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Comparative Study
. 2019 Dec;86(12):1822-1831.
doi: 10.1002/mrd.23276. Epub 2019 Sep 23.

Desiccation and supra-zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

Pei-Chih Lee  1 , Pierre Comizzoli  1
Affiliations
Comparative Study

Desiccation and supra-zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

Pei-Chih Lee et al. Mol Reprod Dev. 2019 Dec.

Abstract

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long-term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes' germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.

Keywords: H3K4me3; H3K9me3; desiccation; germinal vesicle; nuclear envelope.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Nuclear envelope conformation and chromatin configuration in cat germinal vesicles after desiccation and vitrification. (A) Representative micrographs of different categories of the nuclear envelope (NE) conformation (immunostaining of lamin A/C; a–h) and chromatin configurations (DAPI staining; a’–h’). Scale bar = 10 μm. (B) Proportion of different categories of nuclear envelope conformation and chromatin configuration in different treatment groups. Proportions with different letters (spherical NE/reticular chromatin category: capital letters; irregular NE/abnormal chromatin category: lowercase letters) differ within the category (p < .05). Numbers on the top of the bars indicate a total number of oocytes in each treatment group. (C) Areas of nuclear envelope relative to the area of the in oocytes after different treatments. The dotted line represents 1:1 ratio in which the size of chromatin equals that of the nuclear envelope
FIGURE 2
FIGURE 2
Relative fluorescent intensity of epigenetic markers in cat GVs following desiccation or vitrification. (A) Representatives of GVs immunostained with H3K4me3 (a, b) or H3K9me3 (c, d) antibodies. Corresponding DNA was counterstained with DAPI (a’–d’). Scale bar = 20 μm. (B, C) Influence of desiccation and vitrification on the intensity of (B) H3K4me3 and (C) H3K9me3. Mean fluorescent intensity of control, untreated GVs was set as 1. Values are mean ± SEM. Values with different letters differ (p < .05). Numbers at the bottom of the bars indicate total number of oocytes in each group. GV, germinal vesicle; SEM, standard error of the mean
FIGURE 3
FIGURE 3
Influence of main steps of the desiccation procedure and storage temperatures on the relative fluorescent intensity of epigenetic markers (a) H3K4me3 and (B) H3K9me3 in cat GVs. Treatment groups were labeled as illustrated in Figure 1. Mean fluorescent intensity of fresh control GVs was set as 1. Values are mean ± SEM. Values with different letters differ (p < .05). Numbers at the bottom of the bars indicate a total number of oocytes in each group. MW, microwave drying; RT, room temperature; GV, germinal vesicle; SEM, standard error of the mean
FIGURE 4
FIGURE 4
Influence of two rehydration protocols (direct versus sequential) on the relative fluorescent intensity of epigenetic markers (a) H3K4me3 and (b) H3K9me3 in cat GVs. Mean fluorescent intensity of fresh control GVs was set as 1. Values are mean ± SEM. Values with different letters differ (p < .05). Numbers at the bottom of the bars indicate a total number of oocytes in each group. GV, germinal vesicle; SEM, standard error of the mean
FIGURE 5
FIGURE 5
Flow charts for (a) Experiment 1, (b) Experiment 2, and (c) Experiment 3. Open arrows indicate samples collected at the end of the procedure and boxed texts indicate the names of the treatment groups. MW, microwave drying. RT, room temperature
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