Skip to main content
SpringerLink
Log in

Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit)

  • Fertility Preservation
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model.

Methods

Pieces of immature testicular tissue (1 mm3) were inserted into “E.Vit” devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming.

Results

Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response.

Conclusions

Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.

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

Similar content being viewed by others

References

  1. Onofre J, Baert Y, Faes K, Goossens E. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. Hum Reprod Update. 2016;22:744–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Jahnukainen K, Hou M, Petersen C, Setchell B, Söder O. Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia. Cancer Res. 2001;61:706–10.

    CAS  PubMed  Google Scholar 

  3. Fattahi A, Latifi Z, Ghasemnejad T, Nejabati HR, Nouri M. Insights into in vitro spermatogenesis in mammals: past, present, future. Mol Reprod Dev. 2017;84:560–75.

    CAS  PubMed  Google Scholar 

  4. Gassei K, Orwig KE. Experimental methods to preserve male fertility and treat male factor infertility. Fertil Steril. 2016;105:256–66.

    PubMed  Google Scholar 

  5. Ginsberg JP, Li Y, Carlson CA, Gracia CR, Hobbie WL, Miller VA, et al. Testicular tissue cryopreservation in prepubertal male children: an analysis of parental decision-making. Pediatr Blood Cancer. 2014;61:1673–8.

    PubMed  PubMed Central  Google Scholar 

  6. Martinez F, International Society for Fertility Preservation–ESHRE–ASRM Expert Working Group. Update on fertility preservation from the Barcelona International Society for Fertility Preservation-ESHRE-ASRM 2015 expert meeting: indications, results and future perspectives. Fertil Steril. 2017;108:407–415.e11.

    PubMed  Google Scholar 

  7. Unni S, Kasiviswanathan S, D'Souza S, Khavale S, Mukherjee S, Patwardhan S, et al. Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant. Fertil Steril. 2012;97:200–8.e1.

    CAS  PubMed  Google Scholar 

  8. Wyns C, Curaba M, Petit S, Vanabelle B, Laurent P, Wese JF, et al. Management of fertility preservation in prepubertal patients: 5 years’ experience at the Catholic University of Louvain. Hum Reprod. 2011;26:737–47.

    CAS  PubMed  Google Scholar 

  9. Baert Y, Goossens E, van Saen D, Ning L, in’t Veld P, Tournaye H. Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol. Fertil Steril. 2012;97:1152–7.e1–2.

    PubMed  Google Scholar 

  10. Poels J, Van Langendonckt A, Many MC, Wese FX, Wyns C. Vitrification preserves proliferation capacity in human spermatogonia. Hum Reprod. 2013;28:578–89.

    CAS  PubMed  Google Scholar 

  11. Macente BI, Toniollo GH, Apparicio M, Mansano C, Thomé HE, Canella CL, et al. Evaluation of different fragment sizes and cryoprotectants for cryopreservation of feline testicular tissues. Reprod Domest Anim. 2017;52(Suppl 2):242–7.

    CAS  PubMed  Google Scholar 

  12. Curaba M, Verleysen M, Amorim CA, Dolmans MM, Van Langendonckt A, Hovatta O, et al. Cryopreservation of prepubertal mouse testicular tissue by vitrification. Fertil Steril. 2011;95:229–34.e1.

    Google Scholar 

  13. Zarandi NP, Galdon G, Kogan S, Atala A, Sadri-Ardekani H. Cryostorage of immature and mature human testis tissue to preserve spermatogonial stem cells (SSCs): a systematic review of current experiences toward clinical applications. Stem Cells Cloning. 2018;11:23–38.

    PubMed  PubMed Central  Google Scholar 

  14. Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011;471:504–7.

    CAS  PubMed  Google Scholar 

  15. Yokonishi T, Sato T, Komeya M, Katagiri K, Kubota Y, Nakabayashi K, et al. Offspring production with sperm grown in vitro from cryopreserved testis tissues. Nat Commun. 2014;5:4320.

    CAS  PubMed  Google Scholar 

  16. Kaneko H, Kikuchi K, Nakai M, Somfai T, Noguchi J, Tanihara F, et al. Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice. PLoS One. 2013;8:e70989.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Fayomi AP, Peters K, Sukhwani M, Valli-Pulaski H, Shetty G, Meistrich ML, et al. Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring. Science. 2019;363:1314–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gholami M, Hemadi M, Saki G, Zendedel A, Khodadadi A, Mohammadi-Asl J. Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability. J Assist Reprod Genet. 2013;30:1271–7.

    PubMed  PubMed Central  Google Scholar 

  19. Rondanino C, Maouche A, Dumont L, Oblette A, Rives N. Establishment, maintenance and functional integrity of the blood-testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes. Mol Hum Reprod. 2017;23:304–20.

