1. Introduction. Correspondence should be addressed to Marcilio Nichi;

Transcription

1 Hindwi Oxidtive Medicine nd Cellulr Longevity Volume 217, Article ID , 8 pges Reserch Article The Stimulted Glycolytic Pthwy Is Ale to Mintin ATP Levels nd Kinetic Ptterns of Bovine Epididyml Sperm Sujected to Mitochondril Uncoupling João D. A. Losno, 1 Jun Fernndo Pdín, 2,3 Igo Méndez-López, 2 Dniel S. R. Angrimni, 1 Antonio G. Grcí, 2 Vlquiri H. Brne, 1 nd Mrcilio Nichi 1,4 1 Deprtment of Animl Reproduction, Fculty of Veterinry Medicine, University of São Pulo, São Pulo, SP, Brzil 2 Deprtment of Phrmcology, Fculty of Medicine, Autonomous University of Mdrid, Mdrid, Spin 3 Deprtment of Medicl Science, Fculty of Medicine Ciudd Rel, University of Cstill-L Mnch, Ciudd Rel, Spin 4 School of Veterinry Medicine nd Animl Science, Avenue Orlndo Mrques de Piv, No. 87, São Pulo, SP, Brzil Correspondence should e ddressed to Mrcilio Nichi; mnichi@usp.r Received 3 Ferury 217; Accepted 8 Mrch 217; Pulished 9 My 217 Acdemic Editor: Moh H. Mlek Copyright 217 João D. A. Losno et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Studies hve reported the importnce of mitochondri in sperm functionlity. However, for some species, the glycolytic pthwy ppers to e s importnt s oxidtive phosphoryltion in ATP synthesis nd sperm kinetics. These mechnisms hve not een fully elucidted for ovine spermtozo. Therefore, the im of this study ws to evlute the role of mitochondri nd the glycolytic pthwy in ATP synthesis, sperm movement ptterns, nd oxidtive homeostsis of epididyml spermtozo in ovine specimens. We oserved tht mitochondril uncoupling with protonophores significntly reduced ATP levels. However, these levels were reestlished fter stimultion of the glycolytic pthwy. We verified the sme pttern of results for sperm kinetic vriles nd the production of rective oxygen species (ROS). Thus, we suggest tht, fter its pproprite stimultion, the glycolytic pthwy is cple of mintining ATP levels, sperm kinetic ptterns, nd oxidtive lnce of ovine epididyml spermtozo sumitted to mitochondril uncoupling. 1. Introduction Studies hve shown the importnce of mitochondri in sperm functionlity, s they re considered the min source of ATP for cellulr homeostsis nd motility [1, 2]. However, the role of mitochondri in sperm metolism hs een mtter of dete. Muki nd Okuno [3] verified tht ATP levels nd flgellr eting remined constnt when the mitochondri of mouse sperm ws uncoupled concurrently with glycolysis stimultion. However, y inhiiting glycolysis nd stimulting oxidtive phosphoryltion, uthors oserved tht flgellr eting nd ATP levels were quickly reduced. These results indicte tht glycolysis plys n importnt role in murine sperm energy production. In similr study, Nscimento et l. [4] performed inhiitory nd stimultory tretments for oth oxidtive phosphoryltion nd glycolysis in humn sperm. Authors concluded tht oxidtive phosphoryltion, despite contriuting to ATP production, is not sufficient to sustin sperm motility, confirming tht the glycolytic pthwy is the primry energy source for humn sperm. Additionlly, ATP produced y oxidtive phosphoryltion in the sperm midpiece is not efficiently relesed into the distl portions of the til, indicting tht glycolysis plys key role in the flgellr et of such sperm regions [5 7]. Dvil et l. [8] demonstrted tht equine spermtozo require oxidtive phosphoryltion s glycolytic pthwy to mintin motility. Complementry in rm, Losno et l. [9] demonstrted tht mitochondril depolriztion did not ffect totl motility; however, sperm kinetic ptterns were ltered. On the other hnd, they found tht glycolytic pthwy inhiition impired totl motility nd sperm move-

