Effects of the Czech Propolis on Sperm Mitochondrial Function

Research ArticleEffects of the Czech Propolis on Sperm Mitochondrial Function

Miroslava Cedikova12 Michaela Miklikova12 Lenka Stachova3

Martina Grundmanova4 Zdenek Tuma25 Vaclav Vetvicka6 Nicolas Zech7

Milena Kralickova12 and Jitka Kuncova24

1 Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University in Prague 301 00 Pilsen Czech Republic2 Biomedical Centre Faculty of Medicine in Pilsen Charles University in Prague 301 00 Pilsen Czech Republic3 Institute of Pharmacology and Toxicology Faculty of Medicine in Pilsen Charles University in Prague 301 00 Pilsen Czech Republic4Department of Physiology Faculty of Medicine in Pilsen Charles University in Prague 301 00 Pilsen Czech Republic5 1st Internal Department Faculty of Medicine and Teaching Hospital in Pilsen Charles University in Prague301 00 Pilsen Czech Republic

6Department of Pathology University of Louisville Louisville KY 40292 USA7 IVF Centers Prof Zech - Pilsen 301 00 Pilsen Czech Republic

Correspondence should be addressed to Jitka Kuncova jitkakuncovalfpcunicz

Received 11 April 2014 Revised 2 June 2014 Accepted 6 June 2014 Published 1 July 2014

Academic Editor Shi-Biao Wu

Copyright copy 2014 Miroslava Cedikova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Propolis is a natural product that honeybees collect from various plants It is known for its beneficial pharmacological effectsThe aim of our study was to evaluate the impact of propolis on human sperm motility mitochondrial respiratory activityand membrane potential Semen samples from 10 normozoospermic donors were processed according to the World HealthOrganization criteria Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the freshejaculate and purified spermatozoa Propolis preserved progressive motility of spermatozoa in the native semen samples Oxygenconsumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosinediphosphate and substrates of complex I and complex II (state OXPHOSI+II) was significantly increased in the propolis-treatedsamples Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII) and complex IV activity but itdid not influence state LEAK induced by oligomycin Mitochondrial membrane potential was not affected by propolis This studydemonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratorycomplexes II and IV without affecting mitochondrial membrane potential The data suggest that propolis improves the totalmitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility

1 Introduction

Propolis (bee glue) is a natural product that honeybees (Apismellifera) collect from various plants It is used as buildingmaterial (filling of cracks and gaps) or for protection againstintruders (embalms killed invader insects) [1]

Propolis has been used as a remedy for thousands of yearsThe term is derived from Greek word pro- (meaning in frontof) and polis (city community) Its chemical compositiondepends on the place time of collection and plant sources

making it highly variable To date more than 300 compoundshave been detected in various propolis extracts [2 3] It iscomposed mainly of resin (50) and wax (30) Other com-ponents are pollen (5) essential and aromatic oils (10)and minor compounds as flavonoids (quercetin kaempferolpinocembrin apigenin chrysin etc) beta-steroids terpenesminerals and vitamins [1 4ndash6] Propolis is known in folkmedicine for its pharmacological effects antibacterial antivi-ral antifungal antiparasitic anti-inflammatory chemopre-ventive immunomodulatory hepatoprotective antioxidant

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 248768 10 pageshttpdxdoiorg1011552014248768

2 Evidence-Based Complementary and Alternative Medicine

and antitumor [1 7] As a result of these effects over the past30 years propolis has been the subject of intense medicalstudies

Infertility affects 10ndash15 of couples of reproductive ageand plenty of unanswered questions remain concerning thephysiological mechanisms underlying the successful fertility[8] Male factor contributes to about 50 of cases of infer-tility and most of them are still idiopathic [9] More than90 of male infertility cases are due to low sperm count(oligozoospermia) poor sperm motility (asthenozoosper-mia) abnormal spermmorphology (teratozoospermia) or allthree The remaining cases of male infertility can be causedby a number of factors including anatomical problemshormonal imbalances and genetic defects

Appropriate spermmotility is fundamental for reproduc-tive success in mammals since the spermatozoa have to travela relatively long distance through the female reproductivesystem by active flagellar motion The energy for the spermmovement in the form of adenosine triphosphate (ATP)is supplied by two metabolic processes glycolysis takingplace in the cytoplasm or oxidative phosphorylation in themitochondria found only in the sperm midpiece where72ndash80 helically arranged organelles can be detectedHoweverthe relative contributions of both pathways to the spermmotility in different species are a matter of long-standingdebate [10 11] Nevertheless recent experimental data suggestthat reduced efficiency of the mitochondrial respiratoryactivity may contribute to the reduced sperm motility Inasthenozoospermic patients morphological and functionalchanges in sperm mitochondria have been described [12 13]

Until now the effects of propolis on the mitochondrialmorphology and function have been studied particularly inthe somatic cells However the results of these studies arefar from uniform showing both stimulation and inhibitionof various mitochondrial functions including oxygen con-sumption apoptosis andmitochondrial membrane potential[14ndash17] The aim of our study was to assess the effect ofethanolic extract of propolis (EEP) on the human spermmotility mitochondrial respiratory activity and membranepotential to test the putative therapeutic potential of thisnatural product in the treatment of asthenozoospermia

2 Methods

21 Ethanolic Extract of Propolis Preparation Propolis wascollected using plastic nets in region ofWest Bohemia (HorniSlavkovmdash50∘ 810158401726810158401015840 N 12∘ 4810158404899210158401015840 E) in September2012 Propolis was frozen at minus20∘C and ground in a mill Theresulting powder (10 g) was mixed at room temperature with70 ethanol (100mL) for 24 h and then filtered The filtratewas then made up to 100mL with 70 ethanol [18]The sam-plewas kept in darkness at 4∘Cuntil analysis For experimentswith live cells propolis was further diluted resulting in a finalethanol concentration below 1 which is not toxic to cells[19] The final concentration of propolis chosen for furtherexperiments was 001mgmL of the corresponding medium

22 High Performance Liquid Chromatography Analysis(HPLC) Qualitative and quantitative chromatographic anal-yses of phenolics were performed on a HPLC systemequipped with a binary pump (Waters 1525) Waters 717 plusAutosampler and dual UVVIS detector 2487 Separationwas performed on a Symmetry C18 column particle size5 120583m (150mm times 46mm) using a mobile phase of 008acetic acid in methanol (A) and 01 acetic acid and 10methanol in water (B) The gradient was 10ndash47 A (25min)47 A (25ndash40min) 47ndash70 A (40ndash70min) and 70ndash100A (70ndash80min) at a flow rate of mobile phase 05mLminInjection volume was 10 120583L and column temperature was30∘C

