Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes.

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.

A potential use of embryonic stem cell medium for the in vitro culture of preimplantation embryos.

PURPOSE: To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and post-implantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM). METHODS: Development in ESCM versus KSOM was compared in terms of embryo morphology, cleavage, cavitation, hatching, cell number, expression of TE and ICM transcription factors (Cdx2 and Oct4, respectively), implantation, and development in utero. RESULTS: An enriched medium like ESCM can be beneficial for in vitro embryo development when cultured from the 8-cell stage, as evidenced by promotion of blastocyst development with respect to cavity expansion, hatching, and cell division. Such benefits were not observed when embryos were cultured from the 2-cell stage. CONCLUSIONS: ESCM may augment in vitro embryo development from the 8-cell stage. Using different culture media at different stages may be beneficial to achieve more effective human in vitro fertilization.