Skip to main content
SpringerLink
Log in

Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy

  • Gamete Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Investigation of the changes induced by vitrification on the cortical F-actin of in vitro matured ovine oocytes by Raman microspectroscopy (RMS).

Methods

Cumulus-oocyte complexes, recovered from the ovaries of slaughtered sheep, were matured in vitro and vitrified following the Minimum Essential Volume method using cryotops. The cortical region of metaphase II (MII) oocytes (1) exposed to vitrification solutions but not cryopreserved (CPA-exp), (2) vitrified/warmed (VITRI), and (3) untreated (CTR) was analyzed by RMS. A chemical map of one quadrant of single CPA-exp, VITRI and CTR oocytes was, also, performed. In order to identify the region of Raman spectra representative of the cortical F-actin modification, a group of in vitro matured oocytes were incubated with latrunculin–A (LATA), a specific F-actin destabilizing drug, and processed for RMS analysis. Thereafter, all the oocytes were stained with rhodamine phalloidin and evaluated by fluorescence confocal microscopy. Raman spectra of the oocytes were, statistically, analyzed using Principal Component Analysis (PCA).

Results

The PCA score plots showed a marked discrimination between CTR oocytes and CPA-exp/ VITRI groups. The main differences, highlighted by PCA loadings, were referable to proteins (1657, 1440 and 1300 cm−1) and, as indicated by LATA experiments, also included the changes of the F-actin. Analysis by confocal microscopy revealed a clear alteration of the cortical F-actin of CPA-exp and VITRI oocytes confirming RMS results.

Conclusions

Raman microspectroscopy may represent an alternative analytical tool for investigating the biochemical modification of the oocyte cortex, including the F-actin cytoskeleton, during vitrification of in vitro matured ovine oocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Konc J, Kanyó K, Kriston R, Somoskői B, Cseh S. Cryopreservation of embryos and oocytes in human assisted reproduction. Biomed Res Int. 2014;2014:307268.

    Article  PubMed Central  PubMed  Google Scholar 

  2. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011;141:1–19.

    Article  CAS  PubMed  Google Scholar 

  3. Mullen SF, Fahy GM. A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep. Theriogenology. 2012;78:1709–19.

    Article  CAS  PubMed  Google Scholar 

  4. The Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013;99:37–43.

    Article  Google Scholar 

  5. Arav A, Natan Y. Vitrification of oocytes: from basic science to clinical application. Adv Exp Med Biol. 2013;761:69–83.

    Article  PubMed  Google Scholar 

  6. Díez C, Muñoz M, Caamaño JN, Gómez E. Cryopreservation of the bovine oocyte: current status and perspectives. Reprod Domest Anim. 2012;3:76–83.

    Article  Google Scholar 

  7. Vincent C, Johnson MH. Cooling, cryoprotectants, and the cytoskeleton of the mammalian oocyte. Oxf Rev Reprod Biol. 1992;14:73–100.

    CAS  PubMed  Google Scholar 

  8. Moussa M, Shu J, Zhang X, Zeng F. Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives. Sci China Life Sci. 2014;57:903–14.

    Article  CAS  PubMed  Google Scholar 

  9. Li R, Albertini DF. The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte. Nat Rev Mol Cell Biol. 2013;14:141–52.

    Article  CAS  PubMed  Google Scholar 

  10. Simerly C, Navara CS, Wu GJ, Schatten G. Cytoskeletal organization and dynamics in mammalian oocytes during maturation and fertilization. In: Grudzinskas JG, Yovich JL, editors. Gametes - the oocyte. Cambridge: Cambridge University Press; 1995. p. 54–94.

    Google Scholar 

  11. Lei T, Guo N, Liu JQ, Tan MH, Li YF. Vitrification of in vitro matured oocytes: effects on meiotic spindle configuration and mitochondrial function. Int J Clin Exp Pathol. 2014;7:1159–65.

    PubMed Central  PubMed  Google Scholar 

  12. Tamura AN, Huang TT, Marikawa Y. Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes. Biol Reprod. 2013;89:112.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Egerszegi I, Somfai T, Nakai M, Tanihara F, Noguchi J, Kaneko H, et al. Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model. Cryobiology. 2013;67:287–92.

