Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions.
Abstract: Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl beta-cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.
Publication Date: 2002-10-31 PubMed ID: 12606471DOI: 10.1095/biolreprod.102.011106Google Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This research investigates how the process of phosphorylation, a mechanism that regulates sperm function, behaves in fresh and frozen stallion sperm through different conditions. Essentially, the scientists found that frozen sperm may be more sensitive to certain triggers and have a shorter lifespan than fresh sperm.
Objectives and Hypotheses
- The researchers aimed to determine if fresh and thawed stallion sperm exhibit different levels of tyrosine phosphorylation after in vitro capacitation – a process that sperm undergo to be able to fertilize an egg.
- They hypothesized that stallion sperm undergo tyrosine phosphorylation during capacitation, and this process would be different in cryopreserved sperm (frozen and then thawed).
- They also experimented with the idea that tyrosine phosphorylation could be enhanced using certain activators such as dibutyryl cAMP (db cAMP) and caffeine, as well as with methyl beta-cyclodextrin, which causes cholesterol to be released from the sperm.
- The researchers also considered whether the process could be inhibited by a protein kinase A (PK-A) inhibitor called H-89.
Methodology
- Experiments were conducted to compare various parameters, including tyrosine phosphorylation, motility, and viability of sperm before and after in vitro capacitation in both freshly ejaculated and frozen-thawed sperm.
Key Findings
- The study found that the capacitation process in horse sperm is controlled by a signaling mechanism involving cAMP-dependent PK-A and tyrosine kinases.
- Notably, the researchers discovered that cryopreserved sperm may be more responsive to inducers of capacitation. This heightened sensitivity could account for the shortened lifespan of frozen sperm compared to fresh sperm.
Implications
- The results provide insights into the mechanisms that control capacitation in stallion sperm and highlight the potential differences in these processes between fresh and cryopreserved specimens.
- This knowledge could hold implications for the use and storage of sperm in assisted reproduction technologies, like in vitro fertilization.
Cite This Article
APA
Pommer AC, Rutllant J, Meyers SA.
(2002).
Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions.
Biol Reprod, 68(4), 1208-1214.
https://doi.org/10.1095/biolreprod.102.011106 Publication
Researcher Affiliations
- School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, California 95616, USA.
MeSH Terms
- Animals
- Bucladesine / pharmacology
- Caffeine / pharmacology
- Cryopreservation
- Cyclodextrins / pharmacology
- Enzyme Inhibitors / pharmacology
- Fluorescent Antibody Technique
- Horses / physiology
- Isoquinolines / pharmacology
- Male
- Phosphorylation / drug effects
- Sperm Capacitation / physiology
- Spermatozoa / metabolism
- Sulfonamides
- Tyrosine / metabolism
- beta-Cyclodextrins
Citations
This article has been cited 17 times.- Klusackova B, Pilsova A, Zelenkova N, Pilsova Z, Krejcirova R, Chmelikova E, Komrskova K, Simonik O, Postlerova P. Time to revise: impact of methodology on boar sperm capacitation in vitro via phosphotyrosine patterns. BMC Vet Res 2025 Jul 7;21(1):448.
- Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K. Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes. Biol Reprod 2025 May 13;112(5):867-879.
- Ortiz-Vallecillo A, Santamaría-López E, García-Ruiz D, Martín-Lozano D, Candenas L, Pinto FM, Fernández-Sánchez M, González-Ravina C. Influence of BMI, Cigarette Smoking and Cryopreservation on Tyrosine Phosphorylation during Sperm Capacitation. Int J Mol Sci 2024 Jul 10;25(14).
- Shakeel M, Yoon M. Heat stress and stallion fertility. J Anim Sci Technol 2023 Jul;65(4):683-697.
- Felix MR, Turner RM, Dobbie T, Hinrichs K. Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†. Biol Reprod 2022 Dec 10;107(6):1551-1564.
- Carro MLM, Ramírez-Vasquez RRA, Peñalva DA, Buschiazzo J, Hozbor FA. Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility. Front Cell Dev Biol 2021;9:660165.
- Kato Y, Kumar S, Lessard C, Bailey JL. ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model. PLoS One 2021;16(6):e0251973.
- Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE. Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. Animals (Basel) 2021 Jan 3;11(1).
- 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).
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- Martínez-Fresneda L, Castaño C, Bóveda P, Tesfaye D, Schellander K, Santiago-Moreno J, García-Vázquez FA. Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon). Sci Rep 2019 Oct 30;9(1):15659.
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- Tannert A, Kurz A, Erlemann KR, Müller K, Herrmann A, Schiller J, Töpfer-Petersen E, Manjunath P, Müller P. The bovine seminal plasma protein PDC-109 extracts phosphorylcholine-containing lipids from the outer membrane leaflet. Eur Biophys J 2007 Apr;36(4-5):461-75.
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