2 2 Oxidtive Medicine nd Cellulr Longevity ment ptterns. For oth species, glycolytic pthwy seems to e s importnt s oxidtive phosphoryltion for sperm physiology. However, the role of these pthwys on ovine sperm functionlity hs not een fully elucidted. This informtion is extremely importnt for the understnding of ull sperm physiology. In ddition, studies evluting the energy metolism of ovine sperm my contriute to the understnding of possile cuses for the reduction in sperm qulity nd fertiliztion filures relted to these metolic pthwys nd then improve existent iotechnology s, such s rtificil insemintion which cn impct in higher fertility rtes. Sperm collected directly from the epididymis seem to e the idel cellulr model to study energy metolism. This is due to the mny glycolysis, citric cid cycle, nd oxidtive phosphoryltion stimulnts contined in the seminl plsm derived from the ccessory glnds [1 12]. The fct tht epididyml spermtozo hve not een stimulted with these sustnces provides etter in vitro mnipultion of these cells, llowing the stimultion nd inhiition of these pthwys to evlute the role of ech metolic pthwy on sperm functionlity. Therefore, the im of this study ws to evlute the role of mitochondri nd glycolysis in ATP production, genertion of rective oxygen species (ROS), nd kinetic ptterns of epididyml ovine sperm y mens of mitochondril uncoupling nd glycolytic pthwy stimultion. 2. Mteril nd Methods The present experiment ws conducted ccording to ethicl guidelines for niml experiments nd pproved y the Bioethics Committee of the School of Veterinry Medicine nd Animl Science t the University of São Pulo (protocol numer ). In this study, we sumitted ovine epididyml spermtozo to tretment with the oxidtive phosphoryltion uncoupler cronyl cynide 4-(trifluoromethoxy)phenylhydrzone (FCCP) to significntly reduce mitochondril ATP synthesis nd stimulted the glycolytic pthwy y glucose ddition. However, in order to verify the optiml concentrtions of the uncoupler, FCCP, we performed dose-response curve in experiment 1. Thus, the selected concentrtions were used in the susequent experiments. The im of these experiments ws to evlute the contriution of mitochondri to ATP synthesis (experiment 2), ptterns of sperm kinetics (experiment 3), nd oxidtive homeostsis (experiment 4) of ovine epididyml sperm nd verify if stimultion of the glycolytic pthwy would e le to mintin these sperm prmeters tht re proly suppressed y mitochondril uncoupling Smple Collection. Epididyml sperm smples were collected nd then dissecting the epididymis cud with sclpel lde, ccording to previous protocol [13]. To limit lood contmintion, dissection ws performed crefully. The flowing epididyml fluid ws collected with n utomtic pipette. Then, smples were used in ech respective experiment proposed Experiment 1 Concentrtion-Response Curve of Mitochondril Uncoupler, FCCP. To evlute the effect of mitochondril uncoupling y FCCP, spermtozo from 3 ovine epididymides (n =3) were collected nd diluted to concentrtion of 1 million spermtozo per ml in modified TALP. Despite minimum numer of epididymis used, we evluted the mitochondri of ech spermtozoon singly, s experimentl unit. Thus, we used 15 to 26 cells per FCCP concentrtion. Therefter, the spermtozo were incuted in perfusion chmer with mitochondril fluorophore tetrmethylrhodmine-ethyl-ester perchlorte t 5 nm (ThermoFisher Scientific,.5 μl of TMRE in 1 ml of medium) for 5 minutes t 37 C. For the spermtozo to remin ttched during perfusion with FCCP, coverslips of the perfusion chmer were treted with polylysine. After incution, the mount of TMRE fluorescence cptured y ech sperm mitochondrion ws recorded y the softwre LAS AF Lite (Leic Microsystems, Germny) t n emission of 5 nm nd excittion of 6 nm y fluorescence microscope (Leic Microsystems, Germny). Thirty