Spectrophotometric detection was conducted at 280 nmand 330 nm Identification of polyphenolic compounds wasachieved by comparison of retention times with those ofcommercial pure compounds All standards were dissolvedin dimethyl sulfoxide (Sigma-Aldrich St Louis USA) to give10mmolL standard solutions Calibration standards wereprepared by dilution of the standard solution in ethanol

Quantitative analysis was carried out by external standardmethod Calibration curves showed a linear response of 1198772 gt097 over a concentration range of 5ndash100 120583molL Beforethe HPLC analysis the EEP was filtered on teflon syringemicrofilter Separon 045120583mThe propolis extract was dilutedone hundred times for HPLC analysis

Phenolic compounds were purchased from Sigma-Aldrich (St Louis USA) (apigenin chrysin genisteinkaempferol luteolin naringenin pinocembrin galangin andphenolic acids caffeic p-coumaric t-ferulic t-cinnamicbenzoic and gallic acid and caffeic acid phenethyl ester)Vanillin was purchased fromMerck (Darmstadt Germany)

23 Sperm Sample Preparation The study design wasapproved by the Local Ethics Committee of the UniversityHospital in Pilsen and a written informed consent wasobtained from each of the 10 participants included in thestudy (mean age 242 years SEM plusmn 28)

Ejaculates were collected after 3 days of sexual abstinencein IVF Center Prof Zech Pilsen Semen samples were eval-uated by an experienced employee After liquefaction theywere analyzed according to the World Health Organizationcriteria 2010 [20] We investigated semen volume and underthe microscope with phase-contrast optics at magnificationtimes200 concentration of spermatozoa motility of spermato-zoa and pathologies Sperm motility was assessed at roomtemperature in Makler counting chamber Two hundredspermatozoa per replicate were classified into three motilitycategories (progressive nonprogressive and immotile spermcells) All samples were considered normozoospermic ejacu-lates (Table 1)

Fresh ejaculate (01mL) was subjected to experimentwith propolis Propolis or ethanol only (1 120583L) was addedto 01mL of fresh ejaculate (final concentration of propoliswas 001mgmL) and sperm motility was evaluated after 60minutes

The remaining sample was prepared by gradient separa-tion technique and used for experiment with polarographicoxygraph (Oroboros Innsbruck Austria) and sperm flow

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Main sperm parameters of the normozoospermic men(plusmnSEM)

Parameters Normozoospermicmen (119899 = 10)

Volume (mL) 318 plusmn 026Concentration (times106mL) 8122 plusmn 1397Progressive motility () 625 plusmn 44Pathology morphology () 4200 plusmn 226Concentration after separation (times106mL) 15844 plusmn 1809Progressive motility after separation () 8643 plusmn 210

cytometry evaluation Sperm number after separation wasalso determined in the Makler counting chamber

24 The Sperm Density Gradient Separation Technique Thesperm was separated and purified This was performed usinggradient solution media SpermGrad medium (SGmVitrolife Sweden) and SpermRinse medium (SRmVitrolife Sweden) SpermGrad medium was diluted to90 (015mL SGm 135mL SRm) 70 (009mL SGm 021mL SRm) and 50 (015mL SG 015mL SRm) Intoa conical tube 15mL 90 03mL 70 and 03mL of a50 gradient media were layered Full ejaculate was addedand the sample was centrifuged for 20 minutes at 300 gAfter removal of the supernatant 8mL SRm was added andthen the sample was centrifuged 8 minutes at 300 g Afterremoval of supernatant the sample was evaluated for theconcentration and motility of spermatozoa and ready forinjection into the oxygraph

25 High-Resolution Respirometry Oxygen consumption bypurified spermatozoa was measured at 36∘C in 2mL glasschambers of oxygraph Oroboros (Oroboros Innsbruck Aus-tria) connected to the computer with DatLab software fordata acquisition and analysis (Oroboros Innsbruck Austria)The oxygen flux was calculated as a negative time derivativeof the oxygen concentration All values of oxygen fluxeswere corrected for instrumental and chemical backgroundmeasured in separate experiments performed in the samemedium without human gametes

The medium consisting of 05mmolL ethylene gly-col tetraacetic acid 3mmolL MgCl

2sdot6H2O 60mmolL

K-lactobionate 20mmolL taurine 10mmolL KH2PO4

20mmolL HEPES 110mmolL sucrose and 1 gL albu-min essentially fatty acid free [21] was stirred at 750 rpmand equilibrated for 60min with air After equilibrationoxygen concentration in the chamber corresponded to itsconcentration in the atmospheric air and solubility in themedium (092) The chambers were then closed and thesamples of intact spermatozoa were injected into the cham-bers using Hamilton syringe Into one of two chambersrecording in parallel propolis (001mgmL) was injectedand the samples were further incubated at 36∘C for 20minThe spermatozoa cell membrane was permeabilized withdigitonin (Sigma-Aldrich St Louis USA 5120583gmL) andcombination of substrates inhibitors and uncouplers was

sequentially injected into the chambers to measure the res-piration through different segments of the electron transportsystem (Figure 1) (1) Resting respiration with substratesproviding electrons to complex I malate (2mmolL) andglutamate (10mmolL) was measured as a state S2 (non-phosphorylating LEAK state LN) (2) Active respiration wasinduced by 5mmolL adenosine diphosphate (ADP state S3or OXPHOS) (3) Oxygen consumption was further mea-sured with pyruvate (5mmolL) and a substrate of electrontransfer flavoprotein (ETF) palmitoyl carnitine (20120583molL)(4) Integrity of the mitochondrial inner membrane waschecked with cytochrome c (10 120583molL) (5) Mitochondrialrespiration was then increased by succinate complex IIsubstrate (10mmolL) (6) State LEAK was induced againby inhibition of ATP-synthase oligomycin (2120583gmL) (7)Maximum capacity of the electron-transporting system (stateS3u or ETS) was reached by titration of uncoupler triflu-orocarbonylcyanide phenylhydrazone (FCCP 005 120583molLtitration steps) (8) After addition of a complex I inhibitorrotenone the oxygen flux corresponded to maximum capac-ity of the electron-transporting system with the complexII only (9) Then antimycin A (25 120583molL) a complex IIIinhibitor was injected into the chambers to measure residualoxygen consumption (ROX) (10) NNN1015840N1015840-tetramethyl-p-phenylenediamine dihydrochloride (TMPD 05mmolL)and ascorbate (2mmolL) were injected simultaneously forrespirometric assay for cytochrome c oxidase (C IV) activityIn the results oxygen fluxes recorded in the individual titra-tion steps were corrected for residual oxygen consumption

The dose-response relationship between the propolisconcentration and sperm respiratory activity was tested inanother set of experiments where the final concentrationsof propolis 0001 0005 and 001mgmL were used Higherdose of propolis was not tested as in experiments runningin parallel higher concentrations of propolis in the incuba-tion medium (003 and 005mgmL) were toxic for mouseembryonic stem cells reducing their growth survival andproliferation (unpublished observation)

