A naly s is of Relation s hip B etw een S perm atozoa A bility and Re activ e Ox y g en S pe cie s in P orcine : I. S perm Preincubation by X anthine and X anthine Ox idas e

A naly s is of Relation s hip B etw een S perm atozoa A bility and Re activ e Ox y g en S pe cie s in P orcine : I. S perm Preincubation by X anthine and

X anthine Ox idas e

C .K . P ark , H .T . Ch e on g , J .H . K im

, S .C . L e e

, B .K . Y an g an d C .I . K im

College of Anim al Agricultur e, Kangw on Univer sity , Chunchon , 200- 701, Kor ea

*

S aewha Infertility Clinic, Pu san , 607- 062, Kor ea

돼 지 정 자 의 수 정 능 력 과 Re a c tiv e Ox y g en S pe c ie s 의 관 계 분 석 I. X ant hin e과 X anthin e Ox ida s e에 의 한 정 자 의 전 배 양

박 춘 근 ・ 정 희 태 ・ 김 종 흥

・ 이 상 찬

・ 양 부 근 ・ 김 정 익

강원대학교 축산대학,

세화산부인과 불임크리닉

F our t ables

Correspondence ; t o Dr . C.K . P ark (박춘근) 우편번호: 200- 701

강원도 춘천시 효자2동 192- 1 강원대학교 축산대학

T el : 0361- 250- 8627

F ax : 0361- 51- 7719

= 국문초록 =

본 연구는 x anthine과 x anthine oxidase가 첨가된 배양액내에서 전배양된 돼지동결정

액의 수정능력에 있어서 catalase의 영향을 검토하였다. 체외수정을 위한 기본 배양액내

에서 0 또는 30분간 전배양한 정자는 cat alase를 첨가(40 및 15%)한 경우 보다는 무첨가

(66 및 38% )시 유의적으로 높은 정자침입율을 나타냈지만(P < 0.05), 배양액내에 x ant hine 을 첨가해 정자를 0, 30 및 60분간 전배양했을 때에는 catalase 무첨가(33, 41 및 19%)

보다는 첨가시(68, 70 및 49%) 유의적으로 높은 정자침입율이 인정되었다(P < 0.05). 그러

나 x anthine oxidase를 첨가하여 정자의 전배양을 행하지 않은 경우는 catalase의 첨가

(13% ) 보다는 무첨가(51% )시 유의적으로 높은 정자침입율을 나타냈지만(P < 0.01), 전배 양(30 및 60분)후에는 cat alase의 존재유무와 정자전배양시간에 관계없이 매우 낮은 정자

침입율 (10- 21%)을 나타냈다. x anthine과 x anthine oxidase를 동시에 첨가하여 0, 30 및

60분간 정자를 전배양한 경우 cat alase의 무첨가(14, 4 및 8% )보다 첨가(75, 55 및 52% ) 시 유의적으로 높은 정자침입율을 나타냈다(P< 0.001). 한편, 다정자침입율은 x anthine,

x anthine+x anthine oxidase의 첨가시 정자전배양기간이 길어짐에 따라 감소하였으며,

cat alase의 첨가보다는 무첨가시 낮은 다정자침입율을 나타냈다. 본 연구의 결과로부터

x anthine과 x anthine oxidase를 동시에 첨가시 cat alase의 존재는 정자의 전배양후에도 다정자침입을 억제하면서 수정능력의 유지를 위해 매우 효과적인 것으로 추측되었다.

Key w ards : cat alase, porcine IVF , sperm pr eincubation, x anthine, x anthine oxidase

본 연구는 한국과학재단의 연구비지원(과제번호 971- 0606- 048- 1)으로 수행되었음.

IN T R OD U CT ION

Sperm at ozoa acquire t he ability t o successfully fertilize the oocyt e during their

tr an sit in the fem ale genit al tr act . T hus the fem ale r eproductiv e tr act mu st be

r espon sible for supporting m otility t o ext end the fertile life span of sperm in the

fem ale reproductive tract by secr eting m otility fact or (s ) and/ or by reducing sperm

m et abolism in cert ain locations t o allow bett er survial. T he sperm at ozoa , like all cells

living under aer obic condition s, con st antly faces the oxygen paradox . Oxy gen is

clearly requir ed t o support life, but it s m et abolit es m odify cell funct ion s and/ or can

endanger cell survival.

