Impact of Vitrification on the Meiotic Spindle and Components of the Microtubule-Organizing Center in Mouse Mature Oocytes1

Abstract

Cryopreservation of oocytes is becoming a valuable method for fertility preservation in women. However, various unphysiological alterations occur in the oocyte during the course of cryopreservation, one of which is the disappearance of the meiotic spindle. Fortunately, the meiotic spindle does regenerate after thawing the frozen oocytes, which enables completion of meiosis and further development after fertilization. Nonetheless, the mechanistic understanding of the meiotic spindle regeneration after cryopreservation is still scarce. Here, to gain insight into the mechanisms of the spindle disappearance and regeneration, we examined the status of spindle microtubules as well as the key components of the microtubule-organizing center (MTOC), specifically gamma-Tubulin, NEDD1, and Pericentrin, in mature (metaphase II) mouse oocytes at different steps of vitrification, a major cryopreservation technique. We found that the configuration of the spindle microtubules dynamically changed during the process of vitrification and that spindle regeneration was preceded by excessive microtubule polymerization, followed by reduction into the normal size and shape. Also, all three MTOC components exhibited disappearance and reappearance during the vitrification process, although Pericentrin appeared to regenerate in earlier steps compared to the other components. Furthermore, we found that the localization of the MTOC components to the spindle poles persisted even after depolymerization of spindle microtubules, suggesting that the MTOC components are impacted by vitrification independently from the integrity of the microtubules. The present study would set the stage for future investigations on the molecular mechanisms of the meiotic spindle regeneration, which may contribute to further improving protocols for oocyte cryopreservation.

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

“…In agreement with our study, it has been reported that the vitrified/warmed GV stage oocytes in ovine and other species have lower potential to reach MII stage compared with non‐vitrified oocytes (Moawad et al, ). Cryopreservation can disrupt the normal functions of the oocyte, such as re‐initiation and completion of meiosis, reorganization of cytoplasmic components during maturation, fertilization and the subsequent embryo development (Tamura, Huang, & Marikawa, ). Therefore, in our study, the reduction of nuclear maturation in vitrified/warmed oocytes might be one of the reasons for decreasing the developmental competence of these oocytes.…”

A brief history of oocyte cryopreservation: Arguments and facts

“…Although it has been observed that while the freezing procedure takes place, the spindle configuration may be preserved if oocytes are exposed to cryoprotectants, its structure is completely lost during the thawing procedure . However, further evidence has highlighted a recovery ability of the spindle to reassemble following cryopreservation . This de novo reassembly of the spindle microtubules is a requirement for correct chromosome alignment and segregation after fertilization .…”

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary

“…The goal of vitrification is to minimize IIF and dehydration (or freeze concentration) altogether, but the conventional procedure of vitrification requires a high concentration of CPAs (up to ~8 M, high-CPA) that may induce significant osmotic and metabolic damage to cells including oocytes even in a short exposure of a few minutes [11,36,40]. For example, a high concentration of CPA has been shown to induce cytoskeletal alternations [5,35], dispersal of chromosome [30], and disassembly of spindles [32] in oocytes. Consequently, it is necessary to use multiple steps to gradually load and unload the high CPA to minimize osmotic and metabolic damage to cells during high-CPA vitrification, making it a complicated, lengthy, and stressful process [12,13,29].…”