26 Permeability of Cell Membrane in Sperm Sperm wastreated with the impermeable fluorescent dye propidiumiodide to check cell membrane permeability for substratesinhibitors and uncouplers after treatment by digitonin Finalconcentration of propidium iodide was 1120583gmL

27 Sperm Flow Cytometry Evaluation Mitochondrial mem-brane potential (ΔΨ119898) was determined with MitoProbeJC-1 Assay Kit (Life Technologies) Each sperm samplewas resuspended in 37∘C warm phosphate-buffered saline(PBS) at approximately 1 times 106 cellsmL Propolis (1 120583L) wasadded to test samples (100 120583L) to reach final concentra-tion 001mgmL Controls remained without interventionSamples were incubated in propolis for 60min and afterthat were washed with PBS JC-1 (10 120583L of 200120583molL) wasadded for 20min incubation (37∘C 5 CO

2) Wash with PBS

followed (5min 1500 rpm) Samples were resuspended in500120583L PBS andmeasured on BDFACSCANTO II cytometer(BD Biosciences New Jersey USA) Analysis was performedwith BD FACS Diva software with 488 nm excitation using


CytochromecCoenzyme

Q

Complex I

Complex II

GpDH

ETF

Complex III Complex IV O2

Figure 1 A scheme of an electrotransport convergent system System is situated on the inner mitochondrial membrane GpDH = glucose-6-phosphate dehydrogenase complex I = NADH-Q reductase complex II = succinate-Q oxidoreductase complex III = cytochrome reductasecomplex IV = cytochrome oxidase ETF = electron-transporting flavoprotein O

2= oxygen

emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin

28 Citrate Synthase Activity Mitochondrial content in thesamples aspirated from each oxygraph chamber was assayedby determination of the citrate synthase activity [22 23]The assay medium consisted of 01mmolL 55-dithio-bis-(2-nitrobenzoic) acid 025 Triton-X 05mmolL oxalacetate031mmolL acetyl coenzyme A 5 120583moL EDTA 5mmolLtriethanolamine hydrochloride and 01molL Tris-HCl pH81 [22] Two hundred microliters of the mixed and homoge-nized chamber content was added to 800 120583L of the mediumThe enzyme activity was measured spectrophotometrically at412 nm and 30∘C over 200 s and expressed in mIU per 107cells

29 Data Analysis and Statistics Results are presented asmean plusmn SEM Statistical differences were analyzed usingsoftware package STATISTICA Cz 8 (StatSoft Inc PragueCzech Republic) After testing for the normality of distri-bution and homogeneity of variances comparisons weremade using Studentrsquos 119905-test Wilcoxon signed-rank test andanalysis of variance (ANOVA) with post hoc tests correctedformultiple comparisons by BonferronirsquosmethodThe resultswere considered significantly different when 119875 lt 005

3 Results

31 HPLC Analysis Analyzing the propolis by the HPLC wewere able to identify compounds as t-ferulic acid p-coumaricacid vanillin caffeic acid t-cinnamic acid kaempferol api-genin and chrysin Although we analyzed standards of gallicacid benzoic acid quercetin naringenin luteolin genisteinpinocembrin galangin and caffeic acid phenethyl ester theywere not identified in our propolis sample Chromatogramof ethanolic extract of the Czech propolis is presented inFigure 2 Detailed results with concentrations of observedsubstances are shown in Table 2

000

004

008

012

016

020

Caffe

ic ac

id21225

Vani

llin23409

p-co

umar

ic ac

id27573

Cinn

amic

acid

42792

Kaem

pfer

ol51341

Apig

enin

53415

Chry

sin70074

Time (min)

(AU

)

tfer

ulic

acid

28955

15 20 25 30 35 40 45 50 55 60 65 70 75 80

Figure 2 HPLC chromatogram of the Czech propolis extract andthe identified compoundsretention time (120582 = 280 nmmdashblack lineand 120582 = 330 nmmdashgreen line)

32 Semen Parameters and Effect of Propolis on SpermMotilityin Fresh Ejaculate Ten healthy men were included in thisstudy The mean age was 242 years The general spermcharacteristic of the normozoospermic men after ejaculationis shown in Table 1 The effect of propolis on human spermmotility after incubation for 60 minutes is presented inFigure 3 Propolis preserved the progressive motility of sper-matozoa in the native semen samples since the percentage ofthe progressively motile spermatozoa after incubation withpropolis remained nearly the same as in the fresh sampleswhereas in the ejaculates without propolis the progressivemotility significantly declined with time (119875 = 0028) Ethanolalone had no negative effect on sperm motility

33 High Resolution Respirometry Representative traces ofthe oxygen consumption in permeabilized spermatozoawith and without propolis 001mgmL are depicted inFigure 4 Oxygen consumption of intact spermatozoa (013plusmn 001 nmol(ssdotIU)) was significantly enhanced by propolis(027 plusmn 003 nmol(ssdotIU) 119875 = 0006) After permeabilizationwith digitonin state 2 determined in the presence of malate


Table 2 Analysis of the ethanolic extract of propolis by the HPLC

Compound Rt [min] Concentration [mgL of EEP]Gallic acid 60 ndCaffeic acid 212 65 plusmn 11Vanillin 234 65 plusmn 11p-Coumaric acid 275 231 plusmn 10t-Ferulic acid 289 514 plusmn 15Benzoic acid 336 ndQuercetin 423 ndt-Cinnamic acid 427 29 plusmn 1Naringenin 432 ndLuteolin 447 ndGenistein 456 ndKaempferol 513 101 plusmn 45Apigenin 534 73 plusmn 8Chrysin 700 36 plusmn 5Pinocembrin 651 ndGalangin 731 ndCAPE 714 nd(nd = nondetected CAPE = caffeic acid phenethyl ester)

Control Propolis

Prog

ress

ive m

otili

ty at

60

min

()

lowast

100

90

80

70

60

50

40

30

20

10

0

Figure 3 Effect of propolis on sperm motility Columns representprogressive motility of spermatozoa in native ejaculates after 60minincubation with propolis extract (mean plusmn SEM) Dashed line =progressivemotility at time 0 lowast

119875

lt 005 compared to the respectivecontrol value

and glutamate was 019 plusmn 004 nmol(ssdotIU) in the controlsamples and it was significantly higher in the propolis-treatedsperm samples (029 plusmn 005 nmol(ssdotIU) 119875 = 0014) State 3that is oxygen consumption during oxidative phosphoryla-tion determined in the presence of ADP and substrates ofthe complex I ETF and complex II is depicted in Figure 5Propolis significantly increased (by sim50) S3I+II oxygen flux(119875 = 0003) suggesting increased activity of the complex IIPropolis did not influence state 4 induced by oligomycin that

