membrane stress during thawing elicits redistribution of … · 2017. 5. 22. · membrane stress...

Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of

Aquaporin 9 in Boar Spermatozoa

A Vicente-Carrillo, H Ekwall*, M �Alvarez-Rodr�ıguez and H Rodr�ıguez-Mart�ınez

Department of Clinical and Experimental Medicine, Link€oping University, Link€oping, Sweden

ContentsFreezing of boar spermatozoa includes the cryoprotectantglycerol, but renders low cryosurvival, owing to major changesin osmolarity during freezing/thawing. We hypothesize thataquaporins (AQPs) 7 and 9 adapt their membrane domainlocation to these osmotic challenges, thus maintaining spermhomeostasis.Western blotting (WB) and immunocytochemistry(ICC) at light and electron microscope levels with severalcommercial primary antibodies and protocols explored AQPlocation on cauda epididymal and ejaculated spermatozoa(from different fractions of the ejaculate), unprocessed,extended, chilled and frozen-thawed. Although differences inWB and ICC labelling were seen among antibodies, AQP-7 wasconspicuously located in the entire tail and cytoplasmic dropletin caudal spermatozoa, being restricted to the mid-piece andprincipal piece domains in ejaculated spermatozoa. AQP-9 wasmainly localized in the spermhead in both caudal and ejaculatedspermatozoa. While unaffected by chilling (+5�C), freezing andthawing of ejaculated spermatozoa clearly relocated the headlabelling ofAQP-7, but not that ofAQP-9. In vitromimicking ofcell membrane expansion during quick thawing maintained thelocalization of AQP-9 but relocated AQP-7 towards theacrosome. AQP-7, but not AQP-9, appears as a relevant markerfor non-empirical studies of sperm handling.

Introduction

Ejaculated boar spermatozoa sustain poorly conven-tional cryopreservation protocols (reviewed by Rodri-guez-Martinez and Wallgren 2010). These highlydifferentiated gametes are stored – following maturation– quiescent in the cauda epididymis, embedded in anacidic, hyperosmotic (above 390 mOsm/Kg) mediumwith a very low concentration of bicarbonate (10-foldlower than in blood plasma, Rodr�ıguez-Martinez 1991).At ejaculation, spermatozoa are released in caudal fluidalongside a dominating prostatic secretion in a 30–50 mlfraction called sperm rich, where the first 10 ml constitutethe sperm-peak portion of the ejaculate (Rodr�ıguez-Mart�ınez et al. 2009). A concomitant 10-fold (30 mM/l)increase in bicarbonate levels in seminal plasma (SP)induces major changes in sperm motility, which becomesactivated and progressive; osmolality becoming lower(approximately 300 mOsm/Kg). In the female, ejaculatedspermatozoa interact with a variety of environmentsduring their transport towards the site of fertilization,

bathing in fluids displaying varying osmolality and pH(Rodr�ıguez-Martinez 2007). Such environmental changesrequire spermatozoa to have an extremely active andreceptive plasmamembrane, whose stability and capacityto respond to various stimuli is crucial to allow them toundergo the pre-fertilization processes. Spermatozoa arealso collected for in vitro diagnosis and manipulation(extension, chilling, freezing/thawing, in vitro fertilization(IVF), intracytoplasmic sperm injection (ICSI), etc.),thus being exposed to a variety of exogenous artificialmedia. Although the media aim to mimic conditionsin vivo, changes in osmolarity, dehydration and cooling/warming are yet present.During sperm cooling and freezing at fairly low speeds,

water leaves the cells towards the extracellular milieu,while the opposite occurs during thawing, for examplemoving to the site with higher osmolarity (Ortman andRodriguez-Martinez 1994; Gao et al. 1997). These phe-nomena are less apparent when epididymal spermatozoaare processed (handled and cryopreserved, Watson 2000)or during sperm transport in vivo (Rodr�ıguez-Martinez2007). The high dehydration occurring during slowcooling accumulates solutes intracellularly (a cause forcell death), while the compensating quick transport ofwater during thawing causes a substantial rate of mem-brane and organelle damage that leads to impairment ofcell function, a reduced lifespan and, eventually, also celldeath (Ortman and Rodriguez-Martinez 1994).The main constituent of the extracellular media

surrounding spermatozoa is water. Thus, spermatozoaneed to control water transport rates across their cellmembrane to adapt to different osmotic pressures andmaintain homeostasis. Fluid flow across membranesoccurs through two main mechanisms: diffusion bypassive osmotic pressure and transport through waterchannels. Diffusion occurs through all biological mem-branes at relatively low velocity. Transport throughwater channels includes specific membrane proteins, theso-called aquaporins (AQPs) (Verkman et al. 1996;Borgnia et al. 1999; Izumi et al. 2005).The AQPs are divided into three families based on

functionality: one family including AQP-1, AQP-2,AQP-4, AQP-5 and AQP-8, which selectively transportwater; another including AQP-3, AQP-7, AQP-9 andAQP-10, which transport both water and glycerol(aquaglyceroporins), and possibly other small solutes(Wang et al. 2006) and finally, the subfamily of subcel-lular AQPs or super-aquaporins, which includes AQP-

The article was edited by Prof Emilio Arsenio Martinez-Garcia.

Reprod Dom Anim doi: 10.1111/rda.12728

ISSN 0936–6768

© 2016 Blackwell Verlag GmbH

11 and AQP-12 (Ishibashi et al. 2013). Aquaglycero-porins have been described in fat tissue, suggestinginvolvement in glycerol export during lipolysis, animportant and novel function for these polypeptides(Rodr�ıguez et al. 2006). As well, AQP-7 has been foundin human spermatozoa (Moretti et al. 2011) and in theseminiferous epithelium, suggested as associated withvolume regulation in germ cells (Yeung et al. 2010).AQP-7 has been immunocytochemically (ICC) detectedin boar spermatozoa using different commercial anti-bodies (Prieto-Mart�ınez et al. 2014; Vicente-Carrillo2015) and its role related to sperm quality (Prieto-Mart�ınez et al. 2014) and/or capacity to react underdifferent osmotic pressures (Vicente-Carrillo 2015). TheICC of the above studies depicted different localizationof AQP-7, perhaps due to differences in the specificity ofthe antibodies used, a matter for reported concernregarding AQPs (Yeung 2010). A thorough comparisonof available antibodies ought thus to be performed.AQP-9 has been found in liver, epididymis, testis,

spleen and brain (Schimming et al. 2015), as well as inleucocytes and the urinary and genital tract lining of themale (Da Silva et al. 2006). AQP-9 might be involved inthe regulation of the composition of the luminalenvironment where spermatozoa complete their matu-ration (Da Silva et al. 2006) and it has been demon-strated to be sex-linked, presenting a higher hepaticexpression in males than in female rats (Nicchia et al.2001). The presence of AQP-9 has not yet beendemonstrated in boar spermatozoa.Considering that the above aquaglyceroporins not

only transport water but also glycerol, the most com-monly used cryoprotectant agent (CPA) for freezingsemen (Wang et al. 2006), the hypothesis hereby testedwas that AQP-7 and AQP-9 are present in the plas-malemma of boar spermatozoa (retrieved from caudaepididymis or two ejaculate fractions) and that theirdomain localization accommodates to dramatic envi-ronmental challenges during cryopreservation. Themain objective of the study was to study the distributionof AQP-7 and AQP-9 in spermatozoa from differentorigins (epididymal, ejaculated, ejaculate fractions) andalongside cryopreservation. Since specificity of anti-AQP antibodies has been an issue among researchers(Yeung 2010), the study included ICC and WB com-parisons of a battery of commercial antibodies and

protocols, for AQP-7 (n = 3) and AQP-9 (n = 2),respectively.

