MÁSTER INTERUNIVERSITARIO EN MEJORA GENÉTICA
ANIMAL Y BIOTECNOLOGÍA DE LA REPRODUCCIÓN
Telome e Dynamics du ing Bo ine
P eimplan a ion De elopmen
Tesis de Más e
Valencia, Julio 2016
Da id Albe o Ma ínez Co ona
Di ec o es:
Pablo Be mejo Ál a ez
Al onso Gu ié ez Adán
No hing in Biology makes sense excep in he ligh o e olu ion
Theodosius Dobzhansky, 1972
G acias a odos los que hicie on posible la ealización de es e documen o.
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A quienes c ea on las bases cien í icas y ecnológicas sob e las que se cons uyó es e abajo.
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1
CONTENTS
SUMMARY ...................................................................................................................................................... 2
RESUMEN ................................................................................................................................................... 3
INTRODUCTION ................................................................................................................................................ 4
Telome es Role in P o ec ing he Ch omosome Ends, an E olu iona y Pe spec i e .......................... 4
The end eplica ion p oblem and cellula senescence ......................................................................... 5
Mechanisms o p e en elome e sho ening ...................................................................................... 7
Role o elome es in aging and disease ................................................................................................. 9
Telome e main enance ac oss gene a ions ........................................................................................ 10
Telome e leng hening du ing p eimplan a ion de elopmen ........................................................... 11
Ra ionale o he s udy .......................................................................................................................... 12
Objec i es ............................................................................................................................................. 14
MATERIALS AND METHODS .............................................................................................................................. 15
In i o P oduc ion (IVP) o Bo ine Emb yos....................................................................................... 15
Oocy e eco e y and in i o ma u a ion (IVM) ............................................................................. 15
In i o e iliza ion (IVF) ............................................................................................................... 16
In i o Cul u e (IVC) ........................................................................................................................ 16
A e age Rela i e Telome e Leng h Quan i ica ion ............................................................................. 16
TERT O e exp ession .............................................................................. ¡E o ! Ma cado no de inido.
Gene a ion o plasmids o mouse Te and EGFP exp ession in mammalian cells ...................... 18
Mic oinjec ion .................................................................................................................................. 19
S a is ical analysis ................................................................................................................................ 19
RESULTS AND DISCUSSION ................................................................................................................................ 20
Objec i e 1: Telome e leng h dynamics du ing bo ine p eimplan a ion de elopmen .................. 20
Objec i e 2. Analysis o elome e leng h in emb yos injec ed wi h a plasmid exp essing mTe ... 22
CONCLUSIONS ............................................................................................................................................... 23
REFERENCES .................................................................................................................................................. 24
2
SUMMARY
Telome es a e dynamic s uc u es ha p o ec he ends o linea euka yo ic ch omosomes om abe an
usions o om being misiden i ied as DNA b eaks, playing an impo an ole in de elopmen ,
di e en ia ion, senescence and heal h. Al hough linea DNA sho ens a each cell di ision due o
incomple e eplica ion o he 5’-end, elome es ha e wo mechanisms o main ain hei leng h: a
specialized enzyme called elome ase and he Al e na i e Leng hening o Telome es (ALT) mechanism,
based on homologous ecombina ion. In complex o ganisms like mammals, bo h mechanisms a e u ned
o in soma ic cells a e he p eimplan a ion de elopmen , leading o a p og ammed cellula
senescence, whe eas immo al cells such as s em cells and mos cance cells possess mechanisms o
main ain long elome es. Sho elome e leng h (TL) in newbo ns has been ela ed wi h he appea ance
o ce ain diso de s such as Dyske a osis congeni al, and elome e leng h has been p oposed o be ese
du ing p eimplan a ion de elopmen . Telome e leng hening dynamics and mechanisms du ing
p eimplan a ion de elopmen ha e been widely s udied in he labo a o y mouse. Howe e , labo a o y
mice con ain longe elome es han o he mammals, including wild mouse, bo ine and humans, and hus
he e may be also di e ences in elome e leng hening dynamics du ing p eimplan a ion de elopmen .
The objec i es o his Mas e Thesis ha e been o de e mine he dynamics o elome e leng h du ing
bo ine p eimplan a ion de elopmen and o es he e ec o he injec ion o a plasmid exp essing
mouse Te in bo ine zygo es on he elome e leng h o he blas ocys . Telome e leng h was analyzed by
qPCR a di e en s ages (oocy es, zygo es, 2-cell, mo ula and blas ocys ) obse ing ha a signi ican
inc ease occu ed a e emb yonic genome ac i a ion, doubling i s leng h a he mo ula s age and ending
a he blas ocys s age wi h a elome es 10 imes longe han hose o he oocy e. Howe e , he inc ease
in elome e leng h obse ed a he 2-cell s age in mouse emb yos was no no ed in bo ine emb yos,
sugges ing species-speci ic di e ences in he mechanisms in ol ed in elome e leng hening a hese
de elopmen al s ages. Telome e leng h o he blas ocys s de i ed om bo ine zygo es injec ed wi h a
plasmid encoding o mouse Te did no di e om ha o blas ocys s ob ained a e injec ion wi h a
con ol plasmid exp essing EGFP. This esul sugges ha mouse Te was no able o u he elonga e
elome es a he blas ocys s age because ei he i is no be able o o m he elome ase complex wi h
bo ine componen s, o elome e leng h is igh ly egula ed du ing p eimplan a ion de elopmen o
each a maximum leng h ha canno be exceeded by exogenous exp ession o elome ase componen s.
