Follicular Growth-Stimulated Cows Provide Favorable Oocytes for Producing Cloned Embryos

National Livestock Breeding Center, Nishigo, Fukushima, Japan.
Cellular reprogramming 12/2011; 14(1):29-37. DOI: 10.1089/cell.2011.0060
Source: PubMed


We examined the influence of recipient oocytes on in vitro development, oxygen consumption, and gene expression in the resulting cloned bovine embryos. Oocytes derived from slaughterhouse ovaries and ovum pickup (OPU)-derived oocytes were used as recipient cytoplasts for the production of cloned embryos. A series of OPU sessions was conducted on Holstein cows without follicular growth treatment (FGT). In the same cows, we then performed dominant follicle ablation and subsequently administered follicle-stimulating hormone and prostaglandin F(2α) with controlled internal drug release device before a second series of OPU. Cumulus cells collected from single Holstein cows were used as donor cells. After measurement of oxygen consumption at the blastocyst stage with modified scanning electrochemical microscopy, analysis of 10 genes (CDX2, IFN-tau, PLAC8, OCT4, SOX2, NANOG, ATP5A1, GLUT1, AKR1B1, and IGF2R) was performed with real-time RT-PCR. Rates of fusion, cleavage, and blastocyst formation were not different among the treatment groups. Levels of oxygen consumption in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT were significantly lower than in blastocysts derived from artificial insemination (AI). However, oxygen consumption was increased in cloned blastocysts derived from OPU with FGT, depending on the individual oocyte donor. Furthermore, gene expression of IFN-tau and OCT4 in cloned blastocysts derived from OPU with FGT was similar to that in AI-derived blastocysts, whereas expression of those genes in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT was significantly different from that in AI-derived blastocysts. Thus, recipient oocytes collected by OPU in combination with manipulation of follicular growth in donor cows are suitable for producing cloned embryos.

13 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Conventionally, in vitro-fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors--(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage--pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.
    Full-text · Article · May 2012 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial bioenergetics in mammalian oocytes has not been sufficiently characterized. In this study, the function of oxidative phosphorylation (OXPHOS), a major pathway in mitochondria, was investigated in individual bovine oocytes by monitoring oxygen consumption using modified scanning electrochemical microscopy (SECM). At the germinal vesicle (GV) stage, 65% of basal respiration was used for mitochondrial respiration, which was inhibited by complex IV inhibitor. Around 63% of mitochondrial respiration was coupled to ATP synthesis, as determined by sensitivity to an ATP synthase inhibitor, and the remaining 37% was attributed to proton leak. In contrast, 50% and 43% of mitochondrial respiration were used for ATP synthesis in in vivo- and in vitro-derived metaphase II (MII)-stage oocytes, respectively. ATP-linked respiration, in both in vivo- and in vitro-derived MII-stage oocytes, was significantly lower than in GV-stage oocytes, suggesting that OXPHOS in bovine oocytes is more active at the GV stage compared with the MII stage. Interestingly, basal respiration in in vitro-derived MII oocytes was significantly higher than for in vivo-derived oocytes, reflecting an increase in proton leak. Next, we assessed respiration in MII oocytes cultured for 8 h. The aged oocytes had a significantly reduced maximum respiratory capacity, which was stimulated by a mitochondrial uncoupler, and reduced ATP-linked respiration compared with non-aged oocytes. However, the aging-related phenomenon could be prevented by caffeine treatment. We conclude that OXPHOS in bovine oocytes varies in the transition from GV to MII stage, in vitro maturation and the aging process. This approach will be particularly useful for analyzing mitochondrial bioenergetics in individual mammalian oocytes.
    No preview · Article · Jul 2012 · Journal of Reproduction and Development
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.
    Full-text · Article · Nov 2012 · Journal of Reproduction and Development
Show more