Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa.
Abstract: Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.
Publication Date: 1996-11-01 PubMed ID: 9016398
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- 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 focuses on using an ingredient called peanut agglutinin (PNA) to study the changes occurring in horse sperm’s acrosome, a part of the sperm responsible for breaking the outer layer of the egg cell, during interaction with the female egg’s outer coating.
Understanding the Acrosomal Status
- The investigators used horse sperm for this study, utilizing peanut agglutinin (PNA), a substance known to bind specifically with the acrosomal membrane of horse sperm.
- The researchers used Fluorescein isothiocyanate-PNA (FITC-PNA) staining, which glows under a certain wavelength of light, to track changes in the sperm acrosomal during an extended incubation period of 24 hours and a brief 2-hours exposure to calcium ionophore A23187, a substance known to trigger the acrosome reaction.
Evaluating Sperm and Egg Interaction
- Sperm samples were kept in SP-TALP medium—a synthetic fluid mimicking oviductal fluid—for four hours, before being incubated with hemizonae, halves of the zona pellucida, the outer covering of the female egg. This experiment creates a setting where the sperm interacts with the outer lining of the egg cell, enabling the researchers to assess changes occurring in the acrosome.
Method of Acrosome Assessment
- The sperm samples and the sperm-hemizona complexes were fixed on microscope slides. They were then permeabilized, a process that allows staining solutions to penetrate cells, and stained with FITC-PNA. To identify the DNA present, they were counter-stained with ethidium homodimer, a DNA dye.
- Both sperm cells attached to the zona and those not bound showed similar staining results. Intact acrosomes appeared bright green, those undergoing reaction showed disruption in fluorescence. Sperms with completed acrosome reaction were principally stained on their equatorial segment or were not stained at all.
Acrosome Reaction Observation
- It was noticed during the extended incubation period and the calcium ionophore treatment, that the proportion of sperms exhibiting an acrosomal modification increased significantly. Also, complete breakdown of the acrosome increased noticeably.
- There was a noteworthy induction of acrosome reaction observed within 60 minutes of the sperm contacting the zona pellucida.
Significance of the Study
- This study provides a fast and reliable method of assessing the acrosomal status and reaction frequency of horse sperm as it interacts with the zona pellucida, the outer layer of the female egg cell.
Cite This Article
APA
Cheng FP, Fazeli A, Voorhout WF, Marks A, Bevers MM, Colenbrander B.
(1996).
Use of peanut agglutinin to assess the acrosomal status and the zona pellucida-induced acrosome reaction in stallion spermatozoa.
J Androl, 17(6), 674-682.
Publication
Researcher Affiliations
- Department of Herd Health & Reproduction, Veterinary Faculty, Utrecht University, The Netherlands.
MeSH Terms
- Acrosome / chemistry
- Acrosome / physiology
- Animals
- Arachis
- Binding Sites / drug effects
- Binding Sites / physiology
- Calcimycin / pharmacology
- Calcium / metabolism
- Female
- Fluorescein-5-isothiocyanate
- Horses
- Ionophores / pharmacology
- Lectins
- Male
- Peanut Agglutinin
- Plant Lectins
- Sperm-Ovum Interactions / physiology
- Spermatozoa / chemistry
- Spermatozoa / physiology
- Spermatozoa / ultrastructure
- Staining and Labeling
- Time Factors
- Zona Pellucida / physiology
Citations
This article has been cited 31 times.- Egyptien S, Dewals B, Ectors F, Brutinel F, Ponthier J, Deleuze S. Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.. Animals (Basel) 2023 Jun 4;13(11).
- Gardner CC, James PF. The SLC9C2 Gene Product (Na(+)/H(+) Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.. Int J Mol Sci 2023 Mar 10;24(6).
- Xu K, Su X, Fang K, Lv Y, Huang T, Li M, Wang Z, Yin Y, Muhammad T, Liu S, Chen X, Jiang J, Li J, Chan WY, Ma J, Lu G, Chen ZJ, Liu H. The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.. Elife 2023 Mar 21;12.
- Colombo M, Morselli MG, Zahmel J, Luvoni GC. Ultra-Rapid Freezing Preserves Morphofunctional Integrity and Fertilizing Ability of Epididymal Cat Spermatozoa.. Front Vet Sci 2022;9:866953.
- Yang Y, Suwimonteerabutr J, Angkawanish T, Chatdarong K. Serum Insulin-like Growth Factor-1 Is a Biomarker of Testosterone Production and Intact Acrosome in Asian Elephants (Elephas maximus).. Animals (Basel) 2022 Jun 17;12(12).
- Colombo M, Morselli MG, Franchi G, Schäfer-Somi S, Luvoni GC. Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport.. Animals (Basel) 2022 Mar 23;12(7).
- Yuan H, Sun J, Wang S, Xiang Z, Yang F, Yan Y, Duan Y, Li L, Wu X, Si W. Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey.. J Cell Mol Med 2022 Mar;26(5):1567-1578.
- Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I.. Animals (Basel) 2021 Nov 13;11(11).
- Dos Santos DP, Ribeiro DF, Frigoli GF, Erthal RP, da Silva Scarton SR, de Lion Siervo GEM, Seiva FRF, Staurengo-Ferrari L, Verri WA Jr, Deminice R, Fernandes GSA. Voluntary Exercise Attenuates Hyperhomocysteinemia, But Does not Protect Against Hyperhomocysteinemia-Induced Testicular and Epididymal Disturbances.. Reprod Sci 2022 Jan;29(1):277-290.