3
RESUMEN
Los elóme os son es uc u as dinámicas que p o egen los ex emos de los c omosomas lineales
euca ion es de usiona se en e sí o de se con undidos como o u as en el ADN, jugando un papel
impo an e en el desa ollo, di e enciación, senescencia y en la salud. A pesa de que el DNA lineal su e
aco amien o en cada di isión celula debido a la eplicación incomple a del ex emo 5’, los elóme os
ienen dos mecanismos de elongación: una enzima especializada llamada elome asa, y el mecanismo
de Elongación Al e na i a de los Telóme os (EAT), basado en ecombinación homóloga. En o ganismos
complejos como los mamí e os, ambos mecanismos no son uncionales en células somá icas después del
desa ollo p eimplan acional, de i ando en una senescencia celula p og amada, mien as que las
células inmo ales como las células oncales y la mayo ía de las células cance ígenas cons an de
mecanismos pa a man ene elóme os la gos. Se han elacionado longi udes elomé icas co as en
ecién nacidos con la apa ición de cie as en e medades como la disque a osis congéni a, y se ha
p opues o que la longi ud elomé ica se es ablece du an e el desa ollo p eimplan acional. La dinámica
de elongación elomé ica du an e el desa ollo p eimplan acional se ha es udiado ampliamen e en el
a ón de labo a o io. Sin emba go el a ón de labo a o io con iene elóme os más la gos que o os
mamí e os, incluyendo el a ón sal aje, bo inos y humanos, y po ello ambién puede habe di e encias
en la dinámica de elongación elomé ica du an e el desa ollo p eimplan acional. Los obje i os de es a
Tesis de Más e han sido ca ac e iza la dinámica de la longi ud elomé ica du an e el desa ollo
p eimplan acional bo ino y de e mina el e ec o de la mic oinyección de un plásmido de exp esión pa a
la elome asa ansc ip asa in e sa (Te ) de a ón en cigo os bo inos sob e la longi ud elomé ica en el
blas ocis o. La LT se analizó po qPCR en dis in as e apas (o oci os, cigo os, emb iones de 2 células,
mó ulas y blas ocis os), obse ando un aumen o signi ica i o pos e io a la ac i ación del genoma
emb iona io, doblando la LT en el es adio de mó ula y acabando en el es adio de blas ocis o con
elóme os 10 eces más la gos que los del o oci o. Sin emba go, no se obse ó un inc emen o en la
longi ud elomé ica en el es adio de dos células desc i o en a ones, sugi iendo que exis en di e encias
en e especies en los mecanismos implicados en la elongación elomé ica du an e es as e apas de
desa ollo. La longi ud elomé ica de blas ocis os de i ados de cigo os bo inos inyec ados con un
plásmido codi ican e pa a Te de a ón ue simila a la ob enida en blas ocis os de i ados de cigo os
bo inos inyec ados con un plásmido con ol que exp esa EGFP. Es e esul ado sugie e que Te de a ón
no es capaz de aumen a la longi ud elomé ica en el es adio de blas ocis o po que o no puede o ma
el complejo de la elome asa con los componen es bo inos o po que la longi ud elomé ica es á
es echamen e con olada du an e el desa ollo p eimplan acional pa a alcanza un máximo que no
puede supe a se median e la exp esión exógena de componen es de la elome asa.

4
INTRODUCTION
Telome es a e he na u al ends o euka yo ic ch omosomes, whe e hey e ol e o sol e wo majo
p oblems: 1) a p o ec ion p oblem, a oiding ch omosome ends o be ecognized as double-s and b eaks
(DSB) and 2) a eplica ion p oblem, a oiding he p og essi e sho ening o ch omosomes due o he end-
eplica ion p oblem.
Telome es Role in P o ec ing he Ch omosome Ends, an
E olu iona y Pe spec i e
While p oka yo es ha e ci cula DNA, he la e de eloped euka yo es ha e linea ch omosomes, a
ea u e ha aced some cons ains in he p oka yo ic en i onmen . In an en i onmen whe e ci cula
DNA was ubiqui ous, he i s p oblem ha linea ch omosomes aced is being misiden i ied as DNA-
damage and ale he DNA- epai machine y. In mos o ganisms, double s and b eaks (DSB) ac i a e
he ATM-kinase pa hway. When his happens, cell cycle may be a es ed un il he DSB is epai ed, ei he
by Non-Homologous End Joining (NHEJ) o by homology-di ec ed epai (HDR), leading o end- o-end
usions o igge ing cell dead. In o de o a oid his p oblem, euka yo es de eloped elome es,
specialized s uc u es loca ed a hei ch omosome ends ha a oid hem o be ecognized as DSB,
he eby allowing a s able linea iza ion o he ch omosome and hei e ec i e ansmission h ough
gene a ions.
