Fluorescent probes for the detection of reactive oxygen species in human spermatozoa

Asian Jou nal o And ology (2020) 22, 465–471 www.asiaand o.com; www.ajand ology.com ha di ec s i speci ically o he mi ochond ia.14 Once inside he mi ochond ia, dihyd oe hidium will eac wi h O2 ●-, p oducing ano he molecule, 2-hyd oxye hidium.15 In a educed s a e, his compound emi s blue luo escence. Howe e , on oxida ion, i will in e cala e wi h DNA, emi ing ed luo escence.16 Mi oSOX™ Red has been widely used o he de ec ion o mi ochond ial O2 ●- in human spe ma ozoa.14,17–19 Dihyd oe hidium (DHE) can also be used by i sel o de ec ROS. Lacking he TPP ca ion, DHE is con ined o he cy oplasm20 and is commonly used o de ec cy osolic O2 ●- (o igina ing om he nico inamide adenine dinucleo ide phospha e [NADPH]-oxidase 5 [NOX5] sys em).14,20,21 De Iuliis e al.22 i s epo ed he use o his p obe in human spe ma ozoa. On a di e en no e, RedoxSenso ™ Red CC-1 is used as an indica o o he oxida i e ac i i y in li ing cells.23 Fo mo e gene al ROS de ec ion, CellROX® O ange Reagen has been used, and on oxida ion, i p oduces o ange/ ed luo escence in he cy osol.24 Ano he commonly used p obe is Mi oPY1, also including he TPP ca ion, which is selec i ely a ge ed owa d he mi ochond ia.25 This p obe de i es om bo ona e26 and has been used in he cons i u ion o p obes highly sensi i e o H2O2.27 On in e ac ion wi h H2O2, bo ona e is con e ed o phenol and emi s g een luo escence.25 None o hese h ee p obes (RedoxSenso ™ Red CC-1, CellROX® O ange Reagen , and Mi oPY1) has been p e iously es ed in human spe ma ozoa. Finally, o he de ec ion o RNS, DAF-2 DA is he s anda d p obe. Once inside he cell, DAF-2 DA will be hyd olyzed h ough he ac i i y o cy osolic INTRODUCTION F om he a ious easons con ibu ing o s a is ics ega ding male in e ili y,1 de ec i e spe m unc ion has been conside ed he majo cause,2 and in u n, oxida i e s ess (OS) is a big con ibu o o his impai ed unc ion, s emming om he p oduc ion o eac i e oxygen species (ROS) and eac i e ni ogen species (RNS). Spe ma ozoa p oduce bo h cy osolic and mi ochond ial supe oxide and ROS- ela ed hyd ogen pe oxide (H2O2).3,4 RNS a e also p oduced, namely ni ic oxide (NO) syn hesized by NO syn hase (NOS) and pe oxyni i e (ONOO−).5–7 The o e p oduc ion o ROS/RNS has been associa ed wi h memb ane damage, loss o mo ili y, p ema u e capaci a ion and he ac osome eac ion, abno mal mo phology, impai ed oocy e-spe m usion, and apop osis, among o he s.8,9 DNA damage is also a c i ical by-p oduc o OS ha can ul ima ely esul in e iliza ion p oblems, misca iage, and abno mali ies o he o sp ing.10–12 Mo eo e , ano he consequence o ROS p oduc ion is lipid pe oxida ion,8,9 comp omising he in eg i y o he spe m memb ane, impai ing mo ili y, and dec easing iabili y.13 The inc easing in e es in unde s anding he in ol emen o ROS/RNS p oduc ion in male in e ili y highligh s he need o imp o ed de ec ion sys ems o measu e accu a ely he con en o ROS/RNS, mos ly using luo escen p obes. One o he s aple eagen s is Mi oSOX™ Red, a p obe comp ising dihyd oe hidium bound o he iphenylphosphonium (TPP) ca ion ORIGINAL ARTICLE Fluo escen p obes o he de ec ion o eac i e oxygen species in human spe ma ozoa Sa a Escada-Rebelo1,2,3, F ancisca G Mo a2, Ana P Sousa2,4, Te esa Almeida-San os2,4,5, A u Pai a6, João Ramalho-San os2,7 Reac i e oxygen species (ROS) p oduc ion is a by-p oduc o mi ochond ial ac i i y and is necessa y o he acquisi ion o he capaci a ed s a e, a equi emen o