T he generation of reactive oxy gen species in sperm prepar ations becam e a real

concern becau se of the t oxic effect s they hav e at high concentration s on sperm

functions , and of their possible inv olvm ent in m ade idopathic infert ilit y . Reactive

oxygen species pr oducrion in sem en has been associat ed w it h loss of m otility ,

decr eased capacity for sperm - oocyt e fu sion and los s of fertility (Ait ken et al., 1991).

High concentration s of reactive oxygen species pr oduced by sperm at ozoa them selv es

(Aitken & Clarkson , 1987 ; Alv ar ez et al., 1987) or by the combination s of x anthine

plu s x anthine oxidase (Aitken et al., 1993) induce the form ation of t oxic lipid

peroxides (Windsor et al., 1993) and compr omise sperm viability . Reactive oxygen

species also effect the sperm ax onem e as a result of AT P depletion (De Lamirand

and Gagnon , 1992), inhibit mit ochondrial function s, and synthesis of DNA , RNA and

prot ein s (Com porti, 1989), pr oduce cyt oskelet al m odification s (Hindshaw et al., 1986)

and inhibit sperm - oocyt e fusion (Ait ken et al., 1993). How ev er , the sperm at ozoa hav e

enzym atic defence sy st em s such as super oxide dism ut ase, glut athione

peroxida s/ reduct ase and cat alase (Griveau et al., 1995) t o count er act the t oxic effect s

induced by r eact iv e oxygen species . Alt hough correlation s hav e been r eport ed

bet w een t he effectiv eness of reactiv e oxy gen species and t he duration of sperm

m otility (Alvarez & St or ey , 1989 ; De Lam ir ande & Gagnon, 1997), the im port ance of

their action in vit ro ha s not been fully elucidat ed.

It is known that fr esh sperm hav e higher fertilization pot ential than frozen - thaw

sperm (Hunt er , 1990) and a low er surviv al r at e is possibly the principle explanation

for the reduced capacity . P oly sperm y r efer s t o the penetr ation of m or e than one

sperm at ozoon int o the cyt oplasm of oocyt es (Hunt er , 1991), w hich can result in a

polyploid zy got e, a condition that result s in abnorm al em bryonic developm ent

(Birkhead et al., 1993). T her efore, the present st udy w a s undert aken t o det ermine the

w het her incubation of frozen - thaw ed boar sperm w ith x anthine and/ or x anthine

oxida se in m edium w it h or w ithout cat alase w ould allow in vitr o fertilization .

M A T E RIA L S A N D M E T H OD S

Oo cy t e P re paration

P orcine ovaries w er e collect ed fr om a local slaught er - hou se and kept in saline

(NaCl, 0.9% W/ V ; P enicillin 100,000 IU/ L ; St rept omycin 100m g/ L and Amphot ericin

B 250㎍/ L ; Sigm a Chemical, St - Louis, MO, USA ) at 30 t o 32℃. Cumulu s - oocyt es

complex es w er e aspirat ed from 2 t o 6 m m follicles w ith a 10- ml syringe with an

18- G needle. T he collect ed oocyt es w er e w ashed three tim es in Hepes - buffer ed

T yrode ' s m edium (T LH ) and once in m aturat ion m edium , oocyt es w it h a compact

and complet e cumulu s cells w ere intr oduced t o droplet s of m atur ation m edium (10

oocyt es/ 50㎕ droplet ), covered with m iner al oil and w er e cultur ed under an

at m ospher e of 5% CO

in air at 39℃ for 42- 44 h . T he m atur ation m edium con sist ed

of T CM - 199 with Earle ' s salt s (Gibco Lab ., NY, USA ) supplem ent ed w it h 3.05mM

glucose, 0.32mM Ca - lact at e, 2.5mM Hepes (Sigm a ), 10% fet al calf serum (F CS ), 0.2

mM Na - pyruv at e (Sigm a ), 50㎍/ ml gent am ycin (Sigm a ), 1㎍/ ml F SH (Sigm a ), 5㎍/ ml

LH (Sigm a ), 1㎍/ m l est radiol 17β(Sigm a ) and 10% (v/ v ) por cine follicular fluid.