Material and Methods

Experimental design

Spermatozoa from cauda epididymis and ejaculates(whole single sample, or from specific ejaculate fractions)were examined for the presence and temporal distribu-tion of AQP-7 and AQP-9 using western blotting (WB)and immunocytochemistry (ICC). To evaluate the rela-tive specificity of the antibodies against AQP-7 andAQP-9, a battery of different commercial antibodies wastested, three against AQP-7 [bs-2506 (Bioss Inc.,Woburn, MA, USA), NBP1-30862 (Novus Biologicals,Abingdon, UK) and ab15213 (Abcam, Cambridge, UK)]and two against AQP-9 [bs-2060 (Bioss Inc.) and NBP1-30865 (Novus Biologicals)] (for details of species speci-ficity and epitope binding location, see Table 1).Ejaculated spermatozoa were further studied at four

specific stages of cryopreservation: (i) +20–21°C (Roomtemperature (RT), extended in Beltsville thawing solu-tion (BTS), control samples), (ii) at +15°C (in LEYextender, see below), (iii) at +5°C (in LEYGO extender,see below) and (iv) post-thaw. Moreover, the cycle ofdehydration/rehydration occurring during freezing/thawing was mimicked in vitro via exposure to hyper-osmotic (1000 mOsm/Kg) and hypo-osmotic (75 and150 mOsm/kg) solutions.All experiments were performed in accordance with

relevant regulations (European Community Directive2010/63/EU) and compliance with Swedish currentlegislation (SJVFS 2012:26) at the Department ofClinical Sciences, Swedish University of AgriculturalSciences (SLU), Uppsala, and at the Department ofClinical and Experimental Medicine, Link€oping Univer-sity, Link€oping, Sweden. The experimental protocol hadpreviously been reviewed and approved by the LocalEthical Committee for Experimentation with Animals,at Uppsala (nr C-119-4) and Link€oping (nr 74-12),Sweden.

Animals and sperm collection

Mature boars (Swedish Landrace, Swedish Yorkshire,Swedish Hampshire) selected according to normal

Table 1. Reacting species and location of membrane site epitopes for each of the antibodies used against AQP-7 and AQP-9

Antibody Immunoreacts to

Target membrane site

Extracytoplasmic Transmembrane Intracytoplasmic

AQP-7 bs-2506 Human, mouse and rat Yes Yes No

NBP1-30862 Human and mouse No No Yes

ab15123 Rat No No Yes

AQP-9 bs-2060 Human, mouse and rat Yes No No

NBP1-30865 Human Yes No No

2 A Vicente-Carrillo, H Ekwall, M �Alvarez-Rodr�ıguez, and H Rodr�ıguez-Mart�ınez


semen quality and proven fertility and kept on strawbeds in individual pens at the Division of Reproduction,Swedish University of Agricultural Sciences (SLU),Uppsala (n = 4), the Centre for Biomedical Resources(CBR), Link€oping University, Link€oping (n = 5), orQuality Genetics (now Svenska K€ottf€oretagen, SvKF),H�allsta, Sweden (n = 3), were used. The animals werefed with commercial feedstuff (L€antm€annen, Stockholm,Sweden) according to national standards (Simonsson1994) and provided with water ad libitum.The studied spermatozoa were either collected from

the cauda epididymides or ejaculated. Caudal sperma-tozoa were retrieved post-mortem (n = 5) by aspirationfrom a sectioned cauda epididymis from animals thatalso donated ejaculates. Ejaculated spermatozoa weremanually collected (gloved-hand technique) from Swed-ish Landrace (n = 5), Yorkshire (n = 4) and Hampshire(n = 3) boars using a dummy, either as a wholeejaculate, as the sperm-rich fraction (SRF) or asseparated ejaculate fractions, on a weekly basis. Forsplit ejaculate collection, two portions were collected perejaculate: the first 10 ml of the SRF (sperm-peakportion, Portion 1, P1) and the rest of the ejaculate(Portion 2, P2) which was collected in a plastic bag, bothinside an insulated thermos flask. Sperm suspensionsfrom the SRF from three Hampshire breeding boarswere pooled, extended in Beltsville Thawing Solution(Pursel and Johnson 1975, BTS IMV-Technologies,L0Aigle, France) to a concentration of 20 millionspermatozoa/ml and sent to the laboratory as 24-h-oldcommercial artificial insemination (AI) semen doses. Inall cases, only ejaculates with at least 70% motile and75% morphologically normal spermatozoa immediatelyafter collection were used. At least three differentejaculates from each animal were used for theexperiments.

Boar Sperm Cryopreservation

Ejaculated spermatozoa of all sources were firstextended in BTS at various ratios (P1 or SRF= 1 : 3,P2 = 1 : 1) and cryopreserved following the protocol byEriksson and Rodr�ıguez-Mart�ınez (2000). The BTS-extended semen was centrifuged (300 9 g/10 min, roomtemperature, RT, 16–20�C) and the resulting pelletresuspended with a lactose-egg yolk (LEY) extender(80% 310 mM of b-lactose; 20% egg yolk v/v), to a finalconcentration of 1.5 9 109 spermatozoa/ml. The LEY-extended semen was cooled to +5°C for 2 h. Then, thesemen was slowly mixed with a third extender (LEYGO)consisting of 89.5 ml LEY extender, 9 ml glycerol and1.5 ml of Equex STM (Nova Chemicals Sales Inc.,Scituate, MA, USA) or Orvus WA (963–1000; Preserva-tion Equipment Ltd, Diss, UK), at a ratio of two partsof semen to one part of extender to reach a finalconcentration of 1 9 109 spermatozoa/ml and 3%glycerol rate. Spermatozoa were then packaged at+5°C in 0.5-mL plastic straws which were sealed withpolyvinyl chloride (PVC) powder and transferred to a

programmable freezer (IceCube 14 M-A; MinitubeInternational, Tiefenbach, Germany) set at +5�C. Thecooling/freezing rate was as follows: 6�C/min from +5 to�5�C, 1 min for crystallization and thereafter 60�C/minfrom �5�C to �140�C. The straws were then plungedinto liquid nitrogen (LN2, �196�C) for storage untilthawing for analyses. The straws from the same sourcewere thawed in circulating water at 50°C for 12 s andpulled to minimize interanimal variation after thawing.

In vitro sperm exposure to hyper/hypo-osmotic media

BTS-extended spermatozoa were exposed to glycerol-containing extender (LEYGO, hyperosmotic,1000 mOsm/Kg) and thereafter to hypo-osmotic solu-tions (150 mOsm/Kg, composed of 25 mM sodiumcitrate and 75 mM fructose; and 75 mOsm/Kg, com-posed of 12.5 mM sodium citrate and 37.5 mM fructose)controlled with a micro-osmometer (Fiske Assoc.,Norwood, MA, USA) for 30–60 min at 38�C and fixeduntil analysed (see below).