unc ional spe ma ozoa. Howe e , an inc ease in oxida i e s ess, due o an abno mal p oduc ion o ROS, has been shown o be ela ed o loss o spe m unc ion, highligh ing he impo ance o an accu a e de ec ion o spe m ROS, gi en he speci ic na u e o his cell. In his wo k, we es ed a a ie y o comme cially a ailable luo escen p obes o de ec ROS and eac i e ni ogen species (RNS) in human spe m, o de ine hei speci ici y. Using bo h low cy ome y (FC) and luo escence mic oscopy (FM), we con i med ha Mi oSOX™ Red and dihyd oe hidium (DHE) de ec supe oxide anion (as de e mined using an imycin A as a posi i e con ol), while DAF-2A de ec s eac i e ni ogen species (namely, ni ic oxide). Fo he i s ime, we also epo ha RedoxSenso ™ Red CC-1, CellROX® O ange Reagen , and Mi oPY1 seem o be mos ly sensi i e o hyd ogen pe oxide, bu no supe oxide. Fu he mo e, mean luo escence in ensi y (and no pe cen age o labeled cells) is he main pa ame e ha can be ep oducibly moni o ed using his ype o me hodology. Asian Jou nal o And ology (2020) 22, 465–471; doi: 10.4103/aja.aja_132_19; published online: 14 Janua y 2020 Keywo ds: low cy ome y; luo escen p obes; human spe ma ozoa; oxida i e s ess; eac i e oxygen species 1PhD P og amme in Expe imen al Biology and Biomedicine, CNC-Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, Coimb a 3004-504, Po ugal; 2Biology o Rep oduc ion and S em Cell G oup, CNC-Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, Coimb a 3004-504, Po ugal; 3IIIUC - Ins i u e o In e disciplina y Resea ch, Casa Cos a Alemão, Uni e si y o Coimb a, Coimb a 3030-789, Po ugal; 4Rep oduc i e Medicine Uni , Uni e si y Hospi als o Coimb a, Coimb a 3004-561, Po ugal; 5Facul y o Medicine, Uni e si y o Coimb a, Coimb a 3000-370, Po ugal; 6Clinical Pa hology Uni , Uni e si y Hospi als o Coimb a, Coimb a 3004-561, Po ugal; 7Depa men o Li e Sciences, Uni e si y o Coimb a, Coimb a 3000-456, Po ugal. Co espondence: D . J Ramalho-San os ([email p o ec ed]) Recei ed: 09 June 2019; Accep ed: 10 Sep embe 2019 Open Access Spe m Biology Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Asian Jou nal o And ology P obes o ROS de ec ion in human spe m S Escada-Rebelo e al 466 es e ases, p e en ing i s exi om he cell. By eac ing wi h NO●, DAF-2 is con e ed o i s luo escen iazole de i a i e (DAF-2T), ha emi s g een luo escence.28 Ou goal was o compa e hese comme cially a ailable luo escen p obes, o de e mine hei speci ici y owa ds ce ain eac i e species. MATERIALS AND METHODS Reagen s and media Unless s a ed o he wise, all eagen s we e om Sigma–Ald ich (S . Louis, MO, USA) and all p obes we e om Molecula P obes/In i ogen (Eugene, OR, USA). Two spe m suspension media we e used: Spe m P epa a ion Medium (SPM - Medicul -O igio, Jyllinge, Denma k) and a PBS supplemen ed medium.29 Spe m sample collec ion, p ocessing, and analysis All spe m samples we e kindly p o ided by he Rep oduc i e Medicine Uni a he Uni e si y Hospi als o Coimb a, Coimb a, Po ugal. The pa ien s unde going e ili y ea men s signed in o med consen o ms au ho izing he use o he emaining sample, and all human ma e ial was used in acco dance wi h he app op ia e e hical and In e nal Re iew Boa d (IRB) guidelines p o ided by he Uni e si y Hospi als o Coimb a, which app o ed he s udy. The semen samples we e ob ained a e 3–5 days o sexual abs inence and he spe miog am was pe o med acco ding o he Wo ld Heal h O ganiza ion Guidelines (WHO, 2010),30 including pa ame e s such as concen a ion, mo ili y, and mo phology. A e