CSIRO Publishing blank image blank image blank image blank imageBooksblank image blank image blank image blank imageJournalsblank image blank image blank image blank imageAbout Usblank image blank image blank image blank imageShopping Cartblank image blank image blank image You are here: Journals > Reproduction, Fertility and Development   
Reproduction, Fertility and Development
Journal Banner
  Vertebrate reproductive science and technology
blank image Search
blank image blank image
blank image
  Advanced Search

Journal Home
About the Journal
Editorial Structure
Online Early
Current Issue
Just Accepted
All Issues
Special Issues
Research Fronts
Virtual Issues
Sample Issue
For Authors
General Information
Submit Article
Author Instructions
Open Access
Awards and Prizes
For Referees
Referee Guidelines
Review an Article
Annual Referee Index
For Subscribers
Subscription Prices
Customer Service
Print Publication Dates
Library Recommendation

blue arrow e-Alerts
blank image
Subscribe to our email Early Alert or RSS feeds for the latest journal papers.

red arrow Connect with us
blank image
facebook twitter logo LinkedIn

red arrow Connect with SRB
blank image
facebook TwitterIcon

Affiliated Societies

RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.


Article << Previous     |     Next >>   Contents Vol 23(6)

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

Luisa Bogliolo A C, Federica Ariu A, Giovanni Leoni B, Stefania Uccheddu A and Daniela Bebbere A

A Department of Veterinary Clinics and Pathology, University of Sassari, Via Vienna, 07100 Sassari, Italy.
B Department of Animal Biology, University of Sassari, Via Vienna, 07100 Sassari, Italy.
C Corresponding author. Email: luis@uniss.it

Reproduction, Fertility and Development 23(6) 809-817 http://dx.doi.org/10.1071/RD11023
Submitted: 19 January 2011  Accepted: 8 March 2011   Published: 7 July 2011

PDF (254 KB) $25
 Export Citation

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38°C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5 h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P < 0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P < 0.05) and TE (P < 0.01) cell number and a lower percentage of picnotic nuclei (P < 0.05) compared with the control group. Significantly lower abundance for BAX (P < 0.01) and OCT4 (P < 0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Additional keywords: blastocyst cell number, gene expression analysis, picnosis.


Arya, R., Mallik, M., and Lakhotia, S. C. (2007). Heat-shock genes – integrating survival and death. J. Biosci. 32, 595–610.
CrossRef | CAS | PubMed |

Bebbere, D., Bogliolo, L., Ariu, F., Fois, S., Leoni, G. G., Succu, S., Berlinguer, F., and Ledda, S. (2010). Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization. Theriogenology 74, 712–723.
CrossRef | CAS | PubMed |

Bergeron, L., Perez, G. I., Macdonald, G., Shi, L., Sun, Y., Jurisicova, A., Varmuza, S., Latham, K. E., Flaws, J. A., Salter, J. C., Hara, H., Moskowitz, M. A., Li, E., Greenberg, A., Tilly, J. L., and Yuan, J. (1998). Defects in regulation of apoptosis in caspase-2 deficient mice. Genes Dev. 12, 1304–1314.
CrossRef | CAS | PubMed |

Biggers, J. D. (2001). Thoughts on embryo culture conditions. Reprod. Biomed. Online 4, 30–38.

Bock, I., Losonczi, E., Mamo, S., Polgar, Z., Dinnyes, A., Harnos, A., and Pribenszky, C. (2010). Stress tolerance and transcriptional response in mouse embryos treated with high hydrostatic pressure to enhance cryotolerance. Cryo Lett. 31, 401–412.

Boelhauve, M., Sinowatz, F., Wolf, E., and Paula-Lopes, F. F. (2005). Maturation of bovine oocytes in the presence of leptin improves development and reduces apoptosis of in vitro-produced blastocysts. Biol. Reprod. 73, 737–744.
CrossRef | CAS | PubMed |

Boiani, M., and Schöler, H. R. (2005). Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872–881.
CrossRef | CAS | PubMed |

Christians, E. S., Zhou, Q., Renard, J. P., and Benjamin, I. J. (2003). Heat-shock proteins in mammalian development. Semin. Cell Dev. Biol. 14, 283–290.
CrossRef | CAS | PubMed |

Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998). The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79, 129–168.
CrossRef | CAS | PubMed |

Dinnyes, A., Polgar, Z., Pribenszky, Cs., and Pirity, M. K. (2010). Improved embryoid body cryopreservation and cardiomyocyte differentiation following high hydrostatic pressure treatment. In ‘Proceedings of the 1st International Congress on Controversies in Cryopreservation of Stem Cells, Reproductive Cells, Tissue and Organs, Valencia, Spain, 27–30 May 2010’. (CometMed: Israel.)