Western Blotting

Western Blotting (WB) of boar liver (positive control)was performed to test the specificity of the antibodiesused and test the presence of AQP-7 and AQP-9 in boarspermatozoa. Proteins were extracted from boar sper-matozoa at 48 million sperm concentration, incubatingthem with RIPA buffer (Sigma-Aldrich, Stockholm,Sweden) at 4�C for 40 min. Proteins from liver sampleswere extracted sonicating the samples immersed inRIPA buffer in a Skafte Medlab ultrasound bath(Bandelin Sonorex Digitec, Berlin, Germany) at35 kHz for 30 min in ice. Once the proteins wereextracted, the samples were centrifuged 13 000 9 g10 min and the supernatant was collected. Proteinquantification was done using the DC Protein assaykit (Bio-Rad, Hercules, CA, USA) according to man-ufacturer’s instructions and diluted to a final concen-tration of 25 lg protein/10 ll. These proteinsuspensions were denaturalized by heating them to+70�C for 10 min and aliquots of 10 ll of the proteinsuspension were loaded into a NuPAGE 4–12% Bis–Tris SDS-PAGE gel (Life Technologies, Carlsbad, CA,USA). Electrophoresis was run at 180 V for 90 min,followed by transfer to the proteins to a polyvinyldiflu-oride (PVDF) membrane (Invitrolon PVDF filter papersandwich, Life Technologies) for 90 min at 125 mA.Once the proteins were transferred to the membrane, itwas blocked for 1 h at RT with 5% bovine serumalbumin (BSA) in PBST and after three washes of 5 minin PBST incubated overnight at +4�C with a dilution 1/1000 of the primary antibodies. The day after, themembrane was washed three times in PBST andincubated for 60 min with a dilution 1/15000 of thesecondary antibody (anti-rabbit IRDye 800 CW; LI-COR Biosciences, Lincoln, NE, USA, for the polyclonalrabbit antibodies and anti-chicken IRDye 650; LI-COR

AQP-7 and AQP-9 During Boar Sperm Cryopreservation 3


Biosciences, for the polyclonal chicken antibodies)followed by extensive washing in PBST. The membraneswere scanned using the Odyssey CLx (LI-COR Bio-sciences) and images of the blottings were obtainedusing the IMAGE STUDIO 4.0 software (LI-COR Bio-sciences).

Immunocytochemistry

Spermatozoa from all samples described above werefixed in 4% paraformaldehyde at RT during 20 min.Cell suspensions were then centrifuged (1200 9 g/6 min) and the pellet resuspended in phosphate-bufferedsaline (PBS), pH 7.3 to prepare smears on poly-L-lysineslides (LSM, Thermo Scientific, Germany) or on poly-L-lysine-coated coverslips (SEM). The smears wereallowed to dry, washed three times for 5 min withPBS and covered (blocking) with 5% BSA (Sigma-Aldrich, Sweden) in PBS at 4�C for 2 h for LSM andwith 1% BSA in PBS for 10 min at RT for SEM. Afterthree washings of 5 min in PBS, the slides wereincubated with the primary antibodies against AQP-7or AQP-9 with 1% BSA overnight at +4�C (LSM) or atRT for 60 min (SEM) in a wet chamber. For LSMsamples, the antibodies were as follows: AQP-7: poly-clonal rabbit bs-2506 (diluted 1 : 75 in PBS with 1%BSA), AQP-7: polyclonal rabbit NBP1-30862 (diluted 1/500 in PBS with 1% BSA), AQP-7: chicken polyclonalab15123 (diluted 1/500 in PBS with 1% BSA); AQP-9:polyclonal rabbit bs-2060 (diluted 1 : 75 in PBS with1% BSA) and AQP-9: polyclonal rabbit NBP1-30865(diluted 1/300 in PBS with 1% BSA). For SEM samples,the antibodies (AQP-7: chicken polyclonal ab15123;AQP-9: rabbit polyclonal to bs-2060) were diluted1 : 500 and 1 : 75 with 1% BSA in PBS for AQP-7 or9, respectively. After incubation, the smears werewashed three times in PBS for 5 min each time beforeincubation with the secondary antibody (for LSM:polyclonal goat anti-rabbit Alexa Fluor 488, MolecularProbes, Invitrogen, Carlsbad, CA, USA, diluted 1/1000in PBS containing 1% BSA) at RT for 75 min indarkness and mounted with antifade reagent ProlongGold (Molecular Probes; Invitrogen). For SEM, afterseveral rinses in PBS, the smears were incubated indonkey anti-chicken immunoglobulin Y (IgY, H+L) forAQP-7 or donkey anti-rabbit IgG for AQP-9 conju-gated with 4-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1 : 100 inPBS with 1% BSA, at RT for 2 h. The smears wererinsed three times with PBS and finally in distilled waterbefore silver enhancement with IntenSE M (code RPN491; Amersham Biosciences, Buckinghamshire, UK) for5–7 min. After a final 10-min series of distilled waterwashings, the smears were air-dried overnight. Fromeach slide, round pieces of the glass and the incubatedsmear were cut out with a diamond pen and mounted ona SEM metal stub. Mounted smears were coated withplatinum/palladium, 60 mA for 30 s (208HR HighResolution Sputter Coater; Ted Pella, Redding, CA,

USA), to avoid charging of the sample. Negativecontrols for both LSM and SEM consisted in SRFspermatozoa where the incubation with the primaryantibody was omitted.One of the antibodies above listed for AQP-7 (NBP1-

30862) targeting the cytosol site of the membrane wasfurther ICC-tested on a pooled sperm suspension of theejaculates of three boars, comparing the above-described protocol of PFA-fixed/PFA-permeabilizedcells (Fox et al. 1985), with that described by Prieto-Mart�ınez and collaborators (Prieto-Mart�ınez et al.2014) using additional detergent permeabilization ofthe plasma membrane.The fluorescent ICC samples were examined on a

LSM 700 Zeiss confocal microscope (Carl Zeiss, Stock-holm, Sweden) and the images recorded using ZEN

Navigator software (Carl Zeiss). The SEM samples wereexamined on a JEOL SEM 6320F scanning electronmicroscope (Tokyo, Japan) using its backscatter detec-tor (BSD) and/or secondary electron detector (SED).The SEM images were recorded using Semafor (JeolScandinavia, Sundbyberg, Sweden).The labelling across different domains and the sperm

source was visually scored by one and the same operatorin order to provide an overall, albeit semiquantitative,view of the distribution of the LSM or SEM labelling forall antibodies used. The scoring for relative intensity ofthe immunolabelling was as follows: +++: high intensity,++: medium intensity, +: low intensity and -: no labelling.At least 200 cells were counted in each replicate. Labellingwas obvious in between 90 and 95% of the cells.

Assessment of sperm motility

Sperm motility was monitored using Qualisperm (Bio-phos SA, Lausanne, Switzerland) in all ejaculatedsamples and before cryopreservation and post-thawing.

Statistical analysis

Statistical analysis was performed in the R statisticalenvironment (RDevelopment Core Team,RFoundationfor Statistical Computing, Vienna, Austria; ISBN 3-900051-07-0). Analyses of sperm motility post-thawingdata were carried out using linear mixed-effects modelsand with transformation of data in case of not normaldistribution. Models were built with type of extender asfixedeffectsandejaculatesastherandompartofthemodel.Significant fixed effects were further analysed using mul-tiple comparisons of means with Tukey’s contrasts.

Results

The motility of the different ejaculated sperm samplesdecreased solely post-thaw

Initial total sperm motility was similar (ns) amongejaculated spermatozoa derived from the different por-tions or from the whole ejaculate (range, 76–84%).



Sperm motility was maintained during the differentextension steps (LEY, LEYGO) but decreased(p < 0.01) after thawing, yet yielding >40% of cryosur-vival.

Western blotting confirmed the presence of AQP-7 andAQP-9

Both aquaglyceroporins were detected in boar hepato-cytes and spermatozoa (Fig. 1) with all the antibodiestested, albeit displaying differences in band location. Assuch, both in liver and spermatozoa (see Table 2), thebs-2506 antibody detected AQP-7 as two bands of 25and 51 KDa; the NBP1 30862 antibody showed onlyone band at 55 KDa and the ab15123 antibody detectedtwo bands of 28 and 51 KDa. For AQP-9, the bs-2060antibody detected two bands of 28 and 51 KDa in liverand a band of 50 KDa in spermatozoa, while theantibody NBP1-30865 detected two bands of 37 and 38KDa in liver and a band of 51 KDa in spermatozoa.

