Tgp chi Cong ngh? Sinh hgc 12(2): 235-240, 2014
A N H H U d N G C U A N H A N T O iTC C H E B E : N H B A C H C A U N G U d l L E N SlT P H A T T R I E N C U A P H O I B O IN VITRO
Le T h a n h L o n g ' , D o i n Chinh C h u ^ g ^ D6 Minh Si*, H6 Nguyen Quynh Chi^, Hoang NghTa Scm' ' Vien Smh hgc Nhiet d&i, Vien Hdn ldm Khoa hgc vd Cong nghe Viet Nam
'Khoa Sinh hgc, Tru&ng Dgi hgc Khog hgc Tu nhien. Thdnh pho Ho Chi Minh
^Khoa Cdng nghe smh hgc, Dgi hgc Quoc Te, Dgi hgc Qudc gia Thdnh phd Ho Chi Minh N g i y nhin bii: 09.5,2014
Ngay nhan dang: 17.6.2014 TOM TAT
Trong nghien cihi nay, chung toi danh gia anh hudng ciia nhan td ire che benh bach cau ngudi (human leukemia inhibitory factor-hLlF) len qua trinh nudi cay phdi bd. Ket qua cho thay trong qua trinh phan chia ciia phoi bd tir giai doan hop tii sang din giai doan phdi 2 te bao khdng hi anh hudng bdihLlF. Trong qua trinh phan chia tir phdi 2 tebao thanh phdi 4 tebao, viec bd sung hLIF vdi ndng do 10^ lU/ml vao trong mdi trudng nuoi cky giup lam tang ti le phoi phan chia thanh phoi 4 te bao (31,08%), trong khi dd, nhdm ddi chung va nhdm bd sung hLIF 10" lU/ml cd ti Ie phan chia phdi 4 te bao ihap hon, Hieu qua cua vi?c bosung hLIF vdi ndng do iO^ lU/ml trong moi trudng nuoi cay phdi cdn the hienro qua ti lephoi 8 tS bio (28,71%) va phdi dau (18,24%). Ti l^plidi dau Irong moi trudng bo sung hLIF lO'' lU/ml cao hon nhdm ddi chirng (2,04%) vanhdm bd sung hLIF 10^ lU/ml (5,00%) Dilu do chirng td hLIF cd the giup phdi bd vugt qua giai doan block 8 te bao tdi 16 te bao. Ti le phdi nang trong mdi trudng hd sung hLIF 10^ lU/ml cao ban nhdm ddi chimg va nhdm bo sung 10^ lU/ml. Tning chin, phdi 2-4 te bao, phdi 8-16 le bao, va phdi nang deu bieu hien mRNA cua LIF Chat luong ciia phdi nang trong nhdm bo sung hLIF lO' lU/m! tdi hon nhdm ddi chiing va nhdm bo sung hLIF 10" lU/ml Cac kel qua tren cho thay hLIF cd kha nang cai thien rd ret sir phai trien cua phdi bd giai doan tien lam td.
Tu khoa: ICM (inne, phan chia cua phoi
:ell mass), nhdn to uc che benh bach cdu, phai bo. sir bieu hien gem cphie.
GIOI THIEU
Nhan td lie che benh bach cau (Leukemia inhibitory factoi-LlF) cd vai trd thiet yeu trong q u i trinh phai trien phdi giai doan tien lim td cung nhu giai doan lim td (Robb ei al, 2002; Lass et al.
200l).Qua trinh bam cua LIF vdi thu the ciia LIF (LIFr) se hinh thanh nen mgt phiic hgp cd i i luc cao vdi tieu phan gpl30. Qui trinh dimer hda niy se hoat hda cac con dudng tin hieu ndi bio lien quan tdi cac protein trong t l bio chat nhu con dudng Jak-Stat va Shp2-Erk (Niwa, 2001), Phdi bd sin xuit in vitro bilu hien LIFr va gpl30 trong tat ca cac giai doan phit trien tien lam td.Tuy nhien, phdi bd in vivo khdng bieu hien LIFr trong giai doan hinh thanh phdi nang du gpl30 van bieu hien d miic phien ma (Eckert, Niemann, 1998), Phoi bd sin xuit in vitro bieu hien Ligand ciia LIF d mdi giai doan phat trien phdi khic nhau (Wrenzycki et al. 2005), Mdt sd nghien ciru trudc day cho thiy bd sung LIF trong mdi trudng nudi ciy in vitro cd the giiip cii thien su phat trien ciia phdi
ngudi (Dunglison el al, 1996), chugt (Mitchel et al, 1994), ciru (Fry e/a/„ 1992) v i heo (Haraguchi etal.
