Hostname: page-component-cd9895bd7-dzt6s Total loading time: 0 Render date: 2024-12-18T21:08:35.416Z Has data issue: false hasContentIssue false
  • English
  • Français

In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation

Published online by Cambridge University Press:  01 May 2008

Wataru Fujii  and
Hiroaki Funahashi
Wataru Fujii
Affiliation:
Department of Animal Science, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, 700–8530, Japan.
Hiroaki Funahashi*
Affiliation:
Department of Animal Science, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, 700–8530, Japan. Department of Animal Science, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, 700–8530, Japan.
*
All correspondence to: H. Funahashi. Department of Animal Science, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Okayama, 700–8530, Japan. Tel: +81 86 251 8329. Fax: +81 86 251 8388. e-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

The present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocyte–cumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14 h after human chorionic gonadotropin (hCG) injection. Cumulus nuclei were injected into ovulated oocytes and then stimulated in the presence of 5 mM SrCl2 for 20 min at various time points (0–3.5 h) after injection. Some of the reconstituted eggs were cultured to observe the pronuclear formation, cleavage, and blastocyst formation. The incidences of eggs forming at least one pronucleus or containing two pronuclei were not significantly different among the periods (82.4–83.5% and 43.4–51.9%, respectively). Nor did the incidences of eggs cleaving (86.7–97.7%) and developing to the blastocyst stage (0–3.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 1–1.5 h after injection, 71.7% of the eggs caused premature chromatin condensation, but only 46.2% of them formed two spindles around each of maternal and somatic chromatins. However, the morphology of the somatic spindles differed from that of the spindles, which formed around the oocyte chromatins. Only 7.5% of the eggs contained the normal chromosomal number. In many reconstituted oocytes, before activation, an abnormal spindle formation was observed in the somatic chromatins. In conclusion, these results show that non-enucleated rat oocytes injected with cumulus nuclei can form pronuclei and cleave following chemical activation, whereas blastocyst formation is very limited, probably caused by abnormalities in the spindle formation and distribution of somatic chromatids.

Keywords

Cumulus cellIn vitro developmentMicroinjectionOocytesRat
Type
Research Article
Information
Zygote , Volume 16 , Issue 2 , May 2008 , pp. 117 - 125
Copyright
Copyright © Cambridge University Press 2008

