Asian Jou nal o And ology (2022) 24, 139–146
www.asiaand o.com; www.ajand ology.com
in oked in in i o e iliza ion (IVF) o ICSI p ocedu es. Mo eo e , i
is impo an o emphasize ha spe ma ozoa wi h DNA agmen a ion
a e capable o e iliza ion. The e o e, i would seem easonable o
sugges ha assis ed ep oduc ion echniques should compensa e o
de e io a ion in spe m ch oma in in eg i y, especially when ICSI is
used and only one mo phologically no mal spe ma ozoon is selec ed
o mic oinjec ion. In addi ion, sc eening o mo phologically no mal
spe ma ozoa o ICSI and he selec ion o good-quali y emb yos o
ans e in IVF/ICSI will educe he po en ially ad e se e ec s o
spe m DNA damage on he ou come o assis ed ep oduc ion. Thus,
bioma ke s o assis in he p edic ion o assessmen o male in e ili y
would be a g ea enhancemen o i s ea men and could be used
as complemen a y es s o spe miog ams, imp o ing diagnos ic o
p ognos ic powe . Fu he mo e, hey would allow he selec ion o
he bes spe ma ozoa o be used in assis ed ep oduc ion echniques.
To ob ain addi ional in o ma ion ega ding seminal quali y,
i is in e es ing o compa e whe he spe m DNA agmen a ion
is ela ed o possible al e a ions in pep ide me abolism, bo h in
he spe ma ozoon and in he seminal luid, no only o inc ease
knowledge o he molecula mechanisms unde lying spe m
unc ion and egula ion, bu also o desc ibe possible bioma ke s
o spe m in eg i y and unc ion. In his ega d, he ela ionship
INTRODUCTION
In Wes e n socie ies, app oxima ely one o six couples o ep oduc i e
age is in e ile, wi h male ac o in e ili y accoun ing o 50% o all
cases. To esol e such male e ili y p oblems, a la ge numbe o couples
seek medical help, and in acy oplasmic spe m injec ion (ICSI) is
he p ima y app oach used o achie e p egnancy. The s udy o male
in e ili y ac o s has usually been based on he analysis o semen,
using he Wo ld Heal h O ganiza ion (WHO) guidelines.1 Al hough
no included in his manual, DNA agmen a ion has become an
impo an p edic o o spe m quali y in he las decade. Indeed, DNA
agmen a ion has become an impo an ma ke o spe m quali y
because o i s possible ela ionship wi h a ious c i ical pa ame e s,
such as li e and assis ed ep oduc ion, emb yo-zygo e de elopmen
and quali y, implan a ion, abo ion and newbo n heal h.2–8 Al hough
male e ili y s a us and semen quali y can be assessed h ough
seminog am analysis, his me hod does no de ec he p esence o DNA
agmen a ion in spe ma ozoa. I is es ima ed ha , o 10%–15% o
s e ile men, hese al e a ions a e p esen in he gene ic ma e ial o hei
game es, whe eas pa ame e s o concen a ion, mobili y and spe m
mo phology a e wi hin no mal limi s. Du ing he no mal e iliza ion
p ocess, he emale ep oduc i e sys em has selec i e mechanisms
agains DNA-damaged spe ma ozoa. Howe e , his p ope y is no
Local enin angio ensin sys em and spe m DNA
agmen a ion
Ma ía Vic o ia Apa icio P ie o1, Ma ía Vic o ia Rod íguez Gallego2, Asie Valdi ia Palacín3,
Yosu F anco I ia e4, Go zone He ás Ba ba a3, En ique Eche a ía O ella3, Luis Casis Saenz3
The enin angio ensin sys em (RAS) appea s o in luence male e ili y a mul iple le els. In his wo k, we analyzed he ela ionship
be ween he RAS and DNA in eg i y. Fi y male olun ee s we e di ided in o wo g oups (25 each): con ol (DNA agmen a ion ≤20%)
and pa hological (DNA agmen a ion >20%) cases. Ac i i ies o i e pep idases con olling RAS we e measu ed luo ome ically: p olyl
endopep idase (which con e s angio ensin [A] I and A II o A 1–7), neu al endopep idase (NEP/CD10: A I o A 1–7), aminopep idase
N (APN/CD13: A III o A IV), aminopep idase A (A II o A III) and aminopep idase B (A III o A IV). Angio ensin-con e ing
enzyme (A I o A II), APN/CD13 and NEP/CD10 we e also assessed by semiquan i a i e cy ome y and quan i a i e low cy ome y
assays, as we e he ecep o s o all RAS componen s: A II ecep o ype 1 (AT1R), A II ecep o ype 2 (AT2R), A IV ecep o
(AT4R o insulin- egula ed aminopep idase [IRAP]), (p o) enin ecep o (PRR) and A 1–7 ecep o o Mas ecep o (MasR) None
o he enzymes ha egula e le els o RAS componen s, excep o APN/CD13 (dec ease in agmen ed cells), showed signi ican
di e ences be ween bo h g oups. Mic og aphs o RAS ecep o s e ealed no signi ican di e ences in immunolabeling pa e ns
be ween no mozoospe mic and agmen ed cells. Labeling o AT1R (94.3% no mozoospe mic
s
84.1% agmen ed), AT4R
(96.2%
s
95.3%) and MasR (97.4%
s
87.2%) was simila be ween he g oups. AT2R (87.4% no mozoospe mic
s
63.1%
agmen ed) and PRR (96.4%
s
48.2%) we e highe in non- agmen ed spe ma ozoa. These indings sugges ha agmen ed
DNA spe ma ozoa ha e a lowe capaci y o espond o bioac i e RAS pep ides.
