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IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line.

Lin TC, Yen JM, Gong KB, Hsu TT, Chen LR - BMC Cell Biol. (2003)

Bottom Line: We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture.The effect is beneficial for the later establishment of a stem-cell line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynecology, Obstetrics, and Infertility, Kuo General Hospital, No, 22, Section 2, Ming-Sheng Road, Tainan, 70343, Taiwan. tachin@ksts.seed.net.tw

ABSTRACT

Background: Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.

Results: In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively).

Conclusion: IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line.

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The total cell count of the H33342-stained blastocyst. An H33342-stained blastocyst from the control group revealed a total of 77 visible nuclei observed from different planes.
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Figure 3: The total cell count of the H33342-stained blastocyst. An H33342-stained blastocyst from the control group revealed a total of 77 visible nuclei observed from different planes.

Mentions: The results shown below are pooled from three repeats of the culture experiments. The developmental stages for the cultured embryo were determined daily until day 4.1 post-fertilization using an inverted microscope. They were evaluated before hatching on the morning of day five, that is day 4.1 post-fertilization, instead of day 4.3 post-fertilization, because the hatching of the cultured embryos may distort the integrity of their figures and therefore interfere with the counting of the total cell number. In order to enumerate the total cell number of cultured embryos, the embryos that progressed to the blastocyst stage for both groups were cultured in Hoechst H33342 medium (5 μg /ml) for three to five minutes, and then mounted onto a slide and examined under a fluorescence microscope. The results of cell enumeration are shown in Table 4. There appeared to be a significantly-increased total cell count for the blastocysts deriving from the IGFBP-1/IGF-1-supplemented group than was the case for the control group (87.6 ± 5.3 vs. 73.7 ± 7.1 respectively, p < 0.01; Figure 3).


IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line.

Lin TC, Yen JM, Gong KB, Hsu TT, Chen LR - BMC Cell Biol. (2003)

The total cell count of the H33342-stained blastocyst. An H33342-stained blastocyst from the control group revealed a total of 77 visible nuclei observed from different planes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239990&req=5

Figure 3: The total cell count of the H33342-stained blastocyst. An H33342-stained blastocyst from the control group revealed a total of 77 visible nuclei observed from different planes.
Mentions: The results shown below are pooled from three repeats of the culture experiments. The developmental stages for the cultured embryo were determined daily until day 4.1 post-fertilization using an inverted microscope. They were evaluated before hatching on the morning of day five, that is day 4.1 post-fertilization, instead of day 4.3 post-fertilization, because the hatching of the cultured embryos may distort the integrity of their figures and therefore interfere with the counting of the total cell number. In order to enumerate the total cell number of cultured embryos, the embryos that progressed to the blastocyst stage for both groups were cultured in Hoechst H33342 medium (5 μg /ml) for three to five minutes, and then mounted onto a slide and examined under a fluorescence microscope. The results of cell enumeration are shown in Table 4. There appeared to be a significantly-increased total cell count for the blastocysts deriving from the IGFBP-1/IGF-1-supplemented group than was the case for the control group (87.6 ± 5.3 vs. 73.7 ± 7.1 respectively, p < 0.01; Figure 3).

Bottom Line: We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture.The effect is beneficial for the later establishment of a stem-cell line.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynecology, Obstetrics, and Infertility, Kuo General Hospital, No, 22, Section 2, Ming-Sheng Road, Tainan, 70343, Taiwan. tachin@ksts.seed.net.tw

ABSTRACT

Background: Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line.

Results: In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively).

Conclusion: IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line.

Show MeSH
Related in: MedlinePlus