OBJECTIVE: To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. DESIGN: Experimental animal study. SETTING: University animal laboratory. ANIMAL(S): Eight-week-old B6D2F1 mice. INTERVENTION(S): Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). MAIN OUTCOME MEASURE(S): Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. RESULT(S): Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. CONCLUSION(S): Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.
OBJECTIVE: To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. DESIGN: Experimental animal study. SETTING: University animal laboratory. ANIMAL(S): Eight-week-old B6D2F1 mice. INTERVENTION(S): Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). MAIN OUTCOME MEASURE(S): Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. RESULT(S): Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. CONCLUSION(S): Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.
Authors: Clementina Cantatore; Jenny S George; Raffaella Depalo; Giuseppe D'Amato; Molly Moravek; Gary D Smith Journal: J Assist Reprod Genet Date: 2021-05-22 Impact factor: 3.357