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Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

ABSTRACT: Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.

SUBMITTER: Miyamoto K

PROVIDER: S-EPMC3084120 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

Miyamoto Kei K Nagai Kouhei K Kitamura Naoya N Nishikawa Tomoaki T Ikegami Haruka H Binh Nguyen T NT Tsukamoto Satoshi S Matsumoto Mai M Tsukiyama Tomoyuki T Minami Naojiro N Yamada Masayasu M Ariga Hiroyoshi H Miyake Masashi M Kawarasaki Tatsuo T Matsumoto Kazuya K Imai Hiroshi H

Proceedings of the National Academy of Sciences of the United States of America 20110411 17

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and ab ...[more]

PMID: 21482765

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Project description:Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2?h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10??M MG132 for 2?h in the presence of 500?nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15?h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1 ± 7.2%, 62.7 ± 7.2%, and 62.5 ± 7.2%) on day 2, or total cell number (23.1 ± 2.2, 24.0 ± 2.0, and 24.5 ± 2.3 for the 0, 1, and 10??M MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1 ± 4.7%)??M MG132 treatment groups; however, compared with the 10??M MG132 group (14.0 ± 4.7%), more embryos from the 1??M MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1??M MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte-donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.

Dataset Information

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

Publications

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

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