analysis, he samples we e p epa ed by densi y g adien cen i uga ion as desc ibed p e iously,31 which allowed spe m seg ega ion om seminal plasma and ound cells, and his was also con i med isually, al hough no speci ic me hod o emo e leukocy es was used. Finally, he spe ma ozoa we e incuba ed a leas o 3 h in Spe m P epa a ion Medium o allow capaci a ion o occu . Flow cy ome y (FC) Fo all FC expe imen s, a cell suspension o 5 × 106 spe m ml−1 (in a olume o 500 µl) was analyzed using a BD FACSCalibu (Bec on Dickinson, F anklin Lakes, NJ, USA) low cy ome e wi h an a gon lase ha pe o ms wi h an exci a ion wa eleng h o 488 nm coupled wi h he ollowing emission il e s: 530/30 band pass (FL-1 channel/g een), 585/42 band pass (FL-2 channel/ ed) (Supplemen a y Figu e 1). Flow cy ome y da a we e analyzed using he FlowJo® (FlowJo LLC, Ashland, OR, USA) so wa e and 200 000 e en s we e acqui ed pe assay. Mean luo escence in ensi y (MFI) and pe cen age o labeled cells (PLC) we e bo h conside ed, wi h con ols o assu e ha only spe m cells (and no deb is, leukocy es, o o he ound cells) we e being ga ed and analyzed.30 The concen a ion and incuba ion ime o each p obe was as ollows: Mi oSOX™ Red (1 µmol l−1, 15 min), DHE (50 µmol l−1, 15 min), RedoxSenso ™ Red CC-1 (3 µmol l−1, 10 min), CellROX® O ange Reagen (1 µmol l−1, 30 min), Mi oPY1 (10 µmol l−1, 40 min; Toc is Bioscience, B is ol, UK), and DAF-2 DA (1 µmol l−1, 40 min). As posi i e con ols o he p obes agains ROS, we used h ee di e en condi ions: an imycin A (79 µmol l−1), H2O2 (0.006% [ / ]; Me ck Millipo e, Bille ica, MA, USA), and H2O2 (0.006% [ / ]) + EDTA (100 µmol l−1; Bio-Rad; He cules, CA, USA). This las con ol was o de e mine i he e would be an inc ease in he le els o H2O2 wi hou he p oduc ion o cy osolic O2 ●-, since EDTA would p e en NOX5 ac i a ion by chela ing calcium (Ca2+). Finally, o cla i y i DAF-2 DA (Me ck Millipo e) is sensi i e o NO●, spe mine (Spe mine NONOa e; 50 µmol l−1) was used.31-33 We used only samples wi h high iabili y (o e 95% iabili y a he s a ), and a emp ed o moni o simul aneously spe m iabili y in he same samples, by pe o ming pilo expe imen s wi h Mi oSOX and Sy ox G een. Howe e , a leas in ou hands, he p esence o he iabili y s aining signi ican ly in e e ed wi h he Mi oSOX signal (unpublished da a). This is clea ly an impo an poin o add ess in he u u e. S a is ical analyses S a is ical analysis was pe o med wi h IBM SPSS So wa e, e sion 21 (A monk, NY, USA). All a iables we e checked o no mal dis ibu ion h ough he Shapi o–Wilk no mali y es . In his wo k, when he da a p esen ed a nonno mal dis ibu ion, only nonpa ame ic es s we e applied. The e o e, when compa ing wo dependen g oups, we used he Wilcoxon es . When he da a had a no mal dis ibu ion, he S uden ’s - es was used. No mally dis ibu ed da a we e p esen ed as mean ± s anda d de ia ion (s.d.) and nonpa ame ic da a we e ep esen ed h ough box and whiske plo s as qua iles Q1, Q2 (median), and Q3. P ≤ 0.05 was conside ed s a is ically signi ican . RESULTS To cha ac e ize each p obe, he pe cen age o labeled cells in each condi ion is c ucial and we de e mined i his pa ame e changed when moni o ed ia low cy ome y o when manually coun ed using luo escence mic oscopy. S aining ep esen a i e images o all p obes a e shown in Figu e 1a–1 . Mi oSOX™ Red exhibi s, in mos cases, ed luo escence in he head egion o he cell (Figu e 1a), and, as expec ed, simila esul s we e ob ained o DHE (Figu e 1b). A p e alen s aining o he midpiece