Asian Jou nal o And ology (2022) 24, 139–146; doi: 10.4103/aja202150; published online: 07 Sep embe 2021
Keywo ds: DNA agmen a ion; local enin angio ensin sys em; spe m e ili y
1Human Rep oduc ion Uni , C uces Uni e si y Hospi al, Ba akaldo 48903, Spain; 2Human Rep oduc ion Uni , San Ped o Hospi al, Log oño 26006, Spain; 3Depa men o
Physiology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), Leioa 48940, Spain; 4Human Rep oduc ion Uni , Rube In e na ional Hospi al,
Mad id 28034, Spain.
Co espondence: D . MV Apa icio P ie o ([email p o ec ed])
Recei ed: 16 Feb ua y 2021; Accep ed: 29 June 2021
Male In e ili y
Open Access
ORIGINAL ARTICLE
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Asian Jou nal o And ology
RAS and spe m DNA agmen a ion
MV Apa icio P ie o e al
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be ween a ious e ili y pa ame e s (including game es and spe m
mo ili y) and le els and ac i i ies o a ious pep ide sys ems, such
as opioide gic (neu al aminopep idase: aminopep idase N [APN/
CD13]), basic aminopep idase, neu al endopep idase (NEP/CD10),
p olyl endopep idase (PEP), and µ, d, and k-opioid ecep o s9–12
and oxy ocine gic (PEP and cys-aminopep idase)13–16 sys ems, has
been desc ibed. The indings sugges ha hese ac o s may con ol
ep oduc i e unc ion a mul iple si es, and exe di ec ac ion on
spe ma ozoa. In ecen yea s, esea ch has p o en he p esence o a
local enin angio ensin sys em (RAS) in game es, wi h an impo an
ole in he egula ion o bo h male ep oduc ion and emale
ep oduc ion;17,18 ne e heless, he ex en o i s unc ion is no ully
unde s ood. The p esence and dis ibu ion o RAS componen s in
spe ma ozoa and he ac i i y o angio ensin (A) sugges ha his
sys em egula es ep oduc i e unc ion, di ec ly in luencing spe m
e ili y.19 Ou esea ch g oup has ecen ly shown ha emb yos wi h
highe implan a ion po en ial a e de i ed om spe m samples wi h
a highe pe cen age o es icula angio ensin-con e ing enzyme
( ACE)-posi i e cells and ewe enzyme molecules pe spe ma ozoon
on hei su ace memb ane. On he basis o hese indings, we
p opose ha he ACE may be used o help emb yologis s selec
be e semen samples o ob ain high-quali y blas ocys s du ing IVF.20
In his wo k, we p esen he ela ionship be ween he pe cen age
o DNA agmen a ion and di e en componen s o he local
spe m RAS. The componen s analyzed in his s udy a e as ollows:
PEP (which con e s angio ensin I [A I] and angio ensin II [A
II] o angio ensin 1–7 [A 1–7]), NEP (A I o A 1–7), APN/CD13
(angio ensin III [A III] o angio ensin IV [A IV]), aminopep idase
A (APA;A II o A III), aminopep idase B (APB; A III o A IV), and
angio ensin-con e ing enzyme (ACE/CD143; A I o A II), as well
as he A II ecep o ype 1 (AT1R), A II ecep o ype 2 (AT2R),
A 1–7 ecep o o Mas ecep o (MasR), A IV ecep o (AT4R) and
(p o) enin ecep o (PRR). By compa ing le els o comple e RAS in
no mal and agmen ed spe ma ozoa, we could de e mine i any o he
componen s can be used as a diagnos ic bioma ke o ep oduc i e
success.
PARTICIPANTS AND METHODS
E hical conside a ions
This s udy was app o ed by he E hics Commi ee o he Uni e si y
o he Basque Coun y (UPV/EHU), Leioa, Spain (CEISH/61/2011)
and Clinical Resea ch E hics Commi ee (CEIC) o he Basque
Heal h Se ice/Osakide za (CEIC E14/42). All semen samples
we e ob ained om male pa ne s o women who had unde gone
in au e ine insemina ion (IUI) cycles a he Basque Biobank
(Basque Coun y, Spain). Spe m samples o esea ch we e ob ained
a e he pa ien s p o ided w i en in o med consen .
Pa ien s, semen analysis, and seminal p epa a ion
The samples used o he ial we e he le o e s om he IUI
p ocess. Pa ien s wi h insu icien semen olume o spe m
concen a ion o he s udy and IUI we e elimina ed because he
la e was he p io i y. Fi y pa ien s wi h an a e age age o 37.4
(s anda d de ia ion [s.d.]: 4.4, ange: 29–46) yea s we e included
in his s udy and di ided in o wo g oups (n=25 each): no mal
(DNA agmen a ion ≤20%) and pa hological (DNA agmen a ion
>20%) cases. Powe analysis was pe o med (alpha = 0.05; be a =
0.30; powe o 70%).
The spe m DNA agmen a ion s udy was pe o med on aw
samples. The samples used o he s udy o pep idases and RAS
componen s we e ozen. These samples we e analyzed on consecu i e
days bu always unde iden ical expe imen al condi ions. As a con ol
g oup, DNA agmen a ion a ≤20% was used because some s udies show
ha 20% is a good indica o o he cu -o alue o pa hological-deg ee
agmen a ion.21 In he p esen s udy, he deg ee o agmen a ion in he
con ol g oup was low (maximum: 11.5%), whe eas i was always highe
han 20% (minimum: 21.7%, maximum: 29.6%) in he pa hological
g oup. The di e ence be ween he wo g oups was e iden .
The semen samples we e collec ed in s e ile con aine s on he day
o IUI by mas u ba ion in he hospi al a e 2 days o 5 days o sexual
abs inence. Seminal sample lique ac ion was pe o med a 37°C and
in 5% ( / ) CO2 o 10 min be o e p ocessing by densi y g adien
cen i uga ion o insemina ion.
