Fig 1.
Schematic representation of preparation of freeze-dried spermatozoa, rehydration conditions, and subsequent experiments.
(a) Spermatozoa were first collected from male mice and were capacitated before being aliquoted into glass ampoules in aliquots of 50 μL. Ampoules were frozen in liquid nitrogen and freeze-dried using a vacuum freeze-dryer. Glass ampoules were then opened prior to subsequent experiments and rehydration was then performed. (b) Rehydration was conducted using media subjected to various conditions. To decrease infiltration speed, we used HTF medium with a higher osmotic pressure than ultrapure water. To further reduce infiltration speed, we also used PVP solutions with higher viscosity. In addition, low-temperature ultrapure water was used to decrease infiltration speed, while high-temperature ultrapure water was used to increase it. (c) After adding different rehydration solutions to glass ampoules, we conducted the following experiments: (1) a comet assay to evaluate the degree of DNA damage in spermatozoa, (2) an analysis of gamma-H2Ax foci to assess damage at the pronuclear stage after fertilization, (3) measurements of ACS rates in two-cell embryos, (4) an evaluation of the rate of development to the blastocyst stage, and (5) an assessment of offspring rates after transferring two-cell embryos.
Fig 2.
DNA damage, and offspring rates under rehydration conditions under varying osmotic pressure or viscosity.
(a) Glass ampoules containing freeze-dried (FD) spermatozoa. (b) Freeze-dried spermatozoa showing separation of the sperm head during freeze-drying. (c) Comet tail length of FD sperm was compared between ultrapure water control group (top left), the HTF medium experimental group (top right), the 0% PVP solution control group (bottom left), and the 12% PVP solution experimental group (bottom right). (d) DNA damage in FD sperm rehydrated with HTF medium or PVP solution evaluated using comet assays. Background colors on the graph represent different conditions; blue indicates experimental conditions where osmotic pressure was altered using HTF medium, yellow represents conditions where viscosity was modified using PVP solution. Comet tail lengths were normalized to those of FD sperm rehydrated with ultrapure water (control). The vertical axis represents relative DNA damage, with higher values indicating greater damage. Each data point represents a value recorded for an individual spermatozoon, with error bars showing standard deviation. An asterisk denotes statistically significant differences between pairs of samples (p < 0.05). Different letters (a vs. b, c, d; b vs. c, d; c vs. d) indicate statistically significantly different group means (p < 0.05). (e) Offspring rates were evaluated in intracytoplasmic sperm injection (ICSI) embryos using FD sperm rehydrated with HTF medium or PVP solution. Background colors on the graph represent different conditions; blue indicates experimental conditions where osmotic pressure was altered using HTF medium, yellow represents conditions where viscosity was modified using PVP solution. Asterisks denote statistically significant differences between pairs of samples (p < 0.05).
Fig 3.
ACS level and rates of development to the blastocyst stage for FD sperm were rehydrated at different PVP concentrations.
(a) Two-cell embryos derived from FD sperm rehydrated with 6% PVP and stained with DAPI. Upper left image shows a two-cell embryo with normal chromosome segregation (NCS), while the lower left image shows a two-cell embryo with abnormal chromosome segregation (ACS). The enlarged image on the right shows a highlighted portion of the ACS embryo, and the arrowhead indicates the micronucleus. (b) Proportion of embryos with moderate or higher ACS among all embryos derived from FD sperm rehydrated with different PVP concentrations. Different letters indicate statistically significantly different group means (p < 0.05). (c) Blastocyst images. The top panel shows blastocysts derived from embryos originating from FD sperm rehydrated with 0% PVP (i.e., control). The bottom panel shows blastocysts derived from embryos originating from FD sperm rehydrated with 12% PVP (i.e., experimental group). (d) Rate of development to the blastocyst stage for embryos derived from FD sperm rehydrated under different PVP concentrations. Each data point represents an independent measurement of the proportion of embryos reaching each stage relative to normally fertilized embryos. Bars indicate the mean and error bars represent the standard error of the mean (SEM).
Fig 4.
Changes in DNA damage and developmental potential following rehydration using ultrapure water at different temperatures.
(a) Observed DNA damage in FD sperm rehydrated with ultrapure water at different temperatures as evaluated using comet assays. Different letters indicate statistically significantly different group means (p < 0.05). (b) gamma-H2Ax assay of fertilized embryos derived from FD sperm rehydrated with ultrapure water at different temperatures. Images show male and female pronuclei stained with 4′6-diamidino-2-phenylindole (DAPI, blue, top left), gamma-H2Ax signals indicating double-stranded DNA breaks (red, top right), bright-field images (bottom left), and merged images (bottom right). (c) Relative brightness of male pronuclei sourced from fertilized embryos derived from FD sperm rehydrated at different temperatures. Higher brightness values indicate more DNA damage. (d) Two-cell embryos derived from FD sperm rehydrated with ultrapure water at 50°C after staining with DAPI. The upper left image shows a two-cell embryo with normal chromosome segregation (NCS), while the lower left image shows a two-cell embryo with abnormal chromosome segregation (ACS). The enlarged image on the right shows a highlighted portion of the ACS embryo, and the arrowhead indicates the micronucleus. (e) Proportion of embryos with moderate or higher ACS among all embryos derived from FD sperm rehydrated with ultrapure water at different temperatures. (f) Offspring derived from ICSI embryos using FD sperm rehydrated with ultrapure water at 50°C. (g) Normal fertilization rates and offspring rates of ICSI embryos using FD sperm rehydrated with ultrapure water at different temperatures. Asterisks denote statistically significant differences in sample means (p < 0.05).
Table 1.
Full-term development rates of ICSI embryos using FD sperm rehydrated with ultrapure water at different temperatures.