    Article  PubMed  Google Scholar 

  14. Coticchio G, Bromfield JJ, Sciajno R, Gambardella A, Scaravelli G, Borini A, et al. Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes. Reprod Biomed Online. 2009;19:29–34.

    Article  PubMed  Google Scholar 

  15. Chen SU, Yang YS. Slow freezing or vitrification of oocytes: their effects on survival and meiotic spindles, and the time schedule for clinical practice. Taiwan J Obstet Gynecol. 2009;48:15–22.

    Article  PubMed  Google Scholar 

  16. Succu S, Leoni GG, Bebbere D, Berlinguer F, Mossa F, Bogliolo L, et al. Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes. Mol Reprod Dev. 2007;74:1337–44.

    Article  CAS  PubMed  Google Scholar 

  17. Mikołajewska N, Müller K, Niżański W, Jewgenow K. Vitrification of domestic cat oocytes–effect on viability and integrity of subcellular structures. Reprod Domest Anim. 2012;47:295–9.

    Article  PubMed  Google Scholar 

  18. Wu C, Rui R, Dai J, Zhang C, Ju S, Xie B, et al. Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes. Mol Reprod Dev. 2006;73:1454–62.

    Article  CAS  PubMed  Google Scholar 

  19. Rojas C, Palomo MJ, Albarracín JL, Mogas T. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology. 2004;49:211–20.

    Article  CAS  PubMed  Google Scholar 

  20. Hotamisligil S, Toner M, Powers RD. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol Reprod. 1996;55:161–8.

    Article  CAS  PubMed  Google Scholar 

  21. Combelles CM, Ceyhan ST, Wang H, Racowsky C. Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing. J Assist Reprod Genet. 2011;28:1183–92.

    Article  PubMed Central  PubMed  Google Scholar 

  22. Azoury J, Lee KW, Georget V, Hikal P, Verlhac MH. Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization. Development. 2011;138:2903–8.

    Article  CAS  PubMed  Google Scholar 

  23. Khalili MA, Maione M, Palmerini MG, Bianchi S, Macchiarelli G, Nottola SA. Ultrastructure of human mature oocytes after vitrification. Eur J Histochem. 2012;56:e38.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Caamaño JN, Muñoz M, Diez C, Gómez E. Polarized light microscopy in mammalian oocytes. Reprod Domest Anim. 2010;2:49–56.

    Article  Google Scholar 

  25. Coticchio G, Sciajno R, Hutt K, Bromfield J, Borini A, Albertini DF. Comparative analysis of the metaphase II spindle of human oocytes through polarized light and high-performance confocal microscopy. Fertil Steril. 2010;93:2056–64.

    Article  PubMed  Google Scholar 

  26. Notingher I, Hench LL. Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro. Expert Rev Med Devices. 2006;3:215–34.

    Article  CAS  PubMed  Google Scholar 

  27. Matthaus C, Bird B, Miljikovic M, Chemenko T, Romeo M, Diem M. Infrared and Raman microscopy in cell biology. Methods Cell Biol. 2008;89:275–308.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Wood BR, Chernenko T, Matthaus C, Diem M, Chong C, Bernhard U, et al. Shedding new light on the molecular architecture of oocytes using a combination of synchrotron fourier transm-infrared and Raman spectroscopic mapping. Anal Chem. 2008;80:9065–72.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Davidson B, Murray AA, Elfick A, Spears N. Raman micro-spectroscopy can be used to investigate the developmental stage of the mouse oocyte. PLoS One. 2013;8:e67972.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Davidson B, Spears N, Murray A, Elfick A. The changing biochemical composition and organisation of the murine oocyte and early embryo as revealed by Raman spectroscopic mapping. J Raman Spectrosc. 2012;43:24–31.

    Article  CAS  Google Scholar 

  31. Bogliolo L, Ledda S, Innocenzi P, Ariu F, Bebbere D, Rosati I, et al. Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida. Cryobiology. 2012;64:267–72.

    Article  CAS  PubMed  Google Scholar 

  32. Bogliolo L, Murrone O, Di Emidio G, Piccinini M, Ariu F, Ledda S, et al. Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte. J Assist Reprod Genet. 2013;30:877–82.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Spector I, Shochet NR, Blasberger D, Kashman Y. Latrunculins–novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D. Cell Motil Cytoskeleton. 1989;13:127–44.