The semen olume and spe m concen a ion and mo ili y we e
measu ed o each sample, and all he samples we e double-examined o
de e mine he spe m concen a ion and mo ili y in a Makle ® Chambe
(Se i Labo a o ies, Hai a, Is ael) by coun ing a leas 200 spe ma ozoa
pe eplica e. The mean alue o homogeneous eplica es was used o
analysis. Mo ili y was e alua ed acco ding o s anda ds o he WHO.1
In summa y, spe ma ozoa we e classi ied in o h ee di e en g oups:
(1) p og essi e mo ili y (PR), (2) nonp og essi e mo ili y (NP), and
(3) immo ili y (IM). Excess spe ma ozoa emaining a e clinical
use o IUI p ocedu es we e collec ed o molecula analysis by low
cy ome y. The molecula da a ob ained we e ela ed o spe m DNA
agmen a ion (measu ed in esh samples).
Spe m DNA agmen a ion: he spe m ch oma in dispe sion es
(SCD) es
The SCD (Halospe m®; Halo ech, Mad id, Spain) was used o
de e mine spe m DNA agmen a ion. The SCD is an indi ec
me hod o quan i ying he pe cen age o spe ma ozoa wi h DNA
agmen a ion and is based on he di e en ial esponse o spe m nuclei
when exposed o sligh acid dena u a ion and subsequen p o ein lysis.
Nuclea agmen a ion can be es ima ed by quan i ying he dispe sed
nuclei o ch oma in and condensed nuclei.22–24
The o e all p ocess equi es h ee c i ical s eps: s ep a, in eg a ion
o he sample in o ine aga ose; s ep b, acid dena u a ion o agmen ed
DNA, and s ep c, elimina ion o nuclea p o eins by lysis. Fo s ep a,
he spe m sample was dilu ed in phospha e bu e solu ion (PBS) o
a maximum o 20 × 106 ml−1; 50 μl was ans e ed o an Eppendo
ube wi h 100 µl o mel ed aga ose a 37°C and mixed gen ly wi h a
mic opipe e, and he o ma ion o bubbles was p e en ed. Nex , 8 μl
o he cell suspension was placed a he cen e o a sample well (“S”),
and co e ed wi h a co e slip, and he ma e ial was p essed gen ly o
a oid ai bubbles and he slide was held ho izon ally h oughou he
p ocess. The con ol was labeled “C”. The slides we e placed on a cold
su ace and ans e ed o 4°C, o 5 min o solidi y he aga ose, and
hen he co e slip was emo ed by gen ly sliding i o . All subsequen
p ocessing was pe o med a oom empe a u e (22°C). Fo s ep b,
dena u an agen solu ion was applied o he well o co e he sample
and incuba ed o 7 min and hen emo ed by il ing wi hou shaking;
he samples was d ied and placed ho izon ally; an impo an s ep is o
emo e he eac i e ma e ial wi hou shaking. Fo s ep c, lysis solu ion
was applied o he well, imme sing he sample, ollowed by incuba ing
o 20 min, emo ing he eagen s by il ing as abo e, washing he
slide o 5 min wi h dis illed wa e , emo ing he eagen s as abo e,
and dehyd a ing by incuba ing i s wi h 70% e hanol and hen 100%
e hanol, o 2 min each.
A e hese p ocedu es, eosin s aining solu ion (SSA; Halospe m®;
Halo ech) was applied, incuba ed o 7 min, and hen emo ed by
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Asian Jou nal o And ology
RAS and spe m DNA agmen a ion
MV Apa icio P ie o e al
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il ing. Thiazine s aining solu ion (SSB; Halospe m®; Halo ech) was
nex added o he wells, incuba ed o 7 min, emo ed by il ing and
d ied a oom empe a u e. A minimum o 600 spe ma ozoa we e
obse ed by b igh - ield mic oscopy (Scope A1, ZEISS, Obe kochen,
Ge many) o dispe sed ch oma in halos ha co ela e wi h he
pe cen age o agmen a ion. Spe m wi h agmen ed DNA a e hose
wi h a halo ha is smalle han hal o he smalle diame e o he
nucleus, and hose ha lack a halo.25
Magne ic sepa a ion o apop o ic cells by annexin columns
The echnique is based on he binding o supe pa amagne ic
Annexin-mic obeads o ex e nalized phospha idylse ine (PS) a he
ou e lea le o he plasma memb ane o spe m wi h ac i a ed apop osis
signaling o memb ane damage.26 I he sample in e ac s o seconds
wi h a high-powe ed magne , he a ec ed spe m will emain a ached o
he walls o he column while una ec ed spe m will low. This me hod
o sepa a ion o molecula il a ion is called magne -ac i a ed cell
so ing (MACS; Mic obead ki o he ANMB, Mil enyi Bio ec, Be gisch
Gladbach, Ge many).
Pep idase ac i i y measu emen
Ac i i ies o i e pep idase we e measu ed by a luo ome ic assay: PEP
(A I and A II o A 1–7), NEP (A I o A 1–7), APN/CD13 (A III o A IV),
APA (A II o A III), and APB (A III o A IV). The assay is based on
he luo escence o p oduc s gene a ed om hyd olysis o a speci ic
subs a e by each enzyme p esen in he sample. Rega ding APN
(Enzyme Commission [E.C.] No. 3.4.11.2), APB (E.C. No. 3.4.11.6)
and APA (E.C. No. 3.4.11.21) aminopep idase ac i i ies, di e en
speci ic aminoacyl-β-naph hylamide de i a i es (Sigma Ald ich,
S . Louis, MO, USA) we e used as subs a es. PEP endopep idase
ac i i y was measu ed wi h a modi ied me hod om Alpon i e al.27
wi h H-Gly-P o-β-naph hylamide and Z-Gly-P o-β-naph hylamide
(Bachem, To ance, CA, USA), modi ied om Zol agha i e al.28
N-Dansyl-D-Ala-Gly-p-Ni o-Phe-Gly (DAGNPG; Sigma Ald ich,)
was used as a luo ogenic subs a e o NEP ac i i y measu emen ,
ollowing he me hod o Flo en in e al.29 and modi ied by I azus a
e al.30 The subs a e solu ions we e p epa ed in 50 mmol l−1 PBS
(pH 7.4) con aining 0.25 mg ml−1 bo ine se um albumin (BSA; Sigma
Ald ich) o APN (0.5 mmol l−1), APB (0.5 mmol l−1), APA (0.125 mmol
l−1), and PEP (0.125 mmol l−1), and in 50 mmol l−1 T is-HCl bu e
(pH 7.4) con aining 0.25 mg ml−1 BSA o NEP. Because o he high
simila i y be ween NEP and ACE, 0.005 mmol l−1 cap op il (Sigma
Ald ich) was added o he NEP subs a e solu ion o inhibi ACE. The
seminal ac ion samples (10–50 μl, depending on each ac i i y) we e
mixed in iplica e wi h 1 ml o each subs a e solu ion mix u e and
incuba ed o 30 min a 37°C. The enzyma ic eac ion was s opped by
adding 1 ml o 0.1 mol l−1 sodium ace a e bu e (pH 4.2).
