Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils.
Abstract: Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1-0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.
Publication Date: 2011-03-09 PubMed ID: 21389342DOI: 10.1095/biolreprod.110.084491Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates a protein in horse semen named CRISP3 that reduces the adherence between spermatozoa and a type of innate immune white blood cells, polymorphonuclear neutrophils (PMNs), potentially playing a role in sperm survival within the female reproductive tract.
Objective and Methodology
- The main goal of this research was to identify and isolate a protein in the seminal fluid of horses that can prevent the binding between the sperm cells and PMNs, a situation that can trigger an inflammatory response.
- The process involved collecting and pooling semen from five stallions, isolating the proteins and undergoing a series of tests to identify the protein responsible.
- The extracted proteins were precipitated, dialyzed, submitted to a column (Sephacryl S200), and later to ion-exchange chromatography. They were analyzed and tested for their ability to inhibit PMN/sperm binding.
Findings and Identification
- The protein pools presenting biological activity underwent further analysis involving two-dimensional SDS-PAGE, and sequence analysis to identify the protein.
- The identified protein was recognized as equine CRISP3 and this identification was conclusively confirmed by Western blotting.
- Further testing on CRISP3 confirmed that its presence significantly suppresses PMN/sperm binding. The findings were statistically significant (P < 0.0001), further supporting their validity.
Implications and Conclusion
- It was concluded that the presence of CRISP3 in horse seminal fluid suppresses the binding between PMNs and sperm cells, highlighting a potential mechanism by which sperm cells avoid being eliminated by the female immune response.
- This implies that the CRISP3 protein could have significant implications in equine reproduction, specifically in ensuring the survival and successful fertilization of sperm cells within the female uterus.
- Understanding such mechanisms can prove to be valuable in addressing fertility issues in horses and potentially other mammals as well.
Cite This Article
APA
Doty A, Buhi WC, Benson S, Scoggin KE, Pozor M, Macpherson M, Mutz M, Troedsson MH.
(2011).
Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils.
Biol Reprod, 85(1), 157-164.
https://doi.org/10.1095/biolreprod.110.084491 Publication
Researcher Affiliations
- Department of Animal Sciences, College of Agriculture and Life Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
MeSH Terms
- Amino Acid Sequence
- Ammonium Sulfate
- Animals
- Blotting, Western
- Chromatography, Gel
- Chromatography, Ion Exchange
- Flow Cytometry
- Horses / metabolism
- Male
- Molecular Sequence Data
- Neutrophils / metabolism
- Seminal Plasma Proteins / metabolism
- Sequence Analysis, Protein
- Spermatozoa / metabolism
Citations
This article has been cited 10 times.- Alghamdi AS, Fedorka CE, Scoggin KE, Esteller-Vico A, Beatty K, Davolli G, Ball BA, Troedsson MHT. Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa.. Animals (Basel) 2022 Dec 23;13(1).
- Senra RL, Ramírez-López CJ, Magalhães-Júnior MJ, Neves JGDS, Barros E, Waddington B, Guimarães SEF, Guimarães JD, Baracat-Pereira MC. Kallikrein proteoforms and reproductive parameters in stallion are conditioned by climate.. Sci Rep 2022 Nov 4;12(1):18690.
- Morrell JM, Rocha A. A Novel Approach to Minimising Acute Equine Endometritis That May Help to Prevent the Development of the Chronic State.. Front Vet Sci 2021;8:799619.
- Gonzalez SN, Sulzyk V, Weigel Muñoz M, Cuasnicu PS. Cysteine-Rich Secretory Proteins (CRISP) are Key Players in Mammalian Fertilization and Fertility.. Front Cell Dev Biol 2021;9:800351.
- Miró J, Catalán J, Marín H, Yánez-Ortiz I, Yeste M. Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm-PMN Binding in the Donkey.. Animals (Basel) 2021 May 13;11(5).
- Weber KS, Wagener K, Blanco M, Bauersachs S, Bollwein H. A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling.. BMC Genomics 2021 May 22;22(1):377.
- Papas M, Catalán J, Recuero S, Morrell JM, Yeste M, Miró J. Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils.. Animals (Basel) 2020 Nov 16;10(11).
- Bubenickova F, Postlerova P, Simonik O, Sirohi J, Sichtar J. Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation.. Int J Mol Sci 2020 Sep 3;21(17).
- Morrell JM, Johannisson A. Comparison of the Effect of Heterologous and Homologous Seminal Plasma on Motility and Chromatin Integrity of Stallion Spermatozoa Selected by Single Layer Centrifugation.. J Vet Med 2014;2014:325451.
- Volpert M, Mangum JE, Jamsai D, D'Sylva R, O'Bryan MK, McIntyre P. Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse.. Sci Rep 2014 Feb 27;4:4217.
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