    Article  CAS  PubMed  Google Scholar 

  34. Spector I, Shochet NR, Kashman Y, Groweiss A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science. 1983;219:493–5.

    Article  CAS  PubMed  Google Scholar 

  35. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod BioMed Online. 2005;11:300–8.

    Article  PubMed  Google Scholar 

  36. Zoladek AB, Johal RK, Garcia-Nieto S, Pascut F, Shakesheff KM, Ghaemmaghami AM. Notingher Label-free molecular imaging of immunological synapses between dendritic and T cells by Raman micro-spectroscopy. Analyst. 2010;135:3205–12.

    Article  CAS  PubMed  Google Scholar 

  37. Matthäus C, Chernenko T, Newmark JA, Warner CM, Diem M. Label-free detection of mitochondrial distribution in cells by nonresonant Raman microspectroscopy. Biophys J. 2007;93:668–73.

    Article  PubMed Central  PubMed  Google Scholar 

  38. Kim SS, Olsen R, Kim DD, Albertini DF. The impact of vitrification on immature oocyte cell cycle and cytoskeletal integrity in a rat model. J Assist Reprod Genet. 2014;31:739–47.

    Article  PubMed  Google Scholar 

  39. Albarracín JL, Morató R, Rojas C, Mogas T. Effects of vitrification in open pulled straws on the cytology of in vitro matured prepubertal and adult bovine oocytes. Theriogenology. 2005;63:890–901.

    Article  PubMed  Google Scholar 

  40. Lee K, Wang C, Spate L, Murphy CN, Prather RS, Machaty Z. Gynogenetic activation of porcine oocytes. Cell Reprogram. 2014;16:121–9.

    Article  CAS  PubMed  Google Scholar 

  41. Maro B, Johnson MH, Pickering SJ, Flach G. Changes in actin distribution during fertilization of the mouse egg. J Embryol Exp Morphol. 1984;81:211–37.

    CAS  PubMed  Google Scholar 

  42. Lee K, Wang C, Spate L, Murphy CN, Prather RS, Machaty Z. Gynogenetic activation of porcine oocytes. Cell Reprogram. 2014;16:121–9.

    Article  CAS  PubMed  Google Scholar 

  43. Hosu BG, Mullen SF, Critser JK, Forgacs G. Reversible disassembly of the actin cytoskeleton improves the survival rate and developmental competence of cryopreserved mouse oocytes. PLoS One. 2008;30:3–e2787.

    Google Scholar 

  44. Wen Y, Zhao S, Chao L, Yu H, Song C, Shen Y, Chen H, Deng X. The protective role of antifreeze protein 3 on the structure and function of mature mouse oocytes in vitrification. Cryobiology. 2014, in press.

  45. Cooper JA. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987;105:1473–8.

    Article  CAS  PubMed  Google Scholar 

  46. Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods. 2008;5:605–7.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Chaigne A, Campillo C, Gov NS, Voituriez R, Azoury J, Umaña-Diaz C, et al. A soft cortex is essential for asymmetric spindle positioning in mouse oocytes. Nat Cell Biol. 2013;15:958–66.

    Article  CAS  PubMed  Google Scholar 

  48. Downes A, Mouras R, Bagnaninchi P, Elfick A. Raman spectroscopy and CARS microscopy of stem cells and their derivatives. J Raman Spectrosc. 2011;42:1864–70.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Veterinary Medicine, of Sassari, Via Vienna 2, 07100, Sassari, Sardegna, Italy

    Luisa Bogliolo, Ombretta Murrone, Federica Ariu, Sergio Ledda & Sara Tilocca

  2. ENEA C.R., Frascati, Rome, Italy

    Massimo Piccinini

  3. Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA

    David F. Albertini

Authors
  1. Luisa Bogliolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ombretta Murrone
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Massimo Piccinini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Federica Ariu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sergio Ledda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sara Tilocca
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David F. Albertini
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luisa Bogliolo.

Additional information

Capsule Raman microspectroscopy can be used to detect modification of the oocyte cotex following vitrification.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bogliolo, L., Murrone, O., Piccinini, M. et al. Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy. J Assist Reprod Genet 32, 185–193 (2015). https://doi.org/10.1007/s10815-014-0389-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-014-0389-7

Keywords

Search

Navigation