The amoun o β-naph hylamine eleased was de e mined by
measu ing he luo escence in ensi y in he eac ion mix u e a 412 nm
(wi h an exci a ion wa eleng h o 345 nm) o enzymes assayed wi h
β-naph hylamide de i a i es (spec o luo ome e RF540; Shimadzu,
Kyo o, Japan). To de e mine DAGNPG eleased in he NEP ac i i y
assay, he luo escence in ensi y was measu ed a 410 nm and exci ed
a 342 nm. To sub ac backg ound luo escence, 10 mmol l−1 T is
HCl (pH 7.4) was used o he blank ins ead o sample. Rela i e
luo escence was con e ed o p oduc in pmol using a s anda d cu e
o inc easing concen a ions o β-naph hylamine. To con e ela i e
luo escence p oduc in pmol in he NEP ac i i y assay, a s anda d
cu e o inc easing concen a ions o he p oduc and dec easing
concen a ions o DAGNPG was gene a ed. In all cases, he sample
p o ein concen a ion was measu ed wi h he B ad o d31 me hod
using BSA as he s anda d. The measu ed ac i i ies a e exp essed as
uni s o pep idase ac i i y (UP) pe mg p o ein: UP mg−1, whe e UP
is he amoun o enzyme ha hyd olyses in 1 pmol o luo ogenic
subs a e pe min.
De e mina ion o le els o APN/CD13, ACE/CD143, NEP/CD10, and
ecep o s by semiquan i a i e cy ome y
To measu e le els o APN/CD13, ACE/CD143 and NEP/CD10 as
well as hose o AT1R, AT2R, AT4R/IRAP, PRR, and MasR in spe m
samples, we pe o med semiquan i a i e and quan i a i e low
cy ome y assays using he Quan iBRITE™ PE ki (BD Biosciences,
San Jose, CA, USA). The same semen samples we e simul aneously
used o bo h analyses.
Su plus spe m samples ob ained o assaying DNA agmen a ion
we e ixed in suspension wi h 4% (w/ ) pa a o maldehyde
(PFA; Sigma Ald ich), cen i uged (Labo uge 200; Fishe Scien i ic,
Gö ebo g, Sweden) a 3500g o 6 min and washed in PBS. The samples
we e incuba ed in blocking bu e (PBS wi h 10% [w/ ] e al bo ine
se um [FBS]; Bioch om, Camb idge, UK) o 30 min and hen wi h
he ollowing p ima y an ibodies: an i-AT1R (ex acellula ) an ibody
( abbi polyclonal an ibody o AT1R; AAR011; Alomone Labs,
Je usalem, Is ael), an i-AT2R an ibody ( abbi polyclonal; ab19134;
Abcam, Camb idge, UK), an i-AT4R an ibody (Insulin- egula ed
aminopep idase [IRAP]; abbi polyclonal; clone H-133, sc-135229;
San a C uz Bio echnology, San a C uz, CA, USA), an i- enin ecep o
( abbi polyclonal; clone H-85, sc-67390; San a C uz Bio echnology),
an i-MasR ( abbi polyclonal; AAR-013; Alomone Labs), PE-labeled
human ACE monoclonal phycoe y h in (PE mouse an i-human CD143;
344204; BioLegend, San Diego, CA, USA), PE an i-human CD13 (PE
mouse an i-human CD13; 555394; BD Biosciences), and PE mouse an i-
human CD10 (PE mouse an i-human CD10; 555375; BD Biosciences).
Each o he ecep o s and enzymes s udied was dilu ed a 1:200
in PBS and incuba ed o e nigh a 4°C. The nuclei we e s ained wi h
0.5 μg ml−1 Hoechs 33258 (Molecula P obes, Eugene, OR, USA).
The p ima y an ibody speci ici y was e alua ed wi h an iso ype
con ol an ibody (PE mouse IgG1κ iso ype con ol an ibody; 400 112;
BioLegend) a he same concen a ion as ha o he p ima y an ibody.
To pe o m quan i a i e low cy ome y analysis, we plo ed a
calib a ion cu e using he mean luo escence in ensi y (MFI) alues
ob ained om ou di e en popula ions o PE-conjuga ed beads
wi h a known numbe o PE molecules pe bead p o ided by he
Quan iBRITE™ PE ki (BD Biosciences). B ie ly, he Quan iBRITE™
PE beads we e dilu ed in 500 μl o 1× PBS wi h azide plus 0.5% (w/ )
BSA (Sigma Ald ich) and analyzed by low cy ome y. The luo escence
in ensi y alues o he semen samples and di e en popula ions o he
Quan iBRITE™ PE beads we e ob ained a he same ime using he
same se ings o luo escence and compensa ion.