Du, Y., Pribenszky, Cs., Molnár, M., Zhang, X., Yang, H., Kuwayama, M., Pedersen, A. M., Villemoes, K., Bolund, L., and Vajta, G. (2008a). High hydrostatic pressure (HHP): a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction 135, 13–17.
CrossRef | CAS | PubMed |

Du, Y., Lin, L., Schmidt, M., Bogh, I. B., Kragh, P. M., Sorensen, C. B., Li, J., Purup, S., Pribenzsky, C., Molnár, M., Kuwayama, M., Zhang, X., Yang, H., Bolund, L., and Vajta, G. (2008b). High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells 10, 325–330.
CrossRef | CAS | PubMed |

Edwards, J. L., and Hansen, P. J. (1997). Differential responses of bovine oocytes and pre-implantation embryos to heat shock. Mol. Reprod. Dev. 46, 138–145.
CrossRef | CAS | PubMed |

Enright, B. P., Lonergan, P., Dinnyes, A., Fair, T., Ward, F. A., Yang, X., and Boland, M. P. (2000). Culture of in vitro-produced bovine zygotes in vitro vs. in vivo: implications for early embryo development and quality. Theriogenology 54, 659–673.
CrossRef | CAS | PubMed |

Gardner, D. K. (2008). Dissection of culture media for embryos: the most important and less important components and characteristics. Reprod. Fertil. Dev. 20, 9–18.
CrossRef | PubMed |

Garrido, C., Brunet, M., Didelot, C., Zermati, Y., Schmitt, E., and Kroemer, G. (2006). Heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties. Cell Cycle 5, 2592–2601.
CrossRef | CAS | PubMed |

Gjørret, J. O., Knijn, H. M., Dieleman, S. J., Avery, B., Larsson, L. I., and Maddox-Hyttel, P. (2003). Chronology of apoptosis in bovine embryos produced in vivo and in vitro. Biol. Reprod. 69, 1193–1200.
CrossRef | PubMed |

Hardy, K. (1999). Apoptosis in the human embryo. Rev. Reprod. 4, 125–134.
CrossRef | CAS | PubMed |

Hardy, K., Handyside, A. H., and Winstons, R. M. L. (1989). The human blastocyst: cell number, death and allocation during late pre-implantation development in vitro. Development 107, 597–604.
| CAS | PubMed |

Huang, S. Y., Pribenszky, Cs., Kuo, Y. H., Teng, S. H., Chen, Y. H., Chung, M. T., and Chiu, Y. F. (2009). Hydrostatic pressure affects the protein profile of boar sperm before and after freezing–thawing. Anim. Reprod. Sci. 112, 136–149.
CrossRef | CAS | PubMed |

Kaarniranta, K., Elo, M., Sironen, R., Lammi, M. J., Goldring, M. B., Eriksson, J. E., Sistonen, L., and Helminen, H. J. (1998). Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc. Natl. Acad. Sci. USA 95, 2319–2324.
CrossRef | CAS |

Kidson, A., Rubio-Pomar, F. J., Van Knegsel, A., Van Tol, H. T., Hazeleger, W., and Ducro-Steverink, D. W. (2004). Quality of porcine blastocysts produced in vitro in the presence or absence of GH. Reproduction 127, 165–177.
CrossRef | CAS | PubMed |

Kirchhof, N., Carnwath, J. W., Lemme, E., Anastassiadis, K., Schöler, H., and Niemann, H. (2000). Expression pattern of Oct-4 in pre-implantation embryos of different species. Biol. Reprod. 63, 1698–1705.
CrossRef | CAS | PubMed |

Knijn, H. M., Wrenzycki, C., Hendriksen, P. J., Vos, P. L., Herrmann, D., van der Weijden, G. C., Niemann, H., and Dieleman, S. J. (2002). Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo. Reproduction 124, 365–375.
CrossRef | CAS | PubMed |

Kültz, D. (2003). Evolution of the cellular stress proteome: from monophyletic origin to ubiquitous function. J. Exp. Biol. 206, 3119–3124.
CrossRef | PubMed |

Kuo, Y. H., Pribenszky, Cs., and Huang, S. Y. (2008). Higher litter size is achieved by the insemination of high hydrostatic pressure-treated frozen–thawed boar semen. Theriogenology 70, 1395–1396.
CrossRef |

