PRDX5 and PRDX6 in Oxidative Stress Response During Cryopreservation of Bull Sperm

Academic Background

Cryopreservation is a critical step in animal breeding and assisted reproductive technologies, particularly in the preservation of bull sperm. However, oxidative stress generated during cryopreservation significantly reduces sperm quality, leading to issues such as DNA fragmentation, impaired mitochondrial function, and altered membrane fluidity. These problems not only affect sperm motility and viability but may also impact fertilization potential. To counteract oxidative stress, intracellular antioxidant proteins, particularly peroxiredoxins (PRDXs), play a crucial role. PRDXs are a class of antioxidant enzymes that scavenge reactive oxygen species (ROS), protecting cells from oxidative damage.

In this study, the authors focused on the behavior of PRDX5 and PRDX6, two members of the PRDX family, during the cryopreservation of bull sperm. PRDX5 is primarily located in the mitochondrial sheath, protecting mitochondrial function, while PRDX6 is mainly found on the cell membrane and has the ability to repair oxidized phospholipids. During cryopreservation, the localization and function of these proteins may change, thereby affecting the sperm’s antioxidant capacity. Therefore, studying the behavior of PRDX5 and PRDX6 during cryopreservation not only helps to understand the protective mechanisms of sperm under oxidative stress but may also provide new insights for improving cryopreservation techniques.

Source of the Paper

This paper was co-authored by Agnieszka Mostek-Majewska, Magdalena Bossowska-Nowicka, Mariola Słowińska, and Andrzej Ciereszko from the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences. The study was published in 2025 in the journal Cell Communication and Signaling and was funded by the National Science Centre of Poland (Project No. 2021/43/D/NZ9/01916).

Research Process

1. Research Materials and Cryopreservation

The study used sperm samples from eight sexually mature Holstein-Friesian bulls. Semen was collected using an artificial vagina, immediately diluted with protein-free Bioxcell diluent, and transported to the laboratory at 4°C. Before cryopreservation, the semen samples were equilibrated at 4°C for 2 hours and then frozen in liquid nitrogen.

2. Measurement of Sperm Quality Parameters

Sperm concentration and viability were measured using computer-assisted fluorescent microscopy (NucleoCounter SP-100), and sperm motility was assessed using a computer-assisted sperm analysis system (HT CASA II). Additionally, flow cytometry (Guava EasyCyte system) was used to measure mitochondrial membrane potential (MMP), DNA fragmentation, membrane fluidity, and intracellular nitric oxide (NO) and reactive oxygen species (ROS) levels.

3. Localization and Expression of PRDX5 and PRDX6

Fluorescently labeled antibodies were used to detect the localization of PRDX5 and PRDX6 on the sperm surface and their changes before and after cryopreservation using imaging flow cytometry. Furthermore, Western blotting was employed to analyze the expression of PRDX5 and PRDX6 in seminal plasma extracellular vesicles (EVs) and exosome-depleted seminal plasma.

4. Oligomerization of PRDX5 and PRDX6

Non-reducing SDS-PAGE and immunoblotting were used to analyze the oligomerization state of PRDX5 and PRDX6 before and after cryopreservation. The results showed that PRDX5 and PRDX6 formed high-molecular-weight (HMW) oligomers after cryopreservation, stabilized by disulfide bonds.

5. Interaction of PRDX5 and PRDX6 with TLR4

Co-immunoprecipitation was used to detect the interaction between PRDX5, PRDX6, and Toll-like receptor 4 (TLR4). The results revealed that PRDX5 and PRDX6 formed complexes with TLR4, which may be related to their intracellular transport under oxidative stress.

Key Findings

1. Impact of Cryopreservation on Sperm Quality

Cryopreservation significantly increased ROS and NO levels in sperm, leading to decreased mitochondrial membrane potential, increased DNA fragmentation, and elevated membrane fluidity. These changes indicate that cryopreservation-induced oxidative stress has a significant negative impact on sperm quality.

2. Changes in Localization of PRDX5 and PRDX6

After cryopreservation, PRDX5 translocated from the sperm surface to the intracellular region, while PRDX6 moved from the intracellular region to the cell membrane. This change in localization may represent a protective mechanism of sperm in response to oxidative stress.

3. Oligomerization of PRDX5 and PRDX6

After cryopreservation, PRDX5 and PRDX6 formed high-molecular-weight oligomers. These oligomers were stabilized by disulfide bonds, suggesting that cryopreservation-induced oxidative stress prompted PRDX5 and PRDX6 to shift from peroxidase activity to chaperone-like functions.

4. Interaction of PRDX5 and PRDX6 with TLR4

The study confirmed that PRDX5 and PRDX6 formed complexes with TLR4. This interaction may regulate the intracellular transport and function of PRDX5 and PRDX6 under oxidative stress.

Conclusions and Significance

This study highlights the important roles of PRDX5 and PRDX6 in the cryopreservation of bull sperm. Cryopreservation-induced oxidative stress triggers changes in the localization and oligomerization of these proteins, enabling them to function as molecular chaperones and protect sperm from oxidative damage. Additionally, the interaction between PRDX5, PRDX6, and TLR4 may further enhance their antioxidant capabilities.

These findings not only deepen our understanding of the antioxidant mechanisms in sperm but also provide new insights for improving cryopreservation techniques. By regulating the expression and function of PRDX5 and PRDX6, it may be possible to enhance the quality and fertilization potential of cryopreserved sperm, thereby advancing animal breeding and human assisted reproductive technologies.

Research Highlights

1. First Discovery of Localization Changes in PRDX5 and PRDX6 During Cryopreservation: The study is the first to report the re-localization of PRDX5 and PRDX6 from intracellular regions to the cell membrane during cryopreservation, revealing their protective mechanisms under oxidative stress.
2. Oligomerization of PRDX5 and PRDX6: The study is the first to demonstrate that PRDX5 and PRDX6 form high-molecular-weight oligomers after cryopreservation, stabilized by disulfide bonds, indicating a potential shift from peroxidase to chaperone functions.
3. Interaction of PRDX5 and PRDX6 with TLR4: The study is the first to reveal the interaction between PRDX5, PRDX6, and TLR4, which may be related to their intracellular transport and functional regulation under oxidative stress.

Additional Valuable Information

The study also found that PRDX5 and PRDX6 are present in seminal plasma extracellular vesicles (EVs), suggesting that EVs may serve as a transport system to deliver these antioxidant proteins to sperm cells, further enhancing their antioxidant capacity. This discovery provides a new direction for future research on the role of EVs in sperm protection.