seconds of mitochondril sl fluorescence ws recorded, nd then perfusions were performed with incresing FCCP concentrtions (Tocris Bioscience, MN, USA;.3, 1, 3, 1, 3, 6, nd 1 μm) y mens of n electrovlve controller. Stimultion performed with FCCP t 3 seconds ws recorded, nd the percentge of mitochondril depolriztion ws clculted sed on the difference etween the sl fluorescence nd the mount of fluorescence retined in the mitochondri of ech spermtozoon fter 3 seconds of FCCP stimultion. The lower FCCP concentrtions of the dose-response curve (.3, 1, nd 3 μm) nd the concentrtion insufficient for the promotion of mitochondril depolriztion (.1 μm, concentrtion under the curve) were selected to e used in the susequent experiments. We selected these concentrtions in order to significntly reduce the mitochondril ATP synthesis without promoting disruption in this orgnelle Experiment 2 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on ATP Levels. In this experiment, spermtozo from 6 ovine epididymides (n =6) were collected nd diluted to concentrtion of 1 million spermtozo per ml in modified TALP medium. Ech smple ws divided into ten liquots, which were sumitted to 5 2 fctoril design wherein one of the fctors ws the ddition of glucose (5 mm) nd the other fctor ws the tretment with incresing concentrtions of FCCP (.1,.3, 1, nd 3 μm). After 15-minute incution, the tretments were sujected to mesurements of ATP levels y mens of luminescence technique. For this procedure, 5 μl liquots in duplicte from ech tretment contining 1 spermtozo were dded to 5 μl of CellTiter-Glo Luminescent Cell Viility Assy kit (Promeg, USA) nd incuted for 3 minutes t 37 C ccording to the mnufcturer s recommendtions. Immeditely fter this procedure, ATP levels were mesured in luminescence pprtus (ThermoFisher Scientific, MA, USA) in duplicte. The results otined, expressed in ritrry light units (AUL), were interpolted

3 Oxidtive Medicine nd Cellulr Longevity 3 on stndrd curve contining different concentrtions of ATP (1, 1, 1, 5, nd 1 nm) nd were then expressed in nm ATP Experiment 3 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on Sperm Kinetic Ptterns. To evlute the effect of mitochondril uncoupling nd glycolysis stimultion on sperm kinetic ptterns, spermtozo from 7 ovine epididymides (n =7) were collected nd diluted to concentrtion of 1 million spermtozo per ml in modified TALP medium. Ech smple ws divided into ten liquots, which were sumitted to 5 2 fctoril design wherein one of the fctors ws the ddition of glucose (5 mm) nd the other ws the tretment with incresing concentrtions of FCCP (.1,.3, 1, nd 3 μm). After 5 minutes of incution, the sperm smples were sujected to computerized nlysis of sperm kinetics (ISASPBOS, Proiser, Vlenci, Spin). The following vriles were considered: motility (%), progressive motility (%), VAP (verge pth velocity, μm/s), VSL (stright-line velocity, μm/s), VCL (curviliner velocity, μm/s), ALH (mplitude of lterl hed displcement, μm), BCF (et cross-frequency, Hz), STR (strightness, %), nd LIN (linerity, %). In ddition to these prmeters, the sperm were lso divided into four groups sed on velocity: rpid (VAP > 5 μm/s; %), medium (3 μm/s < VAP < 5 μm/s; %), slow (VAP < 3 μm/s or VSL < 15 μm/s; %), nd sttic (%) [14] Experiment 4 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on Rective Oxygen Species Production. To evlute the effect of mitochondril uncoupling nd glycolysis stimultion on rective oxygen species production, spermtozo from 6 ovine epididymides (n =6) were collected nd diluted to concentrtion of 1 million spermtozo per ml in modified TALP. Ech smple ws divided into ten liquots, which were sumitted to 4 2 fctoril design wherein one of the fctors ws the ddition