불 임 학 회
요 약 제 목 : S perm Injection in Pig
A ctiv ation of P orcine Oocy te s F ollow ing Intracy toplas mic Injection of V arious S perm Com ponent s and f oreig n s pe cie s sperm atozoa
S .H . Ju n , J .S . S hin , J .T . D o , J.K . K w on
*, N .H . K im , H .T . L e e , K .S . Ch un g
A nimal R esource R esearch Center, Kon Kuk University,
*
L ab of E lectrom icr os cop y , m ed ical s chool, H anyang un iv ers ity
여 러 가 지 정 자 구 성 성 분 및 이 종 정 자 주 입 에 의 한 돼 지 난 자 의 활 성
전 수 현 ・ 신 지 수 ・ 도 정 태 ・ 권 중 균
*・ 김 남 형 ・ 이 훈 택 ・ 정 길 생
건국대학교 동물자원연구센터, *한양대학교 의과 대학 전자현미경실
요 약
본 연구에서는 돼지 난자내에 돼지정자, 여러 가지 처리된 정자두부(1% T rit on , 0.1%
T r y p sin , 100m M N aOH ) 및 이종정자 (소, 쥐, 사람)를 미세 주입한 후 난 활성과 웅성 전핵 형성, 전핵의 이동 및 배발달을 관찰하였다. 전자현미경으로 관찰한 결과 T rit on X - 100을
처리하였을 때 첨체가 제거되었으나 핵 주변 물질은 제거되지 않았고 T ryp sin 또는 N aOH
를 처리 할 경우 핵주변 물질(perinuclear m at erial)이 제거됨을 볼 수 있었다. 돼지난자는
정자, 정자두부 및 T riton X - 100을 처리한 정자두부의 주입을 통해 난 활성이 유도되었으며
쥐, 소, 사람의 정자를 주입하였을 때 난 활성이 유도되고 정상적인 전핵형성이 이루어졌다.
그러나 정자꼬리나 T ryp sin 또는 NaOH에 의해 정자 핵주변 물질(perinu clear m aterial)이
제거된 정자두부를 주입하였을 때는 난 활성은 야기되지 않았다. 유사분열 및 2- 세포기까지
정상적인 수정은 동종의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었으나 이종정자
를 주입한 난자에서는 관찰되지 않았다. 또한 상실배 및 배반포까지 정상적인 수정은 동종
의 정자 및 정자두부를 주입한 난자에서 관찰할 수 있었다. 이러한 결과는 돼지에서 정자
및 정자두부의 미세주입에 의해 수정이 이루어지는 것을 시사하며 수정시 정자유래의 난활
성인자는 정자 핵주변 물질(perinu clear m at erial)에 존재하며 종특이적이지 않다는 것을 보
여주는 것이다.
(K ey w or d s : P or cin e eg g s , ICS I, F er t ilizat ion , A ct iv at ion , P er in u clear m at er ial )
Ⅰ . Intro du ction
Dur in g fer tilizat ion t h e sp er m cell a ctiv at es oocy t es b y r elea sin g an oocy t e
a ctiv at in g fact or (s ) (OA F ) in t o oocy t e s . P ar r in gt on (1996) r eport ed th at OA F is a 33
k Da pr ot ein r esidin g in th e equ at or ial s egm en t r egion of th e acr osom e. B ecau se
int r acy t opla sm ic sper m inj ect ion of for eig n species , su ch a s h am st er , r abbit , pig , hum an
or sea ur ch in in t o m ou se oocy t e s act iv at es oocy t es , OA F is n ot str ict species specific
for t h e m ou s e oocy t e (W ak ay am a et al., 1997 ; Kim ur a et al. 1998 ). In t h e m ou s e OA F
appear s (or b ecom e act iv e ) in sper m iog en esis an d locat ed in t h e perinu clear m at er ial
(Kim u r a et al., 1998 ). H ow ev er , infor m at ion is lar g ely lackin g on t h e n at ur e of act iv at ion
fact or s for any sp ecie s ot h er t h an th e m ou s e.