2013). Den nay, inh hudng ciia LIF len q u i trinh phat trien cua phdi bd vin cdn dang tranh cai.Trong nghien ciiu niy, chung tdi dinh gii inh hudng ciia hLIF len qua trinh phit trien ciia phdi giai doan tien lam td, ddng thdi danh gii su bieu hien ctia LIF d mire phien ma ciing nhu chat Iugng ciia phdi thdng qua viec xac dinh sd luong te bio trong khdi ICM (inner cell mass) va trong phdi nang. Day la mdt trong nhirng chi tieu quan trgng de dinh gii chit lugng phdi nham phuc vu muc dich cay truyen phdi hay phan lip v i nudi ciy te bao gdc phdi bd.
VAT LIEU VA PHU'ONG PHAP NGHIEN C U t I Nudi chin trirng bo
Trirng bd duac thu nhan tir Id md sau do dugc chuyen ve phdng thi nghiam, Phiic hgp trirng- cumulus (Cumulus-Oocyte complex) dugc thu nhan
235
Le Thinh Long el al bing phuang phap chgc hiit. Phiic hgp tritng-
cumulus dugc nuoi trong mdi trudng TCMI99 (Sigma) bd sung 20% FBS (Gibco), 1% Pen/Strep (Gibco), 2pg/ml |3-estradioI (Sigma), lOlU FSH (Sigma) va lOIU LH (Sigma) d 3 8 . 5 V 5% COj trong vdng 22-24 gid.
Thy tinh in vitro trirng bd
Trimg sau khi dugc nudi chin se duac sii dung cho thu tmh in vitro. Tinh trung duocthu nhan bang phuong phipSwim-up trong mdi trudng TALP- HEPES (chiia 2mM sodium pyruvate, 1% pen/strep v i 3mg/ml BSA) trong I h d 38,5''C, Tning dugc thu tinh vdi tinh trung trong vi gipt thu tmh vdi mat do IxlO'^ tinh trung/ml. Q u i trinh thu tmh dugc dien ra d 38,5''C, 5% CO; hong 8 h. Sau dd tning thu tmh se dugc loai bd tmh trimg va dugc nudi cay trong mdi trudng nudi phdi Sofaa dugc bo sung hLIF tii td hgp (recombinant hLIF) (Sigma) vdi cie ndng dd 10^ va I0^IU/mld38,5''C, 5 % C 0 2 .
RT-PCR
RNA tdng duoc tach chiet bang RNeasy Protect Kit (Qiagen) Trimg chin v i phdi d cie giai doan khic nhau d moi trudng bd sung 10^ lU/ml hLIF dugc sir dung cho viec tach RNA tdng Su bieu hien mRNA cua LIF tit tning va phdi bd dugc xie dinh bang cap mdi dac hieu nhu sau: mdi xudi (5'-CAG GAG TTG TGC CCT TGC TGC TGG TTC T-3') v i mdi ngugc (5'-CAG GGA GGC GCC CAG GTA CGC GAT GAT G-3'). Thanh phin phan img RT- PCR bao gom 25 pi 2x PCRBIO One-step Mix (Biolabs), 2 pi mdi xudi (10 pM), 2 pi mdi ngugc (10 pM), 2,5 pi 20x RTase, 2 pi RNA, bd sung dHjO du 50 pi Chu ki phan iing RT-PCR dugc thuc hien nhu sau: 45°C trong 45 phut, 95''C trong 5 phut, 40 chu ki 95''C trong 30 giay, 64''C trong 30 giay, 72''C trong 40 giay, 72''C trong 10 phut. Sin pham khuech dai cd kich thudc 332 bp.
Nhuom PI (propidiura iodide)
Phdi dugc cd dinh trong dung dich PBS chiia paraformaldehyade 4% trong vdng 30 phut, sau dd dugc li vdi dung dich PBS/PVA (0,1%) chiia Tnton XIOO 0,1% qua dem, phoi dugc nia 2 lan vdi dung dich PBS/PVA (0,1%) radi lin 10 phut Sau dd phdi dugc nhugm vdi PI (30pg/ml) trong 30 phut. Sau do phdi dugc nia 2 ian trong PBS/PVA (0,1%) mdi lin 10 phiit. Phoi dugc cd dinh va quan sit trong phd cho PI. Sd lugng te bao phdi dugc xac dinh bang each quan sat dudi kinh hien vi huynh quang timg Idp.