of glucose (5 mm) nd the other ws the tretment with incresing concentrtions of FCCP (.1,.3, 1, nd 3 μm). These tretments were incuted for 3 minutes t 37 C nd sujected to the detection of rective oxygen species. To perform this technique, 1 sperm were incuted in modified TALP solution contining 1 μm (finl concentrtion) of the fluorescent proe CM-H2DCFDA for 3 minutes (triplicte smples). After incution ws performed, the ROS were detected using fluorometer (Fluostr microplte reder Omeg, Ltec-BMG, Germny) t excittion nm nd emission nm. The fluorescence intensity results otined were interpolted on stndrd curve contining different concentrtions of hydrogen peroxide (H 2 O 2 : 3, 1, 3, 6, 1, 2, nd 3 μm) nd were then expressed in μl ofo 2 generted. Dt were normlized reltive to the control group (untreted smples) Sttisticl Anlysis. The concentrtion-response curve for FCCP (experiment 1) ws performed y nonliner regression using the softwre GrphPd Prism 6. Dt relting to the mesurement of ATP levels nd computerized nlysis of sperm kinetics (experiments 2 nd 3, resp.) were nlyzed using the SAS System for Windows (SAS Institute Inc., Cry, NC, USA). Thus, the interction etween FCCP nd glucose fctors ws determined y PROC GLM. Differences etween tretments were ssessed using prmetric (Student s t-test for ech fctor seprtely or LSD test for the comintion of fctors) nd nonprmetric tests (Wilcoxon) in ccordnce with the normlity of the residuls (Gussin distriution) nd homogeneity of the vrinces. To nlyze the effect of FCCP in the presence or sence of glucose in the ROS production, dt normlized to the control group were compred y ANOVA vrince nlysis (LSD test) using the SAS System for Windows progrm (SAS Institute Inc., Cry, NC, USA). The level of significnce to reject the H (null hypothesis) ws 5%; tht is, the significnce level ws.5. Significnt differences etween clssifictory vriles (tretments) nd specific response vrile were considered. 3. Results 3.1. Experiment 1 Concentrtion-Response Curve of MitochondrilUncouplerFCCP. By using nonliner regression, we found tht the concentrtion-response curve is squre root = 7 nd EC5 = μm. We oserved high percentge of depolriztion with FCCP concentrtions of 3 μm, 6 μm, nd 1 μm (Figure 1). Thus, in order to select points where there is reduction in ATP without promoting disruption in the orgnelle, we selected 3 μm, 1 μm,.3 μm, nd.1 μm for the concentrtions used in the susequent experiments (concentrtion under the curve Figure 1) Experiment 2 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on ATP Levels. There were significnt effects of FCCP, glucose, nd FCCP-y-glucose interction in the ATP (P <1; Tle 1) nlysis. Then, it ws possile to compre the effects of the ddition of glucose in the FCCP smple (Figure 2). We oserved lower ATP production in the FCCP group t concentrtions of.3 μm (18.3 ± 31.9 nm), 1 μm (22.2 ± 4.4 nm), nd 3 μm (272.3 ± 7.4 nm) thn t μm (control ± 63.7 nm) nd.1 μm (422.4 ± 41.5 nm Figure 2). However, in the group treted with FCCP supplemented with glucose, the concentrtions were similr etween the groups treted with.1 μm (61.8 ± 57.8 nm),.3 μm (66.2 ± 64.2 nm), 1 μm (67.9 ± 61.9 nm), nd 3 μm (696.1 ± 68.5 nm) FCCP nd the group treted with glucose without FCCP (577.2 ± 7.4 nm) (Figure 2) Experiment 3 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on Sperm Kinetics Ptterns. There were significnt effects of FCCP, glucose, nd FCCP-y-glucose interction (P <5) on ll CASA prmeters (Tle 1). We oserved decrese in the totl motility etween smples without FCCP (control) nd with glucose (Figure 3()); however, it ws possile to note n increse in motility in the groups treted with.3 μm,.1 μm, 1 μm, nd 3 μm FCCP supplemented with glucose (Figure 3()).