Additional membrane permeabilization is not required forimmunodetection of epitopes located in the cytosolic sideof the plasma membrane

The commercial antibodies chosen for AQP-7 or AQP-9detection were expected to target epitopes located in theouter, the trans- or the cytosol sites of the membrane(see Table 1). One of these antibodies (NBP1-30862)targeting the cytosol site of the membrane was furthertested, using two protocols, one of them described byPrieto-Mart�ınez and collaborators (Prieto-Mart�ınezet al. 2014) using additional detergent permeabilizationof the plasma membrane and the other using PFA-fixed

cells (see Material and Methods), since the fixation initself is proven permeabilizing (Fox et al. 1985). Therewere no differences in AQP-7 immunolabelling (LSM)between protocols, with the labelling always located inthe membrane of the sperm mid-piece (see Figs 2a and2b,).

Evaluation of the relative distribution and specificity ofthe antibodies against AQP-7 and AQP-9

Table 3 provides a summary of the comparative scoringof the relative immunolabelling intensity of AQP-7 andAQP-9 as monitored with LSM or SEM with all testedantibodies on spermatozoa from different sources (epi-didymal vs ejaculated, whole ejaculate vs portions P1 orP2) and stages of cryopreservation/thawing.The antibodies for AQP-7 or AQP-9 immunolabelled

different bands in the WB analysis (Fig. 1) and differentmembrane domains at ICC (Figs 3, 4, 5, 6 and 7).AQP-7 was ICC-detected in the membrane over theprincipal piece with the bs-2506 antibody (Fig. 3), whileit was detected in the plasma membrane of the mid-piecewith the NBP1-30862 (Fig. 4) and the ab15123 (Fig. 5)

(a) (b)

(d) (e)

(c)

Fig. 1. Controls of specificity andcell presence of the antibodies usedagainst AQP-7 and AQP-9. L1:protein ladder; L2: pig liver; L3:spermatozoa. (a) AQP-7 (primaryantibody: bs-2506), detected as twobands of 25 and 51 KDa both inliver and spermatozoa. (b) AQP-7(primary antibody: NBP1-30862),detected as one band of 55 KDaboth in liver and spermatozoa. (c)AQP-7 (primary antibody:ab15123), detected as two bands of28 and 51 KDa both in liver andspermatozoa. (d) AQP-9 (primaryantibody: bs-2506), detected as twobands of 28 and 51 KDa in liverand a band of 50 KDa inspermatozoa. (e) AQP-9 (primaryantibody: NBP1-30865), detectedas two bands of 37 and 38 KDa inliver and a band of 51 KDa inspermatozoa

Table 2. Band patterns of WB for each antibody used

Primary antibody Bands in liver Bands in spermatozoa

AQP-7 bs-2506 25 and 51 KDa 25 and 51 KDa

NBP1-30862 55 KDa 55 KDa

ab15123 28 and 51 KDa 28 and 51 KDa

AQP-9 bs-2060 28 and 51 KDa 50 KDa

NBP1-30865 37 and 38 KDa 51 KDa



antibodies. In post-thawed spermatozoa, all three anti-bodies showed the AQP-7 was relocated towards themembrane over the acrosome (Figs 3h, 4h and 5h) a

relocation confirmed by SEM (Fig. 8), using the anti-body ab15123. AQP-9 was detected on the membraneover the acrosome with the antibody bs-2060 (Fig. 6) or

(a) (b)

Fig. 2. Comparison of immunolabelling of AQP-7 using the antibody NBP1-30862 with and without extra permeabilization. Bar: 10 lm. (a)Using the protocol described in Material and Methods section; (b) using the protocol described by Prieto-Mart�ınez and collaborators (Prieto-Mart�ınez et al. 2014), where extra permeabilization of the cell membrane was applied

Table 3. Relative intensity of the immunolabelling of aquaporins 7 and 9 in boar spermatozoa from different sources (epididymal v. ejaculateportions P1 and P2) and stages of a conventional process of cryopreservation for all and each of the antibodies used: a) AQP-7, primary antibody:bs-2506; b) AQP-7, primary antibody: NBP1-30862; c) AQP-7, primary antibody: ab15123; d) AQP-9, primary antibody: bs-2506; e) AQP-9,primary antibody: NBP1-30865

Sperm Domain

Acrosome Post-acrosome Cytoplasmic droplet Mid-piece Principal piece End piece

AQP-7 Epididymal spermatozoa (cauda) �a,b,c �a,c

+++b+++a,b,c +++a,b,c ++a,c

�b

+a

�b,c

Fresh ejaculated spermatozoa P1 �a,b,c �a,c

+++b�a,b,c +a

++b,c+a

�b,c

�a,b

P2 �a,b,c �a,c

+++b�a,b,c +a

++b,c+a

�b,c

�a,b

Stages of cryopreservation BTS-extended RT �a,b,c �a,c

+++b�a,b,c +a

++b,c+a

�b,c

�a,b

LEY 15�C �a,b,c �a,c

++b�a,b,c ++a,b

+++c+a

�b,c

�a,b

LEYGO 5�C �a,b,c �a,c

+b�a,b,c +a,b

+++c+a

�b,c

�a,b

Post-thaw +++a,b,c �a,b,c �a,b,c �a,b

++c�a

�b,c

�a,b

AQP-9 Epididymal spermatozoa (cauda) +++d

�e

�d

+++e+d,e �d,e �d,e �d,e

Fresh ejaculated spermatozoa P1 +++d

�e

�d

+++e�d �d,e �d,e �d,e

P2 +++d

�e

�d

+++e�d �d,e +d

�e

�d,e

Stages of cryopreservation BTS-extended RT +++d

�e

�d

+++e�d �d,e +d

�e

�d,e

LEY 15�C +++d

�e

�d

+++e�d �d

+e�d,e �d,e

LEYGO 5�C +++d

�e

�d

+++e�d �d

+e�d,e �d,e

Post-thaw +++d

�e

�d

+++e�d �d

+e+d

�e

�d,e

BTS, Beltsville thawing solution; RT, room temperature.

For details on the composition of extenders LEY and LEYGO, as well as the ejaculate portions (P1 and P2) and stages of freezing/thawing, see ‘Animals and semen

collection’. Scoring for relative intensity of the immunolabelling: +++: high intensity, ++: medium intensity, +: low intensity, �: no labelling.



over the post-acrosomal domain (Fig. 7) if using theantibody NBP1-30865. This particular AQP-9 domainlocation was maintained after thawing (Figs 6h and 7h,and confirmed by SEM, bs-2060, Fig. 9).

Immunolabelling for AQP-7 and AQP-9 on spermatozoafrom different sources

AQP-7 location not only varied with the source ofspermatozoa but also with the antibodies used. Among

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 3. Immunolabelling of AQP-7 in boar spermatozoa using the primary antibody bs-2506, LSM. Bar: 10 lm. (a) Negative control (ejaculated,BTS-extended boar spermatozoa), (b) caudal spermatozoa, (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d) of the ejaculate,(e–h) ejaculated spermatozoa at different stages of cryopreservation, (e) extended in BTS at RT, (f) extended in LEY at +15�C, (g) extended inLEYGO at +5�C, (h) post-thawing. The arrows show the staining outside the spermatozoa