The wo d “ elome e” ( om he G eek τελος “end” and μερος “pa ”) was coined by He mann Mulle o
desc ibe he ends o euka yo ic ch omosomes, ha appea ed o ha e dis inc i e p ope ies no p esen
in he es o he ch omosome. Ba ba a McClin ock also obse ed ha ch omosome ends ha e special
ea u es ha p e en hem om using oge he . In pa icula , she used X- ays and mechanical o ces o
induce ch omosomal b eaks and no ed ha b oken ends ended o use one ano he , 2-by-2 o o o m
ci cula ch omosomes, whe eas elome es we e ne e in ol ed in hose usions (McClin ock 1938;
McClin ock 1941). Subsequen expe imen s ca ied ou by Jack Szos ack and Liz Blackbu n showed ha
elome es was a highly conse ed sys em ac oss euka yo es. These esea che s linea ized yeas ci cula
plasmids and inse ed elome es om he ibosomal DNA ( DNA) o Te ahymena py i o mis, a cilia ed
p o ozoan, in he b oken ends. The ans e ed elome es augmen ed, sugges ing ha bo h o ganisms
sha e a common elome e main enance sys em ha included a leng hening mechanism (Szos ak &
Blackbu n 1982).
The s abili y o linea ch omosomes is gi en by he mos basic componen o elome es, he andem
a ay o epe i i e elemen s. I has been p oposed ha du ing euka yo ic e olu ion, he a chaeal genome
could ha e expe ienced a massi e in asion o G oup II in ons, coming om he endosymbio ic
phagocy osis o α-p o eobac e ial cells (Ma in & Koonin 2006). A e he accumula ion o enough sho
sequences in a ci cula ch omosome, a DBS in one o hose epea s is mo e likely o occu . In ha
si ua ion, he DNA epai machine y would ei he liga e he b oken ends by NHEJ o ec ui he HDR
5
sys em. In he second case, he use o o he G oup II in on om he same ch omosome would lead o
a s and in asion, o ming a e minal loop ha no longe can be ecognized as a DSB, as shown in Figu e
1. This s uc u e, known as a elome ic loop ( -Loop), could ha e a iable size and sequence composi ion,
bu allowed a linea ch omosome o be s able (De Lange 2015).
Fig. 1. Linea ch omosome s abilized by a -Loop, media ed by HDR ac o s a e a DBS in a ci cula
ch omosome. S and-in asion be ween homologous epe i ions hides ch omosomal ends om NHEJ o
DNA-damage machine y.
As seen by elec on mic oscopy, he complemen a i y o he elome ic sequence no only allows o he
o ma ion o seconda y s uc u es like -Loops bu also o he con igu a ions like c uci o m o ma ion
ha can hide ch omosomal ends om being misiden i ied as DSB (Ka e e al. 1976).
The end eplica ion p oblem and cellula senescence
Being ecognized as DSB is no he only p oblem a linea DNA aced in he p oka yo ic en i onmen . The
second cons ain ha linea DNA con on ed was he incomple e eplica ion by DNA-polyme ases as
hese enzymes, e ol ed in he p oka yo ic en i onmen o eplica e ci cula DNA, we e unable o
eplica e he e y end o he linea ch omosomes. Russian biologis Alexey Olo niko was he i s o
no e ha a sho ening would occu in linea DNA i only known eplica ion mechanisms we e in ol ed.
DNA-Polyme ases e ol ed in he con ex o ci cula DNA, using a sho RNA p ime wi h a ee 3’-end o
ini ia e eplica ion. A e inishing he linea copy, he emo al o he e minal p ime a he 5’-end lea es
a gap ha canno be illed in. I le un ixed, his gap leads o a sho ening o he molecule a hei e mini.
In ch omosomes, sho ening would happen a a p edic able cons an a e pe cell cycle, depending on
he size o he missing empla e (Olo niko 1973; Olo niko 1996). This sho ening p o ided a molecula
explana ion o cellula senescence, he so called Hay lick limi , which was disco e ed a decade ea lie .
6
Figu e 2. The end eplica ion p oblem. Incomple e eplica ion a he 5’-end o a linea DNA molecule
a e RNA p ime s emo al.
Cellula senescence was ini ially con i med by he disco e y o he Hay lick limi . Be o e his disco e y,
se e al expe imen s claimed ha cell cul u es could be main ained inde ini ely in i o, ne e s opping
doubling hei popula ion, ega dless o hei na u e. In his con ex , when a cell cul u e was no longe
iable, he common explana ion used o be he lack o p ope manipula ion and knowledge on cell
biology du ing in i o cul u es, and he eby he gene al consensus was ha senescence was an a ibu e
o he body as a whole a he han a cellula phenomenon. This pa adigm was shi ed by Leona d Hay lick
who was es ing whe he human cance s ha e a i al e iology. In o de o do so, he es ablished wo
human cell lines, one de i ed om umo ous issue and o he om e us, obse ing ha cance cells
su i ed inde ini e popula ion doublings, whe eas no mal e al cells we e unable o sus ain a cul u e
inde ini ely. No mal cells appea ed o ha e an in e nal eplica ion coun down ha a oided hem o
eplica e beyond passage ~ 50. This coun down, which Hay lick called he eplicome e , s opped i he
cul u e was ozen-s o ed and con inued a e hawing and e en le him p edic he e en ual dead o
speci ic cul u es (Hay lick & Moo head 1961; Ra an 2000).
