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

Ta-Chin Lin¹ , Jui-Mei Yen² , Kun-Bing Gong³ , Teng-Tsao Hsu³ and Lih-Ren Chen⁴

¹Department of Gynecology, Obstetrics, and Infertility, Kuo General Hospital, No. 22, Section 2, Ming-Sheng Road, Tainan, 70343, Taiwan

²Department of Pediatrics, SinLau Christian Hospital, No. 57, Section 1, Eastgate Road, Tainan 701, Taiwan

³Department of Research, Eupro Biotechnology Co., No 56, 5F-2, Section 2, Ming-Sheng Road, Tainan, 70343, Taiwan

⁴Taiwan Livestock Research Institute, Council of Agriculture. 112 Farm Road, Hsinhua, Tainan 71210, Taiwan

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BMC Cell Biology 2003, 4:14doi:10.1186/1471-2121-4-14


Published:	19 September 2003

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.