was obse ed when using RedoxSenso ™ Red CC-1 (Figu e 1c), and o CellROX® O ange Reagen , o ange/ ed luo escence was also seen exclusi ely in he midpiece (Figu e 1d). Mi oPY1, a p obe a ge ed o he mi ochond ia, also emi s g een luo escence in he midpiece (Figu e 1e).25 Finally, DAF-2 DA e ealed g een labeling in he spe m midpiece (Figu e 1 ). When compa ing low cy ome y and manual luo escence mic oscopy coun ing, he e was no di e ence o Mi oSOX™ Red (Figu e 1g) and DHE (Figu e 1h). Figu e 1i shows ha he e a e s a is ically signi ican di e ences o RedoxSenso ™ Red CC-1. This did no happen wi h CellROX® O ange (Figu e 1j), bu was he case o Mi oPY1 (Figu e 1k). These di e ences ound wi h RedoxSenso ™ Red CC-1 and Mi oPY1 a e, in g ea pa , subjec i e, owing o he ope a o s and hei abili y o iden i y co ec ly labeled cells (i.e., no ably wha in ensi y o s aining agains he backg ound cons i u es posi i e s aining). The e o e, h oughou his wo k, we a o ed MFI o e he pe cen age o labeled cells ( ega dless o he me hod used). None heless, g aphs showing he pe cen age o labeled cells (ob ained h ough low cy ome y, and no manual coun ing) we e also included, o ul ima ely be e cha ac e ize he p obes. Fo Mi oSOX™ Red, ou da a show a s a is ically signi ican inc ease in bo h MFI (Figu e 2a–2c) and PLC (Figu e 2d–2 ) in all con ol condi ions, compa ed wi h he ini ial condi ion, Mi oSOX™ Red only. Rega ding DHE, simila ly, he use o posi i e con ols inc eased he le els o MFI (Figu e 2g–2i) and PLC (Figu e 2j–2l), compa ed wi h he ini ial condi ion (only DHE), and his inc ease was s a is ically signi ican h oughou . When using RedoxSenso ™ Red CC-1 and acqui ing MFI, he con ol wi h an imycin A showed no di e ences (Figu e 3a), and only he con ols H2O2 and H2O2 + EDTA showed a s a is ically signi ican inc ease (Figu e 3b and 3c). Howe e , in e ms o PLC, he e we e s a is ically signi ican di e ences o all he con ols compa ed wi h he ini ial condi ion (Figu e 3d–3 ). Fo CellROX® O ange FC, he esul s show a s a is ically signi ican dec ease in MFI upon he use o an imycin A (Figu e 3g), while, on he o he hand, bo h H2O2 and H2O2 + EDTA p oduced a s a is ically Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Asian Jou nal o And ology P obes o ROS de ec ion in human spe m S Escada-Rebelo e al 467 signi ican inc ease (Figu e 3h and 3i). The exac same pa e n wi h he di e en con ols was seen when using PLC (Figu e 3j–3l). Wi h Mi oPY, he use o an imycin A had no e ec when using MFI (Figu e 4a), while a s a is ically signi ican inc ease was seen ollowing he addi ion o H2O2 and H2O2 + EDTA, (Figu e 4b and 4c). In e es ingly, he use o an imycin A signi ican ly dec eased he pe cen age o labeled cells (Figu e 4d), as did bo h he addi ion o H2O2 and H2O2 + EDTA (Figu e 4e and 4 ). The esul s using hese wo con ols we e simila o hose desc ibed o RedoxSenso ™ Red CC-1. Finally, wi h DAF-2 DA, we could obse e an inc ease in bo h pa ame e s when adding spe mine as a con ol (Figu e 4g and 4h). DISCUSSION Al hough unexpec ed, he nuclea s aining obse ed when using Mi oSOX™ Red (p obe speci ically a ge ed owa d he mi ochond ia), has been p e iously epo ed, pa icula ly when using an imycin A as Figu e 1: (a– ) Rep esen a i e images o spe ma ozoa labeled wi h luo escen p obes agains ROS/RNS. In he i s ow, (a) Mi oSOX™ Red (1 μmol l−1) and (b) DHE (50 μmol l−1) showed ed luo escence in he spe m head; (c) RedoxSenso ™ Red CC-1 (3 μmol l−1) s ained mos ly he midpiece o he cell wi h ed luo