The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins.
Abstract: Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex. Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.
© 2018 American Society of Andrology and European Academy of Andrology.
Publication Date: 2018-12-13 PubMed ID: 30549223DOI: 10.1111/andr.12569Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research aims to understand the role of the enzyme serine protease testisin (PRSS21) in stallion spermatozoa (sperm cells), focusing on its part in sperm capacitation, the process that makes sperm capable of fertilizing an egg. The study shows that testisin’s presence increases as these things happen and it is involved in binding to the egg by interacting with specific proteins.
Objective and Methodology
- The study investigates the serine protease testisin (PRSS21) in stallion sperm cells. The enzyme is examined for its surface expression, its origin in the testicle and the epididymis (the tube where sperm matures), and changes in its capacity in response to capacitation (the process of a sperm being capable of fertilizing an egg) and the acrosome reaction (release of enzymes needed to penetrate the egg).
- This is done through a combination of using live cell immunofluorescence (a technique using antibodies to visualise proteins), flow cytometry (a technology used to analyse physical and chemical characteristics of particles), immunohistochemistry (use of antibodies to detect specific proteins in tissues), and mass spectrometry (technique used to measure the mass-to-charge ratio of ions).
Results
- The results demonstrated that the expression of testisin increased significantly in capacitated spermatozoa and it increased even further following the acrosome reaction. The enzyme was also found in the epididymis, suggesting it originates from there.
- Testisin was discovered to interact with a number of proteins involved in zona pellucida-binding, the layer surrounding the egg that sperm must bind to and penetrate in order for fertilization to occur.
Conclusion
- The study concludes that testisin plays a crucial role in preparing the sperm cell for interaction with the egg. Testisin is part of the protein complex that enables binding to the layer of the egg, and may be involved in the enzyme cascade that readies the sperm surface for this interaction.
Cite This Article
APA
Swegen A, Smith ND, Gibb Z, Curry BJ, Aitken RJ.
(2018).
The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins.
Andrology, 7(2), 199-212.
https://doi.org/10.1111/andr.12569 Publication
Researcher Affiliations
- Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW, Australia.
- Analytical and Biomolecular Research Facility, University of Newcastle, Callaghan, NSW, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW, Australia.
MeSH Terms
- Acrosome Reaction / physiology
- Animals
- Female
- Horses
- Male
- Protein Binding
- Serine Endopeptidases / metabolism
- Sperm Capacitation / physiology
- Spermatozoa / enzymology
- Zona Pellucida / metabolism
Grant Funding
- Invasive Animals Cooperative Research Centre
- Australian Research Council
- LP120100219 / Hunter Thoroughbred Breeders Association
Citations
This article has been cited 7 times.- He Z, Xie M, Li QQ, Duan J, Lu X. Research Progress on the Microregulatory Mechanisms of Fertilization: A Review. In Vivo 2022 Sep-Oct;36(5):2002-2013.
- Peroutka RJ, Buzza MS, Mukhopadhyay S, Johnson TA, Driesbaugh KH, Antalis TM. Testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis. PLoS One 2020;15(6):e0234407.
- Kluczynski DF, Nester I, Prine H, Heising K, Gartee E, Adegoke D, Eriksen G, Liber C, Byreddy Y, Dua R, Adkins M, Pan N, Artz B, Doumet N, Salazar H, Warren J, Avidor-Reiss T. The spermatozoon neck role in infertility and intracytoplasmic sperm injection outcomes. J Assist Reprod Genet 2026 Feb;43(2):347-365.
- Manjon AA, Verón GL, Vitale R, Stegmayer G, Gonzalez Echeverria-Raffo F, Lane L, Vazquez-Levin MH. PRSS38 Is a Novel Sperm Serine Protease Involved in Human and Mouse Fertilization. Int J Mol Sci 2025 Dec 2;26(23).
- Lundgren JG, Flynn MG, List K. GPI-anchored serine proteases: essential roles in development, homeostasis, and disease. Biol Chem 2025 Jan 29;406(1-2):1-28.
- Liu Y, Du M, Zhang L, Wang N, He Q, Cao J, Zhao B, Li X, Li B, Bou G, Zhao Y, Dugarjaviin M. Comparative Analysis of mRNA and lncRNA Expression Profiles in Testicular Tissue of Sexually Immature and Sexually Mature Mongolian Horses. Animals (Basel) 2024 Jun 7;14(12).
- Hernández-Avilés C, Ramírez-Agámez L, Weintraub ST, Scoggin CF, Davis BW, Raudsepp T, Varner DD, Love CC. Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis. Sci Rep 2024 May 30;14(1):12446.
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