Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes

Kusakabe, Hirokazu; Yanagimachi, Ryuzo; Kamiguchi, Yujiroh

doi:10.1093/humrep/dem252

Cited by 76 publications

(81 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sperm nuclei with a defective plasma membrane can easily come into contact with endonuclease, leading to the generation of chromosomal nicks. In the mouse, the chromosomes in 30-60% of the freeze-dried spermatozoa were impaired [3,7,15], while the percentage of chromosomally damaged canine spermatozoa was 72.9% in the present study. The chromosomal damage of freeze-dried mouse spermatozoa can be decreased by modifying the pH value of the storage media [17], and by pre-treatment with diamide [3] and EGTA [15].…”

Section: Discussioncontrasting

confidence: 47%

“…The value was obviously higher than in the other groups. A possible reason is that the plasma membrane of the freeze-dried spermatozoa was completely destroyed [7,11,15], which could lead to a more rapid contact between the sperm nuclei and the ooplasm.…”

Section: Discussionmentioning

confidence: 99%

“…As one of the cryopreservation methods in mammalian spermatozoa, freeze-drying has been applied in mouse [1][2][3][4][5][6][7][8], rat [9,10], rabbit [11], bull [12], boar [13,14] and human [15] spermatozoa. An advantage in freeze-drying is to store the spermatozoa without the use of liquid nitrogen for a long period.…”

Section: Introductionmentioning

confidence: 99%

“…However, freeze-dried spermatozoon must be microinjected into an oocyte to be fertilized, because it is no longer motile. Since the chromosomal integrity of these spermatozoa is impaired [7], methodological optimization of freeze-drying is still an essential issue for successful sperm preservation [15][16][17].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Watanabe

Asano

Abe

et al. 2009

J Assist Reprod Genet

View full text Add to dashboard Cite

Purpose The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa. Methods After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated. Results The rates of oocyte activation were comparable (90.6-100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P< 0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freezedried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa. Conclusions These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.

show abstract

Section: Discussioncontrasting

confidence: 47%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Watanabe

Asano

Abe

et al. 2009

J Assist Reprod Genet

View full text Add to dashboard Cite

show abstract

“…A 0.5 ml aliquot was gently placed at the bottom of a small test tube containing 2 ml of Tris-buffered ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid solution 9 that had been prewarmed to 37 uC. The tube was left standing for 10 min at 37 uC to allow spermatozoa to disperse into the solution.…”

Section: Collection and Treatment Of Human Spermatozoamentioning

confidence: 99%

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

Kusakabe

Tateno²

2010

Asian J Androl

View full text Add to dashboard Cite

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitro with the DNA-damaging agents, methyl methanesulfonate (MMS) or hydrogen peroxide (H 2 O 2 ), and then embedded in agarose gel on glass slides. The slides were immersed in alkaline solution (.pH 13) for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H 2 O 2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H 2 O 2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period (1 min) of the alkaline DNA unwinding time. Keywords: alkaline; comet assay; DNA unwinding; human; mice; spermatozoa INTRODUCTION Single-cell gel electrophoresis assay (the comet assay) is a simple and high-throughput technique to detect the cellular DNA damage induced by various types of genotoxicants in situ. The comet assay is performed in one of two versions, alkaline and neutral. Alkaline comet assays visualize single-and double-strand breaks in cellular DNA, whereas the neutral comet assay reveals mainly double-strand breaks. Nowadays, the alkaline comet assay of reproductive cells has been recommended to screen genotoxic hazards. Furthermore, it is more practical in larger studies to evaluate the DNA integrity of cryopreserved mammalian spermatozoa. 1 The standard alkaline comet assay includes a step of alkali treatment to unwind the DNA before electrophoresis under the alkaline condition. In another version of the assay, cells embedded in agarose gel are treated with an alkali (.pH 13) followed by electrophoresis under neutral conditions. This protocol is named the 'A/N protocol'. 2 Lower background levels of DNA damage and better dose responses are obtained by the A/N protocol. [2][3][4] Recently, the comet assay A/N protocol has been reported for murine spermatozoa. [5][6][7] In most cases, the alkaline DNA unwinding time is set at 20 min or more. To our knowledge, there is little information on the optimal time of alkali treatment to detect sperm DNA damage with the A/N protocol. In the present study, the optimal time of the alkali treatment was determined for mouse spermatozoa by the comet assay with A/N protocol.

show abstract

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation

Comizzoli

Amelkina

Lee

2022

Molecular Reproduction Devel

View full text Add to dashboard Cite

Long‐term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long‐term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long‐term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

show abstract

Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes

Cited by 76 publications

References 41 publications

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation

Contact Info

Product

Resources

About