10 20 30 40 50 60

250

200

150

100

50

0

10

8

6

4

2

0

Time (min)

O2

conc

entr

atio

n (n

mol

mL)

O2

flux

per u

nit (

nmol

(slowast

unit)

)

Dig DD Pcar c

S

Omy

FF F Rot

AmA

M G P

Figure 4 Substrate-uncoupler-inhibitor titration protocol inhuman spermatozoa Substrate-uncoupler-inhibitor titration(SUIT) protocol with substrates for complex I complex II andelectron-transferring flavoprotein (ETF) in human spermatozoaRed line = oxygen flux expressed per IU citrate synthase activity inthe control sample green line = oxygen flux expressed per IU citratesynthase activity in the sample treated with propolis 001mgmLand blue line = oxygen concentration in the oxygraph chamber Dig= digitonin M = malate G = glutamate D = ADP P = pyruvatePcar = palmitoylcarnitine c = cytochrome c S = succinate Omy =oligomycin F = FCCP Rot = rotenone and AmA = antimycin A

reached 081plusmn 007 and 094plusmn 009 nmol(ssdotIU) in the controland propolis-treated spermatozoa respectively Maximumcapacity of the electron-transporting system (state S3u) testedfor both complexes I and II and in the presence of the complexI inhibitor rotenone was significantly higher after propolisadministration (Figure 6) In measurements with TMPD +ascorbate complex IV respiration was 1216 plusmn 258 nmolO2(ssdotIU) in the control samples and it was significantly

enhanced by propolis to 154 plusmn 319 nmol O2(ssdotIU) Absolute

ethanol alone (medium for propolis oligomycin antimycinA FCCP and rotenone) in the volume up to 10120583L did notinfluence respirometric parameters measured with substratesof complexes I II and ETF in the presence of ADP

Flux control ratios were calculated to estimate the relativeefficiency of individual interventions and coupling state ofthe sperm mitochondria The ratio OXPHOSI+IIOXPHOSIwas significantly higher in the propolis-treated samples(353 plusmn 042) compared to controls (28 plusmn 028) suggestingincreased efficiency of coupled respiration when complex IIwas stimulated by succinate The LEAK control ratio is theratio of LEAK respiration and ETS capacity it reached 025 plusmn002 in the control samples and it significantly decreased afterpropolis to 021 plusmn 002 (119875 = 0024)

The dose-response relationship tested in separate exper-iments is shown in Figure 7 for state S3 (OXPHOS) whereglutamatemalate pyruvate succinate andADPwere presentin the medium and for state S3u (ETS) where mitochon-dria where uncoupled by FCCP Propolis dose-dependentlyincreased oxygen consumption although the extent of itseffect was greater for state S3 than for state S3u

34 Permeability of Cell Membrane in Sperm Addition of5 120583gmL of digitonin made the sperm cell membrane per-meable to dye propidium iodide and for the substrates


MG MGP MGPPcar MGPPcarS

30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))

Figure 5 Oxygen consumption rates in the state S3 (OXPHOS)measured under control conditions and with propolis Oxygen con-sumption rates in the state S3 (OXPHOS) measured under controlconditions (control) and with propolis extract added (propolis) inthe presence of ADP and substrates providing electrons to complexI ETF and complex II M = malate G = glutamate P = pyruvatePcar = palmitoylcarnitine and S = succinate Oxygen fluxes werecorrected for residual oxygen consumption and expressed per IUcitrate synthase activity lowast

119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))

Figure 6 Oxygen consumption rates in the state S3u in per-meabilized spermatozoa Oxygen consumption rates in the stateS3u (ETS) in permeabilized spermatozoa measured under controlconditions (control) and with propolis extract added (propolis) inthe presence of FCCP and substrates providing electrons to complexI ETF and complex II M = malate G = glutamate P = pyruvatePcar = palmitoylcarnitine S = succinate and complex I inhibitorrotenone (Rot) Oxygen fluxes were corrected for residual oxygenconsumption and expressed per IU citrate synthase activity lowast

119875

lt

005 compared to the respective control value

55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))

Propolis concentration (mgmL)0000 0002 0004 0006 0008 0010

S3S3u

Figure 7 Dose response relationship between oxygen consumptionand final propolis concentration State S3 (OXPHOS) = oxygenconsumption rate in permeabilized spermatozoa measured withmalate glutamate pyruvate succinate and ADP State S3u = oxygenconsumption rate in permeabilized spermatozoa measured aftersequential addition of malate glutamate ADP pyruvate succinateand FCCP Oxygen fluxes were corrected for residual oxygenconsumption and expressed per IU citrate synthase activity lowast

119875

lt

005 compared to the respective value without propolis

inhibitors and uncouplers required to determine high reso-lution respirometry (Figure 8)

35 Sperm Flow Cytometry Evaluation Depolarization ofthe mitochondrial membrane is a sensitive indicator ofmitochondrial damage JC-1 is a membrane-permeable fluo-rescent probe aggregating in the mitochondrial matrix andthen emitting red fluorescence if ΔΨ119898 is high In case ofmitochondrial depolarization the monomeric form of JC-1cannot accumulate in the mitochondrial matrix and producegreen fluorescence in the cytoplasm In the spermatozoaloss of ΔΨ119898 could serve as a marker of early apoptosisand sperm dysfunction [24] In our experiments incubationof the sperm samples with propolis (60 minutes) had nosignificant effect on ΔΨ119898 (Figure 9) In the control samplesthe percentage of cells with high ΔΨ119898 was 9843 plusmn 107 andit even slightly increased in the propolis-treated spermatozoareaching 9933 plusmn 025

36 Citrate Synthase Activity In the control samples citratesynthase activity was 658 plusmn 084mIU107 cells and it was notsignificantly affected by propolis (608 plusmn 084mIU107 cells)and by the substances (substrates inhibitors media) addedduring the respirometric measurements as tested in separatecontrols


(a)

50120583m

(b)

Figure 8 Effect of digitonin treatment on spermatozoa (a) Micrograph of human spermatozoa and (b) fluorescent micrograph of the sameoptical field after treatment with the nonpermeable nuclear dye propidium iodide

Without intervention

Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102

With propolis (60min)

Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102

Figure 9 Typical double fluorescence dot plots of flow cytometry analysis Mitochondrial membrane potential was determined withMitoProbe JC-1 Assay Kit (Life Technologies) R-phycoerythrin = red fluorescence normal cells Alexa Fluor 488 dye = green fluorescencereduced mitochondrial membrane potential

