Hostname: page-component-78c5997874-g7gxr Total loading time: 0 Render date: 2024-11-04T21:39:25.517Z Has data issue: false hasContentIssue false
  • English
  • Français

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Published online by Cambridge University Press:  01 February 2007

M. Nakai ,
N. Kashiwazaki ,
A. Takizawa ,
N. Maedomari ,
M. Ozawa ,
J. Noguchi ,
H. Kaneko ,
M. Shino  and
K. Kikuchi
M. Nakai
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
N. Kashiwazaki
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
A. Takizawa
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
N. Maedomari
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
M. Ozawa
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
J. Noguchi
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
H. Kaneko
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
M. Shino
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
K. Kikuchi*
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
*
All correspondence to: K. Kikuchi, Division of Animal Sciences, Reproductive Biology Research Unit (Kannondai), Kannondai 2–1-2, Tsukuba, Ibaraki 305–8602, Japan, Tel: +81 298 38 7447. Fax: +81 298 38 7408. e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.

The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

Keywords

DNA fragmentationEndonucleaseFreeze-dryICSIPig
Type
Research Article
Information
Zygote , Volume 15 , Issue 1 , February 2007 , pp. 15 - 24
Copyright
Copyright © Cambridge University Press 2007

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Acharyya, S., Kanjilal, S. & Bhattacharyya, A.K. (2005). Does human sperm nuclear DNA integrity affect embryo quality? Indian J. Exp. Biol. 43, 1016–22.Google ScholarPubMed
Alvarez, J.G. & Storey, B.T. (1993). Evidence that membranes stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J. Andol. 14, 199–209.CrossRefGoogle ScholarPubMed
Azuma, T., Kondo, T., Ikeda, S., Imai, H. & Yamada, M. (2002). Effects of EDTA saturated with Ca2+ (Ca–EDTA) on pig, bovine and mouse oocytes at the germinal vesicle stage during maturation culture and the involvement of chelation of Zn2+ in pronuclear formation induction by Ca–EDTA. Reproduction 124, 235–40.CrossRefGoogle ScholarPubMed
Bunge, R.G. & Sherman, J.K. (1953). Fertilization capacity of frozen human spermatozoa. Nature 172, 767–8.CrossRefGoogle ScholarPubMed
Day, J.G. & McLellan, M.R. (eds) (1995). Cryopreservation and freeze-drying protocols. In Methods in Molecular Biology vol. 38. Humana Press: Totowa, NJ.Google Scholar
Fatehi, A.N., Bevers, M.M., Schoevers, E., Roelen, B.A., Colenbrander, B. & Gadella, B.M. (2006). DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages. J. Androl. 27, 176–88.CrossRefGoogle Scholar
Henkel, R., Hajimohammad, M., Stalf, T., Hoogendijk, C., Mehnert, C., Menkveld, R., Gips, H., Schill, W. & Kruger, T. (2004). Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil. Steril. 81, 965–72.CrossRefGoogle ScholarPubMed
Hirabayashi, M., Kato, M., Ito, J. & Hochi, S. (2005). Viable offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads. Zygote 13, 79–85.CrossRefGoogle ScholarPubMed
Kaneko, T., Whittingham, D.G. & Yanagimachi, R. (2003a). Effects of pH value of freeze-drying solution on the chromosome integrity and development ability of mouse spermatozoa. Biol. Reprod. 68, 136–9.CrossRefGoogle ScholarPubMed
Kaneko, T., Whittingham, D.G., Overstreet, J.W. & Yanagimachi, R. (2003b). Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulphide status. Biol. Reprod. 69, 1859–62.CrossRefGoogle Scholar
Kashiwazaki, N., Kikuchi, K., Suzuki, K., Noguchi, J., Nagai, T., Kaneko, H. & Shino, M. (2001). Development in vivo and in vitro to blastocysts of porcine oocytes matured and fertilized in vitro. J. Reprod. Dev. 47, 303–10.CrossRefGoogle Scholar
Keskintepe, L., Pacholczyk, G., Machinicka, A., Norris, K., Curuk, M.A., Khan, L. & Brackett, B.G. (2002). Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol. Reprod. 67, 409–15.CrossRefGoogle ScholarPubMed
Kikuchi, K., Kashiwazaki, N., Noguchi, J., Shimada, A., Takahashi, R., Hirabayashi, M., Shino, M., Ueda, M. & Kaneko, H. (1999). Developmental competence, after transfer to recipients, of porcine matured, fertilized, and cultured in vitro. Biol. Reprod. 60, 336–40.CrossRefGoogle ScholarPubMed
Kikuchi, K., Onishi, A., Kashiwazaki, N., Iwamoto, M., Noguchi, J., Kaneko, H., Akita, T. & Nagai, T. (2002). Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol. Reprod. 66, 1033–41.CrossRefGoogle ScholarPubMed
King, T.J., Dobrinsky, J.R., Zhu, J., Finlayson, H.A., Bosma, W., Harkness, L., Ritchie, W.A., Travers, A., McCorqquodale, C., Day, B.N., Dinnyes, A., De Sousa, P.A. & Wilmut, I. (2002). Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos. Reproduction 123, 507–15.CrossRefGoogle ScholarPubMed