The luo escence da a om a leas 10 000 e en s we e analyzed
wi h a low cy ome e (Gallios™; BD Biosciences). Blue (Hoechs 33258)
and ed (PE) luo escence we e collec ed in he FL9 and FL2 senso s,
espec i ely. To ensu e ha he luo escence da a we e om li e
spe ma ozoa, we used a disc imina ion ame a ound he spe m
popula ion on o wa d (FSC) and side sca e plo s (SSC) and hen
selec ed Hoechs 33258-posi i e e en s. The pe cen age o PE-posi i e
cells and mean luo escence o he spe m samples we e de e mined
by sub ac ing he backg ound luo escence in each his og am om
i s con ol. The PE and Hoechs 33258 luo escence esul s we e
analyzed wi h Summi so wa e ( e sion 4.3; Beckman Coul e Inc.,
Los Angeles, CA, USA).
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Asian Jou nal o And ology
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Finally, we conside ed he pe cen age o ecep o - and
enzyme-posi i e cells measu ed in each semen sample o
semi-quan i a i e low cy ome y analysis. Fo he quan i a i e low
cy ome y assay, we de e mined he a e age numbe o molecules
pe spe ma ozoon, which was ex apola ed om a calib a ion cu e
ob ained om he popula ions o PE-conjuga ed beads, because he
PE:Ab a io o ou p ima y an ibody was 1:1.
De e mina ion o ecep o exp ession by immunocy ochemis y
P o ein de ec ion was pe o med using indi ec immuno luo escence.
This me hod equi es he consecu i e use o wo an ibodies: a p ima y
an ibody ha binds o he a ge p o ein and a seconda y an ibody
bound o a luo och ome, which speci ically de ec s he p ima y
an ibody.
Spe ma ozoa p e iously ixed in 2% pa a o maldehyde ( / ) we e
washed and esuspended in 1× PBS. They we e hen sp ead on co e
slips p e iously ea ed wi h poly-L-lysine and d ied.
The samples we e pe meabilized wi h 1% ( / ) T i on® x-100
(Sigma Ald ich) in 1× PBS o 10 min unde agi a ion. The T i on®
x-100 was emo ed, and he samples we e washed h ee imes o 5
min wi h 1× PBS. Subsequen ly, he samples we e blocked wi h 10%
FBS ( / ) in 1× PBS o 30 min and hen incuba ed o e nigh a 4°C
wi h he co esponding p ima y an ibodies gi en abo e in PBS/5% FBS:
an i-AT1R (ex acellula ) an ibody, an i-AT2R an ibody, an i-AT4R
an ibody (IRAP), and an i-MasR. The p ima y an ibody was emo ed
om he samples, which we e hen washed h ee imes in 1× PBS o
5 min each. The samples we e incuba ed wi h he seconda y an i-IgG
an ibody Alexa Fluo ® 488 ( abbi polyclonal goa IgG seconda y
an ibody conjuga ed o Alexa Fluo ® 488; A11008; In i ogen) in he
da k o 1 h a oom empe a u e. A e ha , he samples we e washed
again h ee imes o 5 min wi h 1× PBS. Hoechs 33258 (Sigma
Ald ich) was added a a concen a ion o 5 μg ml−1 and incuba ed
o 2 min.
The Alexa Fluo ® 488 luo och ome has maximum exci a ion and
abso p ion wa eleng hs o 495 nm and 519 nm, espec i ely. Thus,
he samples we e exci ed wi h an a gon lase a 488 nm and ligh
was collec ed be ween 505 nm and 520 nm. Hoechs 33258 is a DNA
ma ke ha is cell memb ane pe meable and luo esces in blue when
i binds o he mino g oo e o he DNA double s and. Hoechs
33 258 has maximum exci a ion and abso p ion wa eleng hs o 352
nm and 461 nm, espec i ely, and i s combina ion wi h Alexa Fluo ®
488 luo och ome is sui able because he e is no o e lap be ween hei
emission spec a.
Samples we e p epa ed using Fluo omoun G (EMS, Ha ield, UK)
and obse ed unde a con ocal mic oscope (Fluo iew FV 500; Olympus,
Mel ille, NY, USA), allowing he o e lap o images o consecu i e
spe m planes. The images we e p ocessed using Fluo iew e sion
5.0 so wa e, and image analysis was pe o med using ImageJ
(de eloped on Mac OS X, he sou ce code is eely a ailable; Na ional
Ins i u es o Men al Heal h, Be hesda, MD, USA).
S a is ics
We conduc ed desc ip i e s a is ical analysis o he da a (mean,
s anda d e o mean [s.e.m.], median, and minimum and maximum
alues). These ini ial analyses p o ided in o ma ion as a i s
app oxima ion o begin he da a analysis. Conside ing ha he sample
size was less han 50, his analysis was pe o med using he Shapi o–
Wilk no mali y es . S a is ical analysis o he da a was pe o med
using Mic oso Excel and he s a is ical package IBM SPSS S a is ics
e sion 22 (IBM Co p., A monk, NY, USA). The Mann–Whi ney U
es was used o de e mine ela ionships be ween each o enzyme
s udied and spe m DNA agmen a ion. S a is ical signi icance and
high s a is ical signi icance we e de e mined by P < 0.05 and P <
0.01, espec i ely.
RESULTS
The highes le els o spe m pep ide ac i i y we e obse ed o neu al
aminopep idase, ollowed by APB, and APA; he lowes alue was
o neu al endopep idase (Figu e 1). Simila ly, he highes le els o
pep ide ac i i y ound in seminal plasma co esponded o APB and
neu al aminopep idase (wi h e y simila alues), ollowed by aspa yl
aminopep idase and p olyl endopep idase (Figu e 1). NEP in seminal
plasma and PEP ac i i y in spe ma ozoa we e no ound. Each enzyme
was analyzed acco ding o he alue o agmen a ion; ≤20% was
conside ed non- agmen ed, and >20% was conside ed agmen ed.
Highe mean alues o pep idase uni ac i i y we e obse ed o APN,
and minimum alues o NEP. No di e ences we e ound be ween
agmen ed and non- agmen ed spe ma ozoa.