4 Discussion

This is the first study showing chemical composition ofpropolis collected in the Czech Republic Bee glue has a verycomplex chemical composition that depends on a numberof factors including diversity of plants and geographicallocation from which bees collect it Although the regionof collection of the tested sample of propolis is situatedin the northern temperate zone where the ldquopoplarrdquo typepropolis is a typical bee product [3] the tree structurewithin the mentioned locality is composed of coniferoustrees (90) birch (6) alder (2) beech (1) and oak(1) [25] However the tested sample contained chrysin

the reference flavonoid in poplar propolis and phenolic acidsand other flavonoids typically found in bee glue extractsoriginating from similar geographical regions and reported tobe responsible for various beneficial effects of propolis [26]Among them ferulic acid coumaric acid and kaempferolwere present in the highest concentrations in the Czechpropolis extract Caffeic acid phenethyl ester naringenin andquercetinmdashsubstances frequently contained in the poplarpropolis [2 3] and widely tested for their anti-inflammatoryanticancer and antioxidant activities in recent years [27]mdashhave not been detected in the Czech propolisThe factors thatcould contribute to the above mentioned differences couldinclude not only diversity of plant species growing around


the hive but also season illumination altitude collectortype and food availability [28] It should be noted thatthe search for a single substance or a particular substanceclass that could be responsible for the beneficial actions ofpropolis has not been successful to date Scientific studiesdemonstrated that biological activity of propolis could bealmost identical (ie antimicrobial antitumor antioxidantanti-inflammatory etc) in samples from different climaticzones and of completely different chemical composition [3]Most probably a combination of substances is essential forthe biological activity of bee glue [29]

The present study describes the effects of propolis onthe sperm motility mitochondrial membrane potentialand mitochondrial respiratory activity assessed by high-resolution respirometry allowing determination of individualrespiratory states in a sequential manner on the same samplethus allowing complex evaluation of the mitochondrial func-tion close to the situation in vivo In addition high sensitivityof themethod enables determination of respirometric activityof individual mitochondrial complexes in a relatively smallamount of cells [30] In our experiments we have usedthe purified spermatozoa that were subjected to membranepermeabilization by mild nonionic detergent digitonin thedose of which was carefully titrated to permeabilize the cellmembranewithout damaging themitochondrial function Todate oxygen consumption by human sperm mitochondriahas been determined mostly by traditional oxygraphy inthe germ cells subjected to hypotonic swelling [13 31ndash33] However in the spermatozoa of several mammalianspecies hypotonic challenge could substantially influence theactivity of various protein kinases (PK) including PKA PKCand protein-tyrosine kinase via osmosensitive K+ and Clminuschannels in the process called regulatory volume decreaserequiring energy supply [34]

The major finding of the present study is that propolisenhances the activity of mitochondrial respiratory complexesII and IV without affecting the coupling of the electrontransport to ATP synthesis andor mitochondrial membranepotential in permeabilized human spermatozoa in vitro Inthe coupled state propolis enhanced oxygen consumptionwith complex I and complex II substrates by sim50 Thisincrease was attributed to complex II since the activity ofcomplex I alone was not affected by propolis In additionthe ratio 119875I+II119875I was significantly higher in the propolis-treated samples Similarly in the uncoupled state (S3u E)oxygen consumption was significantly (by sim25) higher afterpropolis both in the presence of substrates of complexes I andII and after inhibition of complex I by rotenone Activity ofthe complex IV was increased to the same extent (sim27)

The data available on the effects of propolis and itsmajor compounds onmitochondrial respiration or activity ofindividual mitochondrial enzymes involved in the electron-transport convergent system are scarce The most frequentfinding is that this effect is negligible in normal somatic cells(cardiomyocytes neurons and hepatocytes) but becomesbeneficial in the cells challenged by toxic stimuli whereoxidative phosphorylation is compromised [16 35 36] Incontrast in various types of tumor cells propolis extract orits constituents inhibit oxidative phosphorylation and trigger

release of cytochrome c and subsequent apoptosis [15 17] Inview of this the action of propolis on the cellular respirationis not easily predictable and depends on the cell type

Some studies suggested that propolis or its phenolic con-stituents might influence mitochondrial membrane poten-tial via increased permeability of the inner mitochondrialmembrane [14] This issue was addressed in the experimentswith MitoProbe JC-1 Assay Kit that clearly showed that inthe human spermatozoa mitochondrial membrane potentialwas not affected by propolis In addition oligomycin-inducedrespiratory state (LEAK) that reflects compensation for theproton leak proton slip electron slip and cation cycling[30] was not affected by propolis Thus the decrease in theLEAK control ratio after propolis could be attributed tothe increased efficiency of the electron transport throughcomplex IIThe putative molecular mechanism of complex IIactivation was suggested in study of Cimen et al [37] wherepropolis constituent kaempferol increased deacetylation ofsuccinate dehydrogenase thus increasing its activity

To date the effects of propolis or propolis compounds onthe sperm characteristics have been rarely studiedThewholepropolis extract was used in studies conducted by Yousefand collaborators on rats and rabbits that were treated withpropolis for 10 to 12weeks [38 39] Administration of propolisresulted in an increased sperm count and motility plasmatestosterone levels and a decreased dead and abnormalsperm count A single study documented positive effect ofthe propolis compound chrysin on the spermmotility spermconcentration and serum testosterone levels [40] Ferulicacidwas also reported to enhance spermmotility and viability[41] All these findings were attributed to the activity ofpropolis as antioxidant and none of these studies dealt withthe action of propolis or its constituents on mitochondrialenergy production Our study describes a novel beneficialeffect of propolis on the sperm characteristics

Recent experimental evidence suggests that oxidativephosphorylation in the human spermatozoa plays a cru-cial role in gaining energy for the sperm motility andindicates that asthenozoospermia might be related to theimpaired mitochondrial functionality [42] High-resolutionrespirometry could provide new data in search for sub-stances that could positively affect human spermmotility andthus improve sperm fertilizing ability In addition detailedanalysis of respiratory efficiency of individual mitochondrialenzymatic complexes under coupled and uncoupled condi-tions could provide better insight into pathophysiology ofasthenozoospermia

5 Conclusions

This study demonstrates for the first time that ethanolicextract of propolis increases activities of mitochondrial respi-ratory complexes II and IV without affecting mitochondrialmembrane potential The obtained data suggest that propolisimproves the total mitochondrial respiratory efficiency inthe human spermatozoa in vitro thereby having potential toimprove the sperm motility


Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Miroslava Cedikova and Jitka Kuncova contributed equally tothis work

Acknowledgments

This work was supported by the Project ED2100030076from European Regional Development Fund the CharlesUniversity Research Fund (Project no P36) the SpecificStudent Research Project no 2600482014 of theCharlesUni-versity in Prague and by the Grant from Charles UniversityGrant Agency no 696212

