Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

Abstract

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria-rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN 3 ), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN 3 -treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN 3 inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN 3 will lead to a molecular understanding of cytoplasmic and cortical reorganization.

Microtubule array observed in the posterior‐vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg

“…Previously, we reported that NaN 3 treatment during the second phase of reorganization disturbed cortical tubulin accumulation but not astral microtubule structure (Ishii et al . ), suggesting a specific inhibition of CAMP formation. So we visualized the effects of NaN 3 on the CAMP formation by immunostaining using G1T0 (Fig.…”

“…; Ishii et al . ). In contrast, when the specimens were treated with G1T0, we observed thick microtubule bundles positioned in parallel arrays to the mid‐plane arc (Fig.…”

“…; Ishii et al . ). On the other hand, the sperm aster was appeared to be smaller than that in our previous study (Ishii et al .…”

“…Egg and sperm handling, dechorionation, and fertilization followed previous publications (Ishii et al . , ). Embryos were reared in filtered seawater at 18°C.…”

“…; Ishii et al . ). These relatively weak imaging results prevent us from clearly following myoplasmic movement and thus hamper attempts to clarify regulatory mechanisms of cytoplasmic rearrangement and body‐axis determination.…”