Kure-bayashi, S., Miyake, M., Okabe, K. & Kato, S. (2000). Successful implantation of in vitro matured, electro activated oocytes in the pig. Theriogenology 53, 1105–19.CrossRefGoogle ScholarPubMed
Kusakabe, H., Szczygiel, M.A., Whittingham, D.G. & Yanagimachi, R. (2001). Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc. Natl. Acad. Sci. USA 98, 13501–6.CrossRefGoogle ScholarPubMed
Kwon, IK., Park, K.E. & Niwa, K. (2004). Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa. Biol. Reprod. 71, 1430–6.CrossRefGoogle ScholarPubMed
Liu, J.L., Kusakabe, H., Chang, C.C., Suzuki, H., Schmidt, D.W., Julian, M., Pfeffer, R., Bormann, C.L., Tian, X.C., Yanagimachi, R. & Yang, X. (2004). Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol. Reprod. 70, 1776–81.CrossRefGoogle ScholarPubMed
Lopes, S., Sun, J.G., Jurisicova, A., Meriano, J. & Casper, R.F. (1998). Sperm deoxyribonucleic acid fragmentation is increased in poor quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril. 69, 528–32.CrossRefGoogle ScholarPubMed
Nakai, M., Kashiwazaki, N., Takizawa, A., Hayashi, Y., Nakatsukasa, E., Fuchimoto, D., Noguchi, J., Kaneko, H., Shino, M. & Kikuchi, K. (2003). Viable piglets generated from porcine oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection. Biol. Reprod. 68, 1003–8.CrossRefGoogle ScholarPubMed
Nakai, M., Kashiwazaki, N., Takizawa, A., Maedomari, N., Ozawa, M., Noguchi, J., Kaneko, H., Shino, M. & Kikuchi, K. (2006). Morphological changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes. Reproduction 131, 603–11.CrossRefGoogle ScholarPubMed
Nasr-Esfahani, M.H., Salehi, M., Razavi, S., Anjomshoa, M., Rozbahani, S., Moulavi, F. & Mardani, M. (2005). Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development postICSI. Reprod. Biomed. Online 11, 198–205.Google ScholarPubMed
Petit, F.M., Frydman, N., Benkhalifa, M., Du, A.L., Aboura, A., Fanchin, R., Frydman, R. & Tachdjian, G. (2005). Could sperm aneuploidy rate determination be used as a predictive test before intracytoplasmic sperm injection? J. Androl. 26, 235–41.CrossRefGoogle ScholarPubMed
Petters, R.M. & Wells, K.D. (1993). Cultured of pig embryos. J. Reprod. Fertil. Suppl. 48, 61–73.Google ScholarPubMed
Pursel, V.G., & Johnson, L.A. (1975). Freezing of boar spermatozoa: fertilizing capacity with cocncentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99–102.CrossRefGoogle Scholar
Rubin, H. (1975). Central role for magnesium in coordinate control of metabolism and growth in animal cells. Proc. Nat. Acad. Sci. USA 72, 3551–5.CrossRefGoogle ScholarPubMed
Salamon, G.W. & Lightfoot, R.J. (1967). Fertilization and embryonic loss in sheep after insemination with deep frozen semen. Nature 216, 194–5.CrossRefGoogle ScholarPubMed
Sanui, H. & Pace, N. (1967). Effect of ATP, EDTA and EGTA on the simultaneous binding of Na, K, Mg and Ca by rat liver microsomes. J. Cell. Physiol. 69, 11–20.CrossRefGoogle Scholar
Snedecor, G.W. & Cochran, W.G. (1989). Statistical Methods, 8th ed. pp. 273–296. Ames: The Iowa State University Press.Google Scholar
Sotolongo, B., Huange, T.T.F., Isenberger, E. & Ward, W.S. (2005). An endogenous nuclease in hamster, mouse, and human spermatozoa cleaves DNA into loop-sized fragments. J. Androl. 26, 272–80.CrossRefGoogle ScholarPubMed
Stewart, D.L. (1951). Storage of bull spermatozoa at low temperatures. Vet. Rec. 63, 65–6.Google Scholar
Sun, J.G., Jurisicova, A. & Casper, R.F. (1997). Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod. 56, 602–7.CrossRefGoogle ScholarPubMed
Suzuki, K., Asano, A., Eriksson, B., Niwa, K., Nagai, T. & Rodriguez-Martinez, H. (2002) Capacitation atatus and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus–oocyte-complexes-conditioned medium and hyaluronan. Int. J. Androl. 25, 84–93.CrossRefGoogle Scholar
Tesarik, J., Greco, E. & Mendoza, C. (2004). Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum. Reprod. 19, 611–5.CrossRefGoogle Scholar
Twigg, J.P., Irvine, D.S. & Aitken, R.J. (1998). Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod. 13, 1864–71.CrossRefGoogle ScholarPubMed
Wakayama, T. & Yanagimachi, R. (1998). Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotechnol. 16, 639–41.CrossRefGoogle ScholarPubMed
Ward, M.A., Kaneko, T., Kusakabe, H., Biggers, J.D., Whittingham, D.G. & Yanagimachi, R. (2003). Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection. Biol. Reprod. 69, 2100–8.CrossRefGoogle ScholarPubMed
Weitze, K.F. (1991). Long-term storage of extended boar semen. Reprod. Domes. Anim. Suppl. 1, 231–53.Google Scholar
Westendorf, P., Richter, L. & Treu, H. (1975). Deep freezing of boar semen: laboratory findings and insemination results with the Hulsenberger Pailleten technique. Dtsch. Tierarztl. Wschr. 82, 261–300.Google Scholar