T o g et in sigh t int o t h e n at ur e of sper m b or n oocy t e fact or s , w e det er m in ed th e
in ciden ce of act iv ation , m ale pr on u clear for m at ion an d pr on u clear app osit ion follow in g
inj ect ion of v ariou s p or cin e sper m com p on ent s an d for eig n species sper m at ozoa in t o
p or cin e oocy t es . In t h e pr es ent st u dy w e als o ex am in ed dev elopm en t al ability in v itro
follow in g inj ect ion of a sp er m at ozoon an d an isolat ed sp er m h ead .
Ⅱ . M A T E R IA L S A N D M E T H OD S
1 . I n V it r o M aturation
P r epu b er t al p or cin e ov ar ies w er e collect ed fr om a local slau g ht er h ou se an d
t r an spor t ed t o t h e lab or at or y at 25°C in Du lb ecco ' s ph osph at e bu ffer ed salin e
supplem ent ed w it h 5.54 m M D - glu cose , 0.33 m M sodium py r uv at e, 75 m g/ m l pot a s sium
p enicillin G an d 50 m g/ m l str ept om y cin su lph at e (m DP BS ). Cu m u lu s - Oocy t e com plex es
(COC ) w er e a spir at ed w it h an 18 - g au g e n eedle in t o a disp os able 10- m l sy r in g e fr om
follicles . F ift y por cin e COC w er e m atu r ed in 500 μl of B S A - fr ee N CS U 23 (P et t er s
an d W ells , 1993 ) m ediu m su pplem ent ed w it h 10% p or cin e follicu lar flu id , 0.6 m M
cy st ein e, 10 IU/ m l P M S G (S igm a , S t . Louis , M O, U S A ) an d 10 IU/ m l h CG (S igm a )
u n der par affin oil at 39°C for 40 t o 44 h .
2 . P reparation of s p erm at oz o a an d i s olat e d s perm h e a ds
P or cin e sp er m - r ich fr action (15 m l) w a s collect ed fr om a b oar by g lov ed h an d
m et h od , an d , aft er addin g an tibiot ic - an t im y cotic solu tion (S igm a ), t h e sem en sam ple w a s
k ept at 20°C for 16 h . S em en w a s w a sh ed 3 tim es by cent r ifu g at ion w it h 0.9% (w/ v )
N aCl su pplem ent ed w it h 10 m g/ m l BS A (fr act ion V ; S igm a ). S per m at ozoa w er e
su spen ded in 1.5 m l of T L - HE P E S (P r at h er et al., 1995 ) for 30 m in t o in du ce
capacit ation . T h e isolat ed sper m h ead s w er e obt ain ed b y t h e m et h od r epor t ed b y t h e
Ku r et ak e et al (1996 ). T h e sp er m su sp en sion w a s son icat ed in 9 m l of cold n u cleu s
isolat ion m ediu m (NIM , Ku r et ak e et al., 1996 ) in t h e pr esen ce of 0.05% t rit on X - 100.
T h e sonicat ion w a s con du ct ed in w at er b at h for 30 s ec u sin g 100 % ou tput of an u lt r a
s on ic s on icat or (M odel, Br an son 8210). T h e sonicat ed sper m su spen sion w a s dilu t ed an d
cent r ifu g ed for 5 m in at 1,000 x g t o th e sper m h eads . T h e sperm h ead s w er e on ce
m or e w a sh ed in 10 m l of NIM b y cen t rifu g ation . F or an ot h er ser ies of ex p er im en t s
isolat ed sper m h eads w er e tr eat ed w ith 0.1% tr y p sin or 100 m M N aOH s olut ion for 3 h
in t ris buffer ed salin e. S om e sp er m h ea ds w ith or w it h out tr eat m en t s w er e fix ed in 2 %
g lu t ar aldehy de in Du lb ecco ' s ph osph at e - buffer ed s alin e. T h e sp ecim en s w er e t h en
p ostfix ed in 2% Os O4 for 1 h , dehy dr at ed in a gr aded eth an ol ser ies , an d em b edded in
epox y r esin . Ultr at hin section s w er e cut w it h a diam on d kn ife an d post - st ain ed fir st
w it h 1% u r any l acet at e in 30% et h an ol an d w ith R ey n old s lead cit r at e. A t r an sm is sion
elect r on m icr oscope (Hit achi 600, H it ach i Lt d, K a shiw a , J ap an ) w a s u sed t o det er m in e
t h e pr esen ce an d ab s en ce of per in u clear m at er ial.