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 4. Immunolabelling of AQP-7 in boar spermatozoa using the primary antibody NBP1-30862, LSM. Bar: 10 lm. (a) Negative control(ejaculated, BTS-extended boar spermatozoa). (b) Caudal spermatozoa (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d) of theejaculate, (e–h) ejaculated spermatozoa at different stages of cryopreservation; (e) extended in BTS at RT; (f) extended in LEY at +15�C; (g)extended in LEYGO at +5�C; (h) post-thawing



cauda epididymal spermatozoa, it was conspicuouslylocalized to the membrane domain over the entire tailand the cytoplasmic droplets (antibody bs-2506,Fig. 3b), in the membrane over the post-acrosome andcytoplasmic droplets (antibody NBP1-30862, Fig. 4b)or in the plasmalemma over the cytoplasmic droplet andthe principal piece of the tail (antibody ab15123,Fig. 5b). Among ejaculated spermatozoa, the AQP-7immunostaining was restricted to the principal piecewith the antibody bs-2506, the membrane over the post-acrosome domain with the antibody NBP1-30862 or themembrane over the mid-piece with the antibodyab15123, irrespective of the portion of the ejaculatethe spermatozoa were collected from (Figs 3 c–d, 4c–dand 5c–d). A comparative view for AQP-9 is shown inFigures 6 b–d and 7b–d, where the immunolabellingwas detected in the proximal or distal cytoplasmicdroplets in epididymal spermatozoa (Figs 6b and 7b).Among ejaculated spermatozoa, it evidently covered theacrosome area in either ejaculate portion when using theantibody bs-2060 (Fig. 6c–d) while for the antibodyNBP1-30865, AQP-9 was detected over the post-acrosome domain (Fig. 7c–d).Interestingly, the P1-sperm samples showed a weak

immunolabelling around the spermatozoa, presumablyin the surrounding SP (Figs 3c, 4c, 5c, 6c, 7c, 8c and 9c),while spermatozoa from P2 depicted a heavier back-ground labelling, both in LSM (Figs 3d, 4d, 5d, 6d and7d) and SEM (Figs 8d and 9d).In order to confirm the LSM observations, SEM was

performed for AQP-7 using the antibody ab15123 andfor AQP-9 using the antibody bs-2060. Figures 8 and 9

depict the ultrastructural (SEM) location of AQP-7 andAQP-9 in spermatozoa retrieved from different sources(cauda epididymides or ejaculate/portions P1 and P2),confirming the LSM location already described. Nega-tive controls (primary antibody excluded) showed nolabelling anywhere on the spermatozoa nor the sur-rounding extracellular area (Figs 8a and 9a for AQP-7and AQP-9, respectively). The AQP-7 labelling dis-tinctly appeared in the plasmalemma over the tail(Fig. 8c–d), particularly in epididymal samples (Fig. 8b)where cytoplasmic droplets, either proximal or distal,were strongly labelled. The AQP-9 labelling wasobserved in the membrane covering the acrosome bothin ejaculated and caudal spermatozoa, which includedthe cytoplasmic droplets (Fig. 9b–e).

Freezing/thawing modifies the relative labelling intensityof sperm AQPs: AQP-7, but not AQP-9, relocatestowards the rostral head membrane at thawing

Cooling and subsequent freezing and thawing of theejaculated spermatozoa modified, stepwise, the mem-brane immunolabelling of AQP-7 as shown by theantibodies bs-2506, NBP1-30862 and ab15123 for LSM,and ab15123 for SEM. After extension in BTS (roomtemperature (RT), Figs 3e, 4e and 5e), and at +15°C (inextender LEY, Figs 3f, 4f and 5f), the immunolabellingwas mainly restricted to the membrane covering thesperm principal piece for the antibody bs-2506, or tothe membrane over the mid-piece for the antibodiesNBP1-30862 and ab15123. However, while cooled(+5°C), extended in glycerol-containing media

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 5. Immunolabelling of AQP-7 in boar spermatozoa using the primary antibody ab15123, LSM. Bar: 10 lm. (a) Negative control(ejaculated, BTS-extended boar spermatozoa); (b) caudal spermatozoa; (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d) of theejaculate; (e–h) ejaculated spermatozoa at different stages of cryopreservation; (e) extended in BTS at RT; (f) extended in LEY at +15�C; (g)extended in LEYGO at +5�C; (h) post-thawing



(LEYGO, Figs 3g, 4g and 5g) and post-thaw (Figs 3h,4h and 5h), the labelling was observed over the mid-piece for all antibodies (Figs 3g, 4g and 5g), but also

over the acrosome when thawed (Figs 3h, 4h and 5h).Such relocation of the labelling from mid-piece and necktowards the rostral/acrosomal domain was evident as a

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 7. Immunolabelling of AQP-9 in boar spermatozoa using the primary antibody NBP1-30865, LSM. Bar: 10 lm. (a) Negative control(ejaculated, BTS-extended boar spermatozoa); (b) caudal spermatozoa; (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d) of theejaculate; (e–h) ejaculated spermatozoa at different stages of cryopreservation; (e) extended in BTS at RT; (f) extended in LEY at +15�C; (g)extended in LEYGO at +5�C; (h) post-thawing

(a) (b) (c) (d)

(e) (f) (g) (h)

Fig. 6. Immunolabelling of AQP-9 in boar spermatozoa using the primary antibody bs-2060, LSM. Bar: 10 lm. (a) Negative control (ejaculated,BTS-extended boar spermatozoa); (b) caudal spermatozoa; (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d) of the ejaculate;(e–h) ejaculated spermatozoa at different stages of cryopreservation; (e) extended in BTS at RT; (f) extended in LEY at +15�C; (g) extended inLEYGO at +5�C; (h) post-thawing. The arrows show the staining outside the spermatozoa



(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Fig. 8. Representative scanning electron micrographs of boar spermatozoa subjected to immunolabelling with AQP-7. The primary antibodyused was ab15123. Labelling is marked with arrows in Figure 1 b. Bar: 1 lm. (a) Control sample, primary antibody excluded; (b) spermatozoonfrom cauda epididymis, note the labelling over the plasma membrane in the mid-piece (mp) and cytoplasmic droplet (cd); (c–d) spermatozoacollected in Portion 1 (P1, c) or Portion 2 (P2, d) of the ejaculate, note the change in labelling in the surrounding seminal plasma, stronger in P2;(e–h) ejaculated spermatozoa at different stages of cryopreservation; (e) extended in BTS at RT; (f) extended in LEY at +15�C; (g) extended inLEYGO at +5�C; (h) post-thawing. Note the very distinct labelling over the plasmalemma in the acrosome (*) as well as the maintained labellingover the plasmalemma in the mid-piece (mp)

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Fig. 9. Representative scanning electron (SEM) micrographs of boar spermatozoa subjected to immunolabelling with AQP-9. The primaryantibody used was bs-2060. Bar: 1 lm. (a) Control sample, primary antibody excluded; (b) spermatozoon from cauda epididymis, labelled forAQP-9 over the plasma membrane in the acrosome and cytoplasmic droplet; (c–d) spermatozoa collected in Portion 1 (P1, c) or Portion 2 (P2, d)of the ejaculate, note the change in labelling in the surrounding seminal plasma, stronger in P2; (e–h) ejaculated spermatozoa at different stages ofcryopreservation; (e) extended in BTS at RT (f) in LEY at +15�C; (g) extended in LEYGO at +5�C; (h) post-thawing. Note that the labelling ismaintained over the plasmalemma in the acrosome



very intense immunolabelling at LSM (Fig. 3h, 4h, 5h)or SEM (Fig. 8e–h). On the other hand, the cryopreser-vation protocol did not apparently modify the distribu-tion of AQP-9 (LSM: bs-2060 and NBP1-30865,Figs 6e–h and 7e–h; SEM: bs-2060 Fig. 9e–h). Yet,the specific AQP-9 location varied with the antibodyused, particularly in relation to the acrosomal (bs-2060,Fig. 6e–h) and post-acrosomal domains (NBP1-30865Fig. 7e–h), throughout all cryopreservation steps.