The Mann–Whi ney U es was used o de e mine he exis ence
o a ela ionship exis ed be ween each o he s udied enzymes and
spe m DNA agmen a ion. The maximum and minimum alues,
as well as he mean and s.e.m. wi h o wi hou DNA agmen a ion
in spe m, a e p esen ed in Figu e 1. The highes alues in seminal
plasma we e obse ed o APN and APB; no di e ences depending on
agmen a ion s a us we e ound. Mo eo e , no associa ion was ound
be ween he ac i i y o he enzymes s udied in seminal plasma in he
p esence o absence o spe m DNA agmen a ion.
Enzyma ic ac i i y by low cy ome y
The pe cen age o APN/CD13 in non- agmen ed DNA spe ma ozoa
samples was highe han ha in agmen ed samples (Figu e 2a).
Fu he mo e, he in ensi y in non- agmen ed was highe han ha
in agmen ed spe ma ozoa (P<0.05; Figu e 2b). The pe cen age o
CD143 in non- agmen ed samples was smalle han ha in agmen ed
samples (Figu e 2a), and he in ensi y in agmen ed spe m was also
highe han ha in non- agmen ed spe m (Figu e 2b). The pe cen age
o CD10 in non- agmen ed DNA samples was smalle han ha in
agmen ed DNA samples (P<0.05; Figu e 2a), bu he in ensi y in
non- agmen ed DNA samples was highe han ha in agmen ed
DNA samples (Figu e 2b).
Exp ession o ecep o s o he RAS: low cy ome y analysis
Le els o bo h AT1R and AT2R we e highe in non- agmen ed han in
agmen ed DNA spe ma ozoa. Mo eo e , he in ensi y in non- agmen ed
samples was highe han ha in agmen ed samples. The labeling o AT1R
(94.3% in DNA agmen a ion ≤20% s 84.1% in DNA agmen a ion
>20%), AT2R (87.4% s 63.1%) and PRR (96.4% s 48.2%) we e p ima ily
exp essed in non-DNA- agmen ed spe ma ozoa (Figu e 3a).
Rega ding he p esence o AT4R/IRAP, mo e han 90% o cells
in bo h samples we e posi i e o he ecep o ; hough he in ensi y
o non- agmen ed DNA was 42.4 and ha o agmen ed was 21.3.
The in ensi y pe cen ages obse ed o PRR in non- agmen ed DNA
samples we e highe han hose in agmen ed DNA (96.4% in DNA
agmen a ion ≤20% s 48.2% in DNA agmen a ion >20%). MasR
was simila be ween bo h g oups (97.4% s 87.2%). PRR and MasR
in ensi ies in non- agmen ed DNA spe ma ozoa we e highe han
hose in DNA- agmen ed spe ma ozoa (P < 0.05; Figu e 3b).
The speci ici y o an ibodies was de e mined by h ee di e en
analyses:
1. Use o a sample o which p ima y and seconda y an ibodies we e
no added (blank)
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RAS and spe m DNA agmen a ion
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2. Addi ion o non-speci ic immunoglobulins a iden ical
concen a ions o hose used wi h he p ima y an ibodies
(con ol o p ima y an ibody)
3. Omission o he p ima y an ibody be o e addi ion o he
seconda y an ibody (con ol o seconda y an ibody). The
obse ed luo escence in ensi y pa e ns we e e y simila .
Thus, he luo escence measu ed in he samples was solely due
o ha emi ed by he speci ic an ibody binding o AT1R, AT2R,
AT4R/IRAP, PRR o MasR (Figu e 3b).
Exp ession o ecep o s o he RAS: immunocy ochemical analysis
The AT1R ecep o was de ec ed in he spe m ail, and AT2R was
localized in he equa o ial/pos -ac osomal egion o he spe m
head; a low-in ensi y signal o AT2R was also obse ed along he
ail (Figu e 4). In spe ma ozoa, PRR was ound in he on o he
spe m head, in he ac osomal egion and a he back o he head;
a weake labeling was obse ed along he ail o he spe ma ozoon.
In DNA- agmen ed samples, howe e , he in ensi y was lowe . In
con as , non-speci ic binding was no obse ed in nega i e con ols
in which he speci ic p ima y an ibody was omi ed.
Finally, no co ela ion was obse ed when he DNA agmen a ion
index alues and analysis o RAS pa hway componen s we e pe o med
pe indi idual.
DISCUSSION
Pep idases a e known o play a key ole in g ow h con ol,
di e en ia ion, and he signal ansduc ion in many cell sys ems by
modula ing he ac i i y o bioac i e pep ides. In 2002, Fe nández
e al.10 desc ibed o he i s ime he ac i i y o se e al pep idases
(enkephalin-deg ading enzymes) in human seminal ac ions. The high
alues obse ed in he di e en ac ions (seminal luid, p os asomes,
cy osolic spe m ac ion, and memb ane ac ion) sugges ed ha bo h
pep idases and hei na u al subs a es migh be in ol ed in seminal
physiology by egula ing physiologically ac i e pep ides, no only in he
es icle bu also in he seminal luid and spe ma ozoa.10 Subsequen ly,
hese s udies we e expanded, demons a ing mo e speci ic oles o
hese pep ide- egula ing enzymes.9,11,12 Thus, he egula o y enzyma ic
ac i i ies o pep ide sys ems a e in ol ed in spe m e iliza ion
p ocesses, which ep esen s a no el oppo uni y o ep oduc i e
managemen , by enhancing he p obabili y o e iliza ion o educing
i h ough he de elopmen o no el a ge ed con acep i es.11
O e he yea s, s udies ha e been ex ended o asce ain whe he
any o hese pep ide sys ems can be used as bioma ke s o ep oduc i e
success in assis ed ep oduc ion p ocesses, hough none ha e been
clea ly desc ibed o da e. In gene al, o e 80 million people wo ldwide
expe ience in e ili y and o e one- hi d o in e ili y cases a e due o
male ac o s. The e o e, in addi ion o he s udy o opioide gic le els
and hei ole in ep oduc ion, a no el sys em has been analyzed
in ecen yea s o inc ease knowledge in ep oduc i e unc ion, o
e alua e possible in ol emen in male ep oduc i e pa hologies and
o iden i y bioma ke s ha migh con ibu e o he de elopmen
o he apeu ic s a egies o he ea men o male sub e ili y. We
e e o he componen s o he RAS, pep ide sys em known mainly
o i s impo ance in he main enance o blood p essu e as well as
elec oly e and luid homeos asis. None heless, a en ion has also
been paid o e idence o a widesp ead local RAS in se e al issues
egula ing se e al speci ic unc ions. In his sense, his molecula
sys em is also p esen in he ep oduc i e ac and seems o ac on
male e ili y by ope a ing a mul iple le els in he egula ion o spe m
e ilizing abili y, sugges ing i s po en ial ole as a he apeu ic a ge
o imp o ing assis ed ep oduc ion he apies. Al hough he unc ion
o RAS in human spe ma ozoa is no comple ely unde s ood, i would
Figu e 2: P esence o aminopep idase N (APN/CD13), angio ensin-con e ing
enzyme (ACE/CD143) and neu al endopep idase (NEP/CD10), in spe ma ozoa
wi h DNA agmen a ion ≤20% and DNA agmen a ion >20% by low
cy ome y, shown as (a) pe cen age o posi i e spe m and (b) ma ke in ensi y.