References

[1] M Viuda-Martos Y Ruiz-Navajas J Fernandez-Lopez andJ A Perez-Alvarez ldquoFunctional properties of honey propolisand royal jellyrdquo Journal of Food Science vol 73 no 9 pp R117ndashR124 2008

[2] V Bankova M Popova S Bogdanov and A G SabatinildquoChemical composition of European propolis expected andunexpected resultsrdquo Zeitschrift fur Naturforschung C A Journalof Biosciences vol 57 no 5-6 pp 530ndash533 2002

[3] V Bankova ldquoChemical diversity of propolis and the problem ofstandardizationrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 114ndash117 2005

[4] A Russo R Longo and A Vanella ldquoAntioxidant activity ofpropolis role of caffeic acid phenethyl ester and galanginrdquoFitoterapia vol 73 supplement 1 pp S21ndashS29 2002

[5] A M Gomez-Caravaca M Gomez-Romero D Arraez-Roman A Segura-Carretero and A Fernandez-GutierrezldquoAdvances in the analysis of phenolic compounds in productsderived from beesrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 41 no 4 pp 1220ndash1234 2006

[6] M L Khalil ldquoBiological activity of bee propolis in health anddiseaserdquo Asian Pacific Journal of Cancer Prevention vol 7 no 1pp 22ndash31 2006

[7] A H Banskota Y Tezuka and S Kadota ldquoRecent progress inpharmacological research of propolisrdquo Phytotherapy Researchvol 15 no 7 pp 561ndash571 2001

[8] M Kralıckova P Sıma and Z Rokyta ldquoRole of the leukemia-inhibitory factor gene mutations in infertile women theembryo-endometrial cytokine cross talk during implantationmdasha delicate homeostatic equilibriumrdquo Folia Microbiologica vol50 no 3 pp 179ndash186 2005

[9] J C St John D Sakkas and C L R Barratt ldquoA role formitochondrial DNA and sperm survivalrdquo Journal of Andrologyvol 21 no 2 pp 189ndash199 2000

[10] W C L Ford ldquoGlycolysis and sperm motility does a spoonfulof sugar help the flagellum go roundrdquo Human ReproductionUpdate vol 12 no 3 pp 269ndash274 2006

[11] R M Turner ldquoMoving to the beat a review of mammalianspermmotility regulationrdquo Reproduction Fertility and Develop-ment vol 18 no 1-2 pp 25ndash38 2006

[12] V Y Rawe R Hermes F N Nodar G Fiszbajn and HE Chemes ldquoResults of intracytoplasmic sperm injection intwo infertile patients with abnormal organization of spermmitochondrial sheaths and severe asthenoteratozoospermiardquoFertility and Sterility vol 88 no 3 pp 649ndash653 2007

[13] A Ferramosca R Focarelli P Piomboni L Coppola andV Zara ldquoOxygen uptake by mitochondria in demembranatedhuman spermatozoa a reliable tool for the evaluation of spermrespiratory efficiencyrdquo International Journal of Andrology vol31 no 3 pp 337ndash345 2008

[14] S Das J Das A Samadder N Boujedaini and A R Khuda-Bukhsh ldquoApigenin-induced apoptosis in A375 and A549 cellsthrough selective action and dysfunction of mitochondriardquoExperimental Biology and Medicine vol 237 no 12 pp 1433ndash1448 2012

[15] V Chen R E Staub S Baggett et al ldquoIdentification and analysisof the active phytochemicals from the anti-cancer botanicalextract Beziellerdquo PLoS ONE vol 7 no 1 Article ID e30107 2012

[16] R Lagoa I Graziani C Lopez-Sanchez V Garcia-Martinezand C Gutierrez-Merino ldquoComplex I and cytochrome c aremolecular targets of flavonoids that inhibit hydrogen peroxideproduction by mitochondriardquo Biochimica et Biophysica Actavol 1807 no 12 pp 1562ndash1572 2011

[17] R D Yeh J C Chen T Y Lai et al ldquoGallic acid inducesG0G1phase arrest and apoptosis in human leukemia HL-

60 cells through inhibiting cyclin D and E and activatingmitochondria-dependent pathwayrdquoAnticancer Research vol 31no 9 pp 2821ndash2832 2011

[18] J S Bonvehı and A L Gutierrez ldquoThe antimicrobial effects ofpropolis collected in different regions in the Basque Country(Northern Spain)rdquo World Journal of Microbiology and Biotech-nology vol 28 no 4 pp 1351ndash1358 2012

[19] M Barbaric K Miskovic M Bojic et al ldquoChemical compo-sition of the ethanolic propolis extracts and its effect on HeLacellsrdquo Journal of Ethnopharmacology vol 135 no 3 pp 772ndash7782011

[20] WHO ldquoWHO laboratory manual for the examination andprocessing of human semenrdquo 2010 httpwwwwhointrepro-ductivehealthpublicationsinfertility9789241547789en

[21] E Gnaiger G Mendez and S C Hand ldquoHigh phosphorylationefficiency and depression of uncoupled respiration in mito-chondria under hypoxiardquo Proceedings of the National Academyof Sciences of the United States of America vol 97 no 20 pp11080ndash11085 2000

[22] A V Kuznetsov D Strobl E Ruttmann A KonigsrainerR Margreiter and E Gnaiger ldquoEvaluation of mitochondrialrespiratory function in small biopsies of liverrdquo AnalyticalBiochemistry vol 305 no 2 pp 186ndash194 2002

[23] S Larsen J Nielsen C N Hansen et al ldquoBiomarkers ofmitochondrial content in skeletal muscle of healthy younghuman subjectsrdquoThe Journal of Physiology vol 590 no 14 pp3349ndash3360 2012

[24] G Barroso S Taylor M Morshedi F Manzur F Gavino andS Oehninger ldquoMitochondrial membrane potential integrityand plasma membrane translocation of phosphatidylserine asearly apoptotic markers a comparison of two different spermsubpopulationsrdquo Fertility and Sterility vol 85 no 1 pp 149ndash1542006

[25] C R Lesy s p httpwwwlesycrczls229charakteristika-lesni-spravyStrankydefaultaspx

[26] R D Wojtyczka A Dziedzic D Idzik et al ldquoSusceptibility ofStaphylococcus aureus clinical isolates to propolis extract alone


or in combination with antimicrobial drugsrdquoMolecules vol 18no 8 pp 9623ndash9640 2013

[27] G Ozturk Z Ginis S Akyol G Erden A Gurel and OAkyol ldquoThe anticancer mechanism of caffeic acid phenethylester (CAPE) review of melanomas lung and prostate cancersrdquoEuropean Review for Medical and Pharmacological Sciences vol16 no 15 pp 2064ndash2068 2012

[28] V C Toreti H H Sato G M Pastore and Y K Park ldquoRecentprogress of propolis for its biological and chemical composi-tions and its botanical originrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 697390 13 pages2013