escence, wi h some cases labeling he head as well; (d) CellROX® O ange Reagen (1 μmol l−1) labeled only he midpiece wi h ed luo escence, while (e) Mi oPY1 (10 μmol l−1) and ( ) DAF-2 DA (1 μmol l−1) labeled he same a ea in g een luo escence. Spe m nucleus we e labeled wi h Hoechs (blue luo escence, middle ow). Phase con as images in g ay show he en i e spe m cell (bo om ow). Scale ba s = 5 μm. (g–k) Pe cen age o labeled cells ob ained h ough low cy ome y (FC) e sus luo escence mic oscopy (FM). Pe cen age o labeled cells o he luo escen p obes: (g) Mi oSOX™ Red (n = 5), (h) DHE (n = 4), (i) RedoxSenso ™ Red CC-1 (n = 4), (j) CellROX® O ange Reagen (n = 5) and (k) Mi oPY1 (n = 5). All da a p esen a Gaussian dis ibu ion; he e o e, he S uden ’s - es was he s a is ical es pe o med. Da a a e p esen ed as mean ± s anda d de ia ion. *P < 0.05, **P < 0.01. (l) Do plo ep esen a ion o he ga e se o exclude nonspe m-speci ic e en s. ROS: eac i e oxygen species; RNS: eac i e ni ogen species. h g d c b a e l k j i Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Asian Jou nal o And ology P obes o ROS de ec ion in human spe m S Escada-Rebelo e al 468 a con ol,20 and may ha e se e al specula i e explana ions. Howe e , o he s udies wi h di e en cell ypes wi h a highe cy oplasmic olume and o ganelle s uc u e ha is e y di e en om he speci ic na u e o spe ma ozoa om di e en species ha e demons a ed mi ochond ial s aining.16,34–36 Rega dless, h ough low cy ome ic expe imen s, we can sugges ha Mi oSOX™ Red is sensi i e o bo h mi ochond ial O2 ●-, which ag ees wi h he li e a u e,20 and wi h H2O2, which does no .27 While he esul s migh lead us o belie e ha his p obe is also sensi i e o cy osolic O2 ●-, he e we e no di e ences obse ed be ween he addi ion o H2O2 (con ol used o inc ease he p oduc ion o H2O2 as well as cy osolic O2 ●-) e sus H2O2 + EDTA (con ol used o inc ease he p oduc ion o H2O2 only), sugges ing ha such de ec ion is unlikely, al hough i canno be uled ou (da a no shown). Simila ly, ou esul s sugges ha DHE is also sensi i e o mi ochond ial O2 ●- and H2O2. While o he s ha e s a ed ha his p obe is no speci ic o H2O2, hey do acknowledge ha i migh eac wi h H2O2.27 Howe e , he da a do no seem o suppo he hypo hesis ha DHE can be used o de ec cy osolic O2 ●- speci ically, since he e we e no s a is ically signi ican di e ences be ween he use o H2O2 e sus H2O2 + EDTA (da a no shown). Owing o he cha ac e is ics o his p obe, namely he lack o he TPP ca ion, i would be expec ed ha DHE is only sensi i e o cy osolic O2 ●-. A possible explana ion o why DHE de ec s mi ochond ial O2 ●- migh be ha his ROS can lea e he mi ochond ia h ough he mi ochond ial ol age-dependen Figu e 3: Assessmen o MFI and PLC in he spe m popula ions labeled wi h RedoxSenso ™ Red CC-1 and CellROX® O ange Reagen . (a–c) MFI alues we e ob ained h ough FC o RedoxSenso ™ Red CC-1 and compa ed o he posi i e con ols: (a) RSR CC-1 e sus RSR + AA (n = 29), (b) RSR e sus RSR + H2O2 (n = 31) and (c) RSR e sus RSR + H2O2 + EDTA (n = 18); (d– ) PLC alues we e ob ained h ough FC o RedoxSenso ™ Red CC-1 and compa ed o he posi i e con ols: (d) RSR e sus RSR + AA (n = 29), (e) RSR e sus RSR + H2O2 (n = 31) and ( ) RSR e sus RSR + H2O2 + EDTA (n = 18); (g–i) MFI alues we e ob ained h ough FC o CellROX® O ange Reagen and compa ed o he posi i e con ols: (g) CRO e sus CRO + AA (n = 31), (h) CRO e sus CRO + H2O2 (n = 33) and (i) CRO e sus CRO + H2O2 + EDTA (n = 13); (j–l) PLC alues we e ob ained h ough FC o CRO