(Lonergan et al., 1994),

48 (6) :741 748, 2002 A cta Zoologica S inica 3 33 (, 650223) + dbcamp IBMX + dbcamp, -, 6 h ; 18 h,, : + dbcamp IBMX + dbcam 60 % GV,, GVBD M, camp IBMX,,, ; ;, ( Holm et al., 1994 ; McCaff rey et al., 1991 ; Nagashima et al., 1996 ; Schramm et al., 1994), 24 48 h,,,,, 2 6 mm 4 10 d 15 mm (Sirad et al., 1992) ;, 14 d (Calderon et al., 1993),,,, /, (Cognie et al., 1998), ( Sorensen (Lonergan et al., 1994 ; Pavlok et al., 1992),, ( Goudet et al., 1997) (Motlik et al., 1984) ( Schramm et al., 1993), (Lonergan et al., 1994),,, ( germinal vesicle, et al., 1976) (Jelinkova et al., 1994) GV),, 2001211201, 2002203204 3 G2000016108 KSCXI205202 33, E2mail : wji @mail. kiz. ac. cn,, 41,, : E2mail : xy z72 @hotmail. com

42 7 48,,, 2 h camp, : M199 ( Giboco BRL, ( Eppig et N Y, USA) 10 % (v/ v), FSH al., 1985) camp / L H 10 g/ ml ( Pergona, Serono), ( Shim et al., 1992 ; Xia, 1996) ( Tornell et al., 1990) (100 IU/ ml) (50 g/ ml) ( Terada et al., 1995) (Miyano et al., 1995) (Aktas et al., 1995) ( Warikoo et al., 1989) ( Tornell et al., 1993), camp VandeVoort (Jagiello et al., 1981), (University of California, Davis), Gomez et al.,, : 15 ml Percoll (95 % 45 %), 200 l Percoll dbcamp (dibutryryl cyclic AMP,, 1 500 g 15 min, Percoll ) ( hypoxanthine, HX) IBMX (isobutylmethylxantihine, ( HSOF, 1 ) ( Gomez et al., ) 1997), 600 g 6 min, HSOF 25 10 6 / 1 111, Sigma 1) ( Gomez et al., 1997),, 1 10 6 / ml 4 Nunc ( 104, 500 l BSOF, 15 20 ) 39 (DPBS), 5 %CO 2 39 100 % 2 h (400 IU/ 18 21 h ml) (200 g/ ml) DPBS, 113 Tyrode2Hepes - ( Tyrodeπs lactate2hepes Medium, TL2Hepes) (Bav2, ister et al., 1983) ( Gomez et al., 1997) 2 8 mm ( Gomez et al., 1997, Watson et al., 1994), 5 %CO 2 39 100 %, - (COCs) 8, 35 (OπBrien et al., 1996) TL2Hepes,,, 2 h COCs, 114 TL2Hepes : 1) :, COCs dbcamp) : 3 mmol/ L 5 %CO 2 39 100 % (Sigma, H9377) 110 mmol/ L dbcamp ( Sigma, 4 Nunc, 500 D0627) ; 3) IBMX dbcamp ( IBMX + db2 l, 25 40 COCs (OπBrien et al., 1997) 013 mmol/ L, 1 g/ ml ( Gomez et al., 1997 ; OπBrien et al., 1996 ; Watson et al., 1994) 37 112 (1997), 3 ml Hepes ml (37 ) (BSOF, ( modified synthetic oviduct fluid, MSOF, 1 ), 10 % MSOF mm Petri, 50 l MSOF, ; 2) dbcamp ( HX + camp) : 011 mmol/ L IBMX ( Sig2