- Bhandari S, Sharma J, Rizal S, Yi YJ, Manandhar G. Artemisia vulgaris extract causes precocious acrosome reaction and viability loss but low rate of membrane damage in mouse spermatozoa.. J Anim Sci Technol 2021 Jan;63(1):58-68.
- Hitit M, Ugur MR, Dinh TTN, Sajeev D, Kaya A, Topper E, Tan W, Memili E. Cellular and Functional Physiopathology of Bull Sperm With Altered Sperm Freezability.. Front Vet Sci 2020;7:581137.
- Kang W, Harada Y, Yamatoya K, Kawano N, Kanai S, Miyamoto Y, Nakamura A, Miyado M, Hayashi Y, Kuroki Y, Saito H, Iwao Y, Umezawa A, Miyado K. Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.. Lab Invest 2020 Apr;100(4):583-595.
- Baggelaar MP, den Dulk H, Florea BI, Fazio D, Bernabò N, Raspa M, Janssen APA, Scavizzi F, Barboni B, Overkleeft HS, Maccarrone M, van der Stelt M. ABHD2 Inhibitor Identified by Activity-Based Protein Profiling Reduces Acrosome Reaction.. ACS Chem Biol 2019 Oct 18;14(10):2295-2304.
- Hara-Yokoyama M, Kurihara H, Ichinose S, Matsuda H, Ichinose S, Kurosawa M, Tada N, Iwahara C, Terasawa K, Podyma-Inoue KA, Furukawa K, Iwabuchi K. KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.. J Histochem Cytochem 2019 Nov;67(11):813-824.
- Mundt N, Spehr M, Lishko PV. TRPV4 is the temperature-sensitive ion channel of human sperm.. Elife 2018 Jul 2;7.
- Yu JF, Lai YH, Wang TE, Wei YS, Chang YJ, Li SH, Chin SC, Joshi R, Chang HW, Tsai PS. The effects of type I collagenase on the degelification of chimpanzee (Pan troglodytes) semen plug and sperm quality.. BMC Vet Res 2018 Feb 27;14(1):58.
- Liu J, Mochida K, Hasegawa A, Inoue K, Ogura A. Identification of quantitative trait loci associated with the susceptibility of mouse spermatozoa to cryopreservation.. J Reprod Dev 2018 Apr 13;64(2):117-127.
- Flegel C, Vogel F, Hofreuter A, Wojcik S, Schoeder C, Kieć-Kononowicz K, Brockmeyer NH, Müller CE, Becker C, Altmüller J, Hatt H, Gisselmann G. Characterization of non-olfactory GPCRs in human sperm with a focus on GPR18.. Sci Rep 2016 Aug 30;6:32255.
- Prapaiwan N, Tharasanit T, Punjachaipornpol S, Yamtang D, Roongsitthichai A, Moonarmart W, Kaeoket K, Manee-In S. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.. Asian-Australas J Anim Sci 2016 May;29(5):646-51.
- Park AK, Liegel RP, Ronchetti A, Ebert AD, Geurts A, Sidjanin DJ. Targeted disruption of Tbc1d20 with zinc-finger nucleases causes cataracts and testicular abnormalities in mice.. BMC Genet 2014 Dec 5;15:135.
- Liegel RP, Ronchetti A, Sidjanin DJ. Alkylglycerone phosphate synthase (AGPS) deficient mice: models for rhizomelic chondrodysplasia punctate type 3 (RCDP3) malformation syndrome.. Mol Genet Metab Rep 2014;1:299-311.
- Manee-In S, Parmornsupornvichit S, Kraiprayoon S, Tharasanit T, Chanapiwat P, Kaeoket K. L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility.. Asian-Australas J Anim Sci 2014 Jun;27(6):791-6.
- Aliabadi E, Karimi F, Talaei-Khozani T. Effects of L-carnitine and pentoxifylline on carbohydrate distribution of mouse testicular sperm membrane.. Iran J Med Sci 2013 Jun;38(2):107-15.
- Lin YW, Hsu TH, Yen PH. Mouse sperm acquire a new structure on the apical hook during epididymal maturation.. Asian J Androl 2013 Jul;15(4):523-8.
- Wu Y, Chen X, Wang S, Jiang M, Zheng B, Zhou Q, Bi Y, Zhou Z, Huang X, Sha J. Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis.. J Biomed Res 2012 Jul;26(4):278-87.
- Varesi S, Vernocchi V, Faustini M, Luvoni GC. Morphological and acrosomal changes of canine spermatozoa during epididymal transit.. Acta Vet Scand 2013 Feb 26;55(1):17.
- Chankitisakul V, Am-In N, Tharasanit T, Somfai T, Nagai T, Techakumphu M. Sperm pretreatment with dithiothreitol increases male pronucleus formation rates after intracytoplasmic sperm injection (ICSI) in swamp buffalo oocytes.. J Reprod Dev 2013;59(1):66-71.
- Hirohashi N, Gerton GL, Buffone MG. Video imaging of the sperm acrosome reaction during in vitro fertilization.. Commun Integr Biol 2011 Jul;4(4):471-6.
- Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA, Chiba K, Hirohashi N. Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization.. Proc Natl Acad Sci U S A 2011 Mar 22;108(12):4892-6.
- Chanapiwat P, Kaeoket K, Tummaruk P. Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.. Asian J Androl 2009 Sep;11(5):600-8.
- Katila T. In vitro evaluation of frozen-thawed stallion semen: a review.. Acta Vet Scand 2001;42(2):199-217.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
