOI: 10.1017/S1431927617012740Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't

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 article is centered on the use of Confocal microscopy in examining the various stages of equine zygote development following the injection of sperm into oocytes that were matured either in vivo or in vitro.

Overview of the Study and Its Key Elements

  • The researchers explored the effectiveness of Confocal microscopy, a highly specialized imaging process, in tracking the progress of equine zygote development. This process was carried out after a highly specialized reproductive process called Intracytoplasmic Sperm Injection (ICSI).
  • The oocytes (female reproductive cells) involved in this study had been matured under two different conditions; some in vivo (in the natural environment within the body), and others in vitro (in a lab setting outside the body).
  • The development of the zygotes was then observed at different time intervals after ICSI.

Method Used in the Research

  • After ICSI, the zygotes were preserved at various time intervals: 4, 6, 8, 12, or 16 hours. They were then treated with specific antibodies, washed, and treated again using secondary antibodies, ancillary to either Alexa 488 or Alexa 647. This was a crucial step in preparing the samples for Confocal microscopy.
  • The process then involved the zygotes being treated with 561-Phalloidin and Hoechst 33258 for clear specimen observation. The Olympus IX81 spinning disk confocal microscope was utilized to image the various stages of zygote development.
  • The data gathered were then dissected using χ2 (Chi-square) and Fisher’s exact tests to derive statistical significance from the observed data points.

Findings and Conclusion

  • The study revealed minor differences in developmental stages of oocytes matured in vivo or in vitro.
  • The oocytes matured in vivo seemed to form pronuclei faster than those matured in vitro. By the 6-hour mark, 67% of in vivo matured oocytes formed pronuclei, compared to just 13% of in vitro matured oocytes.
  • A higher percentage (30%) of zygotes had abnormal phenotypes, indicative of inadequate zygote development, when the oocyte maturation occurred in vitro when compared to in vivo (11%). However, even some potential zygotes from oocytes matured in vivo showed normal phenotypes, although development appeared to be slow or stalled.
  • The results serve as an assertion that Confocal microscopy provides a practical way to monitor equine zygote development using a limited number of samples.

Cite This Article

APA
Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Stokes JE, Carnevale EM. (2017). Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro. Microsc Microanal, 23(6), 1197-1206. https://doi.org/10.1017/S1431927617012740

Publication

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
ISSN: 1435-8115
NlmUniqueID: 9712707
Country: England
Language: English
Volume: 23
Issue: 6
Pages: 1197-1206

Researcher Affiliations

Ruggeri, Elena
  • 1Department of Biomedical Sciences,Colorado State University,1693 Campus Delivery,Fort Collins,CO 80523,USA.
DeLuca, Keith F
  • 3Department of Biochemistry and Molecular Biology,Colorado State University,1870 Campus Delivery,Fort Collins,CO 80523,USA.
Galli, Cesare
  • 4Laboratory of Reproductive Technologies,Avantea,Via Porcellasco 7f,26100,Cremona,Italy.
Lazzari, Giovanna
  • 4Laboratory of Reproductive Technologies,Avantea,Via Porcellasco 7f,26100,Cremona,Italy.
DeLuca, Jennifer G
  • 3Department of Biochemistry and Molecular Biology,Colorado State University,1870 Campus Delivery,Fort Collins,CO 80523,USA.
Stokes, Joanne E
  • 1Department of Biomedical Sciences,Colorado State University,1693 Campus Delivery,Fort Collins,CO 80523,USA.
Carnevale, Elaine M
  • 1Department of Biomedical Sciences,Colorado State University,1693 Campus Delivery,Fort Collins,CO 80523,USA.

MeSH Terms

  • Animals
  • Fertilization
  • Horses
  • Microinjections
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods
  • Time Factors
  • Zygote / cytology
  • Zygote / growth & development

