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Home / Equine Research Bank / PLoS One / Autophagy and apoptosis have a role in the survival or death...
PloS one2012; 7(1); e30688; doi: 10.1371/journal.pone.0030688

Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration.

Abstract: Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.
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Publication Date: 2012-01-26 PubMed ID: 22292020PubMed Central: PMC3266901DOI: 10.1371/journal.pone.0030688Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates the impact of apoptosis (programmed cell death) and autophagy (cell recycling process) on the vitality of stallion sperm during refrigerated storage. The study revealed that these two processes greatly influence the quality and survival rate of sperm throughout refrigeration.

Research Methodology

  • The study utilized samples from seven stallions which were divided into two groups. One group underwent a process called single layer centrifugation, while the other was extended without processing.
  • Both groups were stored at 5 degrees Celsius for five days.
  • During the storage period, the researchers evaluated the sperm motility (movement) and membrane integrity (the sperm’s structural health) daily using Computer Assisted Sperm Analysis (CASA) and flow cytometry techniques.
  • Apoptosis indicators (active caspase 3, increased membrane permeability, phosphatidylserine translocation, and mitochondrial membrane potential) were observed using flow cytometry on days 1, 3, and 5.
  • The processing of LC3B, a protein involved in autophagy, was also investigated using a western blotting technique at the beginning and end of the storage period.

Research Findings

  • The decline in sperm quality during the storage period was largely attributed to apoptosis.
  • Single layer centrifugation was found to select non-apoptotic spermatozoa, but this process did not lead to notable differences in sperm motility between selected and unselected sperm.
  • A significant percentage of spermatozoa exhibited active caspase 3, a key protein in the apoptosis process, after ejaculation and this increased during the storage period.
  • No changes in the percentage of live sperm were observed using the SYBR-14/PI assay, a dual staining technique used to differentiate live and dead sperm cells.
  • The protein LC3B displayed differential processing in processed and unprocessed sperm. More LC3B-II, an essential form in autophagy, was present in processed sperm compared to unprocessed ones, indicating an active autophagy process. Moreover, an increase in LC3B-II was observed in unprocessed sperm after five days of refrigerated storage.

Conclusions

  • This study showed that apoptosis plays a significant role in sperm death during refrigerated storage.
  • The results also indicated that autophagy may contribute to the survival of spermatozoa during refrigeration, providing a new understanding of pro-survival mechanisms in sperm cells.

Cite This Article

APA
Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ. (2012). Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration. PLoS One, 7(1), e30688. https://doi.org/10.1371/journal.pone.0030688

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 7
Issue: 1
Pages: e30688
PII: e30688

Researcher Affiliations

Gallardo Bolaños, Juan M
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura Cáceres, Cáceres, Spain.
Miró Morán, Álvaro
    Balao da Silva, Carolina M
      Morillo Rodríguez, Antolín
        Plaza Dávila, María
          Aparicio, Inés M
            Tapia, José A
              Ortega Ferrusola, Cristina
                Peña, Fernando J

                  MeSH Terms

                  • Animals
                  • Apoptosis / physiology
                  • Autophagy / physiology
                  • Biological Transport
                  • Biomechanical Phenomena / physiology
                  • Cell Death
                  • Cell Membrane Permeability / physiology
                  • Cell Survival
                  • Horses / physiology
                  • Male
                  • Phosphatidylserines / metabolism
                  • Refrigeration / veterinary
                  • Semen Analysis
                  • Semen Preservation / methods
                  • Semen Preservation / veterinary
                  • Spermatozoa / physiology

                  Grant Funding

                  • R49 CE001002 / NCIPC CDC HHS

                  Conflict of Interest Statement

                  The authors have declared that no competing interests exist.

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