Hu m an ej a culat ed sem en sam ples w er e ob t ain ed by m a st ur b at ion . S em en w er e
m ix ed w it h an equ al v olum e of H E P E S - bu ffer ed h um an t ub al fluid m ediu m
(H T F - P ep es , Ir v in e S cien t ific, S ant a A n a , CA ) an d cent r ifu g e for 5 t o 10 m in at 1800 g .
T h e pellet w a s su sp en ded in 1.5 m l of H E P E S - b uffer ed H T F - P ep es t o in du ce
capacit ation . M ou s e sp er m at ozoa w er e collect ed fr om a m at ur e m ale m ou se (ICR , 8 t o
13 w eek old ) cau da epididy m is e. S p er m at ozoa w er e w a sh ed in T L - H E P E S an d th en
su spen ded in 1.5 m l of T L HE P E S . Bov in e sp er m at ozoa w er e pr epar ed fr om
fr ozen - th aw ed s em en . S per m at ozoa w er e w a sh ed t w ice w it h T L - H E P E S an d t h en
su spen ded in 1.5 m l of T L - H E P E S . T h e sper m w a s r esu spen ded t o 1 m l w ith
h epar in - cont ainin g (10 u g/ m l) T L - HE P E S in a effen dor f t ub e an d k ept at 39 C for 30
t o 60 m in t o in du ce cap acit at ion .
3 . Oo cy t e A ctiv ation
T h e pr ocedur es for elect r ical st im ulat ion of por cin e oocy t es w er e a s des cr ib ed by
Kim et al (1996a ). E lect r ical st im ulat ion t o in du ce act iv ation w a s deliv er ed w ith a BT X
E lect r o Cell M an ipulat or (Biot ech n ologies an d E x perim ent al Resear ch , In c., S an Dieg o,
CA ) t o a ch am b er w it h t w o par allel plat in um w ir e elect r odes (200 m m o.d .) spa ced 1
m m ap ar t ov er laid . A t 30 t o 60 m in b efor e sper m cell inj ect ion , cu m ulu s cell denu ded
oocy t es w er e st im ulat ed b y a 10 sec pulse at 0.48 KV/ cm A C follow ed by a 30 m s
pulse at 1.26 kV/ cm D C at r oom t em per atu r e (25°C ). T h e oocy t es w er e th en tr an sfer r ed
t o 500 m l of N CS U 23 m edium an d cu lt ur ed at 39°C in an at m osph er e of 5% CO
2in air
u nt il sp er m cell inj ect ion .
4 . S perm at o z oon inje ction int o O oc y te s
S per m at ozoa w er e cent r ifu g ed (400g , 5 m in ) an d r esu spen ded in T L HE P E S : 10%
p oly v in y lpy r r olidon e solu tion (1:1). A m icr odr op (5 m l) of t his su spen sion w a s placed a
slide , an d t h e slide w a s placed in L eit z Differ en t ial In t er fer en ce Cont r a st inv er t ed
m icr os cop e equ ipped w ith L eit z m icr om anipulat or s . T h e oocy t es w er e denu ded of
cum ulu s cells by r epeat ed pipet t in g . Oocy t es w ith v isible polar b ody an d of ex cellen t
m or ph ology w er e u sed for t his ex per im ent . Oocy t e s w er e cen t rifu g ed for 10 m in in an
E ppen dor f cent r ifu g e at 12,000 g in 50 m l T L - HE P E S m edium in 1.2 m l E ppen dor f
cent r ifu g e t ub e. T h e inj ection of sper m at ozoon or is olat ed sp er m h ead in t o th e oocy t e
cy t opla sm w a s per for m ed u sin g t h e m et h od of L ee et al (1998). Br iefly , t h e inj ection
n eedle u s ed w a s of 6 - 7 m inn er an d 8 - 9 m ou t er diam et er . T h e p olar b ody w a s at 6 or
12 o ' clock an d t h e p oint of inj ect ion at 3 o ' clock . A n oocy t e w a s p en etr at ed by t h e
inj ect in g m icr opipett e, a sm all am oun t of cy t opla sm w a s dr aw n in t o t h e m icr opip ett e,
an d t h en th e cy t opla sm t og eth er w it h t h e sperm at id an d a sm all am ou nt of m edium w a s
ex p elled int o t h e oocy t e. Im m ediat ely aft er oopla sm ic inj ect ion , t h e inj ect in g m icr opipett e
w a s w it h dr aw n qu ickly , an d t h e oocy t e s w er e r elea sed fr om t h e h oldin g pip et t e t o
r edu ce th e int r acy t opla sm ic pr es sur e ex er t ed t o t h e oocy t e. A ft er inj ect ion , all of th e
oocy t es w er e t r an sferr ed t o N CS U 23 m ediu m an d cultu r ed for at 39°C u n der 5% CO2 in
air . T h e oocy t e s w er e fix ed at 10 t o 15 h an d 20 t o 24 h follow in g sper m cell inj ect ion .
