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Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.ORCID-id: 0000-0002-0256-1958
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.ORCID-id: 0000-0003-0120-354X
Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.ORCID-id: 0000-0002-5194-2124
2016 (Engelska)Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, nr 5, s. 665-679Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Blackwell Verlag, 2016. Vol. 51, nr 5, s. 665-679
Nationell ämneskategori
Utvecklingsbiologi
Identifikatorer
URN: urn:nbn:se:liu:diva-130228DOI: 10.1111/rda.12728ISI: 000388334100005PubMedID: 27405395OAI: oai:DiVA.org:liu-130228DiVA, id: diva2:949479
Anmärkning

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

Tillgänglig från: 2016-07-19 Skapad: 2016-07-19 Senast uppdaterad: 2018-03-23Bibliografiskt granskad
Ingår i avhandling
1. Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening
Öppna denna publikation i ny flik eller fönster >>Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2016. s. 65
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1526
Nyckelord
Plasma membrane, membrane channels, membrane receptors, kinematics, 3Rprinciples, spermatozoa, boar.
Nationell ämneskategori
Cell- och molekylärbiologi Farmakologi och toxikologi Cellbiologi Farmaceutiska vetenskaper Biokemi och molekylärbiologi Veterinärmedicin
Identifikatorer
urn:nbn:se:liu:diva-131862 (URN)10.3384/diss.diva-131862 (DOI)9789176857267 (ISBN)
Disputation
2016-11-11, Berzeliussalen, Campus US, Linköping, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-10-11 Skapad: 2016-10-11 Senast uppdaterad: 2019-10-29Bibliografiskt granskad

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