Leoni, G. G., Bebbere, D., Succu, S., Berlinguer, F., Mossa, F., Galioto, M., Bogliolo, L., Ledda, S., and Naitana, S. (2007). Relations between relative mRNA abundance and developmental competence of ovine oocytes. Mol. Reprod. Dev. 74, 249–257.
CrossRef | CAS | PubMed |

Leoni, G. G., Berlinguer, F., Succu, S., Bebbere, D., Mossa, F., Madeddu, M., Ledda, S., Bogliolo, L., and Naitana, S. (2008). A new selection criterion to assess good-quality ovine blastocysts after vitrification and to predict their transfer into recipients. Mol. Reprod. Dev. 75, 373–382.
CrossRef | CAS | PubMed |

Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-Delta Delta CT. Method. Methods 25, 402–408.
CrossRef | CAS | PubMed |

Lonergan, P. (2007). State-of-the-art embryo technologies in cattle. Soc. Reprod. Fertil. Suppl. 64, 315–325.
| CAS | PubMed |

Lonergan, P., Rizos, D., Gutiérrez-Adán, A., Fair, T., and Boland, M. P. (2003). Effect of culture environment on embryo quality and gene expression – experience from animal studies. Reprod. Biomed. Online 7, 657–663.
CrossRef | CAS | PubMed |

Luft, J. C, and Dix, D. J. (1999). Hsp70 expression and function during embryogenesis. Cell Stress Chaperones 4, 162–170.
CrossRef | CAS | PubMed |

Melka, M. G., Rings, F., Hölker, M., Tholen, E., Havlicek, V., Besenfelder, U., Schellander, K., and Tesfaye, D. (2010). Expression of apoptosis regulatory genes and incidence of apoptosis in different morphological quality groups of in vitro-produced bovine pre-implantation embryos. Reprod. Domest. Anim. 45, 915–921.
CrossRef | CAS | PubMed |

Moussa, M., Tremoleda, J. L., Duchamp, G., Bruyas, J. F., Colenbrander, B., and Bevers, M. M. (2004). Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5–8°C. Theriogenology 61, 921–932.
CrossRef | CAS | PubMed |

Pomar, F. J., Teerds, K. J., Kidson, A., Colenbrander, B., Tharasanit, T., Aguila, B., and Roelen, B. A. (2005). Differences in the incidence of apoptosis between in vivo- and in vitro-produced blastocysts of farm animal species: a comparative study. Theriogenology 63, 2254–2268.
CrossRef | CAS | PubMed |

Pribenszky, C., and Vajta, G. (2010). Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes’ and embryos’ performance? Reprod. Fertil. Dev. 23, 48–55.
CrossRef |

Pribenszky, C., Molnár, M., Cseh, S., and Solti, L. (2005a). Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim. Reprod. Sci. 87, 143–150.
CrossRef | PubMed |

Pribenszky, C., Molnár, M., Ulrich, P., Barbosa, C., Hatamoto, L., and Santos, C. (2005b). Pressure assisted cryopreservation: a novel possibility for IVP bovine blastocyst cryopreservation. Reprod. Domest. Anim. 40, 338..

Pribenszky, C., Molnár, M., Horváth, A., Harnos, A., and Szenci, O. (2006). Hydrostatic pressure induced increase in post–thaw motility of frozen boar spermatozoa. Reprod. Fertil. Dev. 18, 162–163.
CrossRef |

Pribenszky, C., Molnár, M., Horváth, A., Kútvölgyi, G., Harnos, A., Szenci, O., Dengg, J., and Lederer, J. (2007). Improved post-thaw motility, viability and fertility are achieved by hydrostatic pressure-treated bull semen. Reprod. Fertil. Dev. 19, 181–182.
CrossRef |

Pribenszky, C., Siquiera, E., Molnár, M., and Rumpf, R. (2008a). Improved post-warming developmental competence of open-pulled straw-vitrified in vitro-produced bovine blastocysts by sub-lethal hydrostatic pressure pre-treatment. Reprod. Fertil. Dev. 20, 125.
CrossRef |

Pribenzsky, C., Du, Y., Molnár, M., and Vajta, G. (2008b). Sub-lethal stress on porcine oocytes enhances the efficacy of ART procedures. Hum. Reprod. 23, 161..

