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

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.

Keywords: assisted reproductive technology; cryopreservation; meiotic spindle; oocyte; vitrification.

FIG. 1

Formation of spindle-like microtubule bundles around the metaphase chromosomes induced by D₂O and Taxol. Microtubules (green) are detected by immunocytochemistry for β-Tubulin, and chromosomes (red) are visualized with propidium iodide. A) Meiotic spindle in an unmanipulated MII oocyte. B) Depolymerization of spindle microtubules after incubation on ice for 45 min. C) Repolymerization of microtubule bundles around the metaphase chromosomes in a cold-treated oocyte after incubation in the presence of D₂O (50%) and Taxol (1 μM) for 30 min. D) A cold-treated oocyte after incubation for 30 min in the absence of D₂O or Taxol. E) Repolymerization of microtubule bundles around the metaphase chromosomes in a cold-treated oocyte by D₂O alone. F) Repolymerization of microtubule bundles around the metaphase chromosomes in a cold-treated oocyte by Taxol alone. Bar = 10 μm.

FIG. 2

Disappearance and reappearance of spindle microtubules during the oocyte vitrification process. A) Grades of spindle microtubules: grade 0 = no detectable spindle microtubules; grade 1 = severely diminished spindle that is less than 50% of the normal spindle in size; grade 2 = mildly diminished spindle that is larger than 50% of the normal spindle; grade 3 = equivalent to the normal spindle in size and shape; grade 4 = spindle with excessive microtubule polymerization, namely, widening of the spindle poles, presence of astral microtubules emanating from the spindle poles (arrowheads), or astral microtubules from cytoplasmic foci (arrows); grade 5 = oocyte with all three features of excessive microtubule polymerization. B) A graph showing average grades of spindle microtubules at different steps of the vitrification procedure. Error bars represent standard deviations. Numbers of oocyte examined for each step are UM (unmanipulated; n = 22), V1 (n = 58), V2 (n = 42), V3 (n = 40), V3PV (postvitrification; n = 33), T1 (n = 35), T3 (n = 37), T5 (n = 29), IN (incubation for 2 h at 37°C; n = 27). Distributions of grades are compared between two adjacent groups by Student t-test, and asterisks represent statistically significant changes in average grade (P < 0.05). C) A graph showing comparison of average grades of spindle microtubules between oocytes treated with the vitrification solutions (corresponding to V1, V2, and V3 in B) and those incubated in FHM for the same durations. Error bars represent standard deviations. Distributions of grades are compared between the two treatments at the same incubation time point by Student t-test, and asterisks represent statistically significant differences (P < 0.05). D, top) Robust presence of spindle microtubules around the meiotic chromosomes after incubation in FHM at the ambient temperature for 2 h. D, bottom) Disappearance of spindle microtubules after the V2 step even in the presence of Taxol (1 μM). D, right) A graph showing comparison of average grades of spindle microtubules between unmanipulated (UM) oocytes and those in V2 containing Taxol. Error bars represent standard deviations. Distributions of grades are compared between two groups by Student t-test. Asterisks represent statistically significant difference (P < 0.05). E, left) Robust disappearance of spindle microtubules in oocytes that are treated with the vitrification solution consisting of DMSO and EG. E, right) A graph showing comparison of average grades of spindle microtubules between the two groups, and distributions of grades are compared by Student t-test. Error bars represent standard deviations. Asterisks represent statistically significant difference (P < 0.05). Bar = 10 μm.

FIG. 3

Time-lapse videomicroscopy of vitrified oocytes after parthenogenetic activation. A) Snapshot images of time-lapse recording of activated oocytes that have been vitrified and thawed. The top row shows images of activated oocytes that have been incubated for 2 h in KSOM after T5, and the bottom row shows images of those that have been activated soon after T5 without incubation. Asterisks indicate dead oocytes, and arrowheads point to the second polar body. Bar = 100 μm. B) Time course of survival of activated oocytes that have been incubated in KSOM for different durations. C) Time course of the second polar body (second BP) emission in surviving oocytes after activation. UM, unmanipulated oocytes. No, no incubation or less than 1 min of incubation.

FIG. 4

Disappearance and reappearance of MTOC components during oocyte vitrification process. A) γ-Tubulin. B) NEDD1. C) Pericentrin. Localizations of each MTOC component are scored based on their grades: grade 0 = no detectable localization, grade 1 = diminished and/or diffused localization, grade 2 = normal distribution. Representative images for each grade are shown on the left (green for the MTOC components and red for chromosomes). Graphs showing average grades of each MTOC component are shown on the right. Error bars represent standard deviations. Numbers of oocyte examined for each step are: UM (unmanipulated; n = 25, 53 and 36 [for γ-Tubulin, NEDD1, and Pericentrin, respectively]), V1 (n = 9, 16, and 7), V2 (n = 16, 23, and 22), V3 (n = 28, 32, and 39), V3PV (postvitrification; n = 13, 17, and 13), T1 (n = 25, 25, and 24), T3 (n = 27, 25, and 20), T5 (n = 13, 16, and 13), IN (incubation for 2 h at 37°C; n = 20, 16, and 12). Distributions of grades are compared between two adjacent steps by Student t-test, and asterisks represent statistically significant changes (P < 0.05). Bar = 10 μm.

FIG. 5

A) Protein synthesis-independent reappearance of spindle microtubules and Pericentrin during thawing and warming. CHX, cycloheximide (10 μM). B) Microtubule-independent localization of the MTOC components, γ-Tubulin, NEDD1, and Pericentrin, to the spindle poles. Distributions of the MTOC components (green) and chromosomes (red) are examined in unmanipulated (UM) and cold-treated (Cold) oocytes. C) Disappearance of Pericentrin localization in the vitrification solution consisting of DMSO and EG. Bar = 10 μm.