S perm P reparation

P ool of ej aculat es from boar w er e fr ozen , the straw s w er e thaw ed by imm er sion

in a 35- 37℃ w at erbat h for 30 seconds . T haw ed sperm at ozoa w er e dilut ed with 2ml

of BT S (Belt sville T hawing S olution ) and equilibrat ed in air - tight tubes at 37℃ in a

w at erbath for 10 m inut es . Aft er equilibration , the 2m l sem en w ere placed ov er 2

layer s of per coll (65 and 70% ) and centrifuged at 2000×g for 15 minut es . T he

sperm at ozoa in the 65% percoll layer w ere carefully collect ed, w ashed in

preincubation m edium by su spension and centrifugat ion t w o tim es 250×g for 10

minut es and r esu spended in preincubation m edium . Aft er t he final w a sh , the

concentr ation of m otile sperm at ozoa w as adju st ed t o 25×10

. T he m edium for

fertilization and sperm preincubation w as T CM - 199 supplem ent ed w ith 3 mM

glucose, 3 m M Ca - lact at e, 0.2 mM Na - pyruvat e and 10% F CS . T he final

concentr ation of sperm at ozoa w as adju st ed t o 1×10

cells/ ml m ot ile sperm at ozoa

during fert ilizat ion in vitr o.

E x perim e nt al D e s ig n

In the fir st experim ent , su spen sion s of sperm at ozoa w er e added t o a droplet of

50μl fertilization m edium with or without cat alase (0.1m g/ m l, Sigm a ). Sperm at ozoa

w ere pr eincubat ed for 0, 30 and 60 min then m ix ed with fiv e oocyt es each for

fertilization in vit ro. In the second experim ent , t o evaluat e the effect of x anthine and

cat alase on penetr ation in vit ro, sperm at ozoa w er e pr eincubat ed in 50μl fertilization

m edium w ith or without cat alase in the pr esence of x anthine (0.7m g/ ml) for 0, 30

and 60 min , then mix ed w ith oocyt es . In the third experim ent al, the effect of cat alase

on in vitr o penetr ation of sperm at ozoa preincubat ed with x anthine oxida se (1m g/ m l)

for 0, 30 and 60 m in w as ex amined. In the final experim ent , t o ev aluat ed the effect s

of x anthine plu s x ant hine oxida se on sperm at ozoa penet rat ion during in vitro

fertilization , sperm at ozoa w ere pr eincubat ed in fertilization m edium with or w it hout

cat alase in the presence of x anthine plu s x ant hine oxidase for 0, 30 and 60 min , t hen

mix ed with oocyt es .

E v alu ation of Oo cy t e F ertiliz ation

At 20- 22 h aft er insemination, the oocyt es w ere m ount ed, fix ed (acet ic acid :

ethanol 1:3) for 2- 3 day s and st ained w it h 1% acet o- or cein in 40% acetic acid w at er

solut ion . T he proportion s of penetration and poly spermy w ere ex amined with the light

micr oscope at 200 and 400 × m agnification . Oocyt es w ere con sider ed as penet rat ed

w hen sperm at ozoa with a sw ollen head or pronuclei w er e found in t he vit ellus .

Oocyt es penetrat ed by only one sperm at ozoon w er e judged t o be m onospermic

oocyt es .

S t ati s tic s

Chi- squar e analy sis with the Yat es correction w as u sed t o t est the significance

of individual com parisons for the rat es of penetr ation and poly spermy .

RE S U LT S

A s show n in T able 1, oocyt es w er e inseminat ed w ith sperm at ozoa preincubat ed

in m edium w ith or without cat ala se for v ariou s dur ation . T he penetr ation s r at es w er e

significantly (P < 0.05) higher in sperm at ozoa preincubat ed without (66 and 38% for 0

and 30 min ) than w ith (40 and 15% for 0 and 30 min ) cat alase . T he proport ion s of

poly spermy had a t endency t o decrease a s tim e of sperm preincubation w a s

prolonged. No poly sperm y w as observ ed w hen sperm at ozoa w ere pr eincubat ed for 60

min .