[29] A Kujumgiev I Tsvetkova Y Serkedjieva V Bankova RChristov and S Popov ldquoAntibacterial antifungal and antiviralactivity of propolis of different geographic originrdquo Journal ofEthnopharmacology vol 64 no 3 pp 235ndash240 1999

[30] D Pesta and E Gnaiger ldquoHigh-resolution respirometryOXPHOS protocols for human cells and permeabilized fibersfrom small biopsies of human musclerdquo Methods in MolecularBiology vol 810 pp 25ndash58 2012

[31] M Piasecka and J Kawiak ldquoSperm mitochondria of patientswith normal sperm motility and with asthenozoospermiaMorphological and functional studyrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 125ndash139 2003

[32] M Piasecka M Laszczynska and D Gaczarzewicz ldquoMorpho-logical and functional evaluation of spermatozoa from patientswith asthenoteratozoospermiardquo Folia Morphologica vol 62 no4 pp 479ndash481 2003

[33] A Stendardi R Focarelli P Piomboni et al ldquoEvaluation ofmitochondrial respiratory efficiency during in vitro capacita-tion of human spermatozoardquo International Journal of Andrologyvol 34 no 3 pp 247ndash255 2011

[34] A M Petrunkina R A P Harrison M Tsolova E Jebe and ETopfer-Petersen ldquoSignalling pathways involved in the controlof sperm cell volumerdquo Reproduction vol 133 no 1 pp 61ndash732007

[35] R Barros Silva N A G Santos N M Martins et al ldquoCaf-feic acid phenethyl ester protects against the dopaminergicneuronal loss induced by 6-hydroxydopamine in ratsrdquo Neuro-science vol 233 pp 86ndash94 2013

[36] K S Kumaran and P S M Prince ldquoCaffeic acid protectsrat heart mitochondria against isoproterenol-induced oxidativedamagerdquo Cell Stress and Chaperones vol 15 no 6 pp 791ndash8062010

[37] H Cimen M J Han Y Yang Q Tong H Koc and E CKoc ldquoRegulation of succinate dehydrogenase activity by SIRT3in mammalian mitochondriardquo Biochemistry vol 49 no 2 pp304ndash311 2010

[38] M I Yousef and A F Salama ldquoPropolis protection fromreproductive toxicity caused by aluminium chloride in maleratsrdquo Food and Chemical Toxicology vol 47 no 6 pp 1168ndash11752009

[39] M I Yousef K I Kamel M S Hassan and A M A El-Morsy ldquoProtective role of propolis against reproductive toxicityof triphenyltin in male rabbitsrdquo Food and Chemical Toxicologyvol 48 no 7 pp 1846ndash1852 2010

[40] O Ciftci I Ozdemir M Aydin and A Beytur ldquoBeneficialeffects of chrysin on the reproductive system of adult male ratsrdquoAndrologia vol 44 no 3 pp 181ndash186 2012

[41] R Zheng and H Zhang ldquoEffects of ferulic acid on fertile andasthenozoospermic infertile human sperm motility viability

lipid peroxidation and cyclic nucleotidesrdquo Free Radical Biologyand Medicine vol 22 no 4 pp 581ndash586 1997

[42] A Ferramosca S P Provenzano L Coppola and V ZaraldquoMitochondrial respiratory efficiency is positively correlatedwith human sperm motilityrdquo Urology vol 79 no 4 pp 809ndash814 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and antitumor [1 7] As a result of these effects over the past30 years propolis has been the subject of intense medicalstudies

Infertility affects 10ndash15 of couples of reproductive ageand plenty of unanswered questions remain concerning thephysiological mechanisms underlying the successful fertility[8] Male factor contributes to about 50 of cases of infer-tility and most of them are still idiopathic [9] More than90 of male infertility cases are due to low sperm count(oligozoospermia) poor sperm motility (asthenozoosper-mia) abnormal spermmorphology (teratozoospermia) or allthree The remaining cases of male infertility can be causedby a number of factors including anatomical problemshormonal imbalances and genetic defects

Appropriate spermmotility is fundamental for reproduc-tive success in mammals since the spermatozoa have to travela relatively long distance through the female reproductivesystem by active flagellar motion The energy for the spermmovement in the form of adenosine triphosphate (ATP)is supplied by two metabolic processes glycolysis takingplace in the cytoplasm or oxidative phosphorylation in themitochondria found only in the sperm midpiece where72ndash80 helically arranged organelles can be detectedHoweverthe relative contributions of both pathways to the spermmotility in different species are a matter of long-standingdebate [10 11] Nevertheless recent experimental data suggestthat reduced efficiency of the mitochondrial respiratoryactivity may contribute to the reduced sperm motility Inasthenozoospermic patients morphological and functionalchanges in sperm mitochondria have been described [12 13]

Until now the effects of propolis on the mitochondrialmorphology and function have been studied particularly inthe somatic cells However the results of these studies arefar from uniform showing both stimulation and inhibitionof various mitochondrial functions including oxygen con-sumption apoptosis andmitochondrial membrane potential[14ndash17] The aim of our study was to assess the effect ofethanolic extract of propolis (EEP) on the human spermmotility mitochondrial respiratory activity and membranepotential to test the putative therapeutic potential of thisnatural product in the treatment of asthenozoospermia

2 Methods

21 Ethanolic Extract of Propolis Preparation Propolis wascollected using plastic nets in region ofWest Bohemia (HorniSlavkovmdash50∘ 810158401726810158401015840 N 12∘ 4810158404899210158401015840 E) in September2012 Propolis was frozen at minus20∘C and ground in a mill Theresulting powder (10 g) was mixed at room temperature with70 ethanol (100mL) for 24 h and then filtered The filtratewas then made up to 100mL with 70 ethanol [18]The sam-plewas kept in darkness at 4∘Cuntil analysis For experimentswith live cells propolis was further diluted resulting in a finalethanol concentration below 1 which is not toxic to cells[19] The final concentration of propolis chosen for furtherexperiments was 001mgmL of the corresponding medium

22 High Performance Liquid Chromatography Analysis(HPLC) Qualitative and quantitative chromatographic anal-yses of phenolics were performed on a HPLC systemequipped with a binary pump (Waters 1525) Waters 717 plusAutosampler and dual UVVIS detector 2487 Separationwas performed on a Symmetry C18 column particle size5 120583m (150mm times 46mm) using a mobile phase of 008acetic acid in methanol (A) and 01 acetic acid and 10methanol in water (B) The gradient was 10ndash47 A (25min)47 A (25ndash40min) 47ndash70 A (40ndash70min) and 70ndash100A (70ndash80min) at a flow rate of mobile phase 05mLminInjection volume was 10 120583L and column temperature was30∘C