4 4 Oxidtive Medicine nd Cellulr Longevity 12 I II 1 IV 8 훥휓 m (%) 6 III 4 II III IV 2 I Log (M) FCCP Figure 1: Dose-response curve of FCCP concentrtions (.3, 1, 3, 1, 3, 6, nd 1 μm) in sperm of ovine epididyml smples. Superscript numerls indicte the FCCP concentrtion used nd their respective imges. Mitochondril depolriztion of ovine spermtozo t FCCP concentrtion of μm (I), 1 μm (II), 3 μm (III), nd 6 μm (IV). Arrows indicte mitochondri leled with the TMRE fluorescent proe t different percentges of mitochondril depolriztion. 4x mgnifiction. Tle 1: Proility vlues for the FCCP (,.1,.3, 1, nd 3 μm), glucose, nd their interction on computer-ssisted sperm nlysis (CASA). FCCP Glucose FCCP glucose Totl sperm motility (%) <.1.3 <.1 Sperm progressive motility (%) <.1.5 <.1 Percentge of rpid sperm (%).6.77 <.1 Percentge of medium sperm (%) <.1 Percentge of slow sperm (%) Amplitude of lterl hed displcement (ALH μm) Averge pth velocity (VAP μm/s) <.1.2 <.1 Stright line velocity (VSL μm/s) <.1.2 <.1 Curviliner velocity (VCL μm/s) Bet cross-frequency (BCF Hz) <.1.2 <.1 Sperm strightness (STR %).2.2 <.1 Sperm linerity (LIN %) <.1.3 <.1 Wole (WOB %) <.1.3 <.1 This sme effect ws detected for progressive motility (Figure 3()), VAP, VSL, VCL, nd rpid sperm velocity (see Supplementry Mteril ville online t doi.org/1.1155/217/ ). Next, we exmined the effects of the ddition of glucose in the FCCP smples (Figure 3 nd Supplementry Mteril). In the BCF nlysis, we oserved n increse in the groups with 1 μm nd 3 μm of FCCP supplemented with glucose ut decrese in the glucose group (Supplementry Mteril). Furthermore, we oserved n increse in the slow sperm velocity in the smples supplemented with glucose in the groups treted with 1 μm nd 3 μm of FCCP nd glucose lone ut decrese in the group treted with.3 μm FCCP (Supplementry Mteril). With FCCP tretment, the control nd.1 μm groups hd higher vlues of totl sperm motility, VAP, nd VSL thn the.3 μm group, which ws superior to the 1 μm nd 3 μm smples (Figure 3 nd Supplementry Mteril). However, in the ALH, BCF, strightness, linerity, nd wole nlyses, the control,.1 μm, nd 3 μm groups hd higher rtes thn the 1 μm nd 3 μm groups (Supplementry Mteril). In the VCL nd percentge of medium sperm velocity, we oserved tht the 3 μm nd 1 μm groups hd lower vlues thn the.3 μm group,

5 Oxidtive Medicine nd Cellulr Longevity ATP (nm) Control Glucose FCCP (휇M) FCCP (휇M) + glucose (5 mm) Figure 2: ATP production y ovine epididyml sperm treted with FCCP in different concentrtions ( μm,.1 μm,.3 μm, 1 μm, nd 3 μm) in sence or presence of glucose 5 mm. - superscripts indicte differences etween concentrtions (P <5). indictes differences fter the glucose supplementtion (P <5). which ws similr to the.1 μm group ut lower thn the control (Supplementry Mteril). In progressive motility (PM), the control group hd the highest rtes (Figure 3). However, we oserved lower rtes of PM in the 3 μm nd 1 μm groups thn in the.3 μm group, which ws inferior to the.1 μm group (Figure 3). In the medium sperm velocity, the control group ws superior to the 1 μm nd 3 μm groups (Supplementry Mteril). On the other hnd, in the slow sperm velocity, the control nd 1 μm groups hd lower rtes thn the.1 μm nd.3 μm groups (Supplementry Mteril). When we compred the results etween the concentrtions of FCCP supplemented with glucose, we highlighted the higher vlues of progressive motility, strightness, nd rpid sperm velocity in the groups treted with 3 μm nd.3 μm of FCCP, which were superior to the glucose group (Figure 3 nd Supplementry Mteril). In the totl motility nlysis, the 3 μm group ws superior to the glucose group (Figure 3). However, in the VCL, the.3 μm group hd higher vlues thn the 1 μm group (Supplementry Mteril). The glucose group ws lower thn