    CAS  PubMed  Google Scholar 

  20. Zhang XG, Li H, Hu JH. Effects of various cryoprotectants on the quality of frozen-thawed immature bovine (Qinchuan cattle) calf testicular tissue. Andrologia. 2017;49:9.

    Google Scholar 

  21. Li H, Bian YL, Schreurs N, Zhang XG, Raza SHA, Fang Q, et al. Effects of five cryoprotectants on proliferation and differentiation-related gene expression of frozen-thawed bovine calf testicular tissue. Reprod Domest Anim. 2018;53:1211–8.

    CAS  PubMed  Google Scholar 

  22. Arav A, Natan Y, Kalo D, Komsky-Elbaz A, Roth Z, Levi-Setti PE, et al. A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos. J Assist Reprod Genet. 2018;35:1161–8.

    PubMed  PubMed Central  Google Scholar 

  23. Benvenutti L, Salvador RA, Til D, Senn AP, Tames DR, Amaral NLL, et al. Wistar rats immature testicular tissue vitrification and heterotopic grafting. JBRA Assist Reprod. 2018;22:167–73.

    PubMed  PubMed Central  Google Scholar 

  24. Karlsson JO, Toner M. Long-term storage of tissues by cryopreservation: critical issues. Biomaterials. 1996;17:243–56.

    CAS  PubMed  Google Scholar 

  25. Yavin S, Arav A. Measurement of essential physical properties of vitrification solutions. Theriogenology. 2007;67:81–9.

    CAS  PubMed  Google Scholar 

  26. De Gendt K, Verhoeven G. Tissue- and cell-specific functions of the androgen receptor revealed through conditional knockout models in mice. Mol Cell Endocrinol. 2012;352:13–25.

    PubMed  Google Scholar 

  27. Nishiyama H, Danno S, Kaneko Y, Itoh K, Yokoi H, Fukumoto M, et al. Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature. Am J Pathol. 1998;152:289–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Kliesch S, Penttilä TL, Gromoll J, Saunders PT, Nieschlag E, Parvinen M. FSH receptor mRNA is expressed stage-dependently during rat spermatogenesis. Mol Cell Endocrinol. 1992;84:R45–9.

    CAS  PubMed  Google Scholar 

  29. Gruppi CM, Zakeri ZF, Wolgemuth DJ. Stage and lineage-regulated expression of two hsp90 transcripts during mouse germ cell differentiation and embryogenesis. Mol Reprod Dev. 1991;28:209–17.

    CAS  PubMed  Google Scholar 

  30. Zhang L, Tang J, Haines CJ, Feng HL, Lai L, Teng X, et al. c-kit and its related genes in spermatogonial differentiation. Spermatogenesis. 2011;1:186–94.

    PubMed  PubMed Central  Google Scholar 

  31. Pauls K, Schorle H, Jeske W, Brehm R, Steger K, Wernert N, et al. Spatial expression of germ cell markers during maturation of human fetal male gonads: an immunohistochemical study. Hum Reprod. 2006;21:397–404.

    CAS  PubMed  Google Scholar 

  32. Selvaratnam J, Paul C, Robaire B. Male rat germ cells display age-dependent and cell-specific susceptibility in response to oxidative stress challenges. Biol Reprod. 2015;93:72.

    PubMed  PubMed Central  Google Scholar 

  33. Van Saen D, Pino Sánchez J, Ferster A, van der Werff ten Bosch J, Tournaye H, Goossens E. Is the protein expression window during testicular development affected in patients at risk for stem cell loss? Hum Reprod. 2015;30:2859–70.

    PubMed  Google Scholar 

  34. Ravindranath N, Dalal R, Solomon B, Djakiew D, Dym M. Loss of telomerase activity during male germ cell differentiation. Endocrinology. 1997;138:4026–9.

    CAS  PubMed  Google Scholar 

  35. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet. 2001;36:653–9.

    Google Scholar 

  36. Stolz A, Ertych N, Bastians H. A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation. Cell Cycle. 2015;14:827–37.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Jiang Y, Xie M, Chen W, Talbot R, Maddox JF, Faraut T, et al. The sheep genome illuminates biology of the rumen and lipid metabolism. Science. 2014;344:1168–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Wu H, Yu XL, Guo XF, Zhang F, Pei XZ, Li XX, et al. Effect of liquid helium vitrification on the ultrastructure and related gene expression of mature bovine oocytes after vitrifying at immature stage. Theriogenology. 2017;87:91–9.

    CAS  PubMed  Google Scholar 

  39. Wen Y, Zhao S, Chao L, Yu H, Song C, Shen Y, et al. The protective role of antifreeze protein 3 on the structure and function of mature mouse oocytes in vitrification. Cryobiology. 2014;69:394–401.