Spectrophotometric detection was conducted at 280 nmand 330 nm Identification of polyphenolic compounds wasachieved by comparison of retention times with those ofcommercial pure compounds All standards were dissolvedin dimethyl sulfoxide (Sigma-Aldrich St Louis USA) to give10mmolL standard solutions Calibration standards wereprepared by dilution of the standard solution in ethanol

Quantitative analysis was carried out by external standardmethod Calibration curves showed a linear response of 1198772 gt097 over a concentration range of 5ndash100 120583molL Beforethe HPLC analysis the EEP was filtered on teflon syringemicrofilter Separon 045120583mThe propolis extract was dilutedone hundred times for HPLC analysis

Phenolic compounds were purchased from Sigma-Aldrich (St Louis USA) (apigenin chrysin genisteinkaempferol luteolin naringenin pinocembrin galangin andphenolic acids caffeic p-coumaric t-ferulic t-cinnamicbenzoic and gallic acid and caffeic acid phenethyl ester)Vanillin was purchased fromMerck (Darmstadt Germany)

23 Sperm Sample Preparation The study design wasapproved by the Local Ethics Committee of the UniversityHospital in Pilsen and a written informed consent wasobtained from each of the 10 participants included in thestudy (mean age 242 years SEM plusmn 28)

Ejaculates were collected after 3 days of sexual abstinencein IVF Center Prof Zech Pilsen Semen samples were eval-uated by an experienced employee After liquefaction theywere analyzed according to the World Health Organizationcriteria 2010 [20] We investigated semen volume and underthe microscope with phase-contrast optics at magnificationtimes200 concentration of spermatozoa motility of spermato-zoa and pathologies Sperm motility was assessed at roomtemperature in Makler counting chamber Two hundredspermatozoa per replicate were classified into three motilitycategories (progressive nonprogressive and immotile spermcells) All samples were considered normozoospermic ejacu-lates (Table 1)

Fresh ejaculate (01mL) was subjected to experimentwith propolis Propolis or ethanol only (1 120583L) was addedto 01mL of fresh ejaculate (final concentration of propoliswas 001mgmL) and sperm motility was evaluated after 60minutes

The remaining sample was prepared by gradient separa-tion technique and used for experiment with polarographicoxygraph (Oroboros Innsbruck Austria) and sperm flow









2sdot6H2O 60mmolL







2) Wash with PBS



CytochromecCoenzyme

Q

Complex I

Complex II

GpDH

ETF



2= oxygen




3 Results


000

004

008

012

016

020

Caffe

ic ac

id21225

Vani

llin23409

p-co

umar

ic ac

id27573

Cinn

amic

acid

42792

Kaem

pfer

ol51341

Apig

enin

53415

Chry

sin70074

Time (min)

(AU

)

tfer

ulic

acid

28955

15 20 25 30 35 40 45 50 55 60 65 70 75 80







Control Propolis

Prog

ress

ive m

otili

ty at

60

min

()

lowast

100

90

80

70

60

50

40

30

20

10

0


119875



10 20 30 40 50 60

250

200

150

100

50

0

10

8

6

4

2

0

Time (min)

O2

conc

entr

atio

n (n

mol

mL)

O2

flux

per u

nit (

nmol

(slowast

unit)

)

Dig DD Pcar c

S

Omy

FF F Rot

AmA

M G P










30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875

lt


55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))


S3S3u


119875

lt






(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2sdot6H2O 60mmolL







2) Wash with PBS



CytochromecCoenzyme

Q

Complex I

Complex II

GpDH

ETF



2= oxygen




3 Results


000

004

008

012

016

020

Caffe

ic ac

id21225

Vani

llin23409

p-co

umar

ic ac

id27573

Cinn

amic

acid

42792

Kaem

pfer

ol51341

Apig

enin

53415

Chry

sin70074

Time (min)

(AU

)

tfer

ulic

acid

28955

15 20 25 30 35 40 45 50 55 60 65 70 75 80







Control Propolis

Prog

ress

ive m

otili

ty at

60

min

()

lowast

100

90

80

70

60

50

40

30

20

10

0


119875



10 20 30 40 50 60

250

200

150

100

50

0

10

8

6

4

2

0

Time (min)

O2

conc

entr

atio

n (n

mol

mL)

O2

flux

per u

nit (

nmol

(slowast

unit)

)

Dig DD Pcar c

S

Omy

FF F Rot

AmA

M G P










30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875

lt


55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))


S3S3u


119875

lt






(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CytochromecCoenzyme

Q

Complex I

Complex II

GpDH

ETF



2= oxygen




3 Results


000

004

008

012

016

020

Caffe

ic ac

id21225

Vani

llin23409

p-co

umar

ic ac

id27573

Cinn

amic

acid

42792

Kaem

pfer

ol51341

Apig

enin

53415

Chry

sin70074

Time (min)

(AU

)

tfer

ulic

acid

28955

15 20 25 30 35 40 45 50 55 60 65 70 75 80







Control Propolis

Prog

ress

ive m

otili

ty at

60

min

()

lowast

100

90

80

70

60

50

40

30

20

10

0


119875



10 20 30 40 50 60

250

200

150

100

50

0

10

8

6

4

2

0

Time (min)

O2

conc

entr

atio

n (n

mol

mL)

O2

flux

per u

nit (

nmol

(slowast

unit)

)

Dig DD Pcar c

S

Omy

FF F Rot

AmA

M G P










30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875

lt


55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))


S3S3u


119875

lt






(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Control Propolis

Prog

ress

ive m

otili

ty at

60

min

()

lowast

100

90

80

70

60

50

40

30

20

10

0


119875



10 20 30 40 50 60

250

200

150

100

50

0

10

8

6

4

2

0

Time (min)

O2

conc

entr

atio

n (n

mol

mL)

O2

flux

per u

nit (

nmol

(slowast

unit)

)

Dig DD Pcar c

S

Omy

FF F Rot

AmA

M G P










30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875

lt


55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))


S3S3u


119875

lt






(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















30

25

20

15

10

05

00

lowast

S3 (OXPHOS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875


MGPPcarS S(Rot)

6

5

4

3

2

1

0

lowast

lowast

S3u (ETS)

ControlPropolis

Oxy

gen

flux

(nm

ol(

slowastIU

))


119875

lt


55

50

45

40

35

30

25

20

15

10

05

00

lowast

lowast

lowast

Oxy

gen

cons

umpt

ion

(nm

ol(

slowastIU

))


S3S3u


119875

lt






(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

50120583m

(b)



Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


Green fluorescence

Red

fluor

esce

nce

Living cells

105

104

103

102

105104103102


4 Discussion












5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusions







Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Effects of the Czech Propolis on Sperm Mitochondrial Function

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Transcript of Effects of the Czech Propolis on Sperm Mitochondrial Function