6 : 743 ma, I7018) 110 mmol/ L dbcamp 11411 COCs Table 1 Formulations for main media, HX + dbcamp/ IBMX + dbcamp 6 h, Comporrents 1/ 4, (1 3),, ( GV), (, GVBD) 11412 6 h NaH 2 PO 4, COCs 18 h ( 24 h) ( COCs 1/ 3),, GV GVBD ( Metaphase, M ) (Anaphase, A ) ( Telophase, T ) (Metaphase, M ) (Lonergan et al., 1997) 11413 24 h,, MSOF: 18 21 h, ( ), bovine serum ( FBS) ], ( Gomez et al., 1997) ; 10 % ( T ) SOF, 8 d, dbcamp, IBMX dbcamp,, 35183 % 115 37116 % ( ANOVA,, P < 010001), L SD, ASIN 60 % GV P < 0105, P < 0101 (,, P < 010001),, M 2 1 8, 24 h, ( M 211 ) 71182 % HX + db2 2 6 h camp IBMX + dbcamp,, 82192 % 18 h,, ( GV ), 66122 % M, GVBD, ( 73172 % 74113 %), MSOF (mmol/ L) HSOF (mmol/ L) BSOF (mmol/ L) TL2Hepes (mmol/ L) NaCl 107170 107170 107170 127100 KCl 7116 7116 7116 3116 CaCl 2 2H 2 O 1171 1171 3142 2100 MgCl 2 6H 2 O 0149 0149 0149 0150 H 2 O 0139 0139 0139 0135 NaHCO 3 25100 4100 25100 2100 Hepes (Na Salt) 10150 5100 Hepes(Acid form) 10150 5100 ( Glucose) 2140 5100 (Na2lactate) 25100 2150 7156 10100 ( Pyruvate) 0132 0130 110 ( Glutamine) 0132 0130 110 ( IU/ ml) ( Penicillin) ( g/ ml) (Streptomycin) 100100 100100 100100 100100 50100 50100 50100 50100 BSA (mg/ ml) 3100 3100 ( modified synthetic oviduct fluid) HSOF : Hepes, 10 % [ HSOF buffered with Hepes and supplemented with 10 % of fetal BSOF :, 2 % [ BSOF buffered with bicarbonate and supplemented with 2 % of sheep serum ] (bovine serum album) BSA : 212

44 7 48 2 6 h Table 2 Meiotic progression of ovine oocytes cultured for 6 h with different inhibitory substances Treatment N GV ( % S E) GVBD ( % S E) M A T M > GV (Control) 86 14 (17108 10157) a 56 (66122 26171) a 12 2 2 0 72 (82192 10157) a HX + dbcamp 82 52 (64117 21195) b 28 (33133 22154) b 2 0 0 0 30 (35183 21195) b IBMX + dbcamp 81 51 (62184 14113) b 20 (24177 16108) b 10 0 0 0 30 (37116 14113) b N : (Nunnrt of oocytes cultured) GV : (Oocytes with germinal vesicle) GVBD : (Oocytes with GV breakdown) M : (Oocytes in Metaphase ) A : (Oocytes in Anaphase ) T : (Oocytes in Telephase ) M : (Oocytes in Metaphase ) > GV : GVBD T (All oocytes from stage of GVBD to T ) S E : (Standard error) ab :, [ within a column, values with different superscripts are significantly different, P < 01001 ] ( P > 0105), GV ( P > 0105), 4 ( P > 0105, ( P > 0105) 3) HX + dbcamp IBMX + dbcamp,,, ( 18 h M, 4, P > 0105),,, 213 3 Table 3 Meiotic progression after removal of inhibitory substances ( Treatment) N GV < M M n % S E n % S E n % S E (Control) 84 10 11159 8154 a 14 16159 4114 a 60 71182 13145 a HX + dbcamp 91 4 4118 3193 a 21 22110 9100 a 66 73172 11101 a IBMX + dbcamp 88 7 7139 6192 a 16 18144 9161 a 65 74117 13135 a N : (Number of oocytes cultured) n : (Number of developing to stage shown) < M : GVBD (All oocytes from stage of GVBD to T ) M : (Oocytes in Metaphase ) S E : (Standard error) a :, ( Within a column, values with same superscripts indicate no difference, P > 0105) 4 6 h Table 4 Oocyte Fertilization and development after suppression of meiotic resumption for 6h in culture N Treatment Normal fertilization Polyspermy % S E % S E N M + B Cleavage % S E % S E (Control) 80 64171 6166 (52 / 80) 8180 6107 (7 / 80) 86 61135 15163 (53 / 86) 27178 6186 (15 / 53) HX + dbcamp 81 61174 14138 (50 / 81) 8178 6144 (7 / 81) 86 63139 20110 (56 / 86) 27150 4131 (15 / 56) IBMX + dbcamp 82 58141 9131 (48 / 82) 7116 6158 (6 / 82) 87 55131 25109 (51 / 87) 22105 6122 (13 / 51) N : (Number of oocytes cultured) M + B : (Number of morula plus blastocyst)