and compa ed o he posi i e con ols: (j) CRO e sus CRO + AA (n = 31), (k) CRO e sus CRO + H2O2 (n = 33) and (l) CRO e sus CRO + H2O2 + EDTA (n = 13). All da a p esen a non-Gaussian dis ibu ion; he e o e, he Wilcoxon es was pe o med. Da a a e p esen ed as qua iles, Q1, Q2 (median) and Q3. **P < 0.01, ***P < 0.001. RSR: RedoxSenso ™ Red CC-1; CRO: CellROX® O ange Reagen ; AA: an imycin A; FC: low cy ome y; EDTA: e hylenediamine e a ace ic acid; MFI: mean luo escence in ensi y; PLC: pe cen age o labeled cells. d h c g b a e l k j i Figu e 2: Assessmen o MFI and PLC in he spe m popula ions labeled wi h Mi oSOX™ Red and DHE. (a–c) MFI alues we e ob ained h ough FC o Mi oSOX™ Red and compa ed o he posi i e con ols: (a) MSR e sus MSR + AA (n = 33), (b) MSR e sus MSR + H2O2 (n = 31) and (c) MSR e sus MSR + H2O2 + EDTA (n = 15); (d– ) PLC alues we e ob ained h ough FC o Mi oSOX™ Red and compa ed o he posi i e con ols: (d) MSR e sus MSR + AA (n = 33), (e) MSR e sus MSR + H2O2 (n = 31) and ( ) MSR e sus MSR + H2O2 + EDTA (n = 15); (g–i) MFI alues we e ob ained h ough FC o DHE and compa ed o he posi i e con ols: (g) DHE e sus DHE + AA (n = 37), (h) DHE e sus DHE + H2O2 (n = 36) and (i) DHE e sus DHE + H2O2 + EDTA (n = 19); (j–l) PLC alues we e ob ained h ough FC o DHE and compa ed o he posi i e con ols: (j) DHE e sus DHE + AA (n = 37), (k) DHE e sus DHE + H2O2 (n = 36) and (l) DHE e sus DHE + H2O2 + EDTA (n = 19). All da a p esen a non-Gaussian dis ibu ion; he e o e, he Wilcoxon es was pe o med. Da a a e p esen ed as qua iles, Q1, Q2 (median) and Q3. **P < 0.01, ***P < 0.001. MSR: Mi oSOX™ Red; AA: an imycin A; DHE: dihyd oe hidium; FC: low cy ome y; EDTA: e hylenediamine e a ace ic acid; MFI: mean luo escence in ensi y; PLC: pe cen age o labeled cells. d h c g b a e l k j i Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Asian Jou nal o And ology P obes o ROS de ec ion in human spe m S Escada-Rebelo e al 469 anion channels (VDACs).37 A any a e, in ou hands, esul s wi h Mi oSOX™ Red and DHE we e basically he same, sugges ing ha owing o he s uc u e o he male game e, a speci ic mi ochond ial a ge ing o his DHE- ela ed p obes migh be mo e di icul han wi h o he cell ypes. Acco ding o he li e a u e, RedoxSenso ™ Red CC-1 has been used bo h o he assessmen o he oxida i e ac i i y23,38,39 and o he de ec ion o ROS, al hough o no species in pa icula .40–43 F om he da a ob ained, we sugges ha RedoxSenso ™ Red CC-1 is sensi i e o H2O2. Rega ding cy osolic O2 ●-, we ha e demons a ed ha RedoxSenso ™ Red CC-1 is no sensi i e o i , owing o he lack o s a is ically signi ican di e ences when compa ing he addi ion o H2O2 wi h ha o H2O2 + EDTA (da a no shown). Fo mi ochond ial O2 ●-, he absence o a s a is ically signi ican inc ease in MFI when adding an imycin A (compa ed wi h he condi ion wi h he p obe only) con i ms ha his p obe does no a ge mi ochond ial O2 ●-. In he case o CellROX® O ange Reagen , he no iceable inc ease in signals in he condi ions whe e H2O2 was added and he dec ease wi h he use o an imycin A allow us o conclude ha CellROX® O ange Reagen is oxidized by H2O2 and is no speci ic owa d mi ochond ial O2 ●-. Fu he mo e, despi e i s localiza ion in he cy osol, ou esul s do no show a speci ici y owa d cy osolic O2 ●-. Al hough he li e a u e a ailable is sca ce, CellROX® O ange Reagen has been p e iously used in di e en cell ypes om a ious species o he de ec ion o in acellula ROS44–47 and was also used in a s udy pe o med in adipocy es de i ed om human s em