T he oocyt es w ere in seminat ed with sperm at ozoa pr eincubat ed in m edium w ith

or w it hout cat ala se in the presence of x anthine. T able 2 show s that the penetration

r at es w er e significantly (P < 0.05) higher in sperm at ozoa pr eincubat ed w it h (68, 70 and

49% ) than without (33, 41 and 19% ) cat ala se for 0, 30 and 60 m in . T he pr oportion s

of poly spermy w ere low er in the absence of cat alase r egar dless of preincubation

duration , not significantly different in m edium with or w ithout cat ala se.

A s show n in T able 3, oocyt es cultur ed w ith sperm at ozoa preincubat ed with or

w ithout cat ala se in the presence of x ant hine oxida se. T he penetration r at es had a

t edency t o decr ease as tim e of sperm preincubation w as prolonged. W hen

sperm at ozoa w er e not preincubat ed, the penetr ation r at e w as significantly (P < 0.01)

higher in m edium w ithout cat alase (51% ) than with cat ala se (13% ). How ev er ,

P enetr ation rat es in sperm at ozoa preincubat ed for 30 and 60 min w ere not

significantly differ ent . On the other hand, poly spermy rat es w er e v ery low , but not

significantly different in m edium w ith or w it hout cat ala se during fertilization .

In another experim ent , oocyt es w ere in sem inat ed w ith sperm at ozoa pr eincubat ed

in m edium w ith x ant hine plu s x anthine oxida se in the presence or absence of

cat alase for 0, 30 and 60 min . T able 4 show s that the penetr ation rat es w ere

significantly (P < 0.001) higher in sperm at ozoa preincubat ed with than without cat alase

for variou s periods . T he proportion s of poly spermy w er e also higher in the pr esence

of cat ala se at 0, 30 and 60 min (28 v s 0%, 17 v s 0% and 20 v s 0%, respectiv ely ),

but no differences w er e observ ed in durat ion s of sperm at ozoa preincubation .

D IS CU S S ION

Capacit at ion is now consider ed by m ost investigat or s as an event or a series of

ev ent s preceding the acrosom e r eaction (Bavist er , 1986). T o det erm ine t he t im e

r equir ed for capacit ation of m am m alian sperm at ozoa , they ar e fir st preincubat ed in an

appr opr oat e m edium and ex amined t o see whether or not acrosom e reaction is

induced and penet rat ion has occurred. T her e have been pr eviou s report s of

sperm at ozoa pr eincubation on fert ilizat ion in por cine (Nagai & Moor , 1990 ; P ark &

Sir ar d, 1996). T he present r esult s indicat e t hat preincubation of frozen - thaw ed

sperm at ozoa w ith cat alase for 0- 60 min (T able 1) is not helpful and result s in low

penetration rat es (10- 40% ) of porcine oocyt es in m edium w ith cat alase . Sperm

capacit ation induced by biological fluids is also pr ev ent ed by super oxide dismut ase

(De Lamirande & Gagnon , 1993). Bize et al. (1991) pr oposed t hat H

O

is inv olved in

ham st er sperm capacit at ion . Cat alase, but not super oxide dismut a se, strongly reduces

the r at e of acr osom e reaction of sperm at ozoa incubat ed for 5 h in the presence of

adr enaline, a subst ance known t o st im ulat e this pr ocess, but also t o gener at e H

O

w hen kept in aerobic condition s . F urtherm or e, addition of H

O

, either dir ectly or

through enzym at ic generation by the combination of glucose and glucose oxida se,

st im ulat es t he acr osom e reaction by 85- 150%, depending on the t im e of ob servation

(Bize et al., 1991). Addition of cat alase t o hum an sperm at ozoa incubat ed in B2

Menezo m edium r educes both the hyper activ ation (by 43% ) and the A 23187- induced

acrosom e reaction (by 46% ) w ithout affecting t he percent age of m otile or viable cells

(Griv eau et al., 1994).

