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Phosphopeptide Analysis of Rodent Epididymal Spermatozoa
Published on: December 30, 2014
DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development
Luca Palazzese1, Jaime Gosálvez2, Debora A Anzalone1
1Faculty of Veterinary Medicine, University of Teramo, Teramo 64100, Italy.
Abstract:
Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi's demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA - Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte's repair capacity.
Insights
Sperm freeze-drying shows promise for preserving genetic material. Ram sperm DNA integrity after freeze-drying correlated with successful embryo development via Intracytoplasmic Sperm Injection (ICSI).
Area of Science:
- Reproductive Biology
- Cryobiology
- Genetics
Background:
- Sperm freeze-drying (lyophilization) is an emerging technique for preserving spermatozoa.
- Previous studies demonstrated successful births in mice using freeze-dried epididymal sperm via Intracytoplasmic Sperm Injection (ICSI).
- Most prior research utilized ejaculated sperm; this study focuses on epididymal sperm in rams.
Purpose of the Study:
- To investigate the relationship between DNA integrity in freeze-dried ram epididymal spermatozoa and subsequent embryo development.
- To assess the impact of different types of sperm DNA damage (Single-Strand Breaks and Double-Strand Breaks) on ICSI outcomes.
Main Methods:
- Epididymal spermatozoa from four rams were freeze-dried using a cryoprotective medium containing trehalose, glucose, KCl, HEPES, and Trolox.
- DNA integrity and damage were evaluated using the Sperm Chromatin Dispersion (SCD) test and the Two-Tailed Comet Assay.
- Rehydrated spermatozoa were used for Intracytoplasmic Sperm Injection (ICSI) to assess embryo development to the blastocyst stage.
Main Results:
- Ram #2 exhibited significantly higher intact DNA rates (28%) and lower Single-Strand Break (SSB) rates (70%) compared to other rams.
- Ram #3 showed a higher level of Double-Strand Breaks (DSBs) (4.6%) than Ram #1 (0.33%).
- Embryo development to blastocyst stage post-ICSI was only achieved using sperm from Rams #2 and #4, which had higher intact DNA levels.
Conclusions:
- Sperm DNA integrity is crucial for successful embryo development following freeze-drying and ICSI in rams.
- The extent and type of sperm DNA damage (SSBs vs. DSBs) influence embryonic development outcomes.
- Oocyte's DNA repair capacity may play a role in overcoming sperm DNA damage.
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