Fig 1.
Flow chart of microwave-assisted dehydration procedure and evaluations.
Ovarian tissue pieces were assessed after drying, after drying and immediate rehydration (solid arrows), or after storage and rehydration (dotted arrows).
Fig 2.
Kinetics of water content in tissues during microwave drying.
Ovarian tissues were exposed to 0 M, 0.2 M, 0.5 M, or 1.0 M trehalose before microwaving for 0 to 40 min. Water contents were expressed as (A) water weight (μg) and (B) percentage of water weight over total tissue weight. Values are mean ± SEM. Drying curves were fitted to a logarithmic model Eq.(1).
Fig 3.
Trehalose content in tissues during microwave drying.
Values are mean ± SEM. Two-way ANOVA analysis indicated significant effect (P < 0.05) of trehalose concentration, but not drying time, on trehalose content. Samples treated with same concentration of trehalose exhibited no significant differences (P > 0.05) over different drying time. At each time point, values with different letters differ (P < 0.05) among groups exposed to different concentrations of trehalose at the same drying time.
Fig 4.
Effect of drying and rehydration on trehalose content and gross morphology of ovarian tissue.
(A) Trehalose levels after dehydration and rehydration. Values are mean ± SEM. Values with different letters differ (P < 0.05). (B-E) Gross morphology of whole ovarian cortical pieces that were (B) un-treated, (C) exposed to 0.5 M trehalose for 10 min, (D) dried for 30 min, and (E) rehydrated for 30 min.
Fig 5.
Effect of drying time on ovarian tissue histology.
(A) Representation of morphologically normal (arrows) and abnormal (arrowheads) follicles after fixation and haematoxylin–eosin staining. (B-C) Analyses of (B) follicular morphology and (C) stromal cell density of tissue pieces dried for up to 30 min. Values are mean ± SD. No statistically significant (P > 0.05) differences were observed.
Fig 6.
Effect of drying time on DNA integrity.
(A) Representation of TUNEL-positive staining (brown) indicating DNA damage in the cortical piece. Inset: DNase-treated positive control tissue. Scale bars = 200 μm. (B) Quantification of TUNEL-positive area using ImageJ. Inset: DNase-treated positive control tissue. (C) Analysis of DNA integrity of tissue pieces dried for up to 30 min. Values are mean ± SD. No statistically significant (P > 0.05) differences were observed.
Fig 7.
Effect of drying time on transcriptional activity.
(A) Representation of transcriptional activities after EU fluorescent labelling. White arrows: transcriptionally active follicles, white arrowheads: transcriptionally inactive follicles, yellow arrows: transcriptionally active somatic cells, and yellow arrowheads: transcriptionally inactive somatic cells. (B) Analysis of follicular transcriptional activity in tissue pieces dried for up to 30 min. Values are mean ± SD. Values with different letters differ (P < 0.05).
Fig 8.
Effect of different trehalose concentrations on (A) follicular morphology, (B) stromal cell density, and (C) transcriptional activity after 1d storage at 4°C. Values are mean ± SD. Values with different letters differ (P < 0.05). No statistically significant (P > 0.05) differences were observed in analyses for follicular morphology and cell density.
Fig 9.
Effect of drying time and storage on follicle survival.
(A-D) Representation of live and dead follicles after Calcein AM and trypan blue staining, respectively. (A) Live oocyte (arrow); (B) live granulosa cells (arrowheads); (C) dead oocyte (arrow); (D) dead granulosa cells (arrowhead). (E) Analysis of follicle survival in fresh control tissue and tissue pieces dried for up to 15 min and either assessed immediately after rehydration or after 1-day culture. (F) Analysis of follicle survival in fresh control tissue and tissue pieces dried for 5 or 10 min and storage for 24 h. Values are mean ± SD. Values with different letters differ (P < 0.05).