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

Abbott, A. (2004) Laboratory animals: the renaissance rat. Nature 428, 464–6.CrossRefGoogle ScholarPubMed
Alberio, R., Campbell, K.H. & Johnson, A.D. (2006). Reprogramming somatic cells into stem cells. Reproduction 132, 709–20.CrossRefGoogle ScholarPubMed
Chen, S.U., Chang, C.Y., Lu, C.C., Hsieh, F.J., Ho, H.N. & Yang, Y.S. (2004). Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos. Hum. Reprod. 19, 1181–8.CrossRefGoogle ScholarPubMed
Collas, P. & Robl, J.M. (1991). Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol. Reprod. 45, 455–65.CrossRefGoogle ScholarPubMed
Hauf, S., Waizenegger, I.C. & Peters, J.M. (2001). Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293, 1320–3.CrossRefGoogle ScholarPubMed
Heindryckx, B., Lierman, S., Van Der Elst, J. & Dhont, M. (2004). Chromosome number and development of artificial mouse oocytes and zygotes. Hum. Reprod. 19, 1189–94.CrossRefGoogle ScholarPubMed
Hirabayashi, M., Kato, M., Aoto, T., Ueda, M. & Hochi, S. (2002). Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids. Mol. Reprod. Dev. 62, 295–9.CrossRefGoogle ScholarPubMed
Hirabayashi, M., Kato, M., Takeuchi, A., Ishikawa, A. & Hochi, S. (2003). Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes. J. Reprod. Dev. 49, 121–6.CrossRefGoogle ScholarPubMed
Ito, J., Hirabayashi, M., Kato, M., Takeuchi, A., Ito, M., Shimada, M. & Hochi, S. (2005). Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes. Reproduction 129, 171–80.CrossRefGoogle ScholarPubMed
Kato, M., Hirabayashi, M., Aoto, T., Ito, K., Ueda, M. & Hochi, S. (2001). Strontium-induced activation regimen for rat oocytes in somatic cell nuclear transplantation. J. Reprod. Dev. 47, 407–13.CrossRefGoogle Scholar
Krivokharchenko, A., Popova, E., Zaitseva, I., Vil'ianovich, L., Ganten, D. & Bader, M. (2003). Development of parthenogenetic rat embryos. Biol. Reprod. 68, 829–36.CrossRefGoogle ScholarPubMed
Kubelka, M. & Moor, R.M. (1997). The behaviour of mitotic nuclei after transplantation to early meiotic ooplasts or mitotic cytoplasts. Zygote 5, 219–27.CrossRefGoogle ScholarPubMed
Lacham-Kaplan, O., Daniels, R. & Trounson, A. (2001). Fertilization of mouse oocytes using somatic cells as male germ cells. Reprod. Biomed. Online 3, 205–11.CrossRefGoogle ScholarPubMed
Losada, A., Yokochi, T., Kobayashi, R. & Hirano, T. (2000). Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes. J. Cell Biol. 150, 405–16.CrossRefGoogle ScholarPubMed
Miyoshi, K., Funahashi, H., Okuda, K. & Niwa, K. (1994). Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate and osmolarity. J. Reprod. Fertil. 100, 21–6.CrossRefGoogle Scholar
Mullins, L.J., Wilmut, I. & Mullins, J.J. (2004). Nuclear transfer in rodents. J. Physiol. 554, 4–12.CrossRefGoogle ScholarPubMed
Palermo, G.D., Takeuchi, T. & Rosenwaks, Z. (2002). Oocyte-induced haploidization. Reprod. Biomed. Online 4, 237–42.CrossRefGoogle ScholarPubMed
Reik, W., Dean, W. & Walter, J. (2001). Epigenetic reprogramming in mammalian development. Science 293, 1089–93.CrossRefGoogle ScholarPubMed
Rideout, W.M. 3rd, Eggan, K. & Jaenisch, R. (2001). Nuclear cloning and epigenetic reprogramming of the genome. Science 293, 1093–8.CrossRefGoogle ScholarPubMed
Roh, S., Malakooti, N., Morrison, J.R., Trounson, A.O. & Du, Z.T. (2003). Parthenogenetic activation of rat oocytes and their development (in vitro). Reprod. Fertil. Dev. 15, 135–40.CrossRefGoogle ScholarPubMed
Tateno, H., Akutsu, H., Kamiguchi, Y., Latham, K.E. & Yanagimachi, R. (2003). Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei. Fertil. Steril. 79, 216–8.CrossRefGoogle ScholarPubMed
Tesarik, J., Nagy, Z.P., Sousa, M., Mendoza, C. & Abdelmassih, R. (2001). Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts. Reprod. Biomed. Online 2, 160–4.CrossRefGoogle ScholarPubMed
Tomashov-Matar, R., Tchetchik, D., Eldar, A., Kaplan-Kraicer, R., Oron, Y. & Shalgi, R. (2005). Strontium-induced rat egg activation. Reproduction 130, 467–74.CrossRefGoogle ScholarPubMed
Uhlmann, F. (2003). Chromosome cohesion and separation: from men and molecules. Curr. Biol. 13, R104–R114.CrossRefGoogle Scholar
Van Thuan, N., Wakayama, S., Kishigami, S. & Wakayama, T. (2006). Donor centrosome regulation of initial spindle formation in mouse somatic cell nuclear transfer: Roles of gamma-tubulin and nuclear meiotic apparatus protein 1. Biol. Reprod. 74, 777–87.CrossRefGoogle Scholar
Waizenegger, I.C., Hauf, S., Meinke, A. & Peters, J.M. (2000). Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase. Cell, 103, 399–410.CrossRefGoogle ScholarPubMed
Wakayama, T. (2007). Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency? J. Reprod. Dev., 53, 13–26.CrossRefGoogle ScholarPubMed
Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–74.CrossRefGoogle ScholarPubMed
Watanabe, Y., Nurse, P., Watanabe, Y. & Nurse, P. (1999). Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400, 461–4.CrossRefGoogle ScholarPubMed
Watanabe, Y. (2005). Shugoshin: guardian spirit at the centromere. Curr. Opin. Cell Biol. 17, 590–5.CrossRefGoogle ScholarPubMed
Zhang, L.S., Yao, L.J., Jiang, Y., Jiang, M.X., Lei, Z.L., Sun, Q.Y. & Chen, D.Y. (2005). Transplantation of somatic nuclei into oocyte cytoplasm reveals that the chromosome properties determine the chromosome separation fate in rabbit. Zygote, 13, 109–14.CrossRefGoogle ScholarPubMed
Zhou, Q., Renard, J.P., Le Friec, G., Brochard, V., Beaujean, N., Cherifi, Y., Fraichard, A. & Cozzi, J. (2003). Generation of fertile cloned rats by regulating oocyte activation. Science 302, 1179.CrossRefGoogle ScholarPubMed