MiP2005: Session 3Mitochondrial Physiology Network 10.9: 38-39 (2005) - download pdf
Hideki Igarashi,1 T Takahashi,1 H Kurachi,1 E Takahashi2
1Dept. Obstetrics and Gynecology; 2Dept. Physiology, Yamagata University School of Medicine, Yamagata 990-9585, Japan. - igarashi@med.id.yamagata-u.ac.jp
In mammalian oocytes, attachment of the sperm to the oocyte induces drastic changes in intracellular Ca2+ concentration ([Ca2+]i ) consisting of a single relatively long lasting (~5 min) rise in [Ca2+]i followed by short repetitive transients of [Ca2+]i lasting several hours. These changes in [Ca2+]i at fertilization are called Ca2+ oscillations and appear to be a prerequisite for the normal development of embryos. Delayed fertilization (postovulatory aging) of oocytes significantly affects both embryonic development and Ca2+ oscillations [1,2]. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum (ER) and the latter relies upon ATP availability in the ER, we undertook the present study to address the role of intracellular ATP regulation at fertilization in the delayed fertilized mouse oocyte. Measurement of [Ca2+]i was conducted using fluorescent dye fura-PE3 while intracellular ATP concentration ([ATP]i) was continuously assessed in single oocytes from changes in intracellular free Mg2+ concentration measured by Mg2+ sensitive dye magnesium green (MgG). At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation of [Ca2+]i indicating a relative decrease in [ATP]i. In the fresh oocyte (oocytes recovered from the oviduct 12.5 hrs after hCG injection), it was quickly followed by a significant decrease below the baseline indicating a relative increase in [ATP]i. In contrast, in the aged oocytes (oocytes recovered from the oviduct 18.5 hrs after hCG injection), such a decrease in MgG fluorescence was not observed. In the separate experiment, ATP content in the fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in the unfertilized fresh and aged oocytes. Intracellular ATP content at 5 hrs after fertilization was increased in the both oocytes, where the fresh oocyte showed a significantly higher value than the aged oocyte. From these results, we conclude that fertilization shifts the set point of intracellular ATP regulation in the fresh oocyte so that abrupt increases in ATP utilizations at fertilization are effectively buffered. In contrast, the aged mouse oocytes failed to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization may lead to the altered Ca2+ oscillations pattern and poor developmental potency commonly noted in the aged oocyte [3].
1. Igarashi H, Takahashi E, Hiroi M, Doi K (1997) Aging-related changes in calcium oscillations in fertilized mouse oocytes. Mol. Reprod. Dev. 48: 383-390.
2. Takahashi T, Saito H, Hiroi M, Doi K, Takahashi E (2000) Effects of aging on inositol 1,4,5-triphosphate-induced Ca2+ release in unfertilized mouse oocytes. Mol. Reprod. Dev. 55: 299-306.
3. Igarashi H, Takahashi T, Takahashi E, Tezuka N, Nakahara K, Takahashi K, Kurachi H (2005) Aged mouse oocytes fail to readjust intracellular adenosine triphosphates at fertilization. Biol. Reprod. 72: 1256-1261.
