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Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-0256-1958
Karlbergsvägen 83 B, Stockholm, Sweden.
AstraZeneca Research and Dev, England.
Karolinska Institute, Sweden; Karolinska Institute, Sweden.
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2015 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 29, no 3, 582-591 p.Article in journal (Refereed) Published
Abstract [en]

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker and JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P less than 0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r= 0.559) and proportions of motile (r = 0.55) or progressively motile (r = 0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

Place, publisher, year, edition, pages
Elsevier , 2015. Vol. 29, no 3, 582-591 p.
Keyword [en]
Sperm; Motility; Mitochondria; Drug; Toxicity; Boar
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-117655DOI: 10.1016/j.tiv.2015.01.004ISI: 000352050100019PubMedID: 25624015OAI: oai:DiVA.org:liu-117655DiVA: diva2:811497
Note

Funding Agencies|Swedish Research Council (VR); Research Council Formas, Stockholm, Sweden

Available from: 2015-05-12 Created: 2015-05-06 Last updated: 2016-10-11
In thesis
1. Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening
Open this publication in new window or tab >>Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. 65 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1526
Keyword
Plasma membrane, membrane channels, membrane receptors, kinematics, 3Rprinciples, spermatozoa, boar.
National Category
Cell and Molecular Biology Pharmacology and Toxicology Cell Biology Pharmaceutical Sciences Biochemistry and Molecular Biology Veterinary Science
Identifiers
urn:nbn:se:liu:diva-131862 (URN)10.3384/diss.diva-131862 (DOI)9789176857267 (ISBN)
Public defence
2016-11-11, Berzeliussalen, Campus US, Linköping, 09:00 (English)
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Available from: 2016-10-11 Created: 2016-10-11 Last updated: 2017-05-29Bibliographically approved

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