Calcium Homeostasis in Early Hamster Preimplantation Embryos¹

^a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706

The development in culture of 1-cell hamster embryos prior to the completion of fertilization is not well understood. In this study it was observed that culture for only 6 h of these early 1-cell embryos collected before pronuclei formation (3 h post-egg activation; PEA) significantly increased intracellular free calcium levels (194.3 ± 3.1 nM) compared to levels in similarly aged 1-cell embryos collected from the oviduct at 9 h PEA, after pronuclei formation is complete (134.2 ± 6.8 nM). Not only was the developmental competence of cultured 3-h PEA embryos with elevated intracellular free calcium levels compromised as compared with that of embryos collected from the oviduct at 9 h PEA; these embryos also had impaired cytoplasmic mitochondrial distribution (ratio of 0.62 ± 0.06 for cultured embryos compared to 0.44 ± 0.04 for in vivo-developed embryos) and decreased lactate metabolism (2.93 ± 0.22 pmol/embryo per 3 h for cultured embryos compared to 5.37 ± 0.36 for in vivo-developed embryos). This impairment in mitochondrial distribution and function and reduced development in culture by 3-h PEA embryos appears related to the ability to regulate intracellular calcium homeostasis. Intracellular free calcium levels were reduced by culture with increased medium magnesium concentrations, calcium channel inhibitors nifedipine or verapamil, or an intracellular calcium chelator. All of these treatments also stimulated development of 3-h PEA embryos to the morula/blastocyst stages and prevented impairment in mitochondrial organization and function. Conversely, culture with low medium magnesium and high calcium concentrations that increased intracellular free calcium levels resulted in low development and reduced mitochondrial function. Therefore, it appears that removal of the early embryo from the oviduct results in an inability to regulate intracellular calcium levels. As increased magnesium concentrations, nifedipine, and verapamil inhibit L-gated calcium channels, it may be a loss of regulation of these channels that alters calcium homeostasis resulting in impaired developmental competence.

¹ This research was supported by National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Embryo Development by the National Institute of Child Health and Human Development (NICHD) through Grant HD22023.

² Correspondence: Michelle Lane, Department of Animal Health and Biomedical Sciences, University of Wisconsin, 1655 Linden Drive, Madison, WI 53706. FAX: 608 262 7420; lane{at}ahabs.wisc.edu

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