Cytoplasmic Determination of Meiotic Spindle Size Revealed by a Unique Inter-Species Germinal Vesicle Transfer Model.

Zhong-Wei Wang,John Carroll,Heide Schatten,Qing-Yuan Sun,Guang-Li Zhang

doi:10.1038/srep19827

Abstract

Spindle sizes are different in diverse species and cell types. In frogs, the meiotic spindle size is positively correlated with the egg cell volume. Across species, relatively small mouse oocytes (70–80 μm) have a relatively large spindle while larger pig oocytes (about 120 μm) have a considerably smaller spindle. In this study we investigated whether species-specific oocyte spindle size was determined by cytoplasmic or nuclear factors. By exchanging the germinal vesicle between mouse and pig oocytes, we obtained two kinds of reconstructed oocytes: one with mouse ooplasm and pig GV (mCy-pGV oocyte), and the other with pig ooplasm and mouse GV (pCy-mGV oocyte). We show that the MII spindle size of the mCy-pGV oocyte is similar to that of the mouse meiotic spindle and significantly larger than that of the pig meiotic spindle. The timing of oocyte maturation also followed that of the species from which the oocyte cytoplasm arose, although some impact of the origin of the GV was observed. These data suggest that spindle size and the timing of meiotic progression are governed by cytoplasmic components rather than cytoplasmic volume and GV materials.

Highlights

We have used the germinal vesicle (GV) transfer technique, exchanged the GVs between mouse oocytes and pig oocytes to investigate the nuclear-cytoplasm interactions (Fig. 1), and to determine what controls meiotic time course and meiotic spindle size
Mouse and pig oocytes were enucleated by micro-manipulation, and the pig GV was transferred into the perivitelline space of the enucleated mouse oocyte
Mouse GV and mouse cytoplast were fused as a mock control. mCy-pGV oocytes were cultured in vitro and observed every 1 or 2 hours from 0 h to 14 h

Summary

Results

In vitro developmental time course and spindle size of mouse and pig oocyte. Mouse and pig oocytes were stained by PI and anti-α -tubulin antibody to detect chromosome condensation and spindle assembly. Maturation of the mCy-pGV oocyte reconstructed by fusing mouse cytoplast with pig GV. Spindle size of the mCy-pGV oocyte reconstructed by fusion of mouse cytoplast with pig GV. The size of spindles of mCy-pGV oocytes was similar to that of the mouse oocytes but significantly larger than that of the pig oocytes (Fig. 4A). GVBD of the pCy-mGV oocytes took place at 6–20 h of culture in vitro, which was significantly extended compared to the time course of mouse oocyte GVBD, it was 4 hours earlier than that of the pig oocyte GVBD (10–24 h) (Fig. 5A). Pig GV and pig cytoplast were fused as a mock control (pCy-pGV oocytes), which had a compromised maturational capability, and failed to reach the MII stage (Fig. 5B). Spindle size of pCy-mGV oocyte reconstructed by microinjection of mouse GV into pig GV cytoplast. Spindle size of the pCy-mGV oocyte was 12.80 ± 1.27 μ m, which was similar to that of the pig MI spindle (13.17 ± 0.74 μ m) and significantly shorter than that of the mouse MI spindle (33.33 ± 4.80 μ m) (Fig. 6B)

Discussion

Conclusions

Methods