Mechanism of meiotic spindle disappearance and regeneration during oocyte vitrification

Abstract

As assisted reproductive technologies (ART) become the standard of care for many infertile individuals, there has been a growing demand for the technology to cryopreserve oocytes. A unique cryopreservation method, called vitrification, promises high rates of oocyte survival, fertilization and development. However, a potential problem with oocyte vitrification is the transient disappearance of the meiotic spindle during freezing. Normal development relies on the integrity of the meiotic spindle, and its malformations can lead to aberrant genetic segregation and birth defects. Our goal is to understand the mechanisms behind the meiotic spindle disappearance and reappearance during vitrification. This understanding is essential to develop safer and more effective vitrification procedures. To gain insight into the mechanisms, we examined the integrity of the spindle microtubules, and three centrosome components, NEDD1, pericentrin and γ-tubulin, in mouse oocytes at various steps during vitrification and thawing. The distribution of these components was assessed by immunostaining using specific antibodies. The Tyho-Galileo vitrification protocol was mainly used for this study, as it yielded the highest rate of oocyte survival in our hands. The spindle microtubules started to disappear gradually in the vitrification solutions, because of reduced temperatures and exposure to cryoprotectants, and became absent after exposure to liquid nitrogen. Regeneration of the spindle microtubules initiated during the thawing process, first in an excessive manner, generating a wide spindle and ectopic microtubule asters. However, the spindle was adjusted after the 37ºC incubation for normal size and shape, suggesting a dynamic nature of spindle regeneration. For centrosome components, partial staining of NEDD1 and γ-tubulin, but not pericentrin, was observed throughout the vitrification and thawing process. Thus, NEDD1 and γ-tubulin appear more resilient to the vitrification process than pericentrin, suggesting that they may serve as anchors in the centrosome to enable microtubule repolymerization during thawing. Our present study should lay a foundation for further studies to elucidate the mechanisms of spindle disappearance and regeneration during oocyte vitrification. Such insight would ultimately help improve the oocyte vitrification procedure to preserve fertility in women.