the.3 μm, 1 μm nd 3 μm groups in the BCF prmeter (Supplementry Mteril). However, in the slow sperm velocity, the 1 μm group ws higher thn the.3 μm group (Supplementry Mteril). The remining CASA vriles did not show ny difference etween the groups (Supplementry Mteril) Experiment 4 Effect of Mitochondril Uncoupling nd Glycolysis Stimultion on Rective Oxygen Species Production. In the production of the rective oxygen species, we highlight in Figure 4 the higher ROS generted y sperm treted with 3 μm of FCCP supplemented with glucose (332.9 ± μl) thn tht with FCCP concentrtions of.1 μm (213.2 ± μl), 1 μm ( ± 5.39 μl), nd 3 μm (17.6 ± μl). 4. Discussion The im of this study ws to evlute the role of mitochondri nd the glycolytic pthwy in the mintennce of ATP levels, the prmeters of sperm movement, nd the production of rective oxygen species in epididyml ovine sperm. To perform this experiment, we sumitted ovine sperm to mitochondril uncoupling with FCCP to significntly reduce the synthesis of ATP y the mitochondri nd evlute the effect of this reduction in sperm functionlity. Furthermore, we promoted stimultion of the glycolytic pthwy y glucose ddition concurrently with the mitochondril uncoupling to ssess whether glycolysis would e le to mintin the ATP levels, sperm kinetic ptterns, nd oxidtive homeostsis possily hrmed y mitochondril depolriztion. The mitochondril uncoupler FCCP is lipophilic molecule with protonophore properties; in other words, it is cple of intercting with the inner mitochondril memrne to llow pumped protons to return to the mitochondril mtrix, dissipting the proton grdient nd influencing the mitochondril chemiosmosis [15, 16]. Indeed, in our experiment, we confirmed the depolrizing effect of the uncoupler FCCP. In experiment 2, we oserved significnt reduction in ATP levels in the groups treted with.3, 1, nd 3 μm of FCCP compred to the control group. ATP production in the mitochondri occurs y mens of the coupling of two rections: the trnsport of electrons throughout the respirtory chin nd the proton grdient. This ltest grdient is cple of storing energy, clled proton motive force, which drives the synthesis of ATP through ADP nd inorgnic phosphte [17]. FCCP hs protonophore effect tht will dissipte the proton grdient, therey reducing ATP synthesis, s noted in our results. On the other hnd, the groups tht were treted with these sme FCCP concentrtions ut were supplemented with glucose hd higher levels of ATP, similr to the control group. From these results, we cn suggest tht the glycolytic pthwy, fter eing stimulted, is le to mintin ATP levels in ovine epididyml sperm. In fct, our results were consistent with previous study in ors, which demonstrted tht sperm mitochondri ccount for only 5% of energy production, while the glycolytic pthwy contriutes to 95% [18]. Additionlly, species such s mice my use ATP from glycolysis nd mitochondril respirtion depending on their iologicl conditions without chnging sperm functionlity or sperm ATP levels [19]. In experiment 3, we oserved very similr pttern of results s in experiment 2. The motility nd spermtic movement ptterns were ffected y mitochondril uncoupling. However, stimultion of the glycolytic pthwy mintined sperm kinetic ptterns, even with cells undergoing mitochondril uncoupling. These results suggest tht for ovine sperm, there is close reltionship etween motility nd ATP levels. However, this reltionship is still mtter of controversy. In ccordnce with our study, Muki nd Okuno [3] verified tht ATP levels nd flgellr eting remined constnt when mouse sperm mitochondri were uncoupled concurrently with the supplementtion of sustrtes for glycolysis. 5