Hay lick limi was pa ially explained by he end eplica ion p oblem, whe e elome e leng h could be
he “ eplicome e ”, i.e. he indica o o he numbe o di ision a cell could sus ain be o e senescence. In
ag eemen , elome e leng h dec eased wi h cumula i e popula ion doublings o ib oblas in i o, as
p edic ed by Olo niko (Ha ley e al. 1990). In his pe spec i e, he ac ha cance cells could di ide
inde ini ely sugges ed he exis ence o a mechanism o main enance o de-no o syn hesis o elome ic
DNA, p esen in cance cells bu no in soma ic cells (Olo niko 1973). On he same p emises, such
mechanism should be p esen in he ge m line o du ing emb yogenesis in o de o a oid elome e
a i ion be ween gene a ions.
7
Mechanisms o p e en elome e sho ening
Telome es can main ain o inc ease hei leng h h ough wo mechanisms: 1) elome ase, he main
esponsible o elome e leng hening in mammalian cells and 2) al e na i e leng hening o elome es
(ALT).
I has been p oposed ha he i s mechanism appea ing in e olu ion o compensa ed elome e
sho ening he -Loop, which, as al eady explained, also plays a c ucial ole in p e en ing he elome es
o be ecognized as a DSB. The s and in asion can ac as a eplica ion o k and use he eplica i e
machine y al eady ound in p oka yo es. The -Loop mechanism is based in homologous ecombina ion,
and cons i u es he simples e sion o al e na i e leng hening o elome es (ALT). The ch omosomes
wi h -Loops can su i e and hide om he DNA- epai machine y Fu he mo e, hey can use he DNA-
eplica ion machine y o inc ease hei leng h (De Lange 2015)(De Lange 2015). In his sense, mo e ALT
mechanisms ha e been ound ac oss species (Lundblad & Blackbu n 1993), wi h he common
cha ac e is ic o being media ed by homologous ecombina ion.
Figu e 3. The end eplica ion p oblem can be sol ed by he s and-in asion o he 3’ o e hang in he -
loop, p o iding a empla e o elome e leng hening.
In con as o ALT mechanism, which can be conside ed as an adap a ion o homologous ecombina ion
mechanisms, elome ase is a highly speci ic mechanism speci ically de eloped o main ain o inc ease
elome e leng h. The exis ence o such mechanism was p edic ed based on he e idence ha elome es
we e o med by he same hexame in a high numbe o andem epea s in he cilia ed p o ozoan
Te ahymena Py i o mis (Ka e e al. 1976), o he p o ozoans (Yao e al. 1981) as well as o he
euka yo es. La e , Ca ol G eide and Liz Blackbu n ound an enzyme specialized on de no o syn hesis o
DNA a he end o linea ch omosomes, he elome ase (G eide & Blackbu n 1985).
Telome ase is a ibonucleop o ein unc ionally composed mainly by wo subuni s, he elome ase
Re e se T ansc ip ase (TERT) and he Telome ase RNA Componen (TERC). TERT and TERC o m a igh
14
Objec i es
The main objec i e o his hesis has been o de e mine elome e dynamics du ing p eimplan a ion
bo ine de elopmen by pu suing he ollowing speci ic objec i es:
1) To de e mine by qPCR he dynamics o elome e leng hening du ing p eimplan a ion
de elopmen a he ma u e oocy e, zygo e, 2-cell, 8-cell, mo ula and blas ocys s ages.
2) Analyze he e ec s o TERT o e exp ession by mic oinjec ion o an exp ession plasmid a
he zygo e s age on he elome e leng h o bo ine blas ocys s.

15
MATERIALS AND METHODS
In i o P oduc ion (IVP) o Bo ine Emb yos
The bo ine in i o p oduc ion p o ocol consis s in 3 di e en ia ed s eps: 1) oocy e eco e y and in
i o ma u a ion, 2) in i o e iliza ion and 3) in i o cul u e.
OOCYTE RECOVERY AND IN VITRO MATURATION (IVM)
Bo ine o a ies a e collec ed om hei e s and cows a a local slaugh e house, immedia ely a e emo al
o he in e nal o gans, in he aba oi chain. The o a ies a e anspo ed inside a plas ic bag con aining
0.9% s e ile saline solu ion supplemen ed wi h 1% gen amycin, inside a he mos bo le wi h wa e a
empe a u es anging 34 ºC o 38 ºC. Once in he lab, o a ies a e washed once in wa e a 37 ºC and
wice in saline a 37 ºC and kep in a lask con aining saline in a wa e ba h a 37-38ºC, while wai ing o
be p ocessed. Cumulus-oocy e complexes (COCs) eco e y is pe o med by aspi a ion o 2-8 mm ollicles
wi h an 18 gauge s e ile needle a ached o a 5 ml sy inge, keeping a cons an nega i e p essu e.