X u ly thdng ke
So lieu thu dugc tir cac nhdm thi nghiem dugc phan tich bing "One way Anova" theo "Tukey's multiple range test". Gia tri P nhd hon 0,05 dugc dinh gia la cd y nghTa thdng ke,
KET QUA VA THAO LUAN S u p h i t tri£n cua phoi
Trong viec tao phdi bang phuong phap thu tinh trong dng nghiem, chi nhiing phdi cd chat lugng tdt nhu phdi khdng cd su phan minh te bao chat, ming zona sang, mang bao chat hen hen tuc, te b i o chit den diu khdng xuit cac diem trang mdi dugc chgn lgc cho qui trinh nudi phoi (Hinh I). Qua trinh phan minh cua phdi Ii mgt dac diem hinh thii ciia q u i trinh chet theo chuong trinh (apoptosis) v i qua trinh nay inh hudng ldn tdi su phit trien va chat luong ciia phdi cung nhu viec thiet lap cac ddng te bao gdc phdi.
Trong nghien ciiu nay, chiing tdi danh gii inh hudng ciia nhan td lie che benh bach cau ngudi (human leukemia inhibitory factor-hLIF) len su phit trien cua phdi bd, Ket q u i cho thay, ti le phdi 2 te b i o trong nhdm doi chimg (34,13%), nhdm bd sung 10^ lU/ml hLIF (34,58%) va nhdm 10^ lU/ml hLIF (34,12%) diu bang nhau (Bing I). Dieu dd chimg td k h i ning phan chia ciia te bio phdi bd khdng phu thugc vao hLIF d cac ndng do khac nhau. O giai doan phdi 4 te bio, ti Ie phdi d nhdm ddi chiing (24,11%)) va nhdm bd sung hLIF 10- lU/ml (21,67%) thip hon so vdi nhdm bd sung hLIF 10^ lU/ml (31,08%). Q giai doan phdi 8 tl bio, ti le phdi d nhdm ddi chirng (17,32%)) v i nhdm bd sung hLIF 10^ lU/ml (17,08%) thip hon ti le phdi d nhdm bd sung hLIF lOVml, Kit q u i tren cho thiy viec bd sung hLIF d ndng bg IO"* lU/ml giup lim tang ti le phdi bd phan chia tir giai doan phdi 2 t l bao sang phdi 4 te bao va tu giai doan phdi 4 te bao sang giai doan phdi 8 te bao Nhu vay, hLIF bit diu cd inh hudng tdi khi nang phin chia ciia phdi tir giai doan 4 t l bao din 8 tl bio. Trong giai doan phdi dau, kit q u i cho thiy ti Ie phdi dau d nhdm bd sung hLIF 10^ lU/tnl (18,24%) la cao nhit, v i nhdm ddi chiing (2,04%)) la thip nhit, nhdm bd sung hLIF 10^ lU/ml (5,00%) cd ti le phdi dau cao hon nhdm ddi chiing nhtmg thip hon nhdm bd sung hLIF 10^ lU/ml. Kit qui tren cho thiy, mdi trudng nudi phdi bd bd sung hLIF 10^ lU/ml lam tang ti le phdi diu ding kl. Ngoai ra, viec bd sung hLIF 10^ lU/ml cung lim tang tl le phdi dau so vdi 16 ddi chiing. Dilu dd chiing td mdi trudng nudi phdi bd sung hLIF cd kha nang lim tang ti le phoi dau, hay giiip phdi bd tang kha nang vuot qua giai doan block phdi 8 t l b i o d i n 16 t l bio. Trong giai doan phdi nang, mdi trudng bd sung
Tgp chi Cong nghe Sinh hgc 12(2): 235-240, 2014
hLIF cung cd tic ddng r6 ret din ti le phdi, ti Ie phdi le tren chiing td LIF giup tang ti le phdi bd giai doan nang d mdi trudng bd sung hLIF 10" lU/ml (4,58%)) vi phdi nang. Cac kit qui tren chung td nhin hLIF cd khi mdi trudng bd sung hLIF 10^ lU/ml (12,16%.) diu cao nang tie ddng len phdi bd tii giai doan phdi 4 te bio va hon nhdm ddi chiing khdng bd sung hLIF (1,36%). Ti 8 tl bio den giai doan phdi nang.