A b ou t 10 oocy t es w er e fix ed , st ain ed w ith 1% (w/ v ) or cein an d ex am in ed n u clear
m or ph ology at 200 X an d 320 X m ag nificat ion w it h ph a se cont r a st m icr os copy . T h e
ot h er s w er e cu lt ur ed for N CS U 23 m edium at 39°C un der 5% CO2 in air .
5 . Ch rom o s om al an aly s is
A t 22 h follow in g inj ect ion , t h e oocy t es w er e cu lt ur ed in cu lt ur e m ediu m in th e
pr esen ce of 5 u M colcem id. T h e oocy t es w er e t h en cent r ifu g ed t o r em ov e lipid s in t h e
cy t opla sm , an d placed in a h y pot onic solu tion of sodium cit r at e (1% ) for 5 m in an d
fix ed in div idu ally on m icr oscope slides b y t h e air - dr y in g m et h od (T ar k ow ski, 1966 ).
T h e oocy t e s w er e st ain ed w ith 10% Giem sa at pH 6.8. T h e chr om osom e con stit u en t of
each spr ead w a s det er m in ed at 1000 m a gn ification w it h a Zeis s m icr oscope.
6 . S tati s tic al A n aly s e s
T h e dat a w er e p ooled fr om at lea st four r eplication s . Differ en ces in th e per cent ag e s
of oocy t es dev elopin g t o a p ar ticu lar st ag e w er e det er m in ed by Ch i- squ ar e pr ocedu r es .
Ⅲ . R E S U LT
F e r t iliz a t ion of p or c in e oocy t es f ollow ing inj e c t ion of sp e r m a t oz oa or is ola t e d sp e r m hea d
T h e oocy t es w ith t w o lar g e pr onu clei an d t w o polar b odie s (2 P N + 2 P b ) w er e
cla s sified a s n orm al fert ilization at 10 t o 15 h follow in g ICS I or int r acy t opla sm ic sp er m
h ead inj ect ion (T able 1). T h e in ciden ces (52 t o 56 % ) of n or m al fer tilization w er e n ot
differ en t b et w een follow in g sper m at ozoon an d sp er m h ead inj ection . T able 2 sum m ar ize
chr om at in config ur at ion in por cin e oocy t es at 20 t o 24 h aft er sperm at ozoon or isolat ed
sper m h ead inj ect ion . T h e in ciden ce of pr on u clear apposit ion , m it osis an d t w o cell
div ision w a s con sider ed a s n or m al fer t ilized . F ollow in g ICS I an d h ead inj ect ion , 49% an d
44% w er e n or m ally fer tilized, r esp ect iv ely .