In vitro exposure to specific hypo- or hyperosmotic milieumodifies the location of immunolabelling of AQP-7 butnot of AQP-9

For this experiment, only the antibodies bs-2506 forAQP-7 and bs-2060 for AQP-9 were used. Exposure ofejaculated spermatozoa to a hyperosmotic solution(LEYGO extender) did not modify the location ofAQP-7 or AQP-9 compared to control (BTS-extendedejaculated spermatozoa), as shown for AQP-7 (Figs 3e,3g, 8e and 8g) or AQP-9 (Figs 7e, 7g, 9e and 9g).However, when LEYGO-extended spermatozoa wereexposed to hypo-osmotic media, both AQP-7 and AQP-9 were mainly located in the plasma membrane over theprincipal piece and the acrosome (Figs 10 and 11).Sperm exposure to the 150 mOsm/Kg hypo-osmoticsolution) for 30 or 60 min changed the location of theAQP-7 to the plasmalemma over the acrosome com-pared to control (BTS, isosmotic, 320 mOsm/Kg) irre-spective of exposure time (Fig. 10c–f). AQP-9 showedstaining on the supra-acrosome domain on all solutionsand exposure times (Fig. 11). Yet, the tail domain wasAQP-9-labelled in the 75 mOsm/Kg solution but not inthe 150 mOsm/Kg solution or the BTS control(Fig. 11c–f).

Discussion

During their lifespan, mature spermatozoa interact witha variety of extracellular fluids, starting with the caudaepididymis where they are stored before ejaculation, theseminal plasma after ejaculation and with the intralu-minal fluid in different segments of the internal femalegenital tract during sperm transport post-deposition(Rodr�ıguez-Mart�ınez et al. 2005). In addition, sperma-tozoa are exposed to a variety of artificial media andenvironments for assisted reproduction techniques (AI,IVF, ICSI, etc.), that requires them to have an extremelyactive and receptive plasma membrane and to respond,without bursting, to stimuli exerted by extension,cooling or warming, yet maintaining fertilizing capacity.Water is the main constituent of the extracellular fluidsspermatozoa interact with. Thus, spermatozoa need tocontrol water exchange with the extracellular fluids toadapt to different osmotic pressures (Chen and Duan2011).Boar spermatozoa are well known for not surviving

conventional cryopreservation well (Rodriguez-Marti-nez 2012). Due to high dehydration during the

customary slow cooling and the quick rehydrationduring thawing, defective transmembranous watertransport causes a substantial rate of membrane andorganelle damage that leads to impairment of cellfunction, a reduced lifespan and, eventually, cell death(Ortman and Rodriguez-Martinez 1994). Sperm mem-brane reactivity can easily be tested by in vitro challengewith hypo-osmotic solutions (Petrunkina et al. 2007),including those spermatozoa that sustain freezing and,particularly, thawing.During freezing, water will first abandon the sperm

cytoplasm in response to exposure to freezing extenderswith high extracellular osmolarity owing to the use ofCPA such as the penetrating glycerol (Chen and Duan2011), most commonly used for cryopreservation ofboar spermatozoa (Ekwall et al. 2007). Water will thuschange from liquid to solid state and form ice crystals inthe extracellular space, increasing even more the osmo-larity of the extracellular space (Storey and Storey2013). Since glycerol is toxic, a controlled flow of thismolecule over the plasma membrane is required in orderfor the cells to survive, a matter that is controllable atlow (5°C) temperature. When thawing, ice crystals willmelt and water became liquid again, quickly reducingthe osmolarity of the extracellular media and logicallyentering the cytoplasm to dislocate the glycerol, whichwill move again to the extracellular space (Rodriguez-Martinez and Wallgren 2010). Although the processtends to reach an equilibrium, thawing is quick(>1000°C per minute) and for a moment, the plasmamembrane dramatically expands, risking breakage thatundoubtedly leads to sperm death (Mazur 1984).The control of water flow through the plasma

membrane is regulated via AQPs (Izumi et al. 2005).During sperm cryopreservation, AQPs will also regulateglycerol flow (Chen and Duan 2011). The bidirectionalwater movement through the AQP-7 channel suggests itmodulates water flow during cryopreservation, since itallows water to permeate beyond the levels permeatedthrough the plasma membrane owing to its lipidcomposition (Suzuki-Toyota et al. 1999).The present results clearly indicate that AQP-7 and

AQP-9 are present in boar spermatozoa. AQP-7 wasmainly present in the sperm tail, the segment locationdiffering with the antibody used (bs-2506: principalpiece; NBP1-30862 and ab15123: mid-piece). AQP-9was mainly present in the sperm head, although withsubtle differences depending on the antibody used (bs-2060: acrosome; NBP1-30865: post-acrosome domain).A relevant observation is that the distribution of AQP-7appears in the sperm areas where more intracellularwater is present (Ekwall et al. 2007) and where the celldehydrates the most during freezing and rehydratesmost while thawing (exposure to hypo-osmotic media,Jeyendran et al. 1992).Specificity of anti-AQP antibodies has been an issue

among researchers (Yeung 2010). Differences amongantibodies have already been demonstrated in humanspermatozoa, where AQP-7 has been identified in



(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

Fig. 10. Immunolabelling of AQP-7 when exposed to hyper- or hypo-osmotic conditions at 38�C. Bar: 10 lm. Left column: 30 min ofincubation. Right column: 60 min of incubation. The primary antibodyused was bs-2506. (a–b) Control, BTS-extended; (c–d) BTS-extended +75 mOsm/Kg; (e–f) BTS-extended + 150 mOsm/Kg; (g–h) LEYGO-extended + 75 mOsm/Kg; (i-j) LEYGO-extended + 150 mOsm/Kg

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

Fig. 11. Immunolabelling of AQP-9 when exposed to hyper- or hypo-osmotic conditions at 38�C. Bar: 10 lm. Left column: 30 min ofincubation. Right column: 60 min of incubation. The primaryantibody used was bs-2060; (a–b) Control, BTS-extended; (c–d)BTS-extended + 75 mOsm/Kg; (e–f) BTS-extended + 150 mOsm/Kg.(g–h) LEYGO-extended + 75 mOsm/Kg; (i-j) LEYGO-extended +150 mOsm/Kg



different regions by either of two research groups(Yeung et al. 2010; Moretti et al. 2011). Such scenariocalled for a comparative use of various antibodies, as itwas performed in the present study where a series of fivecommercial antibodies were tested for ICC and WBfollowing one and the same protocol. In boar semen,Prieto-Mart�ınez and collaborators (Prieto-Mart�ınezet al. 2014) found AQP-7 immunolabelling on themembrane over the sperm neck, while we identifiedAQP-7 over the plasmalemma of the mid-piece using thesame commercial antibody for AQP-7 (NBP1-30862), alocation also detected using other commercial antibody(ab15123). These findings indicate that all antibodies, inour hands, depicted a similar ICC and WB, leaving theresults of Prieto-Mart�ınez and collaborators (Prieto-Mart�ınez et al. 2014) unconfirmed, despite testing thesame antibody. Furthermore, as shown in Figure 2,there were no obvious differences in the location ofAQP-7 on spermatozoa from the same boars eithersimply fixed with 4% PFA (see Material and Methods),a procedure that increases membrane permeability (Foxet al. 1985), or after application of additional detergent-driven permeabilization (Prieto-Mart�ınez et al. 2014).Since the results evidently showed no ICC differencesdespite use of additional permeabilization with deter-gents, it might be wise to avoid such extra permeabi-lization (as applied in Prieto-Mart�ınez et al. 2014) ifin vivo situations are to be mimicked, as we report in thepresent study. It remains to disclose the nature of theICC results shown by Prieto-Mart�ınez and collaborators(Prieto-Mart�ınez et al. 2014). We were not able torepeat their findings, even following their protocol.In addition of the different localization of AQP-7 and

