Chromosomal integrity and DNA damage in freeze-dried spermatozoa

Abstract

Freeze-drying technology may one day be used to preserve mammalian spermatozoa indefinitely without cryopreservation. Freeze-dried mouse spermatozoa stored below 4°C for up to 1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures. Freeze-drying, per se, may reduce the integrity of chromosomes in freeze-dried mouse spermatozoa, but induction of chromosomal damage is suppressed if spermatozoa are incubated with divalent cation chelating agents prior to freeze-drying. Nevertheless, chromosomal damage does accumulate in spermatozoa stored at temperatures above 4°C. Currently, no established methods or strategies can prevent or reduce damage accumulation, and damage accumulation during storage is a serious obstacle to advances in freeze-drying technology. Chromosomal integrity of freeze-dried human spermatozoa have roughly background levels of chromosomal damage after storage at 4°C for 1 month, but whether these spermatozoa can produce healthy newborns is unknown. The safety of using freeze-dried human spermatozoa must be evaluated based on the risks of heritable chromosome and DNA damage that accumulates during storage.

Keywords: Chromosome; Cryopreservation; DNA damage; Freeze‐drying; Spermatozoa.

Figure 1

Primary chromosome damage (PCD) induced after freeze‐drying of mouse spermatozoa. Spermatozoa freeze‐dried in modified CZB (mCZB) and modified ETBS (mETBS) were stored at 4°C for up to 63 days [30] or up to 14 days [21], respectively. The PCD decreases as pre‐freeze‐drying incubation time in the mETBS increases. csb chromosome break, cse chromosome exchange, ctb chromatid break, cte chromatid exchange. Aberrations such as chromosome fragmentation and multiple aberrations (10 or more aberrations per zygote) that could not be counted were excluded from the data set

Figure 2

Schematic diagrams of hypothetical explanations of primary chromosome damage (PCD) and accumulative chromosome damage (ACD) in freeze‐dried mouse spermatozoa. a A DNA single‐strand break (SSB) was probably created by enzymatic action before or immediately after intracytoplasmic sperm injection (ICSI) and resulted in initiation of PCD. b Unidentified DNA lesions other than SSBs may accumulate in DNA of freeze‐dried spermatozoa during their storage. Some SSBs created at lesions may not be converted to DNA double strand breaks (DSBs). The SSBs that persisted until DNA replication stage may be responsible for the formation of chromatid‐type aberrations

Figure 3

Chromosomal integrity of zygotes derived from mouse spermatozoa freeze‐dried in ETBS (a) and modified ETBS (b). Frequency of zygotes with normal chromosome constitution was expressed as the integrity per freeze‐dried sample. Freeze‐dried spermatozoa were preserved at 4°C (open circles) or room temperatures of 22–24°C (filled circles) (a) and at 4°C (open circles) or 25°C (filled circles) (b)

Figure 4

Accumulated chromosome damage (ACD) in freeze‐dried mouse spermatozoa. The spermatozoa freeze‐dried in ETBS and modified ETBS (mETBS) were stored at room temperature or at 50°C. When using mETBS, the spermatozoa were freeze‐dried after pre‐freeze‐drying incubation in mETBS at 4 or 25°C for 3–7 days [46]. csb chromosome break, cse chromosome exchange, ctb chromatid break, cte chromatid exchange. Aberrations such as chromosome fragmentation and multiple aberrations (10 or more aberrations per zygote) that could not be counted were excluded from the data set

Figure 5

Post‐implantation development of mouse (B6D2F₁) oocytes microinjected with mouse (B6D2F₁) spermatozoa freeze‐dried after pre‐freeze‐drying incubation in modified ETBS at 4°C for 5–7 days (a) [21] and 25°C for 4–7 days (b) (unpublished). Post‐freeze‐drying samples were stored for 3 and 12 months at the same temperatures as the pre‐freeze‐drying incubation. The embryos were transferred into CD‐1 females (albino) on the first day of pseudopregnancy after being mated with vasectomized CD‐1 males (albino). Number of implants (white bars) is consistent with total number of normal fetuses (black bars) and resorption sites examined on 14‐ or 18‐day gestation. *Significantly different (P < 0.05) from the data obtained from the spermatozoa preserved for 3 months by χ² comparison