Hinh 1. Cie giai dogn phat tnen ciia phdi bd trong mdi trudng nudi ciy bo sung LIF 10''lU/mi. A. giai doan phdi 2 tebao, 6 giai dgan phdi 4 te bdo. C: giai doan phdi 8 te bao, D giai doan phoi dau, E: giai doan phoi nang, F giai doan phdi rang nd rdng.
Bang 1 . B 6 sung trong hLIF trong md Nong dp LIF
(lU/ml) D6i ehifng 10^
10^
Tdng so thi^ linh 589 240 296
trirng
1 trudng nudi cay phdi.
Phdi 2 te bao (%) 201 (34,13^) 83 (34.58^) 101 (34,12^)
Phdi 4 te bao (%) 142(24.11^) 5 2 ( 2 1 , 6 7 ' ) 92(31,08")
Phdi 8 te bao (%) 102 (17,32=) 41(17,08^) 85(28,71")
Phdi dau
(%)
12 (2,04^) 12 (5,00") 54 (18,24'^)
Phdi nang
(%)
8 (1,36') 11 (4,58") 36 (12.16') Ghi ch6:a. b, c: khac bi#t cd y nghTa thong ke (P<0,05)
Nhan td lic che benh bach cau li mdt cytokine cin thilt cho qui trinh lim td vi duy tri qui trinh mang thai (Robb et al. 2002; Lass et al, 2001).
Trudc day, tic dgng ciia LIF len su qui trinh hinh thinh vi phit trien cua phdi bd d cac giai doan khac nhau trong moi trudng nudi cay vin cdn dang tranh cai. Trong nghien ciiu ciia Morten Vejlsted et al, (2005), viec bd sung lO' lU/ml hLIF (human leukemia inhibitory factor) vio trong mdi trudng nudi cay phdi bd in vitro giiip lim ting ti le phdi phit trien den giai doan phdi thoit ming mgt each rd ret (Vejlsted el al, 2006). Viec bd sung hLIF dugc
thuc hien trong tu ngiy thii 4 den ngiy thii 8 tuong ung vdi giai doan phdi 8 tdi phdi nang. Trong mdt sd nghien cthi khic, viec bd sung hLIF vao mdi trudng nudi cay phdi khdng cd su cii thien rd ret din su phit trien ciia phdi so vdi nhdm khdng bd sung hLIF.
Su bd sung hLIF d cie giai doan phdi I te bio, 4 tl bio va 8 te bio khong cd tic ddng din ti le phit triln phdi bd giai doan phdi nd rdng (Fukui, Matsuyama, 1994). Ket qui nghien ciiu ciia Rodriguez (2007) cho thiy, viec bd sung hUF vao giai doan phdi nang vi giai doan phoi nang nd rdng cd the lim giam ti le phoi a cac giai doan niy,
237
Le Thinh Long el ai Trong nghien ciiu niy, chiing tdi dinh gii inh
hudng ciia viec bo sung hLIF vio mdi trudng nudi cay len su phit trien cua phdi bd qua cie giai doan khic nhau. hLIF dugc bd sung ngay vao mdi trudng nudi cay phdi bd. Ket qua cho thiy hLIF khdng anh hudng tdi kha nang phin chia dau tien thanh phdi 2 te bio ciia phdi bd, Ngoii ra, viec bd sung hLIF lam tang ding ke ti le phdi bd giai doan phdi 4 te bio cho tdi giai doan phdi nang. Ket qui niy gidng vdi (Morten Vejlsted et al, 2005). Nhu vay, viec bd sung hLIF vio mdi trudng nudi cay phdi bd cc khi nang cii thien ti le phit trien ciia phdi bd tdi giai doan phdi nang.
Su bieu hien ciia LIF d mire do phien ma dugc xac dinh trong cie giai doan khic nhau cua phdi bd, C) diy chiing tdi danh gii su bieu hien mRNA cua LIF d nhdm ddi chiing vi nhdm bd sung hLIF 10^
lU/ml. Kit qui RT-PCR cho thiy phdi bd d nhdm ddi chimg vi nhdm bd sung hLIF 10^ lU/ml deu bieu hien mRNA cua LIF d cie giai doan tning chin, hgp
tir, phdi 2-4 te bao, phdi 8-16 te bio vi phdi nang (Hinh 2). Su bieu hign ciia LIF trong cie giai doan phat trien khic nhau cua trimg chin vi phoi chirng td LIF cin thiet cho qua trinh phit trien phoi bd trong dieu kien nudi cay in vilro.LVV duac chimg minh la cin cho su phat trien ciia phdi giai doan tien lim td d ddng vat hiru nhii.O bd, su bieu hien ciia LIF a miic mRNA vi protein da dugc xac dinh d ndi mac hi cung bd (Oshima et al, 1998) tuy nhien su bieu hien cua LIF d phdi giai doan tien lam td thi con it dugc biet den.