T he spont aneou s form ation of reactive oxygen species by cells present in sem en

has been as sociat ed with r educed sperm m ot ilit y (Alv ar ez et al., 1987 ; Iw asaki and

Gagnon , 1992)), abnorm al sperm m orphology (Rao et al., 1989)), decrea sed

sperm - oocyt es int eraction (Aitken et al., 1989)), reduced fertility in vivo (Ait ken et

al., 1991). In the present study , when sperm at ozoa w er e pr eincubat ed and in seminat ed

in m edium w ith x anthine, the rat es of sperm penet rat ion decrea sed in the absence of

cat alase (T able 2). T o the contrary , penetr ation rat es in m edium w it h x anthine

oxida se w er e higher in pr esence of cat alase regardless of duration of sperm at ozoa

preincubation (T able 3). T he x anthine plu s x anthine oxidase sy st em known t o

produce reactive oxygen species that are involved in cellular degradation in several

cell types , including sperm at ozoa . In mice, t he combination of x anthine plu s x ant hine

oxida se cau se a significant incr ease in sperm hyper activ ation and capacit ation (De

Lamirande et al., 1997). Individually , superoxide dismut a se or cat ala se com plet ely

prev ent these effect s, w herea s t ogether they decr ease capacit ation t o r at es ev en low er

than those observ ed in contr ol sperm at ozoa . T hese result s suggest that , under these

condition s, O

and H

O

m ay be needed t o pr om ot e m ou se sperm hyperactivation

and capacit ation . How ever , this study show ed that reactive oxygen species can hav e

beneficial effect s on sperm function s cam e from experim ent s in which sperm at ozoa

that w ere incubat ed w it h x anthine plu s x anthine oxidase in the pr esence of cat ala se

(T able 4). T here ar e pr obably m any differ ent capacit ation inducer s in the v ariou s

biological fluids but the dat a pr esent ed in this study suggest that they m ay all act

through a comm on m echanism , pos sibly by prom oting O

generation at the level of

the sperm m embrane.

T he high incidence of poly sperm y in porcine oocyt es m atur ed in vitro hav e been

r epeat edly r eport ed by m any inv estigat or s (W ang et al., 1991 ; Suzuki et al., 1994 ;

P ark & Sir ar d, 1996). In t he pr esent study , the proport ion s of oocyt es penetrat ed with

m or e than one sperm w ere low (0- 37% ) than previou s study (0- 63% ) by

sperm at ozoa preincubat ed with oviduct al cells (P ark & Sirar d, 1996). Since a high

proport ion of the oocyt es in sem inat ed w ith sperm at ozoa preincubat ed w ith x anthine

plu s x anthine oxidase for 0- 60 min w ere poly sperm ic in the m edium with (17- 28% )

v er su s without cat ala se (0% ), it is believ ed t hat cat alase dose hav e a role for

inducing capacit ation and penetr ation of porcine sperm at ozoa in the pr esence of

x anthine and x anthine oxidase. Park et al. (1997) r eport ed that poly spermy r at es w ere

high bet w een in m edium with (22- 40% ) and w it hout (40% ) superoxide dismut a se, but

no differ ence. In our study , when oocyt es w er e in seminat ed in the presence of

x anthine, although not significantly , the poly spermy rat es w er e below t he rat es

observ ed w ithout (21, 10 and 0% ) the w ith (37, 20 and 13% ) cat alase in sperm at ozoa

preincubat ed for 0, 30 and 60 min . On t he other hand, no differ ences w er e observ ed

in poly sperm y r at es in t he pr esence of x anthine oxidase. It seem s that cat alase can

complet e adv ant ageously w ith x anthine plu s x anthine oxidase t o induce high

fertilization rat es and m oder at e poly sperm y . T his property should allow a further

r eduction of the num ber of sperm t o be added at fert ilizat ion . How ev er , t her e ar e no

r eport s t o dat e showing the penetrability of por cine oocyt es in m edium with x anthine

and/ or x anthine oxida se. F uture st udies should be aim ed at dem on str ating how high

poly spermy r at es r educe by sperm at ozoa treat ed in the presence of r eactiv e oxygen

species .

In conclusion , experim ent al approaches of this study w er e u sed t o dem on str at e

the adv ant age of the preincubation with x anthine plu s x anthine oxidase in the

presence of cat ala se t o m aint ain penetration pot ent ial w ith suppr ess in the

poly spermy r at es during in vitro fertilization in por cine .