Pribenszky, C., Molnár, M., Kútvölgyi, G., Harnos, A., Horváth, A., and Héjja, I. (2009). Sub-lethal hydrostatic pressure treatment improves fresh and chilled boar semen quality in vitro and in vivo. Reprod. Fertil. Dev. 21, 107.
CrossRef |

Pribenszky, C., Vajta, G., Molnar, M., Du, Y., Lin, L., and Bolund, J. Y. (2010). Stress for stress tolerance? A fundamentally new approach in mammalian embryology. Biol. Reprod. 83, 690–697.
CrossRef | CAS | PubMed |

Rizos, D., Fair, T., Papadopoulos, S., Boland, M. P., and Lonergan, P. (2002). Developmental, qualitative and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol. Reprod. Dev. 62, 320–327.
CrossRef | CAS | PubMed |

Rizos, D., Gutiérrez-Adán, A., Pérez-Garnelo, S., de la Fuente, J., Boland, M. P., and Lonergan, P. (2003). Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance and messenger RNA expression. Biol. Reprod. 68, 236–243.
CrossRef | CAS | PubMed |

Rizos, D., Gutierrez-Adan, A., Moreira, P., O’Meara, C., Fair, T., Evans, A. C., Boland, M. P., and Lonergan, P. (2004). Species-related differences in blastocyst quality are associated with differences in relative mRNA transcription. Mol. Reprod. Dev. 69, 381–386.
CrossRef | CAS | PubMed |

Saijoh, Y., Fujii, H., Men, C., Sato, M., Hirota, Y., Nagamatsu, S., Ikeda, M., and Hamada, H. (1996). Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells 1, 239–252.
| CAS | PubMed |

Schöler, H. R. (1991). Octamania: the POU factors in murine development. Trends Genet. 7, 323–329.
| PubMed |

Siqueira, F. E., Caixeta, E. S., Molnár, M., Pribenszky, C., Franco, M. M., and Rumpf, R. (2009). Sub-lethal hydrostatic pressure pre-treatment: effect in bovine embryos gene expression. Biol. Reprod. 81, 629..

Söti, C., and Csermely, P. (2007). Protein stress and stress proteins: implications in ageing and disease. J. Biosci. 32, 511–515.
CrossRef | PubMed |

Succu, S., Bebbere, D., Bogliolo, L., Ariu, F., Fois, S., Leoni, G. G., Berlinguer, F., Naitana, S., and Ledda, S. (2008). Vitrification of in vitro-matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance. Mol. Reprod. Dev. 75, 538–546.
CrossRef | CAS | PubMed |

Thompson, J. G. (1997). Comparison between in vivo-derived and in vitro-produced pre-elongation embryos from domestic ruminants. Reprod. Fertil. Dev. 9, 341–354.
CrossRef | CAS | PubMed |

Walker, S. K., Hill, J. L., Kleemann, D. O., and Nancarrow, C. D. (1996). Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations. Biol. Reprod. 55, 703–708.
CrossRef | CAS | PubMed |

Wang, P., Shu, Z., He, L., Cui, X., Wang, Y., and Gao, D. (2005). The pertinence of expression of heat shock proteins (HSPs) to the efficacy of cryopreservation in HELAs. Cryo Lett. 26, 7–16.

Welch, T. J., Farewell, A., Neidhardt, F. C., and Bartlett, D. H. (1993). Stress response of Escherichia coli to elevated hydrostatic pressure. J. Bacteriol. 175, 7171–7177.

Wemekamp-Kamphuis, H. H., Karatzas, A. K., Wouters, J. A., and Abee, T. (2002). Enhanced levels of cold-shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Appl. Environ. Microbiol. 68, 456–463.
CrossRef | CAS | PubMed |

Wrenzycki, C., Herrmann, D., Carnwath, J. W., and Niemann, H. (1999). Alterations in the relative abundance of gene transcripts in pre-implantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev. 53, 8–18.
CrossRef | CAS | PubMed |

Wrenzycki, C., Herrmann, D., Keskintepe, L., Martins, A., Sirisathien, S., Brackett, B., and Niemann, H. (2001). Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum. Reprod. 16, 893–901.
CrossRef | CAS | PubMed |

Wrenzycki, C., Herrmann, D., and Niemann, H. (2003). Timing of blastocyst expansion affects spatial messenger RNA expression patterns of genes in bovine blastocysts produced in vitro. Biol. Reprod. 68, 2073–2080.
CrossRef | CAS | PubMed |

Wrenzycki, C., Herrmann, D., Lucas-Hahn, A., Korsawe, K., Lemme, E., and Niemann, H. (2005). Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development. Reprod. Fertil. Dev. 17, 23–35.
CrossRef | CAS | PubMed |

Zuccotti, M., Merico, V., Sacchi, L., Bellone, M., Brink, T. C., Bellazzi, R., Stefanelli, M., Redi, C. A., Garagna, S., and Adjaye, J. (2008). Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes. BMC Dev. Biol. 6, 88–97.

Subscriber Login

Legal & Privacy | Contact Us | Help


© CSIRO 1996-2016