Skip to main content
SpringerLink
Log in

Offspring Derived From Intracytoplasmic Injection of Transgenic Rat sperm

  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at −20°C until use. When the sperm heads were aspirated into injection pipettes 7–10 μm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4 μm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.

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

Similar content being viewed by others

References

  • Al-Shawai R, Burke J, Jones CT, Simons JP and Bishop JO (1988) A Mup promoter-thymidine kinase reporter gene shows relaxed tissue-specific expression and confers male sterility upon transgenic mice. Mol Cell Biol 8: 4821–4828.

    Google Scholar 

  • Catt SL, Catt JW, Gomez MC, Maxwell WMC and Evans G (1996) The birth of a male lamb derived from an in vitro matured oocyte fertilized by intra-cytoplasmic injection of a single presumptive male sperm. Vet Rec 139: 494–495.

    Google Scholar 

  • Charreau B, Tesson L, Buscail J, Soulillou JP and Anegon I (1996) Analysis of human CDS9 tissue expression directed by the CMV-IE-1 promoter in the transgenic rats. Transgenic Res 5: 443–450.

    Google Scholar 

  • Dozortsev D, Wakaiama T, Ermilov A and Yanagimachi R (1998) Intracytoplasmic sperm injection in the rat. Zygote 6: 143–147.

    Google Scholar 

  • Fujiwara Y, Miwa M, Takahashi R, Hirabayashi M, Suzuki T and Ueda M (1997) Position-independent and high-level expression of human α-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA. Mol Reprod Dev 47: 157–163.

    Google Scholar 

  • Goto K, Kinoshita A, Takuma Y and Ogawa K (1990) Fertilisation of bovine oocytes by the injection of immobilised, killed spermatozoa. Vet Rec 127: 517–520.

    Google Scholar 

  • Hammer RE, Maika SD, Richardson JA, Tang J and Taurog J (1990) Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human β2m: an animal model of HLA-B27-associated human disorders. Cell 63: 1099–1112.

    Google Scholar 

  • Hochi S, Ninomiya T, Honma M and Yuki A (1990) Successful producton of transgenic rats. Anim Biotechnol 1: 175–184.

    Google Scholar 

  • Hochi S, Ninomiya T, Waga-Homma M, Sagara J and Yuki A (1992) Secretion of bovine α-lactalbumin into the milk of transgenic rats. Mol Reprod Dev 33: 160–164.

    Google Scholar 

  • Hosoi Y, Miyake M, Utsumi K and Iritani A (1988) Development of rabbit oocytes after microinjection of spermatozoon. Proc 11th Int Congr Anim Reprod Artif Insemin 3: 331.

    Google Scholar 

  • Iwakura Y, Asano M, Nishimune Y and Kawade Y (1988) Male sterility of transgenic mice carrying exogenous mouse interferon-β gene under the control of the metallothionein enhancer-promoter. EMBO J 7: 3757–3762.

    Google Scholar 

  • Kimura Y and Yanagimachi R (1995) Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development 121: 2397–2405.

    Google Scholar 

  • Kodaira K, Takahashi R, Hirabayashi M, Suzuki T, Obinata M and Ueda M (1996) Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes. Mol Reprod Dev 45: 403–410.

    Google Scholar 

  • Kuretake S, Kimura Y, Hoshi K and Yanagimachi R (1996a) Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod 55: 789–795.

    Google Scholar 

  • Kuretake S, Maleszewski M, Tokumasu A, Fujimoto H and Yanagimachi R (1996b) Inadequate function of sterile tw5 /tw32 spermatozoa overcome by intracytoplasmic sperm injection. Mol Reprod Dev 44: 230–233.

    Google Scholar 

  • Martin MJ (2000) Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection. Biol Reprod 63: 109–112.

    Google Scholar 

  • Miyata T, Okada H, Hashizume R and Ito M (2000) The offspring of intracytoplasmic sperm injection in the rat (in Japanese). Proc 41 Ann Meet Jpn Soc Mamm Ova Res 24.

  • Miyoshi K, Abeydeera LR, Okuda K and Niwa K (1995) Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos. J Reprod Fertil 103: 27–32.

    Google Scholar 

  • Ninomiya T, Hirabayashi M, Sagara J and Yuki A (1994) Function of milk protein gene 5′ flanking regions on human growth hormone gene. Mol Reprod Dev 37: 276–283.

    Google Scholar 

  • Niwa H, Yamamura K and Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–200.

    Google Scholar 

  • Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic sperm injection of a single spermatozoon into an oocyte. Lancet 340: 17–18.

    Google Scholar 

  • Paul M, Wagner J, Hoffmann S, Urata H and Ganten D (1994) Transgenic rats: new experimental models for the study of candidate genes in hypertension research. Annu Rev Physiol 56: 811–829.

    Google Scholar 

  • Perry ACF, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y and Yanagimachi R (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science 284: 1180–1183.

    Google Scholar 

  • Quinn P, Barros C and Whittingham DG (1982) Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil 66: 161–168.

    Google Scholar 

  • Takahashi R, Hirabayashi M and Ueda M (1999) Production of transgenic rats using cryopreserved pronuclear-stage zygotes. Transgenic Res 8: 397–400.

    Google Scholar 

  • Thadani VM (1980) A study of hetero-specific sperm-egg interactions in the rat, mouse, and deer mouse using in vitro fertilization and sperm injection. J Exp Zool 212: 435–453.

    Google Scholar 

  • Toyoda Y and Chang MC (1974) Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J Reprod Fertil 36: 9–22.

    Google Scholar 

  • Uehara T and Yanagimachi R (1976) Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 15: 467–470.

    Google Scholar 

  • Wei H and Fukui Y (2000) Technical improvement in intracytoplasmic sperm injection (ICSI) in cattle. J Reprod Dev 46: 403–407.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. YS New Technology Institute Inc., 519 Shimoishibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi, 329-0512, Japan

    Masumi Hirabayashi, Megumi Kato, Toshihiro Aoto, Akiyo Sekimoto & Masatsugu Ueda

  2. Faculty of Textile Science and Technology, Shinshu University, Tokida, Ueda, Nagano, 386-8567, Japan

    Megumi Kato & Shinichi Hochi

  3. Institute for Animal Experimentation, School of Medicine, Tohoku University, Aoba, Sendai, Miyagi, 980-8577, Japan

    Ichiro Miyoshi & Noriyuki Kasai

Authors
  1. Masumi Hirabayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Megumi Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toshihiro Aoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akiyo Sekimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masatsugu Ueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ichiro Miyoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Noriyuki Kasai
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shinichi Hochi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masumi Hirabayashi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hirabayashi, M., Kato, M., Aoto, T. et al. Offspring Derived From Intracytoplasmic Injection of Transgenic Rat sperm. Transgenic Res 11, 221–228 (2002). https://doi.org/10.1023/A:1015210604906

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1015210604906

Search

Navigation