Follicula liquid p esump i ely con aining he COCs is gen ly pou ed o a 50 ml s e ile lask ha is kep in
a wa e ba h a 37-38 ºC un il all he o a ies o he ba ch a e p ocessed. Ten minu es a e pou ing he
las con en in o he lask, he supe na an is emo ed using a s e ile disposable Pas eu pipe e inside a
lamina - low cabine . The p ecipi a e, con aining he COCs and ollicula deb is and a minimum ollicula
liquid, is esuspended wi h PBS a 39 ºC and pou ed in s e ile pe i dishes. Unde a s e eoscope, COCs
a e eco e ed and selec ed based on he ollowing c i e ia:
Type
Cha ac e is ics
In i o p oduc ion
sui abili y
1
Compac mul ilaye ed cumulus in es men ; homogeneous
ooplasm; ligh and anspa en
Yes
2
Compac , bu less han 4 cumulus cells laye . Da ke zone a he
oocy e pe iphe y
Yes
3
Denuded oocy e
No
4
Expanded cumulus in es men , degene a ed pykno ic cy oplasm
No
Table 1. Mo phological c i e ia o bo ine cumulus-oocy e complexes (COCs) selec ion o in i o
p oduc ion (IVP)
16
Only g ade 1 and 2 COCs a e washed wice in PBS and once in in i o ma u a ion media be o e being
placed in g oups o ~50 pe well in 4-well NUNC® dishes, each well con aining 500 μl o in i o ma u a ion
media. COCs a e allowed o ma u e o 24 hou s a 39 ºC in a humidi y sa u a ed 5% CO2 a mosphe e.
IN VITRO FERTILIZATION (IVF)
A e 24 hou s o IVM, ma u ed oocy es a e washed wice in empe ed PBS and once wi h he in i o
e iliza ion (IVF) medium, and ans e ed o a s e ile 4 well NUNC pla e wi h 250 μl IVF medium. F ozen
semen om a p o en high e ili y bull is p ocessed wi h Bo iPu e®, acco ding o he p o ocol p o ided
by Nidacon, in o de o disca d dead spe ma ozoa. B ie ly, a wo laye g adien o Bo iPu e® is made by
adding a 40% concen a ion laye o e an 80 % concen a ion laye in a 15 ml s e ile plas ic ube. A semen
s aw, s o ed in liquid ni ogen is hawed in a 37 ºC wa e ba h, pou ed gen ly a he e y op o he
g adien and cen i uged o 10 minu es a 1000 g. Ca e ully and keeping he pelle in ac , he
supe na an is ex ac ed and he emaining pelle is washed wi h 1 ml Bo iWash® and cen i uged o 5
mo e minu es. Ca e ully, he supe na an is emo ed un il 300 μl a e le in he ube. The pelle is
homogenized and 5 μl a e ans e ed o an Eppendo ube con aining 95 μl dis illed wa e o de e mine
spe ma ozoa concen a ion. In o de o ha e a inal concen a ion o spe ma ozoa o 1 million spz/ml,
he pu i ied spe ma ozoa a e dilu ed wi h in i o e iliza ion medium o 2 million spz/ml and 250 μl a e
added o he well con aining he ma u ed COCs. Fe iliza ion akes place a 39 ºC in a wa e sa u a ed
a mosphe e o 5 % CO2.
IN VITRO CULTURE (IVC)
P esump i e zygo es a e washed once in PBS and once in IVC medium (HSOF + 5 % Fe al Cal Se um (FCS))
a e cumulus cells emo al by o ex in PBS. G oups o 25 p esump i e zygo es a e ans e ed in o 25
μl cul u e medium mic o d ople s unde a 3 ml laye o mine al oil (NidOil®) in 35 mm cell cul u e dishes
(Co ning), and kep in incuba o a 39 ºC, 5% 02, 5% CO2 and maximum humidi y o 8 days, wi h
de elopmen e alua ion a 2, 6, 7 and 8 days pos - e iliza ion.
A e age Rela i e Telome e Leng h Quan i ica ion
Fo he i s objec i e, bo ine emb yos we e p oduced in i o as p e iously desc ibed and elome e
leng h was analyzed in 20 samples pe s age: ma u ed oocy es (8/sample, collec ed a e IVM), zygo es
(collec ed a 18 hou s pos insemina ion –hpi-, 8/sample), 2-cell emb yos (collec ed a 32-34 hpi,
4/sample), mo ulae (collec ed a 125 hpi, 1/sample) and blas ocys s (collec ed a 200 hpi, 1/sample).
Zona pellucida was emo ed by incuba ing he emb yos in a 0.5 % p onase solu ion in PBS in o de o
imp o e subsequen emb yo diges ion and o elimina e spe ma ozoa bound o he zona pellucida.
Immedia ely a e zona pellucida emo al, emb yos we e s o ed in PCR ubes and ozen a -80 ºC un il
sample analysis. Samples we e diges ed in 8 µl o a 100 µl/ml p o einase K bu e ed solu ion o 1 h a
65 ºC and p o einase K was inac i a ed by incuba ing a 95 ºC o 10 min. A e age ela i e elome e
17
leng h was measu ed by quan i a i e eal- ime PCR, acco ding o he p o ocol epo ed in (Caw hon,
2003) wi h some mino modi ica ions (Be mejo-Al a ez e al. 2008). B ie ly, TL is de e mined by
con as ing he ampli ica ion o he elome ic sequence o he Rn18S genomic sequence, which se ed
as an in e nal con ol ela i e o he ampli ica ion o he elome es o he o al DNA p esen in he lysa e.
The speci ic sequence and he p oduc leng h o he p ime s used o ampli ica ion is de ailed in Table 2.