AQP-9 found with each antibody, the WB also revealeddifferent band patterns for each antibody. For instance,the bs-2506 antibody detected AQP-7 as two bands of25 and 51 KDa, the NBP1 30862 antibody showed onlyone band at 55 KDa and the ab15123 antibody detectedtwo bands of 28 and 51 KDa. For AQP-9, the bs-2060antibody detected a band of 50 KDa, while the antibodyNBP1-30865 detected a band of 51 KDa in spermato-zoa. Such results confirm the differences in specificityamong the available commercial antibodies for AQP-7and AQP-9.A change in domain localization alongside the cryop-

reservation various steps was observed for AQP-7 butnot for AQP-9, where thawing mainly affected AQP-7distribution. This event was further tested by challengewith hyper/hypo-osmotic solutions. The hypo-osmolar-ity of the extracellular media during thawing might berelevant for the roles of AQP-7 and AQP-9, as a moreintense acrosome immunolabelling for AQP-7 wasobserved in the 150 mOsm/Kg solution vs the75 mOsm/Kg solution or the control, and sperm tailimmunolabelling for AQP-9 was observed in the75 mOsm/Kg solution but not in the 150 mOsm/Kgsolution nor the control. These observations mightindicate AQP-7 would relocate faster in the membranewhen small osmotic variations in the extracellular media

appear, while AQP-9 would need stronger hypo-osmoticstress for relocation to occur. These observations are inagreement with the lacking changes in domain localiza-tion of AQP-9 during the freezing/thawing processes,thus confirming that AQP-9 would be less implicatedthan AQP-7 in the regulation of water flux in boarspermatozoa.Caudal epididymal spermatozoa presented a stronger

labelling for AQP-7 and AQP-9 compared to ejaculatedcells. We can only speculate on the reasons for thesedifferences. The epididymal cauda milieu in boars is veryparticular, with reverse electrolyte ratios (Na:Cl andMg:K) compared with the epididymal head, and with aevident acidic luminal pH (Rodr�ıguez-Martinez et al.1990). The electrolyte balance changes dramatically atejaculation, where the exposure to seminal plasmachanges the Na:Cl and Mg:K ratios as well as it restoresthe concentrations of bicarbonate to blood plasmalevels, becoming 10-fold higher in seminal plasma thanin the caudal fluid. It is well known that boar caudalspermatozoa survive freezing and thawing better thanejaculated spermatozoa do, with less damage to theplasma membrane (Alkmin et al. 2014). Whether ahigher amount of AQP-7 is related to this higherresilience to cryopreservation remains to be proven.There were almost no differences in the distribution of

the labelling of AQP-7 and AQP-9 between spermato-zoa derived from an entire ejaculate (artificial, all theejaculate volume in a single flask) vs those retrievedfrom anyone of the two completely different fractions(portions 1 and 2) of the split ejaculate (physiologicalfractions). The only difference observed between P1 vsP2 was the amount of extracellular binding, which washigher around spermatozoa contained in P2. At first, itmay be tempting to think that the difference in labellinghas to do with the differential seminal plasma proteincontents (P2 contents are approximately 10-fold higherthan in P1, Rodr�ıguez-Mart�ınez et al. 2005), which maymask the epitopes and thus influence sperm labelling.An interesting observation is that despite the fact thatthe samples came from different males and breeds, thedistribution of either AQPs was similar among animals.This might indicate the homogeneity and conservedfunction of both AQPs in boar spermatozoa.However, two observations have to be made. Firstly,

epididymal fluid is very viscous and contains highamounts of epididymal proteins. Secondly, the P1examined here contains mostly spermatozoa (from thecauda) and a 10-fold lower content of seminal plasmaproteins (Rodr�ıguez-Mart�ınez et al. 2005). Despite thesefacts, the sperm labelling for both AQPs was virtuallythe same in either portion (P1 vs P2) and did not showdifferences with whole-ejaculate spermatozoa. There-fore, the difference in immunolabelling may also residein a differential distribution of AQP-7 and AQP-9,which is perhaps related to the different environmentspermatozoa are exposed to before and after ejacula-tion. More studies are obviously needed to bring clarityto this question. In particular, an examination of the



interactions between seminal plasma and the antibodiesused is required.Fertility of frozen-thawed boar semen is, owing to this

decrease in sperm viability, notoriously compromised inthe pig (Roca et al. 2006). A comparison of post-thawspermatozoa fortuitously retrieved from the differentseminal plasma portions P1 or P2 showed that whilespermatozoa from P1 had no additional labelling, it wasmainly spermatozoa from P2 that had the strongestlabelling over the acrosome (data not shown), asdescribed for the post-thaw spermatozoa from thewhole ejaculate. This difference may very well have todo with the fact that it is during cooling and exposure toglycerol that AQP-7 becomes more active (and perhapsvisible), since the steps where this was noticed wereduring the cooling and thawing steps. Such changes arenot only interesting to record, but they also areindicative of the fact that the distribution pattern ofAQP-7 and its temporal change may make it useful as amarker in studies of cooling or rewarming effects, owingto the functional relevance of this aquaporin during celldehydration and rehydration (Calamita et al. 2001).In conclusion, AQP-7 and AQP-9 are present in boar

spermatozoa, epididymal or ejaculated, albeit withdifferent domain localization, enhanced by some speci-ficity differences among commercial antibodies andprotocols. Conventional cryopreservation, thawing in

particular, modifies domain localization of AQP-7 butnot of AQP-9, suggesting these aquaporins might playdifferent roles on the osmotic regulation during theprocess.

Acknowledgements

The authors would like to acknowledge Mr. Anders Delleskog forprofessional care of experimental animals and Prof. Karl-Eric Mag-nusson, Dr. Vesa Loitto and Prof. Rudolf Rigler for valuablecomments on the manuscript.

Conflict of interest

None of the authors have any conflict of interest to declare.

Funding sources

The study has been made possible by grants from The SwedishResearch Council VR, Stockholm (Grant 521-2011-6553), TheResearch Council FORMAS (Grant 221-2011-512), Stockholm; andFORSS (Forskningsr�adet i Syd€ostra Sverige, Grant 473121), Sweden.

Author contributions

A.V.C. performed the experiments, made the analyses of generateddata and wrote the first draft of the manuscript. H.E. and M.A.R.actively assisted with the experiments and draft writing. H.R.M.designed the experiment and corrected the manuscript.

ReferencesAlkmin DV, Perez-Pati~no C, Barranco I,

Parrilla I, Vazquez JM, Martinez EA,Rodriguez-Martinez H, Roca J, 2014:Boar sperm cryosurvival is better afterexposure to seminal plasma from selectedfractions than to those from entire ejacu-late. Cryobiology 69, 203–210.

Borgnia M, Nielsen S, Engel A, Agre P,1999: Cellular and molecular biology ofthe aquaporin water channels. Annu RevBiochem 68, 425–458.

Calamita G, Mazzone A, Bizzoca A, SveltoM, 2001: Possible Involvement of Aqua-porin-7 and -8 in Rat Testis Developmentand Spermatogenesis. Biochem BiophysRes Commun 288, 619–625.

Chen Q, Duan E, 2011: Aquaporins insperm osmoadaptation: an emerging rolefor volume regulation. Acta PharmacolSin 32, 721–724.

Da Silva N, Pi�etrement C, Brown D, BretonS, 2006: Segmental and cellular expressionof aquaporins in the male excurrent duct.Biochim Biophys Acta 1758, 1025–1033.

Ekwall H, Hern�andez M, Saravia F,Rodr�ıguez-Mart�ınez H, 2007: Cryo-scan-ning electron microscopy (Cryo-SEM) ofboar semen frozen in medium-straws andMiniFlatPacks. Theriogenology 67, 1463–1472.