T his study w a s support ed by Kor ea S cience & Engineering F oundation

(971- 0606- 048- 1). F or providing frozen sem en, the aut hor s thank Dr . J .H . Lee and

I.C. Kim of Departm ent of Liv est ock Impr ovem ent , National Liv est ock Resear ch

Institut e, Korea .

S U M M A RY

T he obj ectiv e of this study w as t o t est t he effect of cat alase on penetr ation in

vit ro by sperm at ozoa pr eincubat ed with x anthine and/ or x anthine oxida se. T he

penetration rat es w ere significantly (P < 0.05) higher in sperm at ozoa pr eincubat ed

w ithout (66 and 38% ) than with (40 and 15% ) cat alase for 0 and 30 m in . When

sperm at ozoa w er e pr eincubat ed and in seminat ed in m edium with x anthine, the

penetration r at es w er e significantly higher (P < 0.05) in m edium with (68, 70 and 49%

for 0, 30 and 60m in ) than w it hout (33, 41 and 19% for 0, 30 and 60m in ) cat alase .

How ev er , in oocyt es w ere in sem inat ed w ith sperm at ozoa pr eincubat ed w it h or w ithout

cat alase in the pr esence of x anthine oxida se, no decrea se in penet rat ion s r at es w er e

observ ed for up t o 60 min of pr eincubat ion . In another experim ent , the penetration

r at es w ere significantly (P < 0.001) higher in m edium with (75, 55 and 52% ) than

w ithout (14, 4 and 8% ) cat ala se w hen oocyt es w ere in seminat ed w it h sperm at ozoa

preincubat ed for 0, 30 and 60 min in the pr esence of x anthine plus x anthine oxida se.

On the other hand, T he r at e of poly spermy in oocyt es penet rat ed in m edium without

cat alase in the presence of x anthine or x anthine plu s x anthine oxidase decreased w ith

tim e of sperm at ozoa pr eincubat ion . How ev er , no differences w er e ob serv ed in

poly spermy rat es in the m edium with x anthine oxidase alone despit e presence of

cat alase. T hese result s indicat e the advant ages of sperm at ozoa pr eincubat ed w it h

x anthine plu s x ant hine oxida se in the presence of cat alase t o incr ease penetration

pot ent ial and with suppres sed poly sperm y in por cine.

RE F E RE N CE S

Aitken RJ , Clark son JS : Cellular basis of defectiv e sperm function and it s a ssociation

with the genesis of r eact iv e oxygen species by hum an sperm at ozoa . J Reprod

F ertil 1987, 81, 459- 469.

Aitken RJ, Clark son JS , F ishel S : Generation of r eactiv e oxygen species, lipid

per oxidation and hum an sperm function . Biol Reprod 1989, 40, 183- 197.

Aitken RJ , Irvine DS , W u F C: Pr ospect iv e analy sis of sperm - oocyt e fusion and

reactive oxygen species gener ation as crit eria for the diagnosis of infertility . Am J

Obst et Gynecol 1991, 164, 542- 551.

Aitken RJ , Buckingham DW , Harkiss D: U se of x ant hine oxidase fr ee r adical

generating sy st em t o inv est igat e the cyt ot oxic effect s of r eactiv e oxygen species

on hum an sperm at ozoa . J Reprod F ertil 1993, 97, 441- 450.

Alv arez JG, T ouchst one JC, Blasco L, St or ey BT : Spont aneou s lipid peroxidation and

pr oduction of hydr ogen peroxide and superoxide in hum an sperm at ozoa . Superoxide

dism ut ase as m ajor enzym e prot ect ant against oxygen t oxicity . J Androl 1987, 8,

338- 348.

Alv arez JG, St orey BT : Role of glut athione per oxidase in pr ot ecting m amm alian

sperm at ozoa from loss of m otility cau sed by spont aneou s lipid per oxidat ion .

Gam et e Res 1989, 23, 77- 90.

Bavist er BD: Anim al in - vitro fertilization and em bryo developm ent . In Gw atkin ,

R .B .L . (ed), Manipulation of Mamm alian Dev elopm ent : Plenum Pr ess, New York,

1986, 81- 148.