6 : 745 3 (Moor et al., 1981),,,,,,, camp GVBD, ( ) ( Armst rong et al., 1997 ; Pugh et al., 1991), camp ( dbcamp 82Br2cAMP ), camp (OπBrien et al., 1996) camp ( Chen et al., 2001 ; Xia, 1996), camp,,,, ( Fulka et al., 1993),,, camp,,,, (Mullerian inhibiting substance, M IS) et al., 1986),, camp sterol, MAS ) ( Byskov et al., 1997 ;, camp Grondahl et al., 1998), GV ( Tsafriri et al., 1996), camp, camp,,, (Schultz et al., 1977) dbcamp camp,, HX + dbcamp, IBMX + dbcamp camp IBMX, camp camp, ( 4), GVBD camp (Downs et al., 1989),, 24 h 6 8 h GVBD M ( Moor et al., 1985), (Moor et al., 1985),,, 6 h, HX IBMX dbcamp, 18 h M GV ( ) (DMAP), 6 h, HX + dbcamp IB2 MX + dbcamp 2000 ; Moor et al., 1985),, 60 % Anderiesz et al. GV, Jagiello et al. ( Eppig et al., 1985), camp (Jagiello et al., 1981),, ( Takahashi, ( meiosis2activating,,, (Avery et al., 1998) ( Anderiesz et al., (2000), (1981) DMAP,,,,

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48 7 48 ( Abstract) D EVELOPMENTAL COMPETENCE OF OVINE OOCYTES AFTER IN VI TRO INHIBITION OF MEIOTIC RESUMPTION 3 ZHOU Hong2Lin MA J un J I Wei2Zhi 33 ( Kunmi ng Instit ute of Zoology, Chi nese Academy of Sciences, Kunmi ng 650223, Chi na) The aims of the present study were to assess the effect of various inhibitors (hypoxanthine + dbcamp or IBMX + dbcam P) on meiotic resumption and subsequent develop ment to t he blastocyst stage of ovine oocytes. Ovine ovaries were acquired f rom a local abattoir and t he immat ure cumulus2oocyte complexes ( COC) were dis2 sected and cultured for 6 h in 1) Control : M199 supplemented with 10 % fetal calf serum ; 2) HX 6 h : dbcamp 110 mm + hypoxanthine ( HX) 3 mm ; 3) IBMX 6 h : dbcamp 110 mm + IBMX 011 mm. Subsequently, the oocytes from different treatment groups were washed and cultured for an additional 18 h in fresh M199 + FCS. Then, the oocytes were inseminated with thawed spermatozoa and cultured. At different periods, a representa2 tive sample of the oocytes was fixed and stained with carmine to observe the nuclear status. The results showed : 1) bot h hypoxant hine + dbcam P and IBMX + dbcam P could prevent over 60 % of oocytes f rom undergoing meiosis maturation after 6 h culture with an inhibitor ; 2) there was no significant difference in the maturation rate of oocytes among the three groups (71182 %, 73172 % and 74113 % respectively) after the additional 18 h culture, so the initial inhibitory effect was reversible and the timing of polar body extrusion was accelerated fol2 lowing the withdrawal of the inhibitors ; 3) since the fertilization rate (64171 %, 61174 % and 58141 % respec2 tively), cleavage rate (61135 %, 63139 % and 55131 % respectively) and blastocyst yields on day 8 of culture (27178 %, 27150 % and 22105 % respectively) were not significantly different among t reat ment s we conclude t hat ovine oocyte develop ment competence i n vit ro was not affected after meiotic resumption had been inhibited for 6 h. The results suggested that meiotic inhibition in vit ro could be a tool in the study of the mechanisms in2 volved in t he acquisition of meiotic and develop mental competence in ovine oocytes. Key words Ovine, Meiotic Maturation, Oocyte development competence, camp, Hypoxanthine, IBMX, In vit ro 3 This work was sponsored by National Basic Research Priorities Program ( Grant No. G2000016108) and the Innovation Knowledge Project of Chinese Academy of Sciences (No. KSCX1205202). 33 Corresponding author. wji @mail. kiz. ac. cn