Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

Abstract

Several reliable methods to produce transgenic animals use the male genome. After penetration into oocytes, sperm DNA undergoes dramatic conformational changes that might represent an opportunity for exogenous DNA to integrate into the zygote genome. A nuclease, DNase I, with Ca²⁺/Mg²⁺ dependent activity and Zn²⁺ inhibition, is one of the enzymes responsible for sperm DNA remodeling. To date, the effect of different calcium concentrations in manipulation media on porcine intracytoplasmic sperm injection has not been fully investigated. The present study was conducted to examine the effect of calcium in the surrounding media, and we found that the number of embryos expressing green fluorescent protein (EGFP) was increased in media containing Ca²⁺. However, the number did not change over Ca²⁺ concentrations from 2 to 8 mmol·L^–1 (P>0.05). Moreover, free Ca²⁺ concentrations in the media were found to affect the efficiency which is ICSI embryos expressing EGFP protein, which was related to the activation of ooplasmic DNase I. These findings reveal a mechanism and pathway involving EGFP expression in ICSI embryos.

Keywords

ICSI / calcium / DNase I / GFP / porcine

References

[1] Nagashima H, Fujimura T, Takahagi Y, Kurome M, Wako N, Ochiai T, Esaki R, Kano K, Saito S, Okabe M, Murakami H. Development of efficient strategies for the production of genetically modified pigs. Theriogenology, 2003, 59(1): 95-106

[2] Prather R S, Boice M L, Gibson J, Hoffman K E, Parry T W. In vitro development of embryos from Sinclair miniature pigs: a preliminary report. Theriogenology, 1995, 43(6): 1001-1007

[3] The Sir Hans Krebs Lecture. 26th Meeting of the Federation of European Biochemical Societies (FEBS). Nice, France, June 19-24, 1999. Abstracts. Biochimie, 1999, 81(6 Suppl): S11-S454

[4] Wu Y, Liu C J, Wan P C, Hao Z D, Zeng S M. Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method. Animal Reproduction Science, 2009, 113(1-4): 156-166

[5] Szczygiel M A, Moisyadi S, Ward W S. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biology of Reproduction, 2003, 68(5): 1903-1910

[6] Zannoni A, Spinaci M, Bernardini C, Bacci M L, Seren E, Mattioli M, Forni M. DNase I activity in pig MII oocytes: implications in transgenesis. Reproduction, 2006, 131(3): 461-468

[7] Wu Y, Liu C J, Wan P C, Hao Z D, Zeng S M. Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method. Animal Reproduction Science, 2009, 113(1-4): 156-166

[8] Yoshida N, Perry A C. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nature Protocols, 2007, 2(2): 296-304

[9] Liu C, Xie W, Gui C, Du Y. Pronuclear microinjection and oviduct transfer procedures for transgenic mouse production. Methods in Molecular Biology, 2013, 1027: 217-232

[10] Song Y, Hai T, Wang Y, Guo R, Li W, Wang L, Zhou Q. Epigenetic reprogramming, gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor—m-carboxycinnamic acid bishydroxamide (CBHA). Protein & Cell, 2014, 5(5): 382-393

[11] Li F, Li L, Zhong Y, Xie Q, Huang J, Kang X, Wang D, Xu L, Huang T. Relationship between LTR methylation and gag expression of HIV-1 in human spermatozoa and sperm-derived embryos. PLoS ONE, 2013, 8(1): e54801

[12] Moisyadi S, Kaminski J M, Yanagimachi R. Use of intracytoplasmic sperm injection (ICSI) to generate transgenic animals. Comparative Immunology, Microbiology and Infectious Diseases, 2009, 32(2): 47-60

[13] Kurokawa M, Fissore R A. ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development. Molecular Human Reproduction, 2003, 9(9): 523-533

[14] Tesarik J. Oscillin—reopening the hunting season. Molecular Human Reproduction, 1998, 4(11): 1007-1009

[15] Borini A, Tarozzi N, Bizzaro D, Bonu M A, Fava L, Flamigni C, Coticchio G. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Human Reproduction, 2006, 21(11): 2876-2881

[16] Hao Z D, Liu S, Wu Y, Wan P C, Cui M S, Chen H, Zeng S M. Abnormal changes in mitochondria, lipid droplets, ATP and glutathione content, and Ca²⁺ release after electro-activation contribute to poor developmental competence of porcine oocyte during in vitro ageing. Reproduction, Fertility, and Development, 2009, 21(2): 323-332

Acknowledgments

This study was supported by the National Natural Science Foundation of China (30771548) and Beijing Natural Science Foundation (5072026). We greatly thank technical assistance provided by Wentao Zeng.

Compliance with ethics guidelines

Shuaishuai Wu, Heng Chen, Yingzheng Wang, Hui Gao and Shenming Zeng declare that they have no conflict of interest or financial conflicts to disclose.

All applicable institutional and national guidelines for the care and use of animals were followed.

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg