sos. oyalsocie ypublishing.o g
Resea ch
Ci e his a icle: Lindebe g H, Bu chmo e
RJS, Kennedy MW. 2017 Pulse o in lamma o y
p o eins in he p egnan u e us o Eu opean
poleca s (Mus ela pu o ius) leading o he ime
o implan a ion. R. Soc. open sci. 4: 161085.
h p://dx.doi.o g/10.1098/ sos.161085
Recei ed: 4 Janua y 2017
Accep ed: 22 Feb ua y 2017
Subjec Ca ego y:
Biology (whole o ganism)
Subjec A eas:
biochemis y/de elopmen al biology
Keywo ds:
Mus ela pu o ius, e e , u e ine sec e ions,
p egnancy, α2-mac oglobulin, lipocalin-1
Au ho o co espondence:
Malcolm W. Kennedy
e-mail: [email protected]
Elec onic supplemen a y ma e ial is a ailable
online a h ps://dx.doi.o g/10.6084/m9.
igsha e.c.3711982.
Pulse o in lamma o y
p o eins in he p egnan
u e us o Eu opean poleca s
(Mus ela pu o ius) leading
o he ime o implan a ion
Heli Lindebe g1, Richa d J. S. Bu chmo e2and
Malcolm W. Kennedy3
1Na u al Resou ces Ins i u e Finland (Luke), G een Technology, Halolan ie 31 A,
71750 Maaninka, Finland
2Ins i u e o In ec ion, Immuni y and In lamma ion, and Glasgow Polyomics, College
o Medical, Ve e ina y and Li e Sciences, Uni e si y o Glasgow, Ga scube Campus,
Glasgow G61 1QH, Sco land, UK
3Ins i u e o Biodi e si y, Animal Heal h and Compa a i e Medicine, and he Ins i u e
o Molecula , Cell and Sys ems Biology, College o Medical, Ve e ina y and Li e
Sciences, Uni e si y o Glasgow, G aham Ke Building, Glasgow G12 8QQ, Sco land, UK
MWK, 0000-0002-0970-5264
U e ine sec e o y p o eins p o ec he u e us and concep uses
agains in ec ion, acili a e implan a ion, con ol cellula
damage esul ing om implan a ion, and supply p e-
implan a ion emb yos wi h nu ien s. Unlike in humans,
he ea ly concep us o he Eu opean poleca (Mus ela pu o ius;
e e ) g ows and de elops ee in he u e us un il implan ing
a abou 12 days a e ma ing. We ound ha he p o eins
appea ing in poleca u e i changed d ama ically wi h ime
leading o implan a ion. Se e al o hese p o eins ha e also
been ound in p egnan u e i o o he eu he ian mammals.
Howe e , we ound a combina ion o wo inc easingly
abundan p o eins ha ha e no been eco ded be o e in
p e-placen a ion u e i. Fi s , he b oad-spec um p o einase
inhibi o α2-mac oglobulin ose o domina e he p o ein
p o ile by he ime o implan a ion. I s unc ions may be o
limi damage caused by he elease o p o einases du ing
implan a ion o in ec ion, and o con ol o he p ocesses
a ound si es o implan a ion. Second, lipocalin-1 (also known
as ea lipocalin) also inc eased subs an ially in concen a ion.
This p o ein has no p e iously been eco ded as a u e ine
sec e ion in p egnancy in any species. I poleca lipocalin-1
has simila biological p ope ies o ha o humans, hen i
may ha e a combined unc ion in an imic obial p o ec ion and
anspo ing o sca enging lipids. The changes in he u e ine
2017 The Au ho s. Published by he Royal Socie y unde he e ms o he C ea i e Commons
A ibu ion License h p://c ea i ecommons.o g/licenses/by/4.0/, which pe mi s un es ic ed
use, p o ided he o iginal au ho and sou ce a e c edi ed.
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sec e o y p o ein epe oi e o Eu opean poleca s is he e o e unusual, and may be ep esen a i e
o p e-placen a ion suppo i e u e ine sec e ions in mus elids (o e s, weasels, badge s, mink,
wol e ines) in gene al.
1. In oduc ion
A concep us may implan soon a e i s a i al in he u e us (as in humans and mu oid oden s), o
may emain ee in he u e us o se e al weeks be o e implan ing (as in equids) [1]. In e media e o
hese ex emes a e he concep uses o many Ca ni o a and A iodac yla which implan 10–20 days
a e ma ing. The delay is a ibu ed o ime equi ed o ma ing-induced o ula ion, a el h ough
he allopian ubes, and a a iable pe iod du ing which he blas ocys de elops wi hou close cellula
con ac wi h ma e nal issues [1]. In some species o mus elid, u sids, phocids and oe dee , implan a ion
is pos poned o se e al mon hs, du ing which ime he concep us en e s obliga o y diapause a he
blas ocys s age and mus be main ained and p o ec ed un il eac i a ion is pe mi ed by he mo he
[2–5]. The Eu opean poleca , Mus ela pu o ius [6], i s in o he in e media e g oup, wi h an emb yonic
de elopmen pe iod (wi hou emb yonic diapause) o app oxima ely 12 days p eceding implan a ion.
P o eins sec e ed in o he non-p egnan u e i o eu he ian mammals ha e a ange o p esump i e
unc ions such as main enance o he mucocu aneous su ace o he endome ium, an imic obial
p o ec ion, ecep i i y o spe m and he subsequen concep us, and nu i ion o an emb yo. Dis inc
di e ences ha e been ound among mammal g oups in he p o eins sec e ed in o he u e us be o e
placen a ion, al hough he e a e commonali ies [7–12]. These p o eins ha e been di ided mainly in o
hose in ol ed in nu i ional suppo o he concep us, o in acili a ing and con olling implan a ion
e en s. Pe haps, he mos no able example o he nu i ional unc ion is he u e ocalin/P19 p o ein in
ho ses. This p o ein appea s o deli e essen ial lipids such as e inol and polyunsa u a ed a y acids
ac oss he glycop o ein capsule o he equine concep us [13–15]. Lipids p esen a p oblem in hei ans e
om mo he o concep us because hey end o be insoluble, and in some cases suscep ible o oxida ion
damage unless p o ec ed wi hin a p o ein binding si e. U e ocalin is also no able in ha i s p ima y
s uc u e is en iched in essen ial amino acids, which doubles i s unc ion as a nu ien sou ce o he
de eloping emb yo [15]. O he examples o lipid ca ie s in u e ine sec e ions include a modi ied o m
o plasma e inol binding p o ein, and se um albumin which binds a ange o small molecules, a y
acids in pa icula [11,16,17]. O hose p o eins conside ed o in luence implan a ion e en s, he b oad-
spec um p o einase inhibi o α2-mac oglobulin (α2M) is sec e ed in o he p egnan u e us a ound he
ime o implan a ion in se e al species, and, among o he oles, is hough o limi issue damage du ing
implan a ion and o con ol local in lamma o y esponses [7–12,18,19].
O e all, he e is conside able di e si y in he sui e o p o eins sec e ed in o p e-placen a ion u e i by
di e en clades o mammals, as exempli ied by equids, a iodac yles and Ca ni o a, all o which exhibi
dis inc i e epe oi es [7–10,12]. We in es iga ed he p o eins ound in u e ine sec e ions o p egnan
Eu opean poleca s (domes ica ed as e e s) in o de o explo e he p epa a o y e en s ha lead up o,
and a , implan a ion. Analysis o ch onological samples indica ed ha p og essi e and d ama ic changes
in he p o ein p o ile occu as he ime a which implan a ion would occu app oaches. Some o he
p o eins ha e been ound in p e-implan a ion u e ine lushes o o he species (no able among which is
α2-mac oglobulin), al hough no in he same combina ion o ela i e concen a ions. A leas one p o ein,
lipocalin-1, which we ound o peak in abundance a implan a ion, has p e iously no been epo ed
as a u e ine sec e o y p o ein. No only does his s udy demons a e ha mus elids exhibi a di e en
epe oi e o implan a ion- ela ed u e ine sec e ions om o he species g oups, bu i may also p o ide
a gene al amewo k o in es iga ion o wha happens du ing emb yonic quiescence and subsequen
eac i a ion in species ha engage in p olonged emb yonic diapause.
2. Ma e ial and me hods
2.1. Animals and sample collec ion
The subjec s used in his s udy descended om a popula ion o 70 wild Eu opean poleca s (Mus ela
pu o ius) cap u ed in he 1970s and in e b ed wi h 200 cap i e impo ed domes ic e e s (subspecies
Mus ela pu o ius u o). The esul ing popula ion o ms he basis o all poleca s a med o he
u indus y and esea ch in Finland. The animals we e main ained a he Kannus Resea ch Fa m
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Luo a L d., in Kannus, Finland. They we e held in indi idual cages measu ing 70 ×30 ×38 cm
(leng h ×wid h ×heigh ) wi h nes -boxes measu ing 40 ×29 ×32 cm which we e accessible o he
emales bu ex e nal o he main cage a eas. Du ing he b eeding season, he animals we e exposed o
ou doo empe a u es and ligh condi ions: mean 2°C and 15 h ligh in Ap il, 9°C and 16 h ligh in May,
14°C, and 20 h ligh in June. The animals we e ed acco ding o s anda ds o b eeding a med poleca s
in Finland (p epa ed o p o ide a leas 1150–1250 kcal kg−1, 40–50% p o ein, 30–32% a s and 15–25%
ca bohyd a es, supplemen ed wi h i amins and mine als), and had wa e p o ided ad libi um.
The ime o oes us was es ima ed by he physical appea ance o he ul a. Males used o ma ing
we e known o ha e been e ile he p e ious yea , and ma ings we e isually con i med (a ie obse ed).
Animals we e selec ed o sample collec ion on days 4, 6, 7, 9, 12 and 14 a e ma ing. Those sampled on
days 4 and 14 p o ed no o ha e become p egnan bu we e ne e heless included because hey ac ed
as ma ed bu non-p egnan con ols. Each animal was anaes he ized wi h mede omidine (Do bene®;
Labo a o ios sy a s.a., León, Spain) and ke amine (Ke ala ®; P ize Oy, Helsinki, Finland), and he
u e us emo ed unde s e ile su gical condi ions. The animals we e subsequen ly eu hanized wi h T-61
(In e e In e na ional B.V., Boxmee , The Ne he lands) while s ill unde anaes hesia. Once he u e us
was emo ed, he ips o each ho n we e opened, and he cu ends clea ed o blood. The ce ix was
closed wi h o ceps, and he u e us lushed wi h 5 ml s e ile physiological saline h ough one ho n and
ou he o he , wi h ca e o a oid blood con amina ion. The lush luid was il e -s e ilized and ozen a
−20°C in app oxima ely 2 ml aliquo s. P egnancy was con i med by he p esence o de eloping emb yos
in u e ine lushes. Samples we e collec ed om a o al o 14 animals, p o ein gel analysis om all o which
is shown in elec onic supplemen a y ma e ial, igu e S1, and hose ha we e selec ed o u he analysis
a e indica ed in elec onic supplemen a y ma e ial, igu e S1.
2.2. P o ein elec opho esis
One-dimensional e ical sodium dodecyl sul a e polyac ylamide gel elec opho esis (SDS-PAGE) was
ca ied ou using he In i ogen (The mo Scien i ic, Paisley, UK) NuPAGE sys em using p ecas 4–12%
g adien ac ylamide gels, wi h β2-me cap oe hanol (25 µl added o 1 ml sample bu e ) as educing agen
when equi ed. Gels we e s ained o p o ein using colloidal Coomassie Blue (Ins an Blue, Expedeon,
Ha s on, UK) and images o gels we e eco ded using a Kodak image . Elec onic images we e modi ied
only o adjus men o con as and b igh ness. P e-s ained molecula mass/ ela i e mobili y (M )
s anda d p o eins we e ob ained om New England Biolabs, Ipswich, MA, USA (ca . numbe P7708S).
Samples we e un unde non- educing and educing condi ions using β-2 me cap oe hanol as educing
agen . Selec ed gel slices we e aken om non- educed, Coomassie Blue-s ained gels and p ocessed o
applica ion o esh gels unde educing condi ions. Samples we e concen a ed whe e equi ed using
a Vi aspin 3,000 MWCO PES (Sa o ius, Epsom, Su ey, UK) cen i ugal concen a o de ice ope a ed
acco ding o he manu ac u e ’s ins uc ions.
2.3. P o eomics
S ained p o ein bands we e excised om p epa a i e one-dimensional gels and analysed by liquid
ch oma og aphy–mass spec ome y as p e iously desc ibed [20]. P o ein iden i ica ions we e assigned
using he MASCOT sea ch engine o in e oga e p o ein and gene sequences in he NCBI da abases and
he Mus ela pu o ius genome da abase [21] and linked esou ces, allowing a mass ole ance o 0.4 Da o
bo h single and andem mass spec ome y analyses. BLAST sea ches, o sea ches o genome da abases
o o he Ca ni o a (e.g. dog and gian panda), we e ca ied ou o check he anno a ions. P edic ion
o sec e o y leade pep ides and hei clea age si es was ca ied ou using SignalP so wa e (h p://
www.cbs.d u.dk/se ices/SignalP/;[22]), and molecula masses calcula ed using P o Pa am (h p://
web.expasy.o g/p o pa am/).
3. Resul s
3.1. Sequen ial changes in he p o ein p o ile o p e-implan a ion u e ine sec e ions
The p o ein p o iles o all he u e ine lush samples collec ed om days 4 o 14 a e ma ing a e shown in
he SDS-PAGE analysis in elec onic supplemen a y ma e ial, igu e S1. The dispa i ies be ween o e all
p o ein concen a ions among he samples could be due o di e ences in e iciency o lushing, changes
in he o al issue olume, di e ences be ween indi idual animals’ sec e ion olumes o changes in
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M
day a e ma ing day a e ma ing
M
(kDa)
M
(kDa)
4 6 7 9 12 14
A
175
80
58
46
30
25
17
7
non- educed educed
175
80
58
46
30
25
17
7
B
I
D
F
C
E
G
H
K
M
J
O
P
R
X
W
S
U
V
M46791214
Figu e 1. Changes in Eu opean poleca u e ine sec e o y p o eins wi h ime a e ma ing. Animals sampled on days 4 and 14 we e non-
p egnan . The sample olumes we e adjus ed o no malize he in ensi y o he s ong band a app oxima ely 65 kDa (se um albumin).
See elec onic supplemen a y ma e ial, igu e S1, o SDS-PAGE o all o he samples collec ed and upon which he adjus men s we e
based. Gel band codes a e indica ed by le e s and a e e e ed o in he ex and in able 1. The p o eins ha mos clea ly inc eased in
concen a ion wi h ime we e α2-mac oglobulin (uniquely iden i ied in bands A, B, J and K) and lipocalin-1 (uniquely iden i ied in bands
G and U). M, ma ke /calib a ion p o eins wi h ela i e mobili ies (M ) as indica ed in kilodal ons (kDa).
sec e o y ac i i y. In o de o imp o e compa abili y, selec ed samples we e concen a ed as desc ibed
abo e and/o he olume o loaded sample adjus ed o app oxima ely equalize he in ensi y o he band
a ca 65 kDa M (band C; igu e 1), suspec ed (and subsequen ly con i med by mass spec ome y), o be
se um albumin om i s size and slowe mig a ion unde educ ion.
S anda diza ion o p o ein concen a ion o se um albumin e ealed d ama ic changes in he
ch onological p o ein p o ile o p egnan u e ine lushes, in pa icula he p o eins in bands A, B, D,
E, G, U and W ( igu e 1). The p egnan animals on days 6 and 7 showed ew, i any, di e ences om he
ma ed bu non-p egnan animal a day 4, bu by day 9 conside able di e ences we e e iden be ween
p egnan and non-p egnan indi iduals.
In o de o explo e ela ionships be ween some o he p o eins in he mos in ense bands, gel
slices we e excised om a non- educed gel, and he p o eins in hem subjec ed o elec opho esis
unde educing condi ions. This showed ha bands A and B educed o a simila se o p o eins,
sugges ing ha hey we e ela ed o iden ical ( igu e 2). The sizes o he esul ing agmen s we e
no commensu a e wi h pos - educ ion clea age p oduc s o la ge mul i-chain immunoglobulins (IgM
and IgA) which migh be expec ed o appea in u e ine sec e ions. P o ein band C, which was used
o s anda dize he loading p o ein concen a ions o samples o igu e 1, mig a ed mo e slowly when
educed, which is ypical o se um albumin. The change in i s mig a ion is usually a ibu ed o clea age
o i s in amolecula disul ide bond such ha he un olded p o ein is e a ded in i s mig a ion. Simila
beha iou was exhibi ed by band G p o ein, bu he o he s sepa a ed as unde non- educing condi ions.
The educ ion o band C p o ein unco e ed a di e en p o ein ha was subjec ed sepa a ely o p o eomic
analysis (band R).
3.2. P o eomics
P o ein bands whose concen a ions changed d ama ically wi h ime a e ma ing, o whose
concen a ions emained cons an , we e selec ed o p o eomic analysis om bo h educing and
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175
M
(kDa)
MNR M R A B C D
educed
EFG H
A
B
D
F
C
E
G
H
80
58
46
30
25
17
7
Figu e 2. Subuni composi ion o majo p o eins in Eu opean poleca u e ine lush ollowing p o ein educ ion. The labelled bands we e
excised omanon- educingp epa a i eSDS-PAGEgel o au e ine lushsample aken oma p egnan animalon day12 a e ma ingand
e- un unde educing condi ions. The le e codes o each band a e consis en wi h hose in igu e 1.The wop o eins ha mos no ably
inc eased in concen a ion wi h ime we e α2-mac oglobulin (bands A and B) and lipocalin-1 (band G). The o iginal p o ein samples
we e un unde educing (R) o non- educing (NR) condi ions. M, ma ke /calib a ion p o eins wi h ela i e mobili ies (M ) indica ed in
kilodal ons (kDa).
non- educing gels ( igu e 1). The sepa a ion be ween he bands in he SDS-PAGE gels was su icien
o allow p o eomic iden i ica ion o he p o eins wi h high con idence, all di ec ly om he M. pu o ius
genome-de i ed p o ein sequence da abase. The deduc ions we e unchanged upon checking by BLAST
sea ching o genomic and o he da abases o o he Ca ni o a. Table 1 lis s he iden i ica ions and also
he MASCOT sco es and pep ide ma ches o he p o eins, and illus a es he high quali y o he ma ches.
The wo bands wi h he highes M , bands A and B, we e bo h iden i ied as α2-mac oglobulin (α-2M),
as was hei educed o m in band J. These sizes a e la ge han expec ed om he p o ein’s polypep ide
mass wi h i s p edic ed sec e o y signal pep ide emo ed, and may be due o glycosyla ion and/o
co alen linkage wi h o he en i ies. The o he p o eins ha appea ed a high ela i e amoun s a ound
he ime o implan a ion included lipocalin-1 (bands G, U; also known as ea lipocalin, and an enzyme
o he ec onucleo ide py ophospha ase amily, also ep esen ed in bands D, O), ca hepsin L1 (bands
E, W), u e o e in (bands E, H, W) and zinc-α-2-glycop o ein (band W). T ans e in (bands F, P), α1-
an i ypsin (bands C, R, X) and lac o e in (bands F, P) emained a cons an le els ela i e o se um
albumin h oughou he sampling pe iod.
4. Discussion
The p o eins sec e ed in o he u e i o p egnan Eu opean poleca s changed d ama ically in abundance
o e he app oxima ely 12 days be ween ma ing and he ime o implan a ion by he emb yo. The
epe oi e o p o eins shows some simila i ies wi h ha ound in o he g oups o mammals, p esumably
e lec ing a heme inhe i ed om a common ances o , o con e gen e olu ion in o de o sa is y
simila unc ional equi emen s. In Eu opean poleca s, howe e , he appea ance o α2M was pa icula ly
p onounced, and he appea ance o lipocalin-1 has no p e iously been ound.
4.1. α2-mac oglobulin
α2M was he mos no able p o ein o appea in abundance sho ly be o e he ime o implan a ion. I is
closely ela ed o p egnancy zone p o ein (PZP) and h ee membe s o he complemen sys em [23]. I is
he la ges non-immunoglobulin p o ein in ci cula ion in mammalian blood, comp ises an app oxima ely

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Table 1. Iden i ica ion o he p o eins isola ed om bands excised om he p o ein elec opho esis gels indica ed in igu es 1and 2.
bandap o einbmass o p o einc
da abase
accession codedMASCOT sco ee
numbe o pep ides
(unique pep ide
ma ches) unc ion, associa ion, synonyms and commen sg
A, B, H, I, J, K, M, O α2-mac oglobulin 163781.5 (161296.4) XP_004779762 3502 215 (127) inhibi o o all classes o p o einase. Bai and ap mechanism igge s enclosu e o
a ge p o einase. Thio-es e bond-media ed co alen binding o p o einases.
La ges majo non-immunoglobulin p o ein complex in plasma. Acu e-phase
p o ein in a s, mino acu e phase eac an in o he species. Ele a ed in p egnancy
in humans and mice. Con ols e al posi ioning. Func ionally dis inc iso o ms
.............................................................................................................................................................................................................................................................................................................................................................................
C, R, X α1-an i ypsin 46521.2 (44096.2) XP_004754815 663 51 (26) se ine p o einase inhibi o . Acu e-phase eac an
.............................................................................................................................................................................................................................................................................................................................................................................
G, U lipocalin-1 19342.0 (17491.7) XP_004757035 317 23 (11) syn. ea lipocalin, on Ebne ’s gland p o ein. Lipid anspo . Bac e ial
side opho e-binding. Endonuclease. Cys a in–cys einyl p o einase inhibi o
.............................................................................................................................................................................................................................................................................................................................................................................
W zinc-α-2-glycop o ein 36407.5 (34360.9) XP_004781894 310 22(9) lipid me abolism signal; adipokine
.............................................................................................................................................................................................................................................................................................................................................................................
C se um albumin 68597.4 (66474.9) XP_004766346 1425 99 (52) ca ie o a y acids and o he small lipids, o he small molecules and d ugs, osmo ic
egula ion in ci cula o y sys em
.............................................................................................................................................................................................................................................................................................................................................................................
F, P se o ans e in 78378.1 (76408.6) XP_004762537 849 59 (27) syn. ans e in. I on anspo e
.............................................................................................................................................................................................................................................................................................................................................................................
F, P lac o ans e in 77327.0 (75354.4) XP_004761226 509 37 (20) syn. lac o e in. I on cap u e, an imic obial, nega i e egula o in in lamma ion.
Syn hesized and s o ed in neu ophil g anules
.............................................................................................................................................................................................................................................................................................................................................................................
E, H, W ca hepsin L1 37047.3 (35338.2) XP_004782389 343 34 (17) cys einyl p o einase. Lysosomal. An igen p ocessing in MHC II pa hway. Ex acellula
ma ix modi ica ion. Endome ial emodelling
.............................................................................................................................................................................................................................................................................................................................................................................
E, H, W u e o e in 37645.9 (35353.2) XP_004748428 464 24 (13) a a e- esis an acid phospha ase ype 5 (ACP5). P egnancy-associa ed acid
phospha ase. Syn hesized in esponse o p oges e one. Widely conse ed u e ine
p o ein in mammals. Appea s o unc ion in ansplacen al i on anspo and
s imula ion o e y h opoeisis. Human–bone building and emodelling, nega i e
egula o o in lamma o y signals
.............................................................................................................................................................................................................................................................................................................................................................................
D, O ec onucleo ide
py ophospha ase
109707.5 (none) XP_004743546 443 60 (19) ec oenzyme. Bone mine aliza ion and so issue calci ica ion. Appea s o modula e
insulin sensi i i y and unc ion; insulin ecep o binding
.............................................................................................................................................................................................................................................................................................................................................................................
R legumain 49280.8 (47556.7) XP_004739089 276 16 (7) hyd olyses p o eins a -Asn-Xaa- si e. Mul i unc ional. P ocessing o p o eins o MHC
class II an igen p esen a ion in he lysosomal/endosomal sys em
.............................................................................................................................................................................................................................................................................................................................................................................
V haemoglobin β15994.3 (none) XP_004779082 430 24 (13) O2/CO2 anspo and exchange
.............................................................................................................................................................................................................................................................................................................................................................................
(Con inued.)
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Table 1. (Con inued.).
bandap o einbmass o p o einc
da abase
accession codedMASCOT sco ee
numbe o pep ides
(unique pep ide
ma ches) unc ion, associa ion, synonyms and commen sg
.............................................................................................................................................................................................................................................................................................................................................................................
D, O galec in-3-binding
p o ein
62371.8 (60418.3) XP_004748843 281 22 (9) galec in is a galac ose-speci ic lec in equi ed o e minal di e en ia ion o columna
epi helial cells du ing ea ly emb yogenesis; in ol ed in acu e in lamma o y
esponses. P omo es in e g in-media ed cell adhesion. May s imula e hos de ence
agains i uses and umou cells
.............................................................................................................................................................................................................................................................................................................................................................................
D, O complemen C2 82486.9 (80560.4) XP_012905650 267 23 (11) componen o he complemen sys em. Cell lysis. In lamma ion
.............................................................................................................................................................................................................................................................................................................................................................................
S apolipop o ein A-I 30179.2 (28334.9) XP_004749957 458 31 (13) lipid binding. Choles e ol anspo . Majo p o ein componen o high densi y
lipop o ein (HDL) in plasma. Cons i uen o milk
.............................................................................................................................................................................................................................................................................................................................................................................
I, P alkaline phospha ase,
issue-non-speci ic
isozyme iso o m X1
57449.3 (55717.2) XP_004741321 135 12 (3) non-speci ic phosphomonoes e ases possibly in ol ed in cell signalling and bone
mine aliza ion. Enzymes o his class ound widely in li e , bile duc , kidney, bone,
in es inal mucosa and placen a
.............................................................................................................................................................................................................................................................................................................................................................................
I aminopep idase N 110639.6 XP_012917463 127 12 (4) b oad spec um aminopep idase. May be in ol ed in he me abolism o egula o y
pep ides o di e se cell ypes. Responsible o he p ocessing o pep ide ho mones
.............................................................................................................................................................................................................................................................................................................................................................................
K, M IgG Fc-binding p o ein 234543.3 XP_012918171 401 57 (16) binds Fc agmen o immunoglobulin G (IgG). May be in ol ed in he main enance o
mucosal s uc u e as a gel-like componen o he mucosa
.............................................................................................................................................................................................................................................................................................................................................................................
X ca hepsin D 44455.3 (42483.8) XP_004759751 718 54 (25) acid p o ease ac i e in in acellula p o ein b eakdown. Oes ogen- egula ed ansc ip
in b eas cance cells
.............................................................................................................................................................................................................................................................................................................................................................................
X u e o e in-associa ed
basic p o ein 2
48617.4 (45884.0) XP_004754814 307 68 (17) se ine p o einase inhibi o
.............................................................................................................................................................................................................................................................................................................................................................................
aGel band codes as indica ed in igu es 1and 2.
bP o ein iden i ica ions. Pep ides ma ching o ke a in we e excluded.
cThep edic ed molecula masses,as calcula edbyP o Pa am(h p://web.expasy.o g/p o pa am/), a eo he comple epolypep ides encoded be o e emo alo anyleade /signalpep ides o sec e ion.Thep edic edmasses ollowing emo alo suchleade
pep ides a posi ions p edic ed by Signalp is lis ed in b acke s [22]. No e ha pos ansla ional modi ica ions, p incipally glycosyla ion, will al e mobili y in p o ein elec opho esis gels.
dAccession numbe / e e ence sequence om NCBI GenBank da abase o Mus ela pu o ius genome da abase and checked by BLAST sea ching.
eMASCOT (MOWSE) sea ch sco e whe e sco es g ea e han 38 a e aken o be signi ican . The MASCOT sco e is he highes alue ob ained whe e he p o ein was iden i ied in mo e han one band, as we e he pep ide ma ch alues.
Numbe o pep ides ound o ma ch wi h numbe o pep ides unique o his iden i ica ion in pa en heses.
gPu a i e unc ions and commen s a e d awn om li e a u e ci ed, o NCBI and UniP o KB/Swiss-P o da abases.
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160 kDa polypep ide ha is glycosyla ed, and usually occu s in ci cula ion as a e ame comp ising
a pai o non-co alen ly associa ed disul ide-linked dime s [23]. I is syn hesized mainly in he li e ,
bu also in he decidua and endome ium in mice and possibly also in humans, and is hough o be
impo an o implan a ion [12,24–27].
α2M is a mino acu e-phase in lamma ion eac an in se e al species [28]. I ope a es ia wo
mechanisms as a b oad-spec um p o einase inhibi o [23]. In one mechanism, an ac i e p o einase
clea es a bai pep ide ha causes α2M o old a ound and encapsula e he a ge enzyme. In he o he ,
he a ge p o einase is co alen ly bound ia a hioes e bond. These mechanisms ap p o einases o all
classes bu do no necessa ily inac i a e hem, ins ead emo ing hem om access o in ac p o eins.
In acu e-phase and in lamma o y esponses, he p o einases a ge ed by α2M a e hough o be hose
o pa hogens, bu , signi ican ly, also hose eleased by g anulocy es and o he immune cells in po en ially
damaging in lamma o y eac ions [23]. This unc ion could be di ec ly ele an o placen a ion, gi en
ha he si es o placen a ion in some mammals exhibi local in lamma ion-like ma e nal esponses
[29–31]. The in lamma o y esponse may be ampli ied by localized apop osis o epi helial cells, and
p o einase inhibi ion by α2M may minimize damage caused by eleased enzymes [27,32–37]. α2Malso
binds se e al ho mones, g ow h ac o s and cy okines in ol ed in in lamma o y esponses, and can
modi y he biological ac i i y o hese signalling molecules [19,23]. In p egnancy, α2M is ound in u e ine
sec e ions o se e al species, and has been desc ibed o con ol ophoblas posi ioning and o e g ow h
in he mouse [7–12,18,19].
Se e al bands in he gels o poleca u e ine lushes con ained α2M, anging in size om close o
ha o he monomeic glycop o ein (band J) o highe -o de s uc u es (dime , e ame ; bands A and B).
Some ha did no con o m o simple monome s, dime s o e ame s o he p o ein may be co alen
α2M : p o einase complexes ha we e no dissembled by he educing agen ( igu e 2). The p esence
o α2M in he u e ine lumen could be due o inc eased pe meabili y o he u e ine essels o plasma
p o eins o issue dis up ion. I plasma leakage we e o ha e con ibu ed o he p esence o α2M, as
would also be he case o blood con amina ion du ing sampling, hen o he majo plasma p o eins, such
as immunoglobulins and complemen C3, would be p esen in p opo iona ely high amoun s. None o
hese we e obse ed, al hough se um albumin was clea ly p esen a all imes. While his is he mos
abundan plasma p o ein in e ms o ela i e mola i y, i is ound in u e ine sec e ions o o he species in
he absence o o he plasma p o eins [8,12]. Mo eo e , α2M is known o be syn hesized by u e ine issues
in humans and mice (see abo e).
α2M and PZP a e closely ela ed p o eins. The plasma concen a ion o PZP inc eases d ama ically
in he cou se o human p egnancy. I s unc ions a e no unde s ood [38], al hough i is known o ha e
p o einase inhibi o y ac i i ies simila o hose o α2M[38,39]. Despi e PZP’s documen ed syn hesis in
se e al ep oduc i e issues [38], and ha a gene encoding a PZP homologue has been p edic ed in
he Eu opean poleca genome (NCBI accession XP_012906269.1), ou analysis iden i ied no p o ein ha
migh be iden i ied as PZP in he u e ine lushes.
4.2. Lipocalin-1
The appea ance o lipocalin-1 was unexpec ed, as i has no p e iously been desc ibed as a componen
o u e ine sec e ions. Mo eo e , while o he lipocalins ha e been desc ibed in he ep oduc i e issues
o o he species, only lipocalin-1 was ound in his s udy o poleca u e ine sec e ions. Also known as
ea lipocalin and on Ebne ’s gland p o ein, lipocalin-1 is a membe o a la ge amily o p o eins ha
ope a es p edomina ely ex acellula ly. In addi ion o i s eponymous p esence in ea s, lipocalin-1 has
been ound in a wide ange o issues [40], albei no in u e ine sec e ions.
Mos lipocalins bind small hyd ophobic ligands such as e inol, a y acids, odo an s o phe omones,
and some a e enzyma ic [40,41]. O he s a e in ol ed in in ec ion de ence, such as α1-acid glycop o ein
(o osomucoid), which is an acu e-phase p o ein in humans ha binds a ange o small lipophilic
molecules and d ugs. Lipocalin-2 (neu ophil gela inase-associa ed lipocalin) is also an acu e-phase
p o ein and binds bac e ial side opho es [42,43]. Lipocalins associa ed wi h ep oduc ion include
p oges ogen-associa ed endome ial p o ein (glycodelin, p egnancy-associa ed endome ial α2-globulin,
placen al p o ein 14) sec e ed by he human endome ium om mid-lu eal phase o he mens ual cycle
and du ing he i s imes e o p egnancy; a sali a y lipocalin ound in pigs is sec e ed in o hei u e i
a he ime o implan a ion [44]; and equine u e ocalin is sec e ed by he endome ium o ma es in he
p e-placen a ion pe iod, du ing which he equine concep us is con ined wi hin a glycop o ein capsule
[13,14]. Despi e all hese examples o lipocalins ound in ep oduc i e issues in o he species, lipocalin-1
was he only one ound in ou Eu opean poleca samples.
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The biochemical p ope ies o lipocalin-1 a e known only om he human o m, which binds small
lipids such as a y acids and s e ols [40]. The p o ein may he e o e be in ol ed in deli e ing o
sca enging lipids a a ime when he e is signi ican issue eo ganiza ion and po en ial o cellula
dis up ion a implan a ion, as is specula ed o he ole o po cine sali a y lipocalin [8]. Lipocalin-1,
like lipocalin-2, is known o bind bac e ial side opho es, so i may unc ion as pa o he inna e immune
sys em in an an i-bac e ial ole [40,45]. Human lipocalin-1 also has been eco ded as ha ing cys einyl
p o einase inhibi o y ac i i y [46], and nuclease ac i i y ha can ope a e agains i al genomes [47,48].
The poleca p o ein may he e o e ha e mul iple p o ec i e oles in he u e us, as is hough o i s
unc ion in ea s and epi helia in humans [40,49].
Taken oge he , hese obse a ions sugges ha lipocalin-1 may wo k alongside α2M o p o ec he
u e ine en i onmen agains in ec ions, and i s lipid-binding may in ol e seques a ion o oxic lipid
pe oxida ion p oduc s c ea ed unde condi ion o oxida i e s ess [50].
4.3. Tissue-p o ec i e and emb yo suppo p o eins
In addi ion o α-2M and lipocalin-1, we iden i ied se e al addi ional p o eins ha may p o ec he
u e us agains in ec ion and issue damage associa ed wi h implan a ion. These include he i on-binding
p o eins lac o e in (lac o ans e in) and ans e in (se o ans e in), bo h o which we e p esen a
ai ly cons an le els h oughou he sampling pe iod. Lac o e in in pa icula is associa ed wi h
an imic obial ac i i y by seques e ing i on in milk, i is also syn hesized and s o ed in neu ophil
g anules [51], and i di ec ly a acks bac e ial memb anes and has an i- i al ac i i y [52–56].
α1-an i ypsin was also p esen h oughou , and inc eased in concen a ion sligh ly wi h ime. This
is an inhibi o o se ine p o einases, is a majo acu e-phase eac an in humans, and is inc easingly
ecognized as an an i-in lamma o y media o [57]. I s p ima y unc ion appea s o be p o ec ion agains
excessi e p o eolysis o issues (e.g. in he lowe espi a o y ac ) by neu ophil elas ase in in lamma ion
[58,59]. I has been obse ed in p egnan u e i o o he species, possibly o con ol p o einases
eleased du ing issue emodelling, ophoblas in asion, apop osis o in lamma o y p ocesses ha may
accompany placen a ion [60,61].
O he p o eins inc eased wi h ime. Ca hepsin L1, a cys einyl p o einase, is usually con ined o
lysosomes and is in ol ed in MHC class II an igen p ocessing. I s exp ession le el in u e ine issues
is p oges e one-dependen , and i s impo ance in placen a ion has been ecognized [60,62,63]. O he
p oges e one-dependen p o eins ha appea ed in he poleca u e ine luid included a membe o he
ec onucleo ide py ophospha ase amily (a lysophospholipase ha may be in ol ed in he p oduc ion
o pha macologically ac i e lipid media o s), and u e o e in (p egnancy-associa ed acid phospha ase,
which appea s o unc ion in ansplacen al i on anspo and s imula ion o e y h opoeisis) [16,64–70].
Cu iously, we did no ind any dedica ed nu ien ca ie p o ein simila o equine u e ocalin ha
migh ac o suppo a de eloping concep us du ing a p e-placen a ion pe iod. Equine u e ocalin
may, howe e , be a special adap a ion in equids o p o ide esou ces o a la ge, capsule-enclosed
concep us du ing a p olonged p e-placen a ion pe iod. No hing like i has been ound in non-equids.
Mus elid blas ocys s do ha e ex ended placen a ion pe iods, and hey can e en dis end he u e us
be o e hey implan , bu hey ha e no glycop o ein capsule in e ening be ween hei su aces and he
endome ial su ace [6]. Mo eo e , he su ace o olume a ios o mus elid concep uses a e subs an ially
smalle han in equids a maximum g ow h such ha hey may no need a specialized nu ien ca ie .
Equine u e ocalin and lipocalin-1 bind a simila se o small lipids [15,40], bu he o me is a ypically
en iched in essen ial amino acids [15], a ea u e ha is no ue o lipocalin-1. We did, howe e , ind
o he lipid ca ie s ha may supply he poleca concep us wi h lipids, such as se um albumin and
apolipop o ein A-I.
4.4. Conclusion
The p o ein sec e o y esponse o he p e-implan a ion phase o p egnancy ha we obse ed in Eu opean
poleca s appea s p ima ily o p e en in ec ion, acili a e implan a ion and p o ec agains elease o
dele e ious cellula componen s associa ed wi h he issue auma o implan a ion. Some o he lipid-
binding p o eins we ound may also be in ol ed in ma e nal:emb yo communica ion by anspo ing
insoluble signalling molecules such as p oges e one, p os aglandins and leuko ienes, o hei p ecu so s
(as pos ula ed o equine u e ocalin; [13,15]). I has ecen ly been a gued ha emb yonic diapause is
phylogene ically conse ed and no seconda ily acqui ed by emb yos o diapausing species [2]. I is
he e o e concei able ha a common ances o o mus elids ha includes Eu opean poleca s engaged in