T able 1 . Chrom atin c onfig u ration s in pig o o cy t e s at 12 t o 15 h f ollo w in g in tra cy t opla s m ic s perm ato z oa or i s olate d s p erm h e ad
P ar am et er a s se s sed T y pe of cells inj ect ed
S h am S per m at ozoon S per m H ead N o. of oocy t es
asu cces sfu lly inj ect ed (r eplication ) 51 ( 5) 60 ( 5 ) 62 ( 5 )
w it h t w o P N an d t w o P B (% ) 0 ( 0) 31 (52) 35 (56 )
w it h on e P N , sper m h ead an d t w o P B (% ) 0 ( 0) 9 (15 ) 5 ( 8 ) w it h t w o P N , sper m h ead an d on e P B (% ) 0 ( 0) 2 ( 3 ) 4 ( 6 )
w it h th r ee P N + on e P B (% ) 2 ( 4) 5 ( 8 ) 6 (10)
w it h t w o P N + on e P B (% ) 9 (18) 1 ( 2) 0 ( 0)
w it h on e P N + t w o P B (% ) 36 (71) 0 ( 0) 0 ( 0)
w it h oth er s
b4 ( 8) 12 (20) 12 (19 )
a
A bb r ev iat ion s : P N , pr on u cleu s ; P B ; p olar b ody .
b
Ot h er s in clu de t h e oocy t es w it h out or un iden tified m u lt iple n u clei.
T able 2 . Chrom atin c onfig u ration s in pig o o cy t e s at 20 t o 24 h f ollo w in g
in tra cy t opla s m ic s perm ato z oa or i s olate d s p erm h e a
P ar am et er a s se s sed T y p e of cells inj ect ed
S h am S per m at ozoon S per m H ead N o. of oocy t es
asu cces sfu lly inj ect ed (r eplication ) 49 ( 5 ) 6 1 ( 5 ) 62 ( 5 )
closely app osed 2 P N 0 ( 0 ) 20 ( 33 ) 18 ( 29 )
M it osis 1 ( 2 ) 2 ( 3 ) 3 ( 5 )
T w o - cell 2 ( 4 ) 8 ( 13 ) 6 ( 10 )
w it h t w o P N an d t w o P B (% ) 0 ( 0 ) 7 ( 11) 14 ( 23 )
w it h on e P N , sper m h ead an d t w o P B (% ) 0 ( 0 ) 9 ( 15 ) 5 ( 8 ) w it h t w o P N , sper m h ead an d on e P B (% ) 0 ( 0 ) 2 ( 3 ) 4 ( 6 )
w it h t w o P N + on e P B (% ) 10 ( 20 ) 0 ( 0 ) 0 ( 0 )
w it h on e P N + t w o P B (% ) 27 ( 55 ) 0 ( 0 ) 0 ( 0 )
w it h oth er s
b9 ( 18 ) 13 ( 21) 12 ( 19 )
a
A bb r ev iat ion s : P N , pr on u cleu s ; P B ; p olar b ody .
b
Ot h er s in clu de t h e oocy t es w it h out or un iden tified m u lt iple n u clei.
O ocy t e a c t iva t ion and p r on ucl ea r f orm a t ion f ollow ing inj e c t ion of va r io us sp e r m c omp one nts
T ab le 3 su m m ar izes t h e in ciden ce of act iv at ion an d m ale pr on u clear for m at ion
b et w een 10 t o 12 h follow in g inj ection of v ar iou s sp er m com pon ent s . W h en a
sper m at ozoon or T r it on X - 100 t r eat ed por cin e sper m h ead s w er e inj ect ed int o por cin e
oocy t es , th e oocy t es w er e activ at ed , w h er ea s t ail did n ot in du ce activ at ion . Inj ect ion of
eit h er t r y p sin t r eat ed sper m h ead or N aOH t r eat ed sper m at ozoa failed t o in du ce
a ctiv at ion . M ale pr on u cleu s w a s for m ed in th e a ctiv at ed oocy t es follow in g inj ection of
T r it on X - 100 t r eat ed sp er m h ead . N eith er t ry p sin n or N aOH t r eat ed sp er m h ead w a s
decon den sed in por cin e oocy t es w h en t h ey w er e inj ect ed . T r an sm is sion elect r on
m icr os copy sh ow ed sag it t al section s of is olat ed sp er m h eads aft er son ication in t h e
pr esen ce of tr it on X - 100 or t r y p sin (F igu r e 1). W hile T r it on X 100 t r eat m ent left
p er in u clear m at er ial ar ou n d t h e n u cleu s , tr y p sin or N aOH r em ov ed per inu clear m at er ial
ex t en siv ely .
