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International journal of molecular sciences2023; 24(2); 937; doi: 10.3390/ijms24020937

The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa.

Abstract: Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.
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Publication Date: 2023-01-04 PubMed ID: 36674454PubMed Central: PMC9862423DOI: 10.3390/ijms24020937Google Scholar: Lookup
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  • Journal Article

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 article explores how autophagy, the cellular process of “self-eating” or degradation of unnecessary or dysfunctional elements, impacts sperm capacitation and acrosome reaction in stallion spermatozoa. The study primarily analyses how the autophagy marker LC3, is processed and relocated during these biological processes, and the role it may play in their regulation.

Objective and Methodology

  • The main objective of this study was to explore if autophagy and its protein components, such as LC3, are activated during the capacitation and acrosome reaction of stallion spermatozoa and if this activation influences these biological processes.
  • To understand autophagy turnover, the study assessed LC3I and LC3II proteins via western blotting, calculating the ratio between both proteins (LC3II/LC3I) which in somatic cells indicates the activation of autophagy.
  • Autophagy-related protein’s subcellular localization was determined by immunofluorescence, using antibodies that identified Atg16, Beclin-1, and LC3 proteins.

Key Findings

  • LC3 processing in stallion sperm significantly increased when they were incubated in capacitating conditions; this increase was 3-4 times higher post acrosome reaction induction.
  • LC3 was mainly seen in mature ejaculated sperm’s head, showing a clear redistribution from the post-acrosomal region to the acrosome during sperm capacitation.
  • Post acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa.
  • Manipulating the autophagy-related pathways through autophagy activators or inhibitors significantly increased LC3 processing and the percentage of acrosome reacted cells.

Conclusion

  • The findings of the study show that autophagy components may regulate sperm capacitation and acrosome reaction in stallion spermatozoa.
  • This regulation could be independent of the acrosome’s intraluminal pH and depend on LC3 lipidation.
  • The study speculates that a form of noncanonical autophagy in the sperm utilizes parts of autophagy machinery to facilitate the acrosome reaction.

Cite This Article

APA
Aparicio IM, Rojo-Domínguez P, Castillejo-Rufo A, Peña FJ, Tapia JA. (2023). The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa. Int J Mol Sci, 24(2), 937. https://doi.org/10.3390/ijms24020937

Publication

International journal of molecular sciences
ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 24
Issue: 2
PII: 937

Researcher Affiliations

Aparicio, Ines M
  • Department of Physiology, Institute of Molecular Pathology Biomarkers (BICOMCEL), University of Extremadura, 10003 Cáceres, Spain.
Rojo-Domínguez, Patricia
  • Laboratory of Spermatology, Veterinary Teaching Hospital, University of Extremadura, 10003 Cáceres, Spain.
Castillejo-Rufo, Alba
  • Department of Physiology, Institute of Molecular Pathology Biomarkers (BICOMCEL), University of Extremadura, 10003 Cáceres, Spain.
Peña, Fernando J
  • Laboratory of Spermatology, Veterinary Teaching Hospital, University of Extremadura, 10003 Cáceres, Spain.
Tapia, Jose A
  • Department of Physiology, Institute of Molecular Pathology Biomarkers (BICOMCEL), University of Extremadura, 10003 Cáceres, Spain.

MeSH Terms

  • Male
  • Horses
  • Animals
  • Acrosome / physiology
  • Acrosome Reaction / physiology
  • Sperm Capacitation / physiology
  • Semen
  • Spermatozoa / metabolism
  • Autophagy
  • Mammals

Grant Funding

  • GR21037 / Junta de Extremadura-FEDER
  • EQC2019-006152P / Ministerio de Ciencia e Innovaciu00f3n-FEDER
  • EQC2019-006153P / Ministerio de Ciencia e Innovaciu00f3n-FEDER

Conflict of Interest Statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Citations

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