Birkhead T R, Moller AP , Sutherland W J : W hy do fem ales m ake it so difficult for

m ales t o fertilize their egg s ? J T heor Biol 1993, 161, 51- 60.

Bize I, S ant ander G, Cabello P , Driscoll D, Sharpe C: Hydrogen peroxide is inv olv ed

in ham st er sperm capacit ation in vit ro. Biol Reprod 1991, 44, 398- 403.

Com port i M : T hr ee m odels of free radical induced cell injury . Chem ical Biological

Int er action s 1989, 72, 1- 56.

De Lam ir ande E , Gagnon C: Reactive oxygen species and hum an sperm at ozoa Ⅱ.

Depletion of adenosine triphosphat e play s an im port ant role in the inhibition of

sperm m otility . J Andr ol 1992, 13, 379- 386.

De Lamirande E , Gagnon C: Hum an sperm hyper activ ation and capacit ation a s part s

of an oxidative proces s . F ree Rad Biol Med 1993, 14, 157- 163.

De Lamirande E , Jiang H , Zini A , Kom ada H , Gagnon C: React iv e oxygen species

and sperm phy siology . J Repr od F ertil 1997, 2, 48- 54.

Griv eau JF , Renard P , LeLannou D: An in vitr o pr om ot ing r ole for hy drogen peroxide

in hum an sperm capacit ation . Int J Andr o 1994, 17, 300- 307.

Griv eau JF , Dum ont E , Renar d P , Callegari JP , LeLannou D: Reactive oxygen species ,

lipid peroxidation and enzym at ic defence sy st em s in hum an sperm at ozoa . J Repr od

F ertil 1995, 103, 17- 20.

Hindshaw DB, Sklar LA , Bohl B : Cyt oskelet al and m orphologic impact of cellular

oxidant injury . Am J P athology 1986, 123, 454- 464.

Hunt er RHF : F ert ilizat ion of pig egg s in viv o and in vitro. J Reprod F ertil 1990, 40,

211- 226.

Hunt er RHF : Oviduct function in pig s , w it h particular r efer ence t o the pathological

condition of poly spermy . Mol Reprod Dev 1991, 29, 385- 391.

Iw asaki A , Gagnon C: F orm ation of r eactiv e oxy gen species in sperm at ozoa of

infertile m en . F ertil St eril 1992, 57, 409- 418.

Nagai T , Moor RM : Effect of oviduct cells on the incidence of poly spermy in the pig

egg s fertilized in vitr o. Mol Repr od Dev elop 1990, 26, 377- 382.

P ark CK, Sirard MA : T he effect of preincubation of frozen - thaw ed sperm at ozoa w it h

oviduct al cells on the in vit ro penet rat ion of porcine oocyt es . T heriogenology 1996,

46, 1181- 1190.

P ark CK, Lee JH , Cheong HT , Yang BK , Kim CI: Effect of super oxide dismut a se

(SOD) on pronucleu s form ation of por cine oocyt es fertilized in vitr o.

T heriogenology 1997, 48, 1137- 1146.

Rao B, S oufir JC, Martin M , David G: Lipid per oxidation in hum an sperm at ozoa as

relat ed t o midpiece abnorm alities and m otility . Gam et e Res 1989, 24, 127- 134.

Suzuki K , Mori T , Shim izu H : In vitro fertilization of porcine oocyt es in t he

chemically defined m edium . T heriogenology 1994, 42, 1357- 1368.

W ang WH , Niw a K , Okuda K : In - vitr o penetr ation of pig oocyt es m atured in cultur e

by fr ozen - t haw ed ejaculat ed sperm at ozoa . J Reprod F ertil 1991, 93, 491- 496.

W indsor DP , Whit e IG, Selley ML, Sw an MA : Effect s of the lipid peroxidation

pr oduct (E )- 4- hydroxy - 2- nonenal on ram sperm function . J Repr od F ertil 1993, 99,

359- 366.

