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Relationship Between Development, Metabolism, and Mitochondrial Organization in 2-Cell Hamster Embryos in the Presence of Low Levels of Phosphate¹

^a Department of Animal Health and Biomedical Sciences, ^b Laboratory of Molecular Biology, ^c Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

The effect of low concentrations of inorganic phosphate (P_i) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 µM KH₂PO₄. Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P_i did not alter TCA-cycle activity. However, culture with 2.5 µM P_i significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P_i in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of 2.5 µM P_i compared to both control and 1.25 µM P_i. This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P_i in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.

First decision: 3 April 2001.

¹ This work was done as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Embryo Development and was funded by the National Institute of Child Health and Human Development, National Institutes of Health, through cooperative agreement HD-22023.

² Correspondence: Tenneille Ludwig, Department of Animal Health and Biomedical Sciences, 1656 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420;ludwig{at}ahabs.wisc.edu

³ Current address: Department of Biological Sciences, University of New Orleans and the Audubon Institute for Research of Endangered Species, New Orleans, LA 70148.

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