Using Laser-Punched System on IVF of Fresh and Cryopreservated and Post-Thawed Sperm

doi:10.3969/j.issn.1674-5817.2012.03.007

Abstract

Abstract: Objective The IVF rate of the fresh sperm and the post-thawed sperm about C57BL/6J mice were compared, and then C57BL/6J oocytes were laser-punched, and these oocytes were IVF with fresh and post-thawed sperm, their IVF rate were investigated. Method Two inbred mice strains of(C57BL/6J, C57BL/6N) and 5 strains of genetic engineering mice (MKP-5, Sir-168, C57BL/6RLR 3-/-, C57BL/6 TRL 2KO, C57BL/6 TRL 4KO) were chosen to freeze sperms. Firstly, the IVF rate of fresh sperm and post-thawed sperm with normal C57BL/6J oocytes were compared. Secondly, C57BL/6J oocytes were laser-punched, and then the IVF rate of the fresh sperm and post-thawed sperm with these oocytes were compared. Result (1) The IVF rate of fresh sperm and post-thawed sperm of C57BL/6J, C57BL/6N were 50.4% vs 17.7%, 48.6% vs 15.4%; when the oocytes were laser-punched, the IVF rate were 64.6% vs 59.7%, 60.4% vs 15.4%; (2) the IVF rate of fresh and frozen sperm of 5 genetic engineering mice were 91.5% vs 28.6%, 77.4% vs 52.3%, 40.7% vs 12.7%, 56.5% vs 14.6%, 65% vs 6.5%, when those sperm were fertilized with punched C57BL/6J oocytes, the result were 75% vs 50.7%, 74.3% vs 35.6%, 75.7% vs 32.7%, 79.5% vs 48.9%, 59.5% vs 46.3%. Conclusion When oocytes were laser-punched, the IVFrate of post-thawed sperm were increased significantly, it is showed that laser-punched system was a useful and economical way to increase the IVF rate of mice frozen sperm.

Key words: mice, sperm cryopreservation, laser-punched oocytes

CLC Number:

Q95-33

LIU Li-jun, ZHU Quan-feng, YU Li-li, ZHANG Yi-da, SHI Mei-lian. Using Laser-Punched System on IVF of Fresh and Cryopreservated and Post-Thawed Sperm[J]. Laboratory Animal and Comparative Medicine, 2012, 32(3): 203-208.

References

[1] Nakagata N, Taneshima T.High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation[J]. Theriogenology, 1992, 37:1283-1291.
[2] Nakagata N.Cryopreservation of mouse spermatozoa[J]. Mamm Genome, 2000, 11:572-576.
[3] Mouse genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome[J].Nature, 2002, 420:520-562.
[4] Nakagata N, Okamoto M, Ueda O, et al.Positive effect of partia zona-pellucida dissection on the in vitro fertilizing capacity of cryopreserved C57BL/6J transgenic mouse spermatozoa of low motility[J]. Biol Reprod, 1997, 57:1050-1055.
[5] Kaoniko.Mizuno, Kazuhiko, Hoshi and Thomas Huang, Fertilization and embryo develioment in a mouse ICSI model using human and mouse sperm after immobilization in polyvinylpyrrolidone[J]. Hum Reprod, 2002, 17,9:2350-2355.
[6] Fujimoto G, Tsuchiya H, Kobayashi K,et al.Calcium concentration and fertilization by subzonal insemination of a single spermatozoon in mouse oocytes[J].Mol Reprod Dev. 1994, 38(1):54-60.
[7] Quinn P, Kerin J.F, Warners G.M. Improved pregnancy rate in human in vitro fertilization with the use of medium based on the composition of human tubal fluid[J]. Fertil. Steril, 1985, 44:493-498.
[8] Critser, J. K, Mobraaten, L.E.Cryopreservation of murine spermatozoa[J]. ILAR J, 2000. 41:197-206.
[9] Devireddy R. V., Swanlund D.J.,Roberts K.P., et al.Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents[J].Biol Reprod, 1999, 61:764-775.
[10] Dewit, M, Marley, W.S, Graham, J.K.Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs[J]. Cryobiology, 2000, 40:36-45.
[11] An TZ, Iwakiri M, Edashige K, et al.Factors affecting the survival of frozen-thawed mouse spermatozoa[J]. Cryobiology, 2000, 40:237-249.
[12] Nishizono H, Shioda M, Takeo T, et al.Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury[J]. Biol Reprod, 2004, 71(3):973-978.
[13] Snell WJ, White JM.The molecules of mammalian fertilization[J]. Cell, 1996, 85:629-637.
[14] Breitbart H, Naor Z.Protein kinases in mammalian sperm capacitation and the acrosome reaction[J]. Rev Reprod, 1999, 4:151-159.
[15] Sztein JM, Noble K, Farley JS, et al.Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation[J]. Cryobiology. 2001, 42:28-39.
[16] V.J.Hall, D.Compton, P.Stojkovie et al. Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer[J]. Hum Reprod, 2007, 22:52-62.
[17] Willian T Poueymirou, Wojtek Auerbach, David Frendewey et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses[J]. Nat Biotech, 2006, 24:1-9.
[18] Noriko Tanaka, Takumi Takeuchi, Queenie V Neri, et al.Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system:applications and preliminary results in a murine model[J]. J Translational Medicine, 2006, 4:20-33.