Elsevier

Theriogenology

Volume 76, Issue 4, 1 September 2011, Pages 598-606
Theriogenology

Research article
A simple method for producing tetraploid porcine parthenogenetic embryos

https://doi.org/10.1016/j.theriogenology.2011.03.010Get rights and content

Abstract

The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.

Introduction

Tetraploid embryos have been widely used in animal reproduction. In chimeric mouse embryos, reconstructed with diploid and tetraploid blastomeres, diploid cells contributed to the inner cell mass, whereas most tetraploid cells contributed to trophoectoderm [1], [2], [3]. Furthermore, murine chimeras produced with tetraploid embryos and embryonic stem (ES) cells resulted in mice derived completely from ES cells [4], [5], [6], [7]. Most tetraploid cells contributed to the extraembryonic lineage, whereas diploid cells derived from ES cells contributed to both embryonic and extraembryonic lineages [6], [8], [9], [10], [11], [12]. Therefore, tetraploid cells supported differentiation of diploid cells to form an embryo.

In domestic animals, chimeric calves from ES-like cells aggregated with tetraploid embryos were reported to have been born [13]; however, ES like cells contributed to chimera formation only at a very low level. The authors speculated that putative tetraploid embryos produced by electrically fused diploid blastomeres may not have been completely tetraploid, and furthermore, they indicated the need to identify complete tetraploid embryos before aggregation with ES-like cells. However, porcine chimeras constructed by ES like cells and tetraploid embryos have apparently not been reported. Furthermore, utilization of blastomeres in in vitro produced (IVP) porcine embryos makes it more difficult to produce tetraploid embryos, due to a high frequency of polyspermic fertilization.

Thus, in the present study, the first objective was to produce porcine tetraploid embryos using parthenogenetic embryos, whose ploidy was easy to predict compared with IVP embryos. Porcine immature oocytes underwent maturation culture in the presence of cytochalasin B to inhibit first polar body extrusion, were electrically stimulated, and again treated with cytochalasin B. The second objective was to compare early developmental competence of the resulting tetraploid embryos relative to diploid embryos. Fluorescence in situ hybridization (FISH) was used to detect a porcine chromosome 1-specific sequence in porcine parthenogenetic embryos at various stages, and ploidy transition was examined during early development.

Section snippets

In vitro maturation and development of porcine parthenogenetic embryos

Ovaries from prepubertal gilts were obtained at a local abattoir and transported to the laboratory at 30 to 35 °C. Porcine follicular oocytes were aspirated from antral follicles (3 to 6 mm in diameter) with a 20-gauge needle. Cumulus-oocyte complexes (COCs) were washed three times with HEPES-buffered Tyrode's albumin lactate pyruvate medium [14]; only those with uniform ooplasm and a compact cumulus cell mass were selected for in vitro maturation, which was performed by a two-step method [15],

Effects of maturation culture time on meiotic progression and subsequent pronucleus formation after parthenogenetic activation in porcine oocytes

In the first experiment, meiotic progression of porcine oocytes was observed during in vitro maturation in a two step method described previously (Fig. 1A). Immediately after removal of oocytes from ovarian follicles, almost all oocytes (98.2%; 109/111) were in the resting GV stage. Until 24 h after the beginning of culture, most oocytes remained in the GV stage. However, by 27 h, GVBD oocytes were detected, and by 30 to 33 h, more than 90% of oocytes were GVBD, but MII oocytes had not yet

Discussion

In a preliminary study, meiotic progression and pronucleus formation competence of porcine oocytes were examined during maturation culture, using a culture method described previously. Oocytes that had reached MII appeared 36 h after the beginning of culture and became more abundant as duration of culture increased. At 42 to 48 h, the proportion of MII oocytes peaked at approximately 90%. In contrast, only 6.2% (12/193) of oocytes cultured for 42 h formed pronuclei after electrical stimulation.

Acknowledgments

We are grateful to the pig farm staff (National Institute of Livestock and Grassland Science, Japan) for care and maintenance of pigs and the assistance in handling the animals. We also express our gratitude to Dr. T. Nagai of the National Institute of Livestock and Grassland Science for critical reading of the manuscript.

References (42)

  • A. Nagy et al.

    Derivation of completely cell culture-derived mice from early-passage embryonic stem cells

    Proc Natl Acad Sci USA

    (1993)
  • O. Ueda et al.

    Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos

    Exp Anim

    (1995)
  • T. Amano et al.

    Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cell-derived mice

    Zygote

    (2001)
  • A.K. Tarkowski et al.

    Development of cytochalasin B-induced tetraploid and diploid/tetraploid mosaic mouse embryos

    J Embryol Exp Morphol

    (1977)
  • A. Nagy et al.

    Embryonic stem cells alone are able to support fetal development in the mouse

    Development

    (1990)
  • P.C. Tang et al.

    The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

    Zygote

    (2000)
  • Y. Goto et al.

    Developmental potential of mouse tetraploid cells in diploid ↔ tetraploid chimeric embryos

    Int J Dev Biol

    (2002)
  • S. Iwasaki et al.

    Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos

    Biol Reprod

    (2000)
  • B.D. Bavister et al.

    Development of preimplantation embryos of the golden hamster in a defined culture medium

    Biol Reprod

    (1983)
  • K. Kikuchi et al.

    Successful piglet production after transfer of blastocysts produced by a modified in vitro system

    Biol Reprod

    (2002)
  • H. Funahashi et al.

    Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization

    Biol Reprod

    (1997)
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