Grant Funding

  • R01 GM088371 / NIGMS NIH HHS

References

This article includes 52 references
  1. Altermatt JL, Suh TK, Stokes JE, Carnevale EM. Effects of age and equine follicle-stimulating hormone (eFSH) on collection and viability of equine oocytes assessed by morphology and developmental competency after intracytoplasmic sperm injection (ICSI). Reprod Fertil Dev 2009;21(4):615–623.
    pubmed: 19383268
  2. Ambruosi B, Lacalandra GM, Iorga AI, De Santis T, Mugnier S, Matarrese R, Goudet G, Dell’aquila ME. Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI. Theriogenology 2009;71(7):1093–1104.
    pubmed: 19167745
  3. Arlotto T, Schwartz JL, First NL, Leibfried-Rutledge ML. Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes. Theriogenology 1996;45(5):943–956.
    pubmed: 16727855
  4. Barrett SL, Albertini DF. Allocation of gamma-tubulin between oocyte cortex and meiotic spindle influences asymmetric cytokinesis in the mouse oocyte. Biol Reprod 2007;76(6):949–957.
    pubmed: 17287496
  5. Bromfield J, Messamore W, Albertini DF. Epigenetic regulation during mammalian oogenesis. Reprod Fertil Dev 2007;20(1):74–80.
    pubmed: 18154701
  6. Cantone I, Fisher AG. Epigenetic programming and reprogramming during development. Nat Struct Mol Biol 2013;20(3):282–289.
    pubmed: 23463313
  7. Carnevale E, Ginther O. Defective oocytes as a cause of subfertility in old mares. Biol Reprod Mono 1995;1:209–214.
  8. Carnevale EM. Advances in Collection, Transport and Maturation of Equine Oocytes for Assisted Reproductive Techniques. Vet Clin North Am Equine Pract 2016;32(3):379–399.
    pubmed: 27726987
  9. Carnevale EM, da Silva MAC, Maclellan LJ, Seidel GE, Squires EL. Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares. Reproduction 2004;128(5):623–628.
    pubmed: 15509708
  10. Carnevale EM, Maclellan LJ, Coutinho da Silva MA, Scott TJ, Squires EL. Comparison of culture and insemination techniques for equine oocyte transfer. Theriogenology 2000;54(6):981–987.
    pubmed: 11097049
  11. Carnevale EM, Sessions DR. In vitro production of equine embryos. J Equine Vet Sci 2012;32(7):367–371.
  12. Carnevale EM, Stokes J, Squires EL, Campos-Chillon LF, Altermatt J, Suh TK. Clinical use of intracytoplasmic sperm injection in horses. Proc Am Assoc Equine Pract 2007;53:560.
  13. Choi YH, Love LB, Varner DD, Hinrichs K. Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection. Reproduction 2004;127(2):187–194.
    pubmed: 15056784
  14. Colleoni S, Barbacini S, Necchi D, Duchi R, Lazzari G, Galli C. Application of ovum pick-up, intracytoplasmic sperm injection and embryo culture in equine practice. 2007:554–559.
  15. Colleoni S, Lagutina I, Lazzari G, Rodriguez-Martinez H, Galli C, Morrell JM. New methods for selecting stallion spermatozoa for assisted reproduction. J Equine Vet Sci 2011;31(9):536–541.
  16. Courtois A, Schuh M, Ellenberg J, Hiiragi T. The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development. J Cell Biol 2012;198(3):357–370.
    pmc: PMC3413348pubmed: 22851319
  17. Deng M, Li R. Sperm chromatin-induced ectopic polar body extrusion in mouse eggs after ICSI and delayed egg activation. PLoS One 2009;4(9):e7171.
    pmc: PMC2746308pubmed: 19787051
  18. Edirisinghe WR, Murch A, Junk S, Yovich JL. Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study. Hum Reprod 1997;12(12):2784–2791.
    pubmed: 9455853
  19. Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965;208(5008):349–351.
    pubmed: 4957259
  20. El Hajj N, Haaf T. Epigenetic disturbances in in vitro cultured gametes and embryos: implications for human assisted reproduction. Fertil Steril 2013;99(3):632–641.
    pubmed: 23357453
  21. Enders AC, Liu IK, Bowers J, Lantz KC, Schlafke S, Suarez S. The ovulated ovum of the horse: cytology of nonfertilized ova to pronuclear stage ova. Biol Reprod 1987;37(2):453–466.
    pubmed: 3676399
  22. Figueiredo T, Paiva R, Kozicki LE, Kaercher F, Weiss RR, Santos IWd, Muradas PR. Induction of ovulation in quarter horse mares through the use of deslorelin acetate and human chorionic gonadotrophin (hCG). Brazilian Archives of Biology and Technology 2011;54(3):517–521.
  23. Freeman DA, Weber JA, Geary RT, Woods GL. Time of embryo transport through the mare oviduct. Theriogenology 1991;36(5):823–830.
    pubmed: 16727051
  24. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci 2007;98(1–2):39–55.
    pubmed: 17101246
  25. Galli C, Colleoni S, Duchi R, Lagutina I, Lazzari G. Equine assisted reproduction and embryo technologies. Anim Reprod 2013;10:334–343.
  26. Galli C, Duchi R, Colleoni S, Lagutina I, Lazzari G. Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. Theriogenology 2014;81(1):138–151.
