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Effect of protein synthesis inhibition for varying periods before or during in vitro maturation on development of bovine oocytes
290
Theriogendogy
EFFECT OF PROTEIN SYNTHES IS INHIBITION FOR VARYING PERIODS BEFORE OR DURING IN VII-R0 MATURATION ON DEVELOPMENT OF BOVINE OGCYI-ES P. Lonergan, H. Khatir and P. Mermillod INRA-PRMD, 37380 Nouzilly, France We have recently shown that it is possible to reversibly inhibit meiotic resumption in bovine oocytes for 24 h using cycloheximide (CX), an inhibitor of protein synthesis, with over 80% of blocked oocytes reaching metaphase II after removal of the inhibitory conditions and about 20% developing to the blastocyst stage (Lonergan et al., 1997; J Reprod Fert, 105: 355365). While encouraging, these developmental rates are still inferior to those obtained with unblocked oocytes. The aim of the work presented here was to extend these results by examining the effect of inhibition of meiotic resumption for varying periods after initiation of culture (6, 12, 18,24 h) by protein synthesis inhibition on subsequent oocyte development to identify at what point development becomes compromised. In addition, the effect of inhibiting protein synthesis for a short period (6 h) at various times during the process of maturation was examined. Development rates were analyzed by Chi-square and cell number by Student’s t test. In exneriment 1, immature cumulus oocyte complexes (n=l156) were randomly allocated to one of the following treatment groups: (1) Ml99 alone; (2) +lO% FCS; (3) +lO pg CX ml-l 6h; (4) +CX 12 h; (5) +CX 18 h; (6) +CX 24 h; (7) +CX+FCS 24 h. For the control groups (1) and (2), following 24 h culture COCs were inseminated and cultured using standard techniques. For groups (3)-(7), following incubation for the specified times in the presence of CX, COCs were washed to remove residual CX, before being cultured for a further 24 h in M199+10% FCS. In experiment 2, CGCs (n=l117) were randomly allocated to one of the following treatment groups: (1) M199+10% FCS 24 h; (2) +lO pg CX ml-l 6 h, +FCS 24 h; (3) +FCS 6 h, +CX 6 h, -+FCS 18 h; (4) +FCS 12 h, +CX 6 h, +FCS 12 h; (5) +FCS 18 h, +CX 6 h, +FCS 6 h; (6) +FCS 30 h. Oocytes in group (1) were inseminated 24 h after initiation of culture, while all other groups were inseminated 30 h after initiation of culture. Thus, all groups spent 24 h in conditions permissive to normal maturation (M199+10% FCS). The presence of FCS during in vitro maturation resulted in a significantly higher cleavage rate (90 vs 73%, PcO.O002), % 5-8 cells (64 vs 47%, P
Theriogendogy
EFFECT OF PROTEIN SYNTHES IS INHIBITION FOR VARYING PERIODS BEFORE OR DURING IN VII-R0 MATURATION ON DEVELOPMENT OF BOVINE OGCYI-ES P. Lonergan, H. Khatir and P. Mermillod INRA-PRMD, 37380 Nouzilly, France We have recently shown that it is possible to reversibly inhibit meiotic resumption in bovine oocytes for 24 h using cycloheximide (CX), an inhibitor of protein synthesis, with over 80% of blocked oocytes reaching metaphase II after removal of the inhibitory conditions and about 20% developing to the blastocyst stage (Lonergan et al., 1997; J Reprod Fert, 105: 355365). While encouraging, these developmental rates are still inferior to those obtained with unblocked oocytes. The aim of the work presented here was to extend these results by examining the effect of inhibition of meiotic resumption for varying periods after initiation of culture (6, 12, 18,24 h) by protein synthesis inhibition on subsequent oocyte development to identify at what point development becomes compromised. In addition, the effect of inhibiting protein synthesis for a short period (6 h) at various times during the process of maturation was examined. Development rates were analyzed by Chi-square and cell number by Student’s t test. In exneriment 1, immature cumulus oocyte complexes (n=l156) were randomly allocated to one of the following treatment groups: (1) Ml99 alone; (2) +lO% FCS; (3) +lO pg CX ml-l 6h; (4) +CX 12 h; (5) +CX 18 h; (6) +CX 24 h; (7) +CX+FCS 24 h. For the control groups (1) and (2), following 24 h culture COCs were inseminated and cultured using standard techniques. For groups (3)-(7), following incubation for the specified times in the presence of CX, COCs were washed to remove residual CX, before being cultured for a further 24 h in M199+10% FCS. In experiment 2, CGCs (n=l117) were randomly allocated to one of the following treatment groups: (1) M199+10% FCS 24 h; (2) +lO pg CX ml-l 6 h, +FCS 24 h; (3) +FCS 6 h, +CX 6 h, -+FCS 18 h; (4) +FCS 12 h, +CX 6 h, +FCS 12 h; (5) +FCS 18 h, +CX 6 h, +FCS 6 h; (6) +FCS 30 h. Oocytes in group (1) were inseminated 24 h after initiation of culture, while all other groups were inseminated 30 h after initiation of culture. Thus, all groups spent 24 h in conditions permissive to normal maturation (M199+10% FCS). The presence of FCS during in vitro maturation resulted in a significantly higher cleavage rate (90 vs 73%, PcO.O002), % 5-8 cells (64 vs 47%, P