*P<0.05.
b
a
Figu e 1: Tes ed ac i i ies included (a) aminopep idase N (APN/CD13; A III o A IV) and aminopep idase B (APB; A III o A IV), (b) aminopep idase A
(APA; A II o A III), neu al endopep idase (NEP; A I o A 1–7), and p olyl endopep idase (PEP; which con e s angio ensin [A] I and A II o A 1–7). NEP in
seminal plasma and PEP ac i i y in spe m we e no ound. A: angio ensin
b
a
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MV Apa icio P ie o e al
144
be in e es ing o de e mine whe he some RAS componen s can be
conside ed bioma ke s o spe m unc ion and selec ion.
In ac , one o he componen s o his sys em has ecen ly been
desc ibed a he es icula le el: he spe m p o ein ACE. The amoun
o his enzyme on spe m su ace memb anes sugges s ha i migh play
a ole du ing emb yo de elopmen , e en be o e he ac i a ion o he
ansla ional machine y o he emb yo. These esul s indica e ha no
only he p esence o absence o spe m p o eins bu also he numbe o
molecules pe spe ma ozoon may p o ide e y aluable in o ma ion
ega ding emb yo de elopmen , quali y and iabili y. Thus, ACE may
be use ul as a bioma ke o con ibu e in o ma ion o emb yologis s in
he selec ion o a spe m popula ion wi h s ong po en ial o p oduce
high-quali y emb yos du ing ICSI.20
Acco dingly, we epo he le els o di e en enzymes in bo h
spe m and seminal luids ha (in g ea e o lesse p opo ion) egula e
le els o pep ide componen s o RAS. Speci ically, he enzymes
e alua ed we e: PEP (A I and A II o A 1–7), NEP (A I o A 1–7),
APN (A III o A IV), APA (A II o A III), APB (A III o A IV), and
ACE (A I o A II). Because i is es ima ed ha 10%–15% o men wi h
s e ili y ha e game e gene ic ma e ial al e a ions, e en hough spe m
concen a ion, mobili y and mo phology a e no mal, we also examined
le els o hese enzyme in spe m samples wi h DNA agmen a ion o
de e mine whe he spe m DNA agmen a ion is ela ed o al e a ions
in pep ide me abolism and o desc ibe bioma ke s o spe m in eg i y
and unc ion, when compa ing spe ma ozoa wi h and wi hou DNA
agmen a ion.
Enzymes ha egula e he le els o di e en RAS componen s
(e.g., egula o y aminopep idases) es ed in his s udy showed no
signi ican al e a ions in ac i i y be ween non- agmen ed- and
agmen ed-DNA samples in seminal luid o spe ma ozoa. This
non- a ia ion o seminal ac i i y seems o demons a e ha , in
his s udy, he seminal luid ha con ains he game es emains
s able. Howe e , a he cellula le el, we obse ed di e ences in he
APN/CD13 and ACE/CD143 le els by bo h immuno luo escence
and low cy ome y. A III/A IV/AT4R pa hway ac i i y, egula ed by
APN, was educed in DNA- agmen ed spe ma ozoa, hough in his
case, he e was no a ia ion in exp ession o he ecep o . I is di icul
o in e p e hese esul s, because only he p esence o APN has been
p e iously desc ibed in spe ma ozoa, whe eas A IV/AT4R has no
ye been de ec ed, and his is he i s ime ha he p esence o he
AT4 ecep o on spe m has been desc ibed. The A I/A II/AT2R axis
egula ed by ACE inc eases in agmen ed samples due o ecep o
exp ession a ia ion. I can he e o e be deduced ha his main RAS
pa hway is a ec ed in spe ma ozoa wi h DNA agmen a ion. Owing
o he p incipal loca ion o he enzymes and ecep o s al e ed in his
p incipal axis o RAS ( he ail o spe ma ozoa), i may be sugges ed
ha in cells wi h DNA agmen a ion, spe m mo ili y is mo e al e ed
han he ac osome eac ion o he spe ma ozoon.
