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Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0002-0256-1958
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 [en]
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: urn:nbn:se:liu:diva-131862DOI: 10.3384/diss.diva-131862ISBN: 9789176857267 (print)OAI: oai:DiVA.org:liu-131862DiVA: diva2:1034136
Public defence
2016-11-11, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2016-10-11 Created: 2016-10-11 Last updated: 2017-05-29Bibliographically approved
List of papers
1. Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa
Open this publication in new window or tab >>Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa
2016 (English)In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, no 5, 665-679 p.Article in journal (Refereed) Published
Abstract [en]

Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

Place, publisher, year, edition, pages
Blackwell Verlag, 2016
National Category
Developmental Biology
Identifiers
urn:nbn:se:liu:diva-130228 (URN)10.1111/rda.12728 (DOI)000388334100005 ()27405395 (PubMedID)
Note

Funding agencies: Swedish Research Council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Stockholm [221-2011-512]; FORSS (Forskningsradet i Sydostra Sverige), Sweden [473121]

Available from: 2016-07-19 Created: 2016-07-19 Last updated: 2017-11-28Bibliographically approved
2. The CatSper channel modulates boar sperm motility during capacitation.
Open this publication in new window or tab >>The CatSper channel modulates boar sperm motility during capacitation.
2017 (English)In: Reproductive biology, ISSN 2300-732X, Vol. 17, no 1, 69-78 p.Article in journal (Refereed) Published
Abstract [en]

The cation channel of sperm (CatSper) comprises four transmembrane subunits specifically expressed in human, equine, murine and ovine spermatozoa, apparently implicated in capacitation, hyperactivation and acrosome exocytosis. Western blotting and immunocytochemistry showed hereby that CatSper subunits are also present in boar spermatozoa, primarily over the sperm neck, tail and cytoplasmic droplets; albeit CatSper -1 presented in addition some distribution over the membrane of the acrosome and CatSper -2 and -4 over the membrane of the post-acrosome. The role of the Catsper channel in boar spermatozoa was investigated by extending the spermatozoa in media containing different calcium (Ca(2+)) availability and exposure to the capacitation-trigger bicarbonate, to progesterone or CatSper inhibitors (Mibefradil and NNC 55-0396), separately or sequentially, at physiological and toxicological doses. Extracellular Ca(2+) availability, combined with bicarbonate exposure (capacitation-inducing conditions) decreased sperm motility, similarly to when spermatozoa incubated in capacitation-inducing conditions was exposed to Mibefradil and NNC 55-0396. Exposure of these spermatozoa to progesterone did not cause significant changes in sperm motility and nor did it revert its decrease induced by CatSper antagonists. In conclusion, the CatSper channel regulates sperm motility during porcine capacitation-related events in vitro.

Place, publisher, year, edition, pages
Elsevier, 2017
Keyword
CatSper, Spermatozoa, Capacitation, Sperm motility, Boar
National Category
Biological Sciences
Identifiers
urn:nbn:se:liu:diva-134398 (URN)10.1016/j.repbio.2017.01.001 (DOI)000397370900009 ()28077244 (PubMedID)2-s2.0-85009212156 (Scopus ID)
Note

Funding agencies: The Swedish Research council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Stockholm [221-2011-512]; FORSS (Forskningsradet i Sydostra Sverige, Sweden [473121]

Available from: 2017-02-23 Created: 2017-02-23 Last updated: 2017-05-29Bibliographically approved
3. The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility
Open this publication in new window or tab >>The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility
2016 (English)In: Molecular Reproduction and Development, ISSN 1040-452X, E-ISSN 1098-2795, Vol. 83, no 8, 724-734 p.Article in journal (Refereed) Published
Abstract [en]

Endogenous and exogenous opioids modulate reproductive functions in target cells via opioid receptors (µ, δ, and κ). Sperm motility is a metric of gamete functionality, and serves as a suitable parameter for in vitro drug-induced toxicity assays. This study identifies the presence and location of opioid receptors in pig spermatozoa as well as their functional response after in vitro challenge with known agonists (morphine [µ]; [D-Pen 2,5]-enkephanile [δ]; and U 50488 [κ]) and antagonists (naloxone [µ]; naltrindole [δ]; and nor-binaltrorphimine [κ]). Only the µ- and δ-opioid receptors were present in the sperm plasma membrane, overlying the acrosome, neck, and principal piece. Challenge experiments with agonists and antagonists identified both µ- and δ-opioid receptors as regulators of sperm kinematics, wherein µ maintains or increases sperm movement whereas δ decreases sperm motility over time. This article is protected by copyright. All rights reserved.

Place, publisher, year, edition, pages
John Wiley & Sons, 2016
Keyword
opioids, membrane receptors, kinematics, pig
National Category
Developmental Biology
Identifiers
urn:nbn:se:liu:diva-130181 (URN)10.1002/mrd.22675 (DOI)000387014800007 ()27391529 (PubMedID)
Note

Funding agencies: Swedish Research Council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Sweden [221-2011-512]; Research Council in Southeast Sweden (FORSS), Sweden [378091/31297]

Available from: 2016-07-14 Created: 2016-07-14 Last updated: 2017-11-28Bibliographically approved
4. Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
Open this publication in new window or tab >>Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
Show others...
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
Keyword
Sperm; Motility; Mitochondria; Drug; Toxicity; Boar
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-117655 (URN)10.1016/j.tiv.2015.01.004 (DOI)000352050100019 ()25624015 (PubMedID)
Note

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

Available from: 2015-05-12 Created: 2015-05-06 Last updated: 2017-12-04

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