The qPCR was pe o med in a Ro o gene 6000 Real Time Cycle (Co be Resea ch), incuba ing o 3 min
a 94 ºC, ollowed by 40 cycles o 10 sec a 94 ºC, 30 sec a 60 ºC and 30 sec a 72 ºC. Quan i ica ion was
pe o med by he compa a i e C Me hod (Schmi gen & Li ak 2008): Fluo escence was acqui ed in each
cycle o de e mine he h eshold cycle o he cycle du ing he log-linea phase o he eac ion a which
luo escence inc eased abo e backg ound o each sample. Wi hin his egion o he ampli ica ion cu e,
a di e ence o one cycle is equi alen o doubling o he ampli ied PCR p oduc . Acco ding o he
compa a i e CT me hod, he ΔC alue was de e mined by sub ac ing he Rn18S C alue o he
Telome e CT alue o each sample. Fo ep esen a ion pu poses, ΔΔC was calcula ed by no malizing
each C alue o he highes C obse ed alue (i.e. he lowes ela i e elome e leng h). Fold changes in
he ela i e elome e leng h we e de e mined using he o mula 2-ΔΔC .
Gene
P ime sequence (5´-3´)
F agmen
Size, bp
Gene Bank
Accession No.
Telome e
F:CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
R:GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT
79
NT_039202.7
Rn18s
F:AGAAACGGCTACCACATCCAA
R:CCTGTATTGTTATTTTTCGTCACTACCT
91
NR_003278.1
Mouse
Te
F: GGATTGCCACTGGCTCCG
R: TGCCTGACCTCCTCTTGTGAC
279
NM_009354.1
Table 2: De ails o p ime s used o elome e leng h de e mina ion and plasmid injec ion e i ica ion
18
TERT O e exp ession
GENERATION OF PLASMIDS FOR MOUSE TERT AND EGFP EXPRESSION IN
MAMMALIAN CELLS
Fo he second objec i e, a plasmid o exogenous TERT exp ession was gene a ed by cloning he
comple e mouse Te cDNA sequence in o he EcoRI si e o he pCAGGs ec o (Hi oshi e al. 1991). The
plasmid con ains he CAG p omo e , a s ong syn he ic p omo e widely used in mammals. The CAG
p omo e con ains he cy omegalo i us (CMV) ea ly enhance elemen (C), he p omo e , i s exon and
i s in on o he chicken β-ac in gene (A) and he abbi β-globin (G) splice accep o . As a mic oinjec ion
con ol, a comme cial plasmid (pEGFP, Clon ech) ha codes o he enhanced g een luo escen p o ein
(EGFP) was also used. Plasmids we e mul iplied by ans o ming DH5α compe en bac e ia. B ie ly, ~50
ng o plasmid-con aining solu ion is added an aliquo o compe en E. coli p e iously hawed in ice o
i e minu es. A e 30 min incuba ion in ice, a 30 seconds 42 ºC hea -shock ollows. Bac e ia is allowed
o eco e in ice o 5 min and 500 µl SOC media is added be o e 1 hou 37 ºC shaking incuba ion. The
aliquo is cen i uged a 800 g o 7 min, bac e ia a e pla ed in o selec i e (ampicillin) LB aga pla es and
incuba ed o e nigh a 37 ºC. In o de o eco e he plasmids, a MiniP ep was pe o med using he
Fa o P ep™ Plasmid Ex ac ion Mini Ki , acco dingly o he manu ac u e ’s ins uc ions (FAVORGEN).
Plasmids we e pu i ied by phenol-chlo o o m ex ac ion ollowed by isop opanol p ecipi a ion.
Figu e 5. Schema ic ep esen a ion o pCAGGsmTe .
19
MICROINJECTION
In o de o achie e a TERT o e exp ession be o e emb yonic genome ac i a ion, we mic oinjec ed he
CAG TERT plasmid in bo ine zygo es. As a con ol, o he g oup was mic oinjec ed wi h pEGFP, a
comme cial plasmid ha codes o he enhanced g een luo escen p o ein (EGFP). P io o
mic oinjec ion, he holding pipe e and mic oinjec ion pipe es need o be c a ed. The holding pipe es
we e made om TW100-6, hin-wall capilla ies, 0.6”” 1.0 mm (Wo ld P ecision Ins umen s) in a P-97
Pipe e Pulle (Su e Ins umen CO.) unde a p og am wi h he ollowing se ings: Hea 800, Pull 75,
Veloci y 130, Time 100. The pipe e was b oken by hea and i e-polished in a mic o o ge (Mic o o ge de
Fon b une BEAUDOIN 5262) o ob ain an in e nal diame e o a ound 50 µm. The mic oinjec ion pipe es
we e made om TW100F-4 Glass hin 1.0 mm 4 inches wi h an in e nal ilamen (Wo ld P ecision
Ins umen s) in he same Pipe e Pulle , bu wi h di e en se ings (Hea 775, Pull 150, Veloci y 90, and
Time 120). The wo king d op was made by placing 20 µl o PBS a he cen e o a mic oscope slide and
co e ing i by enough mine al oil.
Figu e 6. Rep esen a i e image o he mic oinjec ion se ing o cy oplasmic injec ion.