cells speci ically o de ec H2O2,24 which suppo s ou esul s. Mi oPY1 also p o ed o be sensi i e owa d H2O2. This con i ms he li e a u e a ailable since Mi oPY1 has been p e iously used speci ically o he de ec ion o H2O2 in a ious cell ypes om di e en species,25,34,35,48 including mouse spe ma ozoa26 bu ne e in human spe ma ozoa. We we e also able o conclude ha Mi oPY1 does no de ec mi ochond ial O2 ●-, owing o he lack o esponse in bo h pa ame e s upon use o an imycin A, no cy osolic O2 ●-, since he e we e no s a is ically signi ican di e ences be ween Mi oPY1 + H2O2 and Mi oPY1 + H2O2 + EDTA (da a no shown). Finally, FC expe imen s wi h DAF-2 DA allowed o conclude ha i is speci ic o NO●. This kind o labeling has been p e iously shown49 in bo ine spe ma ozoa and human game es.28 The p esen esul s co obo a e all he epo s men ioned, enhancing he conclusion ha DAF-2 DA labels he spe m midpiece in he p esence o NO●. CONCLUSIONS Wi h he p esen wo k, we show ha Mi oSOX™ Red, CellROX® O ange Reagen , DHE, and Mi oPY1 can be employed o he de ec ion o ROS wi h each p obe showing dis inc speci ici ies owa d supe oxide (Mi oSOX™ Red and DHE), hyd ogen pe oxide (CellROX® O ange Reagen and Mi oPY1), o bo h (Mi oSOX™ Red and DHE) while DAF-2 DA can be used o de ec RNS. The use o RedoxSenso ™ Red CC-1, CellROX® O ange Reagen , and Mi oPY1 in human spe ma ozoa is epo ed o he i s ime. We no e ha MFI (and no PLC) is he main pa ame e ha can be ep oducibly moni o ed by his ype o me hodology. In his wo k, i was, howe e , impossible in p ac ice o dis inguish mi ochond ial om cy osolic con ibu ions. Al hough Mi oSOX™ Red and DHE de ec H2O2, hese p obes should no be used only o his speci ic pu pose. All he ROS p obes es ed, Mi oSOX™ Red and Mi oPY1 a e hose we ad ise o be used o he accu a e de ec ion o supe oxide and hyd ogen pe oxide in human spe ma ozoa, espec i ely, owing o he cla i y o he esul s when obse ing/analyzing he cells, whe he h ough low cy ome y (whe e we can, oge he wi h he con ol condi ions, see a clea speci ici y owa d he espec i e ROS p obe) o luo escence mic oscopy (we can easily dis inguish labeled cells om nonlabeled as well as obse e he expec ed cell egion emi ing luo escence). In u u e, i would be in e es ing o co ela e he con en o ROS/RNS de ec ed by hese p obes wi h unc ional cellula pa ame e s ha a e known o be a ec ed by oxida i e s ess (such as capaci a ion, he ac osome eac ion, DNA damage, among o he s) o in e game e quali y h ough ROS/RNS con en . AUTHOR CONTRIBUTIONS JRS, APS, and SER designed he s udy. SER and FGM pe o med expe imen s and da a analysis and con ibu ed equally o his wo k. TAS and APS p o ided all human spe m samples and pe o med basic spe m analysis, and AP p o ided low cy ome e expe ise. All au ho s c i ically analyzed he da a. SER, FGM, APS, and JRS w o e he manusc ip . All au ho s ead and app o ed he inal manusc ip . COMPETING INTERESTS All au ho s decla ed no compe ing in e es s. ACKNOWLEDGMENTS We would like o hank o all he membe s o he Biology o Rep oduc ion and S em cell esea ch g oup (CNC) o help ul discussions. SER is Figu e 4: Assessmen o MFI and pe cen age o labeled cells in he spe m popula ions labeled wi h Mi oPY1 and DAF-2 DA. (a–c) MFI alues we e ob ained h ough FC o Mi oPY1 and compa ed o he posi i e con ols: (a) MPY1 e sus MPY1 + AA (n = 28), (b) MPY1 e sus MPY1 + H2O2 (n = 30) and (c) MPY1 e sus MPY1 + H2O2 + EDTA (n = 14); (d– ) PLC alues we e ob ained h ough FC o Mi oPY1 and compa ed o he posi i e con