6 6 Oxidtive Medicine nd Cellulr Longevity 1 Totl motility (%) c c Progressive motility (%) c d d Control Glucose FCCP (휇M) FCCP (휇M) + glucose (5 mm) Control Glucose FCCP (휇M) FCCP (휇M) + glucose ( 5 mm) () () Figure 3: Totl () nd progressive () motility in ovine epididyml sperm treted with FCCP in different concentrtions ( μm,.1 μm,.3 μm, 1 μm, nd 3 μm) in sence or presence of glucose 5 mm. d superscripts indicte differences etween concentrtions (P <5). indictes differences fter the glucose supplementtion (P <5). Amount of O 2 generted (휇L) cd Glucose cd cd cd Additionlly, Krzyzosik et l. [2] lso oserved tht ovine sperm re cple of mintining similr motility ptterns in oth eroic nd neroic conditions, ssuming tht glycolysis is cple of mintining sperm motility. On the other hnd, Rmió-Lluch et l. [21] demonstrted tht the inhiition of ATP synthse impirs sperm motility, while intrcellulr ATP levels remin unchnged. Therefore, the uthor suggested n unknown essentil mitochondril mechnism responsile for motility mintennce tht does not rely only on the mintennce of ATP levels. The vritions in the results of the different experiments seem to e relted to the d c FCCP (휇M) FCCP (휇M) + glucose (5 mm) Figure 4: Amount of O 2 generted y ovine epididyml sperm treted with FCCCP in different concentrtions ( μm,.1 μm,.3 μm, 1 μm, nd 3 μm) in sence or presence of glucose 5 mm. d superscripts indicte differences etween concentrtions (P <5). species involved nd the iologicl conditions to which such cells hve een sujected [22, 23]. Therefore, there is need for further studies to elucidte these mechnisms. Regrding experiment 4, we oserved tht the groups treted with FCCP t 1 nd 3 μm in the sence of glucose hd lower production of rective oxygen species (ROS). The rective oxygen species produced y sperm ply key role in mny physiologicl processes such s hyperctivtion [24], cpcittion [25], nd the interction etween the sperm nd oocyte [26]. The fct tht the groups treted with FCCP nd glucose did not differ from the control group suggests tht glycolysis stimultion is le to mintin the physiologicl ROS production nd, ultimtely, oxidtive lnce. Moreover, the ility of FCCP in the sence of glucose to reduce ROS production revels possile therpeutic potentil for preventing the relese of excessive rective oxygen species. This ility to prevent ROS production my e due to the increse of the electron trnsport rtes ccompnied y reduction in mitochondril intermedite sttes which is le to donte electrons to oxygen [27]. Furthermore, studies hve demonstrted tht the reduction in ATP synthesis y mitochondri is ccompnied y reduction in ROS production [28]. In fct, studies hve shown this ility of mitochondril uncouplers in somtic cells [29, 3]. Therefore, knowledge of the role of ech metolic pthwy on sperm functionlity my trget therpies using sustrtes to stimulte these pthwys in reproduction iotechnologies. Furthermore, the dt of mitochondril uncoupling FCCP reduces the rective oxygen species production nd suggests tht this molecule cn e used to prevent seminl oxidtive stress during procedures tht induce this stress, such s cryopreservtion. Thus, such procedure cn improve reproductive indexes y mens of ssisted reproduction techniques in cttle, especilly

7 Oxidtive Medicine nd Cellulr Longevity 7 intruterine rtificil insemintion. Nevertheless, this therpeutic effect should e further studied in spermtozo. 