Universität zu Köln

Peltier, Manon ORCID: 0009-0008-3832-4111 (2024). Effect of D-aspartate and storage temperature on cryopreserved sperm quality and fertility in mice. PhD thesis, Universität zu Köln.

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Abstract

Recent advances in genetic engineering have resulted in the generation of thousands of transgenic mouse lines. Assisted reproductive technologies have become essential for managing and archiving all these lines in repositories. Sperm cryopreservation appears to be the most economical way to archive mouse lines compared to embryo cryopreservation. However, there are still major challenges in using cryopreserved spermatozoa. In particular, cryodamage can compromise sperm integrity and fertility. Another challenge relates to the use of liquid nitrogen (LN2, -196°C) due to the inherent risk of injury, cost and potential difficulty in accessing this resource. The amino acid D-aspartate (D-Asp) improves in vitro fertilisation (IVF) rates with cryopreserved sperm in several mammalian species. However, how this compound exerts these effects remains poorly understood. In parallel, the use of -80°C freezers is an attractive alternative to LN2. It has already shown promising results with B6N sperm, but how this temperature affects the sperm integrity in other mouse strains has not been investigated. The results show that D-Asp administrated orally to B6N males improves sperm motility, morphology and maturation after cryopreservation in LN2. Some of these effects were recapitulated in sperm treated in vitro with D-Asp, showing that this compound can act directly on spermatozoa. D-Asp treatment was associated with a signature of oxidative stress, suggesting that this amino acid acts through reactive oxygen species (ROS) to exert its cryoprotective effects. In parallel, the response of a panel of widely used mouse strains to sperm cryopreservation at -80°C was assessed. IVF rates were similar when spermatozoa from B6N, CD-1, FVB and 129 were cryopreserved and stored in LN2 or in -80°C freezers. However, sperm from B6J and BALB/c mice showed a dramatic decrease in fertility when cryopreserved at -80°C, which was associated with a decline in viability. This shows variations in the sensitivity to cryopreservation at -80°C among mouse strains. Given the beneficial role of D-Asp on sperm stored in LN2, I have tested whether this compound could improve the quality of sperm cryopreserved at -80°C. D-Asp improved the motility, morphology and maturation of B6J and BALB/c sperm under this condition, showing that D-Asp alleviates the physiological limitations of cryopreservation at -80°C in these strains. D-Asp treatment could therefore be used in combination with long-term storage at -80°C to facilitate sperm cryopreservation and greatly improve the workflow of mouse repositories.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Peltier, Manon peltiermanon@gmail.com orcid.org/0009-0008-3832-4111 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-731260
Date:	2024
Language:	English
Faculty:	Faculty of Medicine
Divisions:	Zentrum für Molekulare Medizin
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Mouse, spermatozoa, cryopreservation, fertility, D-aspartate UNSPECIFIED
Date of oral exam:	5 June 2024
Referee:	Name Academic Title Polidori, Maria Cristina Prof. Dr. Lemberg, Marius Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/73126