ols: (d) MPY1 e sus MPY1 + AA (n = 28), (e) MPY1 e sus MPY1 + H2O2 (n = 30) and ( ) Mi oPY1 e sus MPY1 + H2O2 + EDTA (n = 14); (g) MFI and (h) PLC alues we e ob ained h ough FC o DAF-2 DA and compa ed o he posi i e con ol: (g) D2D e sus D2D + Spe mine (n = 28), (h) D2D e sus D2D + Spe mine (n = 28). All da a p esen a non-Gaussian dis ibu ion; he e o e, he Wilcoxon es was pe o med. Da a a e p esen ed as qua iles, Q1, Q2 (median) and Q3. ***P < 0.001. MPY1: Mi oPY1; D2D: DAF-2 DA; AA: an imycin A; FC: low cy ome y; EDTA: e hylenediamine e a ace ic acid; MFI: mean luo escence in ensi y; PLC: pe cen age o labeled cells. d h c g b a e Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Asian Jou nal o And ology P obes o ROS de ec ion in human spe m S Escada-Rebelo e al 470 suppo ed by he Po uguese unding agency o science and echnology (PD/BD/128237/2016 – PhD P og amme in Expe imen al Biology and Biomedicine). CNC is unded by FEDER, h ough P og ama Ope acional Fac o es de Compe i i idade – COMPETE 2020 and Na ional unds ia FCT unde he p ojec POCI-01-0145-FEDER-007440. This wo k was also pa ially unded by he Eu opean Regional De elopmen Fund (ERDF), h ough he Cen o 2020 Regional Ope a ional P og amme: p ojec CENTRO-01-0145-FEDER-000012-Heal hyAging2020, he COMPETE 2020 – Ope a ional P og amme o Compe i i eness and In e na ionalisa ion, and he Po uguese na ional unds ia FCT – Founda ion o Science and Technology I.P.: p ojec POCI-01-0145-FEDER-007440 and UID/NEU/04539/2019. Supplemen a y In o ma ion is linked o he online e sion o he pape on he Asian Jou nal o And ology websi e. REFERENCES 1 Ja ow JP, Sha lip ID, Belke AM, Lipshul z LI, Sigman M, e al. Bes p ac ice policies o male in e ili y. J U ol 2002; 167: 2138–44. 2 Hull MG, Glazene CM, Kelly NJ, Conway DI, Fos e PA, e al. Popula ion s udy o causes, ea men , and ou come o in e ili y. B Med J (Clin Res Ed) 1985; 291: 1693–7. 3 Aga wal A, Vi k G, Ong C, du Plessis SS. E ec o oxida i e s ess on male ep oduc ion. Wo ld J Mens Heal 2014; 32: 1–17. 4 Goossens E, Tou naye H. Spe ma ogenesis. 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This is an open access jou nal, and a icles a e dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-Sha eAlike 4.0 License, which allows o he s o emix, weak, and build upon he wo k non-comme cially, as long as app op ia e c edi is gi en and he new c ea ions a e licensed unde he iden ical e ms. ©The Au ho (s)(2020) Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023 Supplemen a y Figu e 1: Rep esen a i e do plo s ob ained h ough FC o he spe m popula ions labeled wi h he p obes agains ROS/RNS. (a) Mi oSOX™ Red, (b) DHE, (c) Mi oPY1 and (d) DAF-2 DA. FC expe imen s, we e conduc ed using a BD FACSCalibu (Bec on Dickinson; NJ, USA) low cy ome e wi h an a gon lase ha pe o ms wi h an exci a ion wa eleng h o 488 nm coupled wi h he ollowing emission il e s: 530/30 band pass (FL-1 channel/g een), 585/42 band pass (FL-2 channel/ ed). The ol age used o each de ec o was as ollows: FSC – 0 V, SSC – 381 V, FL1 – 605 V and FL2 – 510 V. The compensa ion used was FL1- 0.6% FL2 and FL2 – 45.3% FL1. The ob ained da a we e analyzed using he FlowJo® (FlowJo LLC, Ashland, OR, USA) so wa e, as shown he e. FC: low cy ome y; ROS: eac i e oxygen species; RNS: eac i e ni ogen species; DHE: dihyd oe hidium. d c b a Downloaded om h p://jou nals.lww.com/ajand ology by BhDM 5ePHKa 1zEoum1 Q N4a+kJLhEZgbsIHo4XMi0hC ywCX1AWnYQp/IlQ HD3i3D0OdRyi7T SFl4C 3VC4/OAVpDDa8K2+Ya6H515kE= on 03/08/2023