T able 1. E ffect of cat ala s e on p en etr at ion in v itr o by sper m at ozoa pr ein cu b at ed for v ariou s per iod s in fer t ilizat ion m edium

P er iods of P r e sen ce of N o. of N o. of oocy t es pen etr at ed w it h N o. of sperm at ozoa cat ala se oocy t es T ot al en lar g ed b ot h p oly sper m ic pr ein cub at ion (m in ) (0.1m g/ m l) ex am in ed (% ) sper m h ead pr on u clei oocy t es (% )†

0 ＋ 63 25 (40)＊ 25 0 6 (24 )

－ 67 44 (66) 39 3 15 (34 )

30 ＋ 68 10 (15)＊ 10 0 1(10)

－ 55 21(38) 19 2 2 (10)

60 ＋ 87 9 (10) 9 0 0 ( 0)

－ 68 14 (21) 13 1 0 ( 0)

† P er centag e of t ot al num ber of oocyt es penetr at ed

＊ P < 0.05, differ en ces bet w een w ith an d w ith out catalase

T able 2. T h e r ole of cat ala se on p en et r at ion in v it r o b y sp er m at ozoa pr ein cu b at ed for v ar iou s du r ation in fer t ilizat ion m edium w it h x ant hin e

P er iods of P r e sen ce of N o. of N o. of oocy t es pen etr at ed w it h N o. of sperm at ozoa cat ala se oocy t es T ot al en lar g ed b oth p oly sper m ic pr ein cub at ion (m in ) (0.1m g/ m l) ex am in ed (% ) sper m h ead pr on u clei oocy t es (% )†

0 ＋ 56 38 (68)＊ 31 7 14 (37 )

－ 43 14 (33) 14 0 3 (21)

30 ＋ 57 40 (70)＊ 29 11 8 (20)

－ 51 21(41) 20 1 2 (10)

60 ＋ 49 24 (49)＊ 2 1 3 (13 )

－ 47 9 (19) 7 2 0 ( 0)

† P er centag e of t ot al num ber of oocyt es penetr aqt ed

＊ P < 0.05, differ en ces bet w een w ith an d w ith out catalase

T able 3. E ffect of cat ala s e on p en etr at ion in v itr o by sper m at ozoa pr ein cu b at ed for v ariou s du r ation in fer t ilizat ion m edium w it h x ant hin e ox ida se

P er iods of P r e sen ce of N o. of N o. of oocy t es pen etr at ed w it h N o. of sperm at ozoa cat ala se oocy t es T ot al en lar g ed b ot h p oly sperm ic pr ein cub at ion (m in ) (0.1m g/ m l) ex am in ed (% ) sper m h ead pr on u clei oocy t es (% )†

0 ＋ 75 10 (13)＊ 10 0 1(10)

－ 65 33 (51) 31 2 4 (12)

30 ＋ 79 8 (10) 8 0 0 ( 0)

－ 66 14 (21) 14 0 1( 7 )

60 ＋ 83 7 ( 8) 7 0 1(14 )

－ 67 12 (18) 10 2 1( 8 )

† P er centag e of t ot al num ber of oocyt es penetr at ed

＊ P < 0.01, differ en ces bet w een w ith an d w ith out catalase

T able 4. E ffect of cat ala s e on p en etr at ion in v itr o by sper m at ozoa pr ein cu b at ed for v ariou s du r ation in fer t ilizat ion m edium w it h x ant hin e plu s x ant hin e ox ida se

P er iods of P r e sen ce of N o. of N o. of oocy t es pen etr at ed w it h N o. of sperm at ozoa cat ala se oocy t es T ot al en lar g ed b ot h p oly sperm ic pr ein cub at ion (m in ) (0.1m g/ m l) ex am in ed (% ) sper m h ead pr on u clei oocy t es (% )†

0 ＋ 53 40 (75 )＊ 29 11 11(28 )

－ 57 8 (14 ) 8 0 0( 0)

30 ＋ 53 29 (55 )＊ 24 5 5 (17 )

－ 55 2 ( 4 ) 2 0 0( 0)

60 ＋ 57 30 (52)＊ 30 0 6 (20)

－ 59 4 ( 8 ) 4 0 0( 0)

† P er centag e of t ot al num ber of oocyt es penetr at ed

＊ P < 0.001, differ ences bet w een w ith an d w ith out cat alase