5. Conclusion In conclusion, fter its stimultion, the glycolytic pthwy is cple of mintining ATP levels, sperm kinetic ptterns, nd oxidtive lnce of ovine epididyml spermtozo sumitted to mitochondril uncoupling. Arevitions ALH: Amplitude of lterl hed displcement ATP: Adenosine triphosphte BCF: Bet cross-frequency CASA: Computer-ssisted sperm nlysis FCCP: Cronyl cynide 4-(trifluoromethoxy) phenylhydrzone LIN: Sperm linerity LSD: Lest significnt difference ROS: Rective oxygen species STR: Sperm strightness TALP: Tyrode lumin lctte pyruvte VAP: Averge pth velocity VCL: Curviliner velocity VSL: Stright line velocity WOB: Wole. Conflicts of Interest The uthors declre tht they hve no competing interests. Acknowledgments This reserch ws finncilly supported y Instituto de Frmcologí Teófilo Hernndo. Also, the uthors thnk The Brzilin Ntionl Council for Scientific nd Technologicl Development (CNPq, Project no /214-4) for the finncil ssistnce nd Frncisco J. Blsco (Integrted Semen Anlysis System, Proiser R+D Systems, Vlenci, Spin) for the computerized nlysis of the motility. References [1] A. J. Trvis, J. A. Foster, N. A. Rosenum et l., Trgeting of germ cell-specific type 1 hexokinse lcking porin-inding domin to the mitochondri s well s to the hed nd firous sheth of murine spermtozo, Moleculr Biology of the Cell, vol. 9, no. 2, pp , [2] J. C. S. John, The trnsmission of mitochondril DNA following ssisted reproductive techniques, Theriogenology, vol. 57, no. 1, pp , 22. [3] C. Muki nd M. Okuno, Glycolysis plys mjor role for denosine triphosphte supplementtion in mouse sperm flgellr movement, Biology of Reproduction, vol. 71, no. 2, pp , 24. [4] J. M. Nscimento, L. Z. Shi, J. 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8 8 Oxidtive Medicine nd Cellulr Longevity [21] L. Rmió-Lluch, M. Yeste, J. M. Fernández-Novell et l., Oligomycin A-induced inhiition of mitochondril ATPsynthse ctivity suppresses or sperm motility nd in vitro cpcittion chievement without modifying overll sperm energy levels, Reproduction, Fertility nd Development, vol. 26, no. 6, pp , 214. [22] B. T. Storey, Mmmlin sperm metolism: oxygen nd sugr, friend nd foe, Interntionl Journl of Developmentl Biology, vol. 52, no. 5-6, pp , 24. [23] A. Amrl, B. Lourenço, M. Mrques, nd J. Rmlho-Sntos, Mitochondri functionlity nd sperm qulity, Reproduction, vol. 146, no. 5, pp. R163 R174, 213. [24] E. de Lmirnde nd C. Cgnon, Humn sperm hyperctivtion nd cpcittion s prts of n oxidtive process, Free Rdicl Biology nd Medicine, vol. 14, no. 2, pp , [25] R. J. Aitken, A. L. Ryn, M. A. Bker, nd E. A. McLughlin, Redox ctivity ssocited with the mturtion nd cpcittion of mmmlin spermtozo, Free Rdicl Biology nd Medicine, vol. 36, no. 8, pp , 24. [26] R. J. Aitken, M. Pterson, H. Fisher, D. W. Buckinghm, nd M. vn Duin, Redox regultion of tyrosine phosphoryltion in humn spermtozo nd its role in the control of humn sperm function, Journl of Cell Science, vol. 18, Prt 5, pp , [27] M. F. Cunh, C. Cldeir d Silv, M. F. Cerqueir, nd J. A. Kowltowski, Mild mitochondril uncoupling s therpeutic strtegy, Current Drug Trgets, vol. 12, no. 6, pp , 211. [28] P. Newsholme, E. Her, S. Hirr et l., Dietes ssocited cell stress nd dysfunction: role of mitochondril nd non-mitochondril ROS production nd ctivity, The Journl of Physiology, vol. 583, Prt 1, pp. 9 24, 27. [29] A. M. Vincent, J. A. Olzmnn, M. Brownlee, W. I. Sivitz, nd J. W. Russell, Uncoupling proteins prevent glucose-induced neuronl oxidtive stress nd progrmmed cell deth, Dietes, vol. 53, no. 3, pp , 24. [3] R. J. Milloux nd M. E. Hrper, Uncoupling proteins nd the control of mitochondril rective oxygen species production, Free Rdicl Biology nd Medicine, vol. 51, no. 6, pp , 211.

9 MEDIATORS of INFLAMMATION The Scientific World Journl Gstroenterology Reserch nd Prctice Journl of Dietes Reserch Interntionl Journl of Journl of Endocrinology Immunology Reserch Disese Mrkers Sumit your mnuscripts t BioMed Reserch Interntionl PPAR Reserch Journl of Oesity Journl of Ophthlmology Evidence-Bsed Complementry nd Alterntive Medicine Stem Cells Interntionl Journl of Oncology Prkinson s Disese Computtionl nd Mthemticl Methods in Medicine AIDS Behviourl Neurology Reserch nd Tretment Oxidtive Medicine nd Cellulr Longevity