Following mic oinjec ion, emb yos we e washed in IVC medium and ans e ed in o IVC dishes o
cul u e a 39 ºC, 5% 02, 5% CO2 and maximum humidi y. Blas ocys s we e eco e ed a day 7 and
p ocessed o analyze ela i e elome e leng h as p e iously desc ibed o objec i e 1. P ope deli e y o
he plasmid was analyzed in wel e blas ocys ob ained a e zygo e injec ion o pEGFP and 9 blas ocys s
ob ained a e injec ion o pCAGGsmTe . Emb yos injec ed wi h EGFP we e es ed posi i e by de ec ing
EGFP by epi luo escence mic oscope. Emb yo injec ed wi h pCAGGsmTe we e es ed by a PCR speci ic
o he mTe sequence ollowing he diges ion p o ocol p e iously desc ibed o elome e leng h
analysis. Telome e leng h was analyzed as p e iously desc ibed.
S a is ical analysis
Da a we e analyzed using he SigmaS a (Jandel Scien i ic, San Ra ael, CA, USA) so wa e package. One-
way analysis o a iance (ANOVA) was pe o med o analyze di e ences be ween g oups.

20
RESULTS AND DISCUSSION
Objec i e 1:
Telome e leng h dynamics du ing bo ine p eimplan a ion
de elopmen
Rela i e elome e leng h was analyzed be o e e iliza ion (oocy es), be ween e iliza ion and
emb yonic genome ac i a ion –EGA- (zygo es and 2-cell emb yos) and a e EGA (mo ulae and
blas ocys s), Fig. 6. Oocy es con ained he sho es elome es o all he s ages analyzed (1±0.15,
mean±s anda d e o o he mean). A e e iliza ion elome es seem o be longe han in oocy es
(1.4±0.17, meaning ~1.4 imes longe han hose o he oocy es based on 2-ΔΔC ), bu his inc ease was
no signi ican ly di e en . Two cell emb yos con ained simila elome es o hose o oocy es o zygo es
(1.13±0.1). A e EGA, a s a is ically signi ican inc ease in elome e leng h was obse ed a he mo ula
s age (2.3±0.33) ollowed by a sha pe inc ease in he mo ula o blas ocys ansc ip ion (10.37±1.37).
Figu e 7. Rela i e elome e leng h be o e e iliza ion (oocy es), be ween e iliza ion and EGA (zygo es
and 2-cell emb yos) and a e EGA (mo ulae and blas ocys s). Di e en le e s indica e s a is ical
di e ences based on ANOVA (p<0.05).
These esul s sugges ha , in con as o he mouse model (Liu e al. 2007), he e is no a signi ican
inc ease in elome e leng h be o e EGA. Righ a e e iliza ion, a small inc ease in elome e leng h was
no iced, bu was no s a is ically signi ican . This small nume ic inc ease may be he consequence o he
21
spe ma ozoa elome es being longe han hose o he oocy es, as epo ed o he mouse model (De
F u os e al. 2016). Howe e , in he absence o s a is ical signi icance and wi hou conduc ing a s udy o
de e mine he di e ences be ween game es we canno es ha hypo hesis. The absence o an inc ease
in elome e leng h in he ansi ion om zygo e o he 2-cell emb yo sugges s ha , in con as o he
mouse model (De F u os e al. 2016; Liu e al. 2007) ALT mechanisms do no elonga e elome es be o e
EGA. A e EGA, a signi ican inc ease was no ed in he mo ula s age concluding wi h a sha p elome e
leng hening occu ing in he mo ula- o-blas ocys ansi ion, esul ing in elome es ~10 imes longe
han hose a he oocy e s age. These indings a e in ag eemen wi h p e ious indings ha obse ed a
sha p inc ease in elome ase ac i i y a he blas ocys s age in di e en mammalian species, including
bo ine (Liu e al. 2007; Schae zlein e al. 2004; W igh e al. 2001). In ag eemen wi h ou esul s,
elome e leng h de ec ed by qFISH has been obse ed o signi ican ly inc ease om he mo ula o he
blas ocys s age (Schae zlein e al. 2004) al hough he leng hening was less e iden han in ou s udy
(~14 kb in mo ula s ~20 kb in blas ocys ). The nume ic di e ences be ween s udies may be caused by
he di e en echniques used o de e mine elome e leng h. In pa icula , qFISH elies on he in ensi y
o a luo escen p obe agains he elome ic sequence and ha in ensi y does no only depend on
elome e leng h, as he con o ma ion o elome es and sub elome ic egions do a ec p obe accessibili y
and he eby luo escence in ensi y. In his pe spec i e, qFISH end o unde es ima e elome e leng h
di e ences, as an example, human spe ma ozoa elome e leng h quan i ied by qFISH (Tu ne &
Ha sho ne 2013) a e hal o hose ob ained using elome e es ic ion agmen (TRF) analysis (Bai d e
al. 2006; K. Kimu a e al. 2008; Kozik e al. 1998; Picke e al. 2011), and in mouse emb yos elome e
leng h analyzed by qFISH yielded inconsis en esul s depending on he s ain, in con as o qPCR (Liu e
al. 2007).
