In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

Im, Gi‐Sun; Lai, Liangxue; Liu, Zhong Hua; Hao, Yanhong; Wax, David; Bonk, Aaron J.; Prather, Randall S.

doi:10.1016/j.theriogenology.2003.06.006

Cited by 89 publications

(69 citation statements)

References 33 publications

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“…Oocytes were activated for parthenogenesis with 5 μM Ca 2+ ionophore (Sigma) for 5 min. After 3 h of culture in porcine zygote medium 3 (PZM3) [36] containing 7.5 μg/ml cytochalasin B (Sigma) embryos were washed three times in PZM3 medium containing 0.4% (w/v) BSA and cultured in the same medium at 39 C in an atmosphere of 5% CO 2 and 95% air. MII oocytes (MII) were collected before parthenogenetic activation, and One-cell (1C, 3 h or 6 h after activation)-, 2-cell (2C)-, 4-cell (4C)-, morula (MO)-, and blastocyst (BL)-stage embryos were collected at 3, 6, 24, 48, 120 and 168 h after parthenogenetic activation, respectively.…”

Section: In Vitro Porcine Oocyte Maturation and Parthenogenetic Activmentioning

confidence: 99%

Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing <i>In Vitro</i>

Shen

Lee

et al. 2012

Journal of Reproduction and Development

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Abstract. Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig. Key words: Apoptosis, Autophagy, LC3, Maternal gene, 3-MA, Rapamycin (J. Reprod. Dev. 58: [576][577][578][579][580][581][582][583][584] 2012) A utophagy is an intracellular, bulk degradation process in which a portion of the cytoplasm is sequestered in an autophagosome and subsequently degraded upon fusion with a lysosome [1][2][3]. Autophagy plays a critical role during fertilization [4] and an essential role in differentiation and development, as well as in the cellular response to stress. Our previous study was the first to report that mitochondrial stress influences autophagy in porcine parthenotes developing in vitro [5]. However, the role of autophagy in early porcine parthenotes is still poorly understood.A balance between the formation and degradation of cellular proteins is required for cell survival. In mammals, protein degradation is accelerated shortly after fertilization and is apparent by the early two-cell stage in mice [6] and the four-cell stage in pigs. Early embryogenesis may rely on maternal protein stores as nutrients. After fertilization, maternal proteins in oocytes are...

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Section: In Vitro Porcine Oocyte Maturation and Parthenogenetic Activmentioning

confidence: 99%

Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing <i>In Vitro</i>

Shen

Lee

et al. 2012

Journal of Reproduction and Development

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“…Since the first cloned pigs were generated using somatic cell cloning [4], substantial improvement for this technique has been realized [5,6]. However, the efficiency of pig cloning has been lower than that of other domestic animals, with only 1%-5% of the embryos reconstructed by nuclear transfer surviving to term [7,8].…”

Section: Introductionmentioning

confidence: 99%

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

Ren

Lü

et al. 2010

J Assist Reprod Genet

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Purpose The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation. Methods The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR. Results Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2-and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P <0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4-and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05).Conclusions The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.

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“…. Although NCSU-23 has been widely used and is one of the most successful media for the culture of porcine embryos after IVF or SCNT, in recent studies, it has reported that PZM-3 is superior to NCSU-23 in supporting development of the embryo to the blastocyst stage of IVF [18], parthenogenetic activation (PA) [19] or SCNT embryos [20]. Furthermore, Suzuki et al developed a porcine in vitro culture medium, namely, 22].…”

mentioning

confidence: 99%

“…For IVC of pigs, several media have been developed, including North Carolina State University (NCSU)-23 and NCSU-37 media [16], Beltsville Embryo Culture Medium (BECM)-3 [17] and Porcine Zygote Medium (PZM)-3 and PZM-4 [18]. Although NCSU-23 has been widely used and is one of the most successful media for the culture of porcine embryos after IVF or SCNT, in recent studies, it has reported that PZM-3 is superior to NCSU-23 in supporting development of the embryo to the blastocyst stage of IVF [18], parthenogenetic activation (PA) [19] or SCNT embryos [20]. Furthermore, Suzuki et al developed a porcine in vitro culture medium, namely, PZM-5 [21,22].…”

mentioning

confidence: 99%

Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs

Yamanaka

Sugimura

Wakai

et al. 2009

Journal of Reproduction and Development

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Abstract. We evaluated the developmental competence of somatic cell nuclear transfer (SCNT) embryos using in vitro embryo culture systems. Embryos were cultured in NCSU-23, NCSU-23 supplemented with essential and non-essential amino acids (NCSU-23aa) or modified PZM-5 supplemented with BSA instead of PVA (mPZM-5). The rates of blastocyst formation were significantly higher in the mPZM-5 group than in the other groups, regardless of the method of embryo production (38.0 vs. 25.3 or 29.1% for IVF, 18.2 vs. 8.7 or 9.4% for SCNT, respectively). The mean cell numbers of IVF and SCNT blastocysts were also significantly higher in mPZM-5 than in the other groups (62.0 vs. 42.3 or 43.0 for IVF, 46.5 vs. 29.4 or 31.3 for SCNT, respectively). Next, the embryos were cultured in mPZM-5 from days 0 to 4 and then in mPZM-5 (P/P), NCSU-23 (P/N) or NCSU-23aa (P/Naa) until day 6. The rates of blastocyst formation were similar among the 3 two-step culture systems in both embryo groups (36.2, 34.2, and 33.6% for IVF, 20.8,14.1, and 17.2% for SCNT, respectively). The mean cell number in the IVF and SCNT blastocysts was significantly lower in P/N than in P/ P and P/Naa (46.5 vs. 63.5 and 68.7 for IVF, 29.3 vs. 45.5 and 39.7 for SCNT, respectively). Next, we examined the effect of media on apoptosis in IVF and SCNT blastocysts. The apoptosis indices in the blastocysts derived from either NCSU-23 or mPZM-5 were analyzed by TUNEL assay. The apoptosis index of the SCNT blastocysts was significantly lower in mPZM-5 than in NCSU-23 (8.8 vs. 13.6%), whereas no such difference was observed between groups in the IVF embryos (5.1 vs. 4.4%). These data suggested that SCNT embryos were more easily affected by culture environment compared with IVF embryos, offering the possibility to further enhance the developmental competence of SCNT embryos by developing more appropriate culture conditions in pigs. Key words: Apoptosis, In vitro embryo culture, Miniature pig, Nuclear transfer (J. Reprod. Dev. 55: [299][300][301][302][303][304] 2009) loned pigs by somatic cell nuclear transfer (SCNT) might be used as improved domestic livestock. However, their use is not limited to this. Owing to the many similarities in between the anatomy and physiology of pigs and humans, these cloned pigs can be used for xenotransplantation and as improved models for studying human physiology and disease [1,2]. Moreover, in particular, miniature pigs offer several advantages: their small body size enables space-saving and facilitates better control of specific pathogens. They therefore represent attractive options for use in medical applications and related technologies [3]. Thus, miniature pig cloning by SCNT might also prove useful in many biomedical applications [4]. Although it has been successful recently, its efficiency is still low, as is the case in other animals [5]. One of the reasons for the very low success rate of miniature pig cloning is the limitations of the in vitro production (IVP) system for pigs, including in vitro maturation (IVM) and their subsequen...

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In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

Cited by 89 publications

References 33 publications

Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing <i>In Vitro</i>

Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing <i>In Vitro</i>

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

Difference in Sensitivity to Culture Condition Between In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos in Pigs

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