Figu e 4: Resul s by immunocy ochemis y o enin-angio ensin ecep o s in spe ma ozoa wi h DNA agmen a ion ≤20% and wi h DNA agmen a ion >20%:
(a) angio ensin (A) II ecep o ype 1 (AT1R), (b) A II ype 2 ecep o (AT2R), (c) A IV ecep o (AT4R/IRAP), (d) (p o) enin ecep o (PRR), and (e) A 1–7
ecep o o Mas ecep o (MasR). H258: Hoechs 33258; Ac2: seconda y an ibody
d
b c
a
e
Figu e 3: P esence and in ensi y o angio ensin (A) II ype 1 ecep o
(AT1R), A II ype 2 ecep o (AT2R), A IV ecep o (AT4R/IRAP), (p o) enin
ecep o (PRR) and A 1–7 ecep o (MasR) in human spe ma ozoa wi h DNA
agmen a ion ≤20% and DNA agmen a ion >20% by low cy ome y, shown
as (a) pe cen age o posi i e spe m and (b) ma ke in ensi y.
b
a
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Asian Jou nal o And ology
RAS and spe m DNA agmen a ion
MV Apa icio P ie o e al
145
ACE is p esen in ma u e spe ma ozoa o di e en species, and a
ecen s udy32 ha e demons a ed he in ol emen o ACE in spe m
mo ili y, capaci a ion, he ac osome eac ion and spe m-oocy e
usion. Thus, he ep oduc i e capaci y o agmen ed spe m may be
al e ed by his inc ease. ACE could hus be use ul as a bioma ke o
aid emb yologis s in he selec ion o a spe m popula ion. Addi ional
s udies should be ca ied ou o conclude ha i can also se e as a
bioma ke o DNA agmen a ion. To acili a e he moni o ing o ou
esul s, in Figu e 5, we ep esen he local spe ma ozoon RAS axis and
he a ia ions ha occu in agmen ed spe ma ozoa.
AT1R was de ec ed in he spe m ail, a inding ha ag ees wi h he
epo by Vinson e al.33,34 Conside ing ha he loca ion o mos RAS
ecep o s (excep pe haps PRR) is in he spe m ail, as sugges ed by
Vinson e al.,35 we in e ha hese ecep o s a e ela ed o he egula ion
o spe m mobili y mo e han o he ac osome eac ion. Conside ing
he loca ion o PRR, i migh be in ol ed in he ac osome eac ion,
a inding ha coincides wi h ha sugges ed by di e en au ho s
ega ding he ela ionship be ween his possibili y and RAS.36–41 In
addi ion, as p e iously sugges ed, bo h enin p ecu so p o ein and
he enin p o ein i sel migh ac on PRR ega dless o he classical
axis.42 Rega ding ou mic og aphs wi h RAS spe m ecep o s, we
could no disc imina e he immunolabeling pa e n o non- agmen ed
and agmen ed samples; ha is, he loca ion in he cell memb ane
pe sis ed ega dless o he spe m DNA agmen a ion s a us. In
ac , we obse ed ha AT1R labeling, AT4R and MasR we e simila
be ween he spe m g oups analyzed (wi h highe le els in spe m wi h
non- agmen ed DNA). Ne e heless, AT1R, AT2R and PRR showed
highe exp ession in spe ma ozoa wi h agmen ed DNA. In addi ion,
he labeling in ensi y, which was lowe in spe ma ozoa wi h agmen ed
DNA in all cases, should be conside ed. Rega dless, i is impossible
o de e mine whe he he di e ence was due o he g ea e o lowe
numbe o ecep o s p esen o mo e spe ma ozoa exp essing hem.
AT2R has al eady been desc ibed in human spe ma ozoa,43 and i may
also play an impo an ole in spe m mo ili y, as i has been obse ed
ha AT2R is ela ed o human spe m concen a ion and mo ili y.
The p esence o PRR has ecen ly been desc ibed,32 sugges ing
ha his ecep o plays a ole in spe m mo ili y, as ligh e s aining was
obse ed along he ail o human spe ma ozoa.
Finally, he ewe immunolabeled spe ma ozoa when DNA
agmen a ion was high and he in ensi y was low could be due o wo
ci cums ances: (1) spe ma ozoa wi h agmen ed DNA ha e a lowe
esponsi eness o ac i e RAS pep ides when hey ha e a lowe labeling
in ensi y; o (2) ewe spe ma ozoa exp ess he ecep o s, leading o
lowe immunolabeling in ensi y.
Al hough he la e possibili y may seem he mos logical,
conside ing he esul s o his s udy and he li e a u e, we sugges ha
he o me is mo e likely. Thus, he ecep o s ha e a lowe capaci y o
induce a esponse because he e a e ewe o hem. This conclusion
can be clea ly in e ed by compa ing he pe cen age o game es wi h
and wi hou DNA agmen a ion ha a e posi i e o AT4R labeling.
Howe e , he in ensi y in spe ma ozoa wi h agmen ed DNA was
app oxima ely hal o ha in spe ma ozoa wi h non- agmen ed DNA.
In conclusion, RAS is in ol ed in he cellula esponse o spe m DNA
agmen a ion. Owing o he limi a ions o he s udy (i.e., powe
analysis o 70%, and isual de e mina ion o he agmen a ion deg ee),
u he s udies a e needed o de e mine he ac ual ole o he RAS
acili y in spe m agmen a ion p ocesses and he possible alida ion
o i s componen s as bioma ke s o ep oduc i e success.
AUTHOR CONTRIBUTIONS
MVAP and LCS concei ed o he s udy, designed he expe imen s,
pe o med he s a is ical analysis, and w o e he manusc ip . MVAP,
MVRG and YFI ca ied ou all he e ili y p ocedu es. AVP, GHB, and
EEO conduc ed luo ome ic, low cy ome y, and immunocy ochemis y
expe imen s. All au ho s ead and app o ed he inal manusc ip .
COMPETING INTERESTS
All au ho s decla e no compe ing in e es s.
ACKNOWLEDGMENTS
The au ho s wish o hank A an za Pé ez Doba an (UPV/EHU) o he
echnical con ibu ion o his s udy. This wo k was suppo ed by g an s om
he Uni e si y o he Basque Coun y (UPV/EHU GIU 17/19) and he Gangoi i
Ba e a Founda ion (Basque Coun y).
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