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Bovine blastocyst production in vitro following inhibition of oocyte meiotic resumption for 24 hours
Theriogenology
293
BOVINE BLASTOCYST PRODUCTION IN VITRO FOLLOWING INHlBlTION OF OOCYTE MEIOTIC RESUMPTION FOR 24 HOURS P. Lonergan, H. Khatir, C. Carolan, P. Mermillod Station de Physiologie de la Reproduction, INRA, 37380 Nouzilly, France. The production of viable embryos in vitro plateaus out at 30-40% of matured oocytes. It would appear that inadequate cytoplasmic maturation underlies this developmental shortfall. A prematuration treatment may be necessary in order to allow oocytes from smaller follicles to ‘catch up’ with those from larger follicles or those matured totally in vivo. Such small follicles constitute the vast majority of the surface visible follicle population and removal of the oocyte may shorten the final phase of folliculogenesis. This necessitates a culture system that reproduces the ovarian follicular environment, in which nuclear maturation is prevented. The aims of this study were to assess the effect of various GVBD-inhibiting substances on meiotic resumption of bovine oocytes and then to examine the developmental potential of such blocked oocytes following removal of the inhibitory conditions and reinstatement of conditions conducive to normal IVM. Concentrations of inhibitors were chosen on the basis of preliminary dose response trials. Immature cumulus oocyte complexes (COCs) were cultured for 24 h in (a) Ml99 alone, or supplemented with (b) 10% FCS, (c) ll.tg/ml cycloheximide (CX), or (d) 2mM 6dimethylaminopurine (6-DMAP). Following 24h, groups (a) and (b) were inseminated with frozen-thawed sperm and subsequently cultured, while groups (c) and (d) were washed in PBS and cultured for a second 24 h period in M 199+FCS, following which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status. To test whether maintenance of meiotic arrest affected oocyte protein neosynthesis, COCs were labeled with [35S]-methionine at (a) 0 h, (b) following 21 h maturation in M199, (c) following 21 h culture in the presence of 2mM 6DMAP or ll.tg/ml CX, (d) following 24 h culture in the presence of 6-DMAP or CX and a second period of culture in M199+FCS. Data were analyzed by chi-square analysis. Incubation with 6-DMAP or CX completely blocked GVBD with most oocytes remaining at GV after 24 h culture (64/72, 89% and 54/54, lOO%, for 6-DMAP and CX, respectively). The inhibitory effect of both was fully reversible. Following a second period of incubation GVBD occured in almost all cases (6-DMAP: 104/105,99%; CX: 60/60, 100%). The majority of these oocytes reached metaphase II (6-DMAP: 89/105, 85%; CX: 50/60,83%). Cleavage rates and day 8 blastocyst yields were, respectively : (a) M199,72% (71198) and 34% (33/98); (b) +FCS, 80% (74/93) and 45% (42/93); (c) +CX, 81% (SWlO5) and 19% (20/105, PcO.05); (d) +6-DMAP, 77% (80/104) andl4% (15/104, PcO.05). In general, the pattern of protein neosynthesis observed was linked to the nuclear stage of the oocytes rather than to conditions of culture (although the latter dictated the former). 6DMAP did not modify methionine incorporation. However, CX completely blocked protein synthesis when present during the period of labeling. When CX was omitted from the labelling medium, blocked oocytes resumed protein synthesis in a manner similar to oocytes at the GV stage immediately after removal from the follicle. In conclusion, our results show that it is possible to reversibly inhibit meiotic resumption in bovine oocytes using CX or 6-DMAP, with over 80% of blocked oocytes reaching metaphase II after removal of the inhibitory conditions and about lo-20% developing to the blastocyst stage. These results give an indication of the feasability of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in competence aquisition. Improved knowledge of the mechanisms involved in vivo in the follicle will allow the establishment of techniques which better preserve the viability of the oocyte.
293
BOVINE BLASTOCYST PRODUCTION IN VITRO FOLLOWING INHlBlTION OF OOCYTE MEIOTIC RESUMPTION FOR 24 HOURS P. Lonergan, H. Khatir, C. Carolan, P. Mermillod Station de Physiologie de la Reproduction, INRA, 37380 Nouzilly, France. The production of viable embryos in vitro plateaus out at 30-40% of matured oocytes. It would appear that inadequate cytoplasmic maturation underlies this developmental shortfall. A prematuration treatment may be necessary in order to allow oocytes from smaller follicles to ‘catch up’ with those from larger follicles or