    CAS  PubMed  Google Scholar 

  40. Yan W, Suominen J, Samson M, Jegou B, Toppari J. Involvement of Bcl-2 family proteins in germ cell apoptosis during testicular development in the rat and pro-survival effect of stem cell factor on germ cells in vitro. Mol Cell Endocrinol. 2000;165:115–29.

    CAS  PubMed  Google Scholar 

  41. Song YS, Narasimhan P, Kim GS, Jung JE, Park EH, Chan PH. The role of Akt signaling in oxidative stress mediates NF-kappaB activation in mild transient focal cerebral ischemia. J Cereb Blood Flow Metab. 2008;28:1917–26.

    CAS  PubMed  Google Scholar 

  42. Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J. A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J Cell Biol. 1997;137:899–908.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Fujita J. Cold shock response in mammalian cells. J Mol Microbiol Biotechnol. 1999;1:243–55.

    CAS  PubMed  Google Scholar 

  44. Wellmann S, Bührer C, Moderegger E, Zelmer A, Kirschner R, Koehne P, et al. Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism. J Cell Sci. 2004;117:1785–94.

    CAS  PubMed  Google Scholar 

  45. Liu DH, Yuan HY, Cao CY, Gao ZP, Zhu BY, Huang HL, et al. Heat shock protein 90 acts as a molecular chaperone in late-phase activation of extracellular signal-regulated kinase 1/2 stimulated by oxidative stress in vascular smooth muscle cells. Acta Pharmacol Sin. 2007;28:1907–13.

    CAS  PubMed  Google Scholar 

  46. Wang XH, Kang L. Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera: Acridiidae). J Insect Physiol. 2005;51:1277–85.

    CAS  PubMed  Google Scholar 

  47. Haase M, Fitze G. HSP90AB1: helping the good and the bad. Gene. 2016;575(2 Pt 1):171–86.

    CAS  PubMed  Google Scholar 

  48. Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997;90:785–95.

    CAS  PubMed  Google Scholar 

  49. Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985;43(2 Pt 1):405–13.

    CAS  PubMed  Google Scholar 

  50. Kent D, Copley M, Benz C, Dykstra B, Bowie M, Eaves C. Regulation of hematopoietic stem cells by the steel factor/KIT signaling pathway. Clin Cancer Res. 2008;14:1926–30.

    CAS  PubMed  Google Scholar 

  51. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122:947–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Zaehres H, Lensch MW, Daheron L, Stewart SA, Itskovitz-Eldor J, Daley GQ. High-efficiency RNA interference in human embryonic stem cells. Stem Cells. 2005;23:299–305.

    CAS  PubMed  Google Scholar 

  53. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76.

    CAS  PubMed  Google Scholar 

  54. Wang X, Zhao Y, Xiao Z, Chen B, Wei Z, Wang B, et al. Alternative translation of OCT4 by an internal ribosome entry site and its novel function in stress response. Stem Cells. 2009;27:1265–75.

    CAS  PubMed  Google Scholar 

  55. Byun K, Kim TK, Oh J, Bayarsaikhan E, Kim D, Lee MY, et al. Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway. Stem Cell Res. 2013;11(3):1323–34.

    CAS  PubMed  Google Scholar 

  56. Miki T, Wong W, Zhou E, Gonzalez A, Garcia I, Grubbs BH. Biological impact of xeno-free chemically defined cryopreservation medium on amniotic epithelial cells. Stem Cell Res Ther. 2016;7:8.

    PubMed  PubMed Central  Google Scholar 

  57. Idda A, Bebbere D, Corona G, Masala L, Casula E, Cincotti A, et al. Insights on cryopreserved sheep fibroblasts by cryomicroscopy and gene expression analysis. Biopreserv Biobank. 2017;15:310–20.

    CAS  PubMed  Google Scholar 

Download references

Funding

Research was supported by Regione Autonoma della Sardegna.- L.R. 7-MIGLIOVINGENSAR Project and by Bando Competitivo Fondazione di Sardegna – 2016, CUP J86C18000800005.

DB is the recipient of an RTDa contract at the University of Sassari, Italy, granted by “P.O.R. SARDEGNA F.S.E. 2014–2020” – CUP J86C18000270002.

Author information

Authors and Affiliations

  1. Department of Veterinary Medicine, University of Sassari, Sassari, Italy

    Daniela Bebbere, Sara Pinna, Stefano Nieddu & Sergio Ledda

  2. FertileSAFE Ltd, 11 Haharash St, 7403118, Ness Ziona, Israel

    Dity Natan & Amir Arav

Authors
  1. Daniela Bebbere
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sara Pinna
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefano Nieddu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dity Natan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amir Arav
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sergio Ledda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniela Bebbere.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bebbere, D., Pinna, S., Nieddu, S. et al. Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit). J Assist Reprod Genet 36, 2145–2154 (2019). https://doi.org/10.1007/s10815-019-01559-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-019-01559-x

Keywords

Search

Navigation