182 INTERACTION OF MICROTUBULES AND MICROFILAMENTS WITH DISTRIBUTION OF MITOCHONDRIA IN TWO-CELL HAMSTER EMBRYOS

Abstract

During early development, mitochondrial distribution in the ooplasm is dramatically changed. In mice and pigs, it has been reported that translocation of mitochondria is mediated by microtubules, but not by microfilaments. However, the study using hamster embryos suggested that microfilaments played a role in the mitochondrial distribution. In this study, the role of microtubules and microfilaments on distribution of mitochondria was evaluated in 2-cell hamster embryos. Female golden hamsters were superovulated on the day of post-estrus discharge by PMSG in a weight-dependent manner [Mackiernan and Bavister 2000 Hum. Reprod. 15, 157–164], and mated with males in the evening 3 days later. Two-cell embryos were collected from the oviducts at 0900–1000 on Day 2 of pregnancy. Embryos were cultured in HECM-10 medium with or without nocodazole (a microtubule assembly inhibitor), and/or cytochalasin D (a microfilament assembly inhibitor) for 8–10 h in a humidified atmosphere of 10% CO₂, 5% O₂, and 85% N₂ at 37.5°C. After incubation of embryos with or without inhibitors, mitochondria were stained with rhodamine 123. Some of them were stained for microtubules, microfilaments, and DNA, as reported previously (Suzuki et al. 1999 Biol. Reprod. 61, 521–526). In 2-cell hamster embryos (controls, n = 44), most mitochondria were accumulated at the perinuclear region, while little mitochondria were noted in the cell cortex. Microtubules were found around the nuclei, and distribution of the mitochondria was somewhat correlated with that of the microtubules. In contrast, microfilaments were located in a high density just under the cell membrane. After nocodazole treatment (n = 22), mitochondria had extended into the subcortical (intermediate) region in various degrees, where mitochondria were aggregated into large masses. After cytochalasin D treatment (n = 17), distributional density of mitochondria at the peripheral region was decreased, and mitochondria moved back around the nucleus. After treatment by both inhibitors (n = 21), a distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. The present results suggest that the distribution of mitochondria at the perinuclear region is mediated by microtubules, whereas the movement of mitochondria to the cell cortex is regulated by microfilaments. Microfilaments seemed to play a role as the anchor of mitochondria at the cell cortex.