Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.
Abstract: Nonviable freeze-dried sperm have intact chromatin and can be used for fertilization via intracytoplasmic sperm injection. Freeze-dried sperm preferably should be stored at 4°C or lower, because DNA damage accumulates during storage at room temperature. Disaccharides are known to protect biomolecules both during freezing and drying, by forming a glassy state. Their use is challenging because cellular membranes are normally impermeable for disaccharides. In the current study, we demonstrate that membrane impermeable compounds, including lucifer yellow and trehalose, are taken up by stallion sperm when exposed to freezing. Trehalose uptake likely occurs during freezing-induced membrane phase transitions. Stallion sperm was freeze-dried in various formulations consisting of reducing or nonreducing sugars combined with albumin as bulking agent. Chromatin stability was studied during storage at 37°C, using the flow cytometric sperm chromatin structure assay and microscopic assessment of chromatin dispersion and DNA fragmentation after electrophoresis. Freeze-drying did not affect sperm chromatin, irrespective of the formulation that was used. DNA fragmentation index (DFI) values ranged from 5 to 8%. If sperm was freeze-dried without protectants or in a combination of glucose and proteins, DNA damage rapidly accumulated during storage at 37°C, reaching DFI values of respectively 95 ± 4 and 64 ± 42% after 1 month. DFI values of sperm freeze-dried with sucrose or trehalose ranged between 9-11% and 33-52% after 1 and 3 months storage, respectively. In conclusion, freeze-drying sperm with disaccharides results in uptake during freezing, which greatly reduces chromatin degradation during dried storage.
© The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Publication Date: 2017-11-10 PubMed ID: 29121172DOI: 10.1093/biolre/iox142Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the use of disaccharides in preserving chromatin in freeze-dried stallion sperm during accelerated aging. It found that freezing sperm with these sugars significantly reduced chromatin degradation during storage, ensuring the integrity of the genetic material in the cell.
Introduction and Methodology
- The research aimed to explore the potential of disaccharides, such as trehalose, in preserving the chromatin structure in freeze-dried stallion sperm during storage at higher temperature (37°C), which is known as accelerated aging. Chromatin is the material in the nucleus of a cell that contains DNA and proteins.
- While natural cellular membranes usually make it impossible for disaccharides to enter cells, the study found that freezing-induced changes in the sperm membrane allowed for disaccharides uptake.
- The researchers used stallion sperm for the study. These were subjected to freezing temperatures, thereby triggering membrane changes and enabling disaccharides to enter the cells.
- They used various mixtures of reducing or nonreducing sugars together with albumin as bulking agents, and freeze-dried the prepared sperms.
Findings and Observations
- The freeze-drying process, irrespective of the nature of the formulation, did not impact the sperm chromatin.
- The DNA fragmentation index (DFI), a measure of genetic material damage, varied from 5 to 8%, which is considered very low.
- Without using any protectants or when using a combination of glucose and proteins, the DFI values attained high levels very rapidly during storage at 37°C, reaching 95 ± 4 and 64 ± 42% respectively after one month of storage.
- Samples preserved using sucrose or trehalose had DFI values much lower between 9-11% and 33-52% after 1 and 3 months of storage respectively, showing significantly lesser DNA damage.
Conclusion
- The results showed that freeze-drying stallion sperm in the presence of disaccharides allowed the uptake of these sugars during the freezing process.
- This, in term, effectively minimized chromatin degradation in the dried sperm during storage, preserving the integrity of its genetic material.
- The approaching could promisingly contribute to the long-term preservation of genetic material in sperm for fertility purposes.
Cite This Article
APA
Oldenhof H, Zhang M, Narten K, Bigalk J, Sydykov B, Wolkers WF, Sieme H.
(2017).
Freezing-induced uptake of disaccharides for preservation of chromatin in freeze-dried stallion sperm during accelerated aging.
Biol Reprod, 97(6), 892-901.
https://doi.org/10.1093/biolre/iox142 Publication
Researcher Affiliations
- Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
- Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
- Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
- National Stud Lower Saxony, Celle, Germany.
- Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
- Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
- Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
- Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
MeSH Terms
- Animals
- Chromatin / ultrastructure
- Cryopreservation
- DNA Fragmentation
- Disaccharides / metabolism
- Freeze Drying
- Freezing
- Horses
- Male
- Semen Preservation / methods
- Spermatozoa
- Sucrose / metabolism
- Trehalose / metabolism
Citations
This article has been cited 11 times.- Sharafi M, Borghei-Rad SM, Hezavehei M, Shahverdi A, Benson JD. Cryopreservation of Semen in Domestic Animals: A Review of Current Challenges, Applications, and Prospective Strategies. Animals (Basel) 2022 Nov 24;12(23).
- Janati Idrissi S, Le Bourhis D, Lefevre A, Emond P, Le Berre L, Desnoës O, Joly T, Buff S, Freret S, Schibler L, Salvetti P, Elis S. Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile†. Biol Reprod 2022 Mar 19;106(3):597-612.
- Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H. Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations. Sci Rep 2021 Jul 7;11(1):14076.
- Jeong SJ, Stitham J, Evans TD, Zhang X, Rodriguez-Velez A, Yeh YS, Tao J, Takabatake K, Epelman S, Lodhi IJ, Schilling JD, DeBosch BJ, Diwan A, Razani B. Trehalose causes low-grade lysosomal stress to activate TFEB and the autophagy-lysosome biogenesis response. Autophagy 2021 Nov;17(11):3740-3752.
- Palazzese L, Anzalone DA, Turri F, Faieta M, Donnadio A, Pizzi F, Pittia P, Matsukawa K, Loi P. Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature. Sci Rep 2020 Nov 2;10(1):18873.
- Keskintepe L, Eroglu A. Preservation of Mammalian Sperm by Freeze-Drying. Methods Mol Biol 2021;2180:721-730.
- Wolkers WF, Oldenhof H. Principles Underlying Cryopreservation and Freeze-Drying of Cells and Tissues. Methods Mol Biol 2021;2180:3-25.
- Palazzese L, Gosálvez J, Anzalone DA, Loi P, Saragusty J. DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development. J Reprod Dev 2018 Oct 12;64(5):393-400.
- Esmaeili A, Esmaeili V, Shahverdi A, Eslaminejad MB. Engineered extracellular vesicles: a breakthrough approach to overcoming sperm cryopreservation challenges. Reprod Biol Endocrinol 2025 May 21;23(1):75.
- Murray A, Kilbride P, Gibson MI. Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities. RSC Med Chem 2024 Sep 19;15(9):2980-2995.
- Luther AM, Varzandeh M, Beckermann C, Feyer L, Maaßen IK, Oldenhof H, Hackbarth S, Waberski D. Fertility after photodynamic inactivation of bacteria in extended boar semen. Front Microbiol 2024;15:1429749.
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