Trong nghien ciru ciia Eckert, Niemann (1998), mRNA ciia LIF da dugc xac djnh bieu hien trong trung, phdi bd giai doan 2-4 tl bao, 8-16 tl bao va phdi nang, 6 nghien ciiu nay, ket qui tuang tu cung dugc nhan thay d cie phdi bd dugc nudi ciy trong dieu kien invtlro. Nhu vay, ben csinh khi nang LIF ndi tai ciia phdi bd, vi^c bd sung hLIF cung gdp phan tang cudng viec sir dung LIF giiip phdi phat trien vi ting ti le phdi bd d cie giai doan khic nhau.
Phoi Ph6i Thang Trimg Hop 2 4 8-161^ Ph6i
DNA chin tn te bao bao
332bp
lO^Il/mlLIF
Hinh 2. Su bieu hien mRNA cua UP d trung va phdi bd d cac giai doan khac nhau trong mdi trudng nudi civ bo suna va khdng bd sung LIF a a / a
Dinh gia chat lugng ciia phoi
Chit iugng phdi nang ddng vai trd rit quan trgng trong qui trinh phin lap va nudi ciy tl bio gdc phdi tir phdi nguyen hay tir khdi ICM. 0 diy chiing tdi dinh gia chat Iugng phdi bd giai doan phdi nang thdng qua viec xac djnh sd lugng te bao trong khdi ICM vi trong phdi bing phuong phap nhugm nhan vdi PI (Hinh 3).
Ket qui dugc md ta trong bang 2. Sd tl bio trung binh hong phdi nang d nhdm bd sung hLIF 10^ lU/ml li cao nhit (51,75 te bao/phdi) va cao hon nhdm ddi chiing va nhdm bd sung hLIF 10" lU/ml. Khdng cd su khic biet ve mat sd lugng tl bio frung binh trong mdt phdi nang giQa nhdm ddi chimg (42,25 tl bio/phdi) vi nhdm bd sung hLIF 10^ lU/ml (41,50 tl bio/phdi) Nhu vay viec
238bd sung hLIF vdi ndng dd lO' lU/ml cd tic dung dang ke trong viec tang sd lugng tl bio hung binh trong phdi bd giai doan phdi nang. Sd lugng tl bio tnDng khdi ICM ciia nhdm bd sung hLIF lO' lU/ml (23,25 tl bao/ICM) cung cao hon hin so vdi nhdm ddi chiing (14,75 tl bio/ICM) va nhdm bd sung (13,25 te bio/ICM). Dieu dd chung td viec bd sung hLIF vio mdi trudng nudi ciy lim tang ti Ie tl bio trong khdi ICM, day li mdt dilu quan trgng giiip ting khi ning thiet lap cie tl bao gdc phoi tir khdi ICM hoac tir nudi ciy phdi nguyen d bd.
Anh huong cua LIF len sd Iugng tl bio trong
khdi ICM van cdn tranh cai. Trong nghien ciru ciia
tic gii Sirisathjen vi cdng su cho ring viec bd sung
Tgp chi Cong nghe Sinh hgc 12(2): 235-240,2014 hLIF vdi ndng do 100 ng/ml khdng nhQng cii thiSn ti le phdi bd phat trien tdi giai doan phdi nang hay phdi thoit nang m i cdn cd t h i lim ting ding ke sd lugng t l b i o trong khdi ICM (Sirisathien el al, 2003). Trong khi dd, trong nghien ciiu cua Morten Vejlsted et al, (2005) cho ring viec bd sung hLIF vdi ndng do 10^ lU/ml tuy cd the lim ting ti le phdi bd phit trien den giai doan phdi nang nhung lai klidng lim tSng sd lugng te bao trong khdi ICM, sd lugng t l b i o trong klidi ICM ciia phdi dugc nuoi ciy uong phdi trudng bo sung hLIF voi ndng do 10^
lU/m! thap ban sd te b i o trong nhdm ddi chiing khdng bd sung hLIF (Vejlsted el al, 2006). Khdng nhimg the, sd iugng te b i o trong khdi ICM cdn phu thugc v i o loai LIF dugc sii dung. Trong nghien ciiu cua Rodriguez et al., (2005) cho thiy sd lugng tl b i o trong khdi ICM giim khi sir dung mLIF (mouse LIF) trong khi dd viec su dung hLIF lai khdng lim giim sd lugng cua t l b i o trong khdi ICM (Rodriguez el al, 2007). Trong nghien ciiu niy, chiing tdi thiy Bang 2 Sd lu'gng te bSo trong khoi ICM v ^ phdi nang.