T able 3 A ctiv ation an d m ale pron u cle ar f orm ation in porcin e oo cy t e s at 10 to 15 h f ollo w in g v ariou s s perm c om pon ent s
S per m h ea ds t r eat ed
N o. (% ) of oocy t es su cces sfully
inj ect ed (r ) act iv at ed M P N
n on e 42 (5 ) 7 (17 ) -
sp er m at ozoa 25 (4 ) 19 (76 ) 15 (60)
sp er m h ead
1% t r it on 46 (5 ) 41 (89 ) 29 (63 )
0.1% t r y p sin 44 (5 ) 10 (23 ) 6 (14 )
100 m M N aOH 35 (4 ) 3 (9) 0 (0)
sp er m t ail 19 (3 ) 2 (11) -
M P N : m ale pr on u cleu s
F ig 1. S ag it t al section s of por cin e sper m h ead follow in g v ariou s t r eatm en t s . A ) In t a ct
sper m at ozoa , B ) S onicat d sperm h ead in th e pr es en ce of tr it on X , C ) T h e sper m h ead
cultu r ed in 100 m M N a OH .
O ocy t es a c t iva t ion and m a le p r onuclea r f orm a t ion f ollow ing inj e c t ion of f or e ig n sp e c ies sp e rm a t oz oa
M ost oocy t es w er e activ at ed at 10 t o 12 h follow in g inj ect ion of sperm cell
r eg ar dles s of elect r ical st im u lat ion (T able 4 ). A lt h ou g h som e oocy t es (23% ) w er e
a ctiv at ed in t h e oocy t es follow in g sh am inj ection , t h e r at es is v er y low . T h e in ciden ce
of a ctiv at ion an d pr on u clear for m ation w a s n ot differ en t in oocy t es follow in g inj ection of
p or cin e, b ov in e, m ou s e or h um an sp er m at ozoa (T ab le 4 ) in t h e ab sen ce of electr ical
stim ulat ion . A ddition al electr ical st im ulat ion did n ot in cr ea se t h e in ciden ce of m ale
pr on u clear for m at ion (P > 0.1). A t 36 h follow in g inj ection , m aj or ity oocy t es , w h ich
w er e inj ect ed por cin e sper m at ozoa , w er e cleav ed t o t h e 2- cell st ag e (T able 5 ). H ow ev er ,
n on e of oocy t es w er e n or m ally div ided t o t h e t w o cell st ag e follow in g for eig n species
sper m inj ection . A lth ou gh som e oocy t es w er e div ided t o t h e t w o- cell st a g e follow in g
sh am , m ou s e, b ov in e or h um an sper m cell inj ection , it app ear s t o b e du e t o
p ar th en og en et ic a ctiv at ion .
T able 4 . A c tiv ation an d m ale pronu cle ar form ation in porcin e o oc y te s at 10 t o 15
h f ollo w in g m ou s e , b ov in e , hu m an an d porcin e s perm inje ction
S ou r ce of sp er m at ozoa
elect r ical st im ulat ion
N o. (% ) of oocy t es su cces sfu lly
inj ect ed (r ) activ at ed M P N n on e
(sh am inj ect ion )
+ 28 (4 ) 26 (93) 0 ( 0)
- 26 (4 ) 6 (23) 0 ( 0)
pig + 55 (6 ) 53 (96) 34 (62)
- 50 (6 ) 42 (84) 29 (58)
cow + 52 (6 ) 50 (96) 31 (60)
- 52 (6 ) 43 (83) 23 (44)
hu m an + 54 (6 ) 48 (89) 25 (46)
- 59 (6 ) 39 (66) 31 (53)
m ou se + 50 (6 ) 49 (98) 36 (72)
- 51 (6 ) 37 (73) 29 (57)
T able 5 . cle av a g e an d nu cle ar s tatu s of porcin e o oc y te s at 36h f ollo w in g in tra cy t opla s m ic inje c tion of p orc in e , b ov in e , m ou s e or h um an s p erm at oz o a .