Eriksson BM, Rodr�ıguez-Mart�ınez H, 2000:Effect of freezing and thawing rates on thepost-thaw viability of boar spermatozoa

frozen in large 5 ml packages (FlatPack).Anim Reprod Sci 63, 205–220.

Fox CH, Johnson FB, Whiting J, Roller PP,1985: Formaldehyde fixation. J His-tochem Cytochem 33, 845–853.

Gao D, Mazur P, Critser JK, 1997: Funda-mental cryobiology of mammalian sper-matozoa. In: Karow A, Critser JKM(eds), Reproductive Tissue Banking. Aca-demic Press, San Diego, CA, pp. 263–328.

Ishibashi K, Tanaka Y, Morishita Y, 2013:The role of mammalian superaquaporinsinside the cell. Biochim Biophys Acta1840, 1507–1512.

Izumi Y, Sonoda S, Yoshida H, Danks H,Tsumuki H, 2005: Role of membranetransport of water and glycerol in thefreeze tolerance of the rice stem borer,Chilo suppressalis Walker (Lepidoptera:Pyralidae). J Insect Physiol 52, 215–220.

Jeyendran RS, van der Ven HH, ZaneveldLJ, 1992: The hypoosmotic swelling test:an update. Arch Androl 29, 105–116.

Mazur P, 1984: Freezing of living cells:mechanisms and implications. Am J Phys-iol 247, C125–C142.

Moretti E, Terzuoli G, Mazzi L, Iacoponi F,Collodel G, 2011: Immunolocalization ofaquaporin 7 in human sperm and itsrelationship with semen parameters. SystBiol Reprod Med 58, 129–135.

Nicchia G, Frigeri A, Nico B, Ribatti D,Svelto M, 2001: Tissue distribution andmembrane localization of aquaporin-9water channel: evidence for sex-linked

differences in liver. J Histochem Cyto-chem 49, 1547–1556.

Ortman K, Rodriguez-Martinez H, 1994:Membrane damage during dilution, cool-ing and freezing-thawing of boar sperma-tozoa packaged in plastic bags. ZentralblVeterinarmed A 41, 37–47.

Petrunkina A, Harrison R, Tsolova M, JebeE, T€opfer-Petersen E, 2007: Signallingpathways involved in the control of spermcell volume. Reproduction 133, 61–73.

Prieto-Mart�ınez N, Vilagran I, Morat�o R,Rodr�ıguez-Gil JE, YesteM, Bonet S, 2014:Aquaporins 7 and 11 in boar spermatozoa:detection, localisation and relationshipwith sperm quality. Reprod Fertil Dev 28,663–672.

Pursel V, Johnson L, 1975: Freezing of boarspermatozoa: fertilizing capacity withconcentrated semen and a new thawingprocedure. J Anim Sci 40, 99–102.

Roca J, Rodr�ıguez-Mart�ınez H, V�azquezJM, Bolar�ın A, Hern�andez M, Saravia F,Wallgren M, Mart�ınez EA, 2006: Strate-gies to improve the fertility of frozen–thawed boar semen for artificial insemina-tion. In: Ashworth CJ, Kraeling RR (eds),Control of Pig Reproduction. VII. ManorFarm.. Nottingham University Press,Nottingham, pp. 261–275.

Rodr�ıguez A, Catal�an V, G�omez-Ambrosi J,Fr€uhbeck G, 2006: Role of aquaporin-7 inthe pathophysiological control of fataccumulation in mice. FEBS Lett 580,4771–4776.



Rodriguez-Martinez H, 2012: Cryopreserva-tion of porcine gametes, embryos andgenital tissues: state of the art. In: KatkovI (ed.), Current Frontiers in Cryobiology.InTech – Open Access Publisher, Rijeka,Croatia, pp. 231–258.

Rodr�ıguez-Martinez H, 1991: Aspects of theelectrolytic composition of boar epididy-mal fluid with reference to sperm matu-ration and storage. Reprod Domest AnimSuppl 1 1, 13–27.

Rodr�ıguez-Martinez H, 2007: Role of theoviduct in sperm capacitation. Theri-ogenology Suppl 68(Suppl 1), S138–S146.

Rodriguez-Martinez H, Wallgren M, 2010:Advances in boar semen cryopreservation.Vet Med Int 2010. doi:10.4061/2011/396181.

Rodr�ıguez-Martinez H, Ekstedt E, EinarssonS, 1990: Acidification of the epididymalfluid in the boar. Int J Androl 13, 238–243.

Rodr�ıguez-Mart�ınez H, Saravia F, WallgrenM, Tienthai P, Johannisson A, V�azquezJM, Mart�ınez E, Roca J, Sanz L, CalveteJJ, 2005: Boar spermatozoa in the ovi-duct. Theriogenology 63, 514–535.

Rodr�ıguez-Mart�ınez H, Kvist U, Saravia F,Wallgren M, Johannisson A, Sanz L,Pe~na FJ, Mart�ınez EA, Roca J, V�azquezJM, Calvete JJ, 2009: The physiologicalroles of the boar ejaculate. Soc ReprodFertil (Suppl 66), 1–21.

SchimmingB,PinheiroP,MatteisR,MachadoC, Domeniconi R, 2015: Immunolocaliza-tion of Aquaporins 1 and 9 in the RamEfferent Ducts and Epididymis. ReprodDomest Anim 50, 617–624.

Simonsson A, 1994: N€aringsrekommenda-tioner och fodermedelstabeller till svin[Nutrient and metabolizable energy rec-ommendations for swine, in Swedish].Swedish University of AgriculturalSciences, SLU Info. Rapporter, Husdjur,p. 75.

Storey KB, Storey JM, 2013: Molecularbiology of freezing tolerance. ComprPhysiol 3, 1283–1308.

Suzuki-Toyota F, Ishibashi K, Yuasa S,1999: Immunohistochemical localizationof a water channel, aquaporin 7 (AQP-7),in the rat testis. Cell Tissue Res 295,279–285.

Verkman AS, van Hoek AN, Ma T, FrigeriA, Skach WR, Mitra A, Tamarappoo BK,Farinas J, 1996: Water transport acrossmammalian cell membranes. Am J Phys-iol 270, C12–C30.

Vicente-Carrillo A, 2015: Hypo-osmotic stressis regulated by Aquaporins 7 and 9 in boarspermatozoa. Available: http://www.liu.se/forskning/scientium/abstracts?l=en (ac-cessed 12 May 2016).

Wang F, Feng X, Li Y, Yang H, Ma T,2006: Aquaporins as potential drug tar-gets. Acta Pharmacol Sin 27, 395–401.

Watson P, 2000: The causes of reducedfertility with cryopreserved semen. AnimReprod Sci 60, 481–492.

Yeung CH, 2010: Aquaporins in spermato-zoa and testicular germ cells: identifica-tion and potential role. Asian J Androl 12,490–499.

Yeung CH, Callies C, T€uttelmann F, KlieschS, Cooper TG, 2010: Aquaporins in thehuman testis and spermatozoa – identifi-cation, involvement in sperm volumeregulation and clinical relevance. Int JAndrol 33, 629–641.

Submitted: 12 May 2016; Accepted: 1 Jun

2016

Author’s address (for correspondence):

Alejandro Vicente-Carrillo, Department ofClinical and Experimental Medicine (IKE),Clinical Sciences/O&G (Campus US, Lab 1,Plan 12), Link€oping University, SE-581 85Link€oping, Sweden. E-mail: [email protected]*Present address: Nyckelaxet 10, 75 756Uppsala, Sweden.



http://dx.doi.org/10.4061/2011/396181

http://www.liu.se/forskning/scientium/abstracts?l=en

http://www.liu.se/forskning/scientium/abstracts?l=en

membrane stress during thawing elicits redistribution of … · 2017. 5. 22. · membrane stress...

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