rang viec bd sung hLIF vdi ndng do 10^ lU/ml khdng nhiing cd tic ddng duong tinh tdi ti le phit trien ciia phoi bo den giai doan phdi nang m i cdn cd tac ddng cii thien sd lugng te bao trong khdi ICM ciia phdi bd giai doan phdi nang.
KET KUAN
Chiing tdi da dinh gii duoc inh hucmg ciia nhin td lie che benh bach ciu ngudi (hLIF) len su phit trien ciia phdi bd trong dieu kien nudi ciy in V/7/-O.LIF khdng nhirng lim tang ti le phat trien ciia phdi bd in vitro m i cdn cii thien chat lugng cua phdi bd thdng qua viec tam tang sd lugng le b i o phdi va sd lugng te bao trong khdi ICM.
Ldi c i m on; Chung toi chdn thdnh cdm on So Khoa hg vd Cong nghe Thdnh phd Ho Chi Minh da cung cdp kmh phi cho nghien cuu nay.
Ndng d d hLIF (lU/mi) Sd te b a o t r o n g phdi nang Sd te bao t r o n g ICM (%) f)di chung
10' 10^
42,25' 41,50"
51,75"
14,75(34,91°) 13,25(31,93") 23,25 (44,93") Ghi c h u : a, b khac bi§t cd y nghia thong ke (PS0.05)
n i l l l l d . I - I I U I u u ^ ) d l UUI^ p i l U I llCIJiy I M I U V I I I vu/l ^1U[JIUIUIII IU sung LIF 10' lU/ml, C: phdi nang nhdm bo sung LIF l O ' lU/ml
TAI LIEU THAM KHAO
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Eckert J and Niemann H (1998) mRNA expression of Fukui Y and Matsuyama K (1994) Development of in vitro
Le Thinh Long et al.
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E F F E C T O F L E U K E M I A I N H I B I T O R Y F A C T O R O N D E V E L O P M E N T O F B O V I N E IN VITRO E M B R Y O S
Le Thanh Long''*, Doan Chinh Chung^ Do Minh S i \ Ho Nguyen Quynh Chi^, Hoang Nghia Son' 'institute of Tropical Biology, Vielnam Academy ofScience and Technology
^Biology Department, University ofScience, Ho Chi Minh city
^Biotechnology Department, Inlemalional University, Ho Cht Mmh city
SUMMARY
In this study, we accessed the effect of human leukemia inhibitory factor (hLIF) on invilro development of bovine embryo m preimplantation stages. The number of bovine embryos at different stages was determined to evaluate the development of embryos and the cell number of bovine blastocyst embryos and inner cell mass were counted to estimate the quality of blastocyst embryos The results showed that hLIF did not effect on bovme embryo cleavage from zygote stage to 2-cell stage. 10^ lU/ml LIF supplementation m bovine embryo culture medium supported to embryo division from 2-cell stage to 4-cell stage (31,085). The ratio of embryo division from 2-cell stage to 4-cell stage of control group and 10^ lU/ml LIF supplemented group was lower than that of lO' lU/ml LIF supplemented group. In 10^ lU/ml LIF supplemented group, the ratio of bovine embryo in 8-cel! stage (28,71%) and morula stage (18,24%) was higher than those of control group and 10^
lU/ml LIF supplemented group. Thus, LIF could rescue to bovine embryo block from 8-cell stage to I6-cell stage. The ratio of blatocysl embryo in lO' lU/ml LIF supplemented group was higher than control group and 10^ lU/ml LIF supplemented group. The mature oocytes, 2 and 4-ceII stage embryos, 8 and 16-cell stage embryos, and blastocyst embryo expressed LIF transcription. The bovine blastocyst embryo quality in 10^
lU/ml LIF supplemented was higher than control group and 10^ lU/mi H F supplemented group by accessing cell number in inner cell mass and blastocyst embryo. These results showed that LIF could positively support to bovine in vitro development in preimplantation stage.
Keywords: Bovine embryo, embryo cleavage. ICM (inner cell mass), leukemia inhibitory factor, transcription
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