S our ce of sperm at ozoa
N o. (% ) of oocy t es su cces sfully
inj ect ed (r ) on e cell n or m al t w o cell
aabn or m al t w o cell
bsh am inj ection 15 (2) 11 (73 ) 1 ( 7 ) 4 (27 )
pig 26 (3) 5 (19 ) 17 (65 ) 4 (15 )
cow 27 (3) 17 (63 ) 0 ( 0) 10 (37 )
hu m an 25 (3) 16 (64 ) 0 ( 0) 9 (36 )
m ou se 29 (3) 19 (66 ) 0 ( 0) 10 (34 )
a
E m br y os w it h t w o int er ph a se nu clei
b
T w o t o fou r cell st a g e eg g s w it h a bla st om er e w hich h av e n o or m ultiple nu clei.
I n V itr o d e v e lop m en t of p or c in e o ocy t es f ollow ing inj e c t ion of a sp e rm a t oz oon or
is ola t e d sp e r m hea d
Chr om osom e an aly sis r ev ealed t h at m ost oocy t es am on g ident ified t h em (73% in
ICS I ; 70% in h ead inj ect ion ) w er e diploid follow in g ICS I an d isolat ed sperm h ead
inj ect ion . T h er e is n o diploid in oocy t es at 22 h aft er sh am inj ect ion (T able 6 ). T h e
m ost oocy t e s cleav ed at 24 h an d div ided t o 4 - cell at 48 h follow in g ICS I or isolat ed
sper m h ead inj ect ion (T ab le 7 ). H ow ev er , t h e sh am oocy t es cleav ed t o t h e 2- cell aft er
24 h . A t 7 day s t h e in ciden ces of b la st ocoel for m ation follow in g ICS I (38% ) an d is olat ed
sper m h ea d inj ection (22% ) w er e h ig h er t h at follow in g sh am inj ect ion cont r ol (3% , T able
8 ). S om e oocy t es follow in g sh am inj ect ion w er e dev elop ed t o b la st ocy st s at 9 day s .
T able 6 . Chrom o s om al an aly s i s of porcin e o oc y te s at 22 h f ollow in g s perm at o z oon or i s olat e d s pe rm h e ad inje c tion
T y pe of cell inj ect ed N o. (% ) of eg g s
t est ed ident ified h aploid diploid > t et r aploid
n on e 22 9 9 (100) 0 ( 0) 0 ( 0)
sperm at ozoon 26 15 2 ( 13 )
*11 (73 )
*2 (13 )
*sperm h ead 25 10 2 ( 20)
*7 (70)
*1 (10)
**
P 〈 0.05.
T able 7 . Cle av ag e of porcin e o o cy t e s at 24 an d 48 h after s p erm at oz o a or
i s olat e d s perm h e a d inje ction
T im es (h ) aft er inj ect ion
T y pe of cell inj ect ed
T ot al n o. of oocy t es inj ect ed
N o. (% ) of eg g s cleav ed t o
1- cell 2- cell 4 - cell fr ag m ent at ion
24
n on e 65 50 (77 ) 7 (11) 1 (2) 7 (11)
sper m at ozoa 65 13 (20) 45 (69 )
*6 (9 ) 1 (2)
sper m h ead 65 16 (25 ) 38 (58 )
*8 (12) 5 (8 )
48
n on e 65 15 (23 ) 32 (49 ) 6 (18 ) 12 (9 )
sper m at ozoa 65 8 (12) 10 (15 ) 38 (58 )
*9 (14 )
sper m h ead 65 11 (17 ) 16 (25 ) 41 (63 )
*7 (11)
*
P 〈 0.05.
T able 8 . In v itro dev elopm ent of pig zy g ot e s cu lture d for 7 an d 9 day s f ollo w in g s perm at o z oon or s p erm h e ad inje ction
T y p e of cell inj ect ed
N o. of em br y os ex am in ed (r )
N o. (% ) of oocy t es dev eloped t o bla st ocy st s at 7 day s 9 day s of cultu r e
n on e 65 (5) 2 ( 3 ) 8 (12)
sperm at ozoon 65 (5) 25 (38 )
*29 (45)
*is olat ed sp er m h ead 65 (5) 14 (22)
*20 (31)
**