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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 in vivo fertilized in vitro grown embryos examined for apoptosis and necrosis. (A) A day five in vitro cultured embryo arrested at six-cell stage from the control group is observed before and after epifluorescent technique. Green fluorescence by Annexin V is observed over the outer cell membrane and means that apoptosis is underway. There is negative PI fluorescence at the time of harvest. (B) An arrested embryo at the morula stage becomes severely fragmented. Green fluorescence can be noted following Annexin V staining with simultaneous red fluroscence by PI staining. (C) A blastocyst cultured from the control group appears to be normal looking prior to epifluorescent observation. The same blastocyst under epifluorescent microscope shows apoptotic changes with Annexin V fluorescence and absence of PI fluorescence. (D) An apoptotic blastocyst, following Annexin V and PI staining, revealed green fluorescence. Under epifluoresvent microscope, Annexin V staining revealed a well fluorescent membrance with central clearing.
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Figure 2: The in vivo fertilized in vitro grown embryos examined for apoptosis and necrosis. (A) A day five in vitro cultured embryo arrested at six-cell stage from the control group is observed before and after epifluorescent technique. Green fluorescence by Annexin V is observed over the outer cell membrane and means that apoptosis is underway. There is negative PI fluorescence at the time of harvest. (B) An arrested embryo at the morula stage becomes severely fragmented. Green fluorescence can be noted following Annexin V staining with simultaneous red fluroscence by PI staining. (C) A blastocyst cultured from the control group appears to be normal looking prior to epifluorescent observation. The same blastocyst under epifluorescent microscope shows apoptotic changes with Annexin V fluorescence and absence of PI fluorescence. (D) An apoptotic blastocyst, following Annexin V and PI staining, revealed green fluorescence. Under epifluoresvent microscope, Annexin V staining revealed a well fluorescent membrance with central clearing.

Mentions: Amongst the control group, there were 15 embryos which arrested prior to the blastocyst stage, of which about one half (7/15) were PI-positive (Figure 2B) whilst the other half (8/15) were Annexin V-positive and PI-negative (Figure 2A). Beside these arrested embryos, five blastocysts from the control group stained positively with Annexin V but did not express PI fluorescence (Figure 2C).


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 in vivo fertilized in vitro grown embryos examined for apoptosis and necrosis. (A) A day five in vitro cultured embryo arrested at six-cell stage from the control group is observed before and after epifluorescent technique. Green fluorescence by Annexin V is observed over the outer cell membrane and means that apoptosis is underway. There is negative PI fluorescence at the time of harvest. (B) An arrested embryo at the morula stage becomes severely fragmented. Green fluorescence can be noted following Annexin V staining with simultaneous red fluroscence by PI staining. (C) A blastocyst cultured from the control group appears to be normal looking prior to epifluorescent observation. The same blastocyst under epifluorescent microscope shows apoptotic changes with Annexin V fluorescence and absence of PI fluorescence. (D) An apoptotic blastocyst, following Annexin V and PI staining, revealed green fluorescence. Under epifluoresvent microscope, Annexin V staining revealed a well fluorescent membrance with central clearing.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The in vivo fertilized in vitro grown embryos examined for apoptosis and necrosis. (A) A day five in vitro cultured embryo arrested at six-cell stage from the control group is observed before and after epifluorescent technique. Green fluorescence by Annexin V is observed over the outer cell membrane and means that apoptosis is underway. There is negative PI fluorescence at the time of harvest. (B) An arrested embryo at the morula stage becomes severely fragmented. Green fluorescence can be noted following Annexin V staining with simultaneous red fluroscence by PI staining. (C) A blastocyst cultured from the control group appears to be normal looking prior to epifluorescent observation. The same blastocyst under epifluorescent microscope shows apoptotic changes with Annexin V fluorescence and absence of PI fluorescence. (D) An apoptotic blastocyst, following Annexin V and PI staining, revealed green fluorescence. Under epifluoresvent microscope, Annexin V staining revealed a well fluorescent membrance with central clearing.
Mentions: Amongst the control group, there were 15 embryos which arrested prior to the blastocyst stage, of which about one half (7/15) were PI-positive (Figure 2B) whilst the other half (8/15) were Annexin V-positive and PI-negative (Figure 2A). Beside these arrested embryos, five blastocysts from the control group stained positively with Annexin V but did not express PI fluorescence (Figure 2C).

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