A ecen s udy using qPCR is he only a icle published so a analyzing elome e leng hening in bo ine
emb yos om he oocy e o he blas ocys s age (Gilch is e al. 2015). In con as o ou s udy, hey
obse ed no signi ican di e ences be ween oocy es, zygo es, 2-cell, 4-cell, 8-cell, mo ula and blas ocys
s ages, e en hough hey poin ed a nume ic inc ease om ~0.9 a bi a y uni s in he zygo e o ~2.4 in
he blas ocys s. Al hough he endency was simila o ou s udy, he lack o signi ican di e ences
be ween s ages and he highe s anda d e o s ob ained by hese au ho s may be explained by he
e e ence sequence used o con as he CT ob ained o he elome ic sequence o he amoun o DNA.
In pa icula , hese au ho s used a single copy gene (ZAR1) whose CT le els a e e y a om hose o
he Telome e sequence, which exponen ially enhance he calcula ion e o s ollowing 2-ΔΔC .
Fu he mo e, a single copy gene con ains oo ew copies in he samples analyzed con aining pools o
maximum 15 single zygo es (i.e. 30 copies in he o al lysa e) o a eliable PCR ampli ica ion. This
app oach can be used when a signi ican numbe o genomes is p esen in he sample (such as in
blas ocys s (Be mejo-Al a ez e al. 2008)), bu a mul icopy sequence should be used o ea lie s ages o
a oid hese p oblems (De F u os e al. 2016).
22
Objec i e 2.
Analysis o elome e leng h in emb yos injec ed wi h a
plasmid exp essing mTe
Mic oinjec ion o bo ine zygo es esul ed in success ul deli e y o he plasmids. Emb yos injec ed wi h
pEGFP showed g een luo escence ha was main ained un il he blas ocys s age (Fig. 8A), as expec ed,
no emb yo injec ed wi h pCAGGsmTe showed g een luo escence. The p esence o mTe sequence
was de ec ed by PCR in all 9 blas ocys de i ed om zygo es injec ed wi h pCAGGsmTe , whe eas no
mTe sequence was de ec ed in he pEGFP injec ed g oup. Howe e , elome e leng h was simila
be ween bo h g oups (Fig. 7B). This esul sugges s ha he exogenous exp ession o mouse Te does
no elonga e elome es du ing bo ine p eimplan a ion de elopmen .
Figu e 8. E ec s o mic oinjec ion o he mTERT and pEGFP exp ession plasmids on blas ocys a 200 hpi.
(A) Pho og aphs o a bo ine blas ocys mic oinjec ed wi h pEGFP in an epi luo escence mic oscope
unde whi e ligh (abo e) and epi luo escence (below). (B) Rela i e elome e leng h de e mined by qPCR
in blas ocys s de i ed om zygo es injec ed wi h ei he pEGFP o CAGGsmTERT (1 ± 0.741). The
di e ences be ween g oups we e no signi ican based on ANOVA (p>0.05).
T ans ec ion o TERT exp ession cons uc s in elome ase-nega i e cells elonga es elome es a oiding
senescence (Bodna e al. 1998; Yang e al. 1999). In con as , TERC is ubiqui ously and cons i u i ely (i.e.
non egula ed) exp essed in mos soma ic cells which a e indeed elome ase nega i e (A andi 2002; Fu
e al. 2003) s ongly sugges ing ha TERC is no a limi ing ac o o elome ase ac i i y. Howe e , in ou
s udy, he exogenous exp ession o mouse Te in bo ine emb yos did no inc ease elome e leng h.
Se e al easons may explain he absence o an e ec on elome e leng hening. Fi s , i is possible ha
mouse Te do no ecognize he endogenous bo ine Te c o o he p o eins in ol ed in he ac i e
elome ase complex. Howe e , TERT is e olu iona y conse ed ac oss species (Sandin & Rhodes 2014)
23
and he exogenous exp ession o human TERT has been e ec i e o elonga e elome es in abbi s (Xiang
e al. 2000) and bo ine (Iqbal e al. 2011). In pa icula , i has been epo ed ha he injec ion o a
plasmid encoding o human TERT in bo ine zygo es esul ed in inc eased elome ase ac i i y and
elome e leng h a he blas ocys s age (Iqbal e al. 2011). The same g oup also obse ed ha he
exogenous exp ession o human TERC alone was able o inc ease elome e leng h, al hough hey ailed
o obse e a signi ican inc ease in elome ase ac i i y (Ga els e al. 2012). The la e inding is su p ising
as TERT is supposed o be he limi ing ac o o elome ase ac i i y, being TERC ubiqui ously exp essed
(Chiang, Hemann, e al. 2004). TERT p o ein sequence and mo i s a e e y simila be ween mouse,
human and bo ine, bu sligh changes may ha e impeded o o m an ac i e elome ase complex wi h
bo ine p o eins. Ano he possibili y ha may explain he absence o di e ences is ha elome e
leng hening mechanisms du ing p eimplan a ion de elopmen may be igh ly egula ed o a each a
maximum leng h which canno be modi ied by he exogenous exp ession o elome ase componen s.
CONCLUSIONS
1) Du ing bo ine p eimplan a ion de elopmen elome es elonga es a e emb yonic genome
ac i a ion, doubling i s leng h a he mo ula s age and ending a he blas ocys s age wi h
elome es 10 imes longe han hose o he oocy e.
2) The zygo e injec ion o a plasmid encoding o mouse Te does no inc ease elome e leng h in
bo ine blas ocys s.
30
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