    pubmed: 24274418
  27. Gilchrist RB, Thompson JG. Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro. Theriogenology 2007;67(1):6–15.
    pubmed: 17092551
  28. Grondahl C, Hansen TH, Hossaini A, Heinze I, Greve T, Hyttel P. Intracytoplasmic sperm injection of in vitro-matured equine oocytes. Biol Reprod 1997;57(6):1495–1501.
    pubmed: 9408260
  29. Grondahl C, Hyttel P. Nucleologenesis and ribonucleic acid synthesis in preimplantation equine embryos. Biol Reprod 1996;55(4):769–774.
    pubmed: 8879488
  30. Hinrichs K. The relationship of follicle atresia to follicle size, oocyte recovery rate on aspiration, and oocyte morphology in the mare. Theriogenology 1991;36(2):157–168.
    pubmed: 16726989
  31. Hinrichs K. Assisted reproduction techniques in the horse. Reprod Fertil Dev 2013;25(1):80–93.
    pubmed: 23244831
  32. Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG. In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. Biol Reprod 1993;48(2):363–370.
    pubmed: 8439626
  33. Hyttel P, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology 1997;47(1):23–32.
  34. 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;67(3):767–775.
    pubmed: 12193383
  35. Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A. Why doesn’t conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization. Reproduction 2016;152(6):R233–R245.
    pubmed: 27651517
  36. Lonergan P, Fair T. Maturation of Oocytes in Vitro. Annu Rev Anim Biosci 2016;4:255–268.
    pubmed: 26566159
  37. Mamo S, Carter F, Lonergan P, Leal CL, Al Naib A, McGettigan P, Mehta JP, Evans AC, Fair T. Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation. BMC Genomics 2011;12:151.
    pmc: PMC3068982pubmed: 21410957
  38. Manandhar G, Toshimori K. Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after sperm head microinjection and its role in oocyte activation in mice. Biol Reprod 2003;68(2):655–663.
    pubmed: 12533432
  39. Marchetti F, Bishop J, Gingerich J, Wyrobek AJ. Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair. Sci Rep 2015;5:7689.
    pmc: PMC4286742pubmed: 25567288
  40. Marchetti F, Wyrobek AJ. Mechanisms and consequences of paternally509 transmitted chromosomal abnormalities. Birth Defects Res C Embryo Today 2005;75(2):112–129.
    pubmed: 16035041
  41. Messinger SM, Albertini DF. Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci 1991;100(Pt 2):289–298.
    pubmed: 1721916
  42. Moghbelinejad S, Mozdarani H, Rezaeian Z. The rates of premature chromosome condensation and embryo development after injection of irradiated sperms into hamster oocytes. Iran J Reprod Med 2013;11(5):391–398.
    pmc: PMC3941419pubmed: 24639771
  43. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293(5532):1089–1093.
    pubmed: 11498579
  44. Rinaudo P, Schultz RM. Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction 2004;128(3):301–311.
    pubmed: 15333781
  45. Rosenbusch B. Digynic triploidy: possible mechanisms. Prenat Diagn 2001;21(3):234.
    pubmed: 11260614
  46. Sanfins A, Plancha CE, Albertini DF. Pre-implantation developmental potential from in vivo and in vitro matured mouse oocytes: a cytoskeletal perspective on oocyte quality. J Assist Reprod Genet 2015;32(1):127–136.
    pmc: PMC4294880pubmed: 25381620
  47. Schmiady H, Tandler-Schneider A, Kentenich H. Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection. Human reproduction 1996;11(10):2239–2245.
    pubmed: 8943536
  48. Sluder G, Thompson EA, Miller FJ, Hayes J, Rieder CL. The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry. J Cell Sci 1997;110(Pt 4):421–429.
    pubmed: 9067594
  49. Smitz JE, Thompson JG, Gilchrist RB. The promise of in vitro maturation in assisted reproduction and fertility preservation. Semin Reprod Med 2011;29(1):24–37.
    pubmed: 21207332
  50. Squires E, Carnevale E, McCue P, Bruemmer J. Embryo technologies in the horse. Theriogenology 2003;59(1):151–170.
    pubmed: 12499026
  51. Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update 2003;9(1):35–48.
    pubmed: 12638780
  52. Tremoleda JL, Van Haeften T, Stout TA, Colenbrander B, Bevers MM. Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization. Biol Reprod 2003;69(1):186–194.
    pubmed: 12646492

Citations

This article has been cited 1 times.
  1. Salgado RM, Brom-de-Luna JG, Resende HL, Canesin HS, Hinrichs K. Lower blastocyst quality after conventional vs. Piezo ICSI in the horse reflects delayed sperm component remodeling and oocyte activation. J Assist Reprod Genet 2018 May;35(5):825-840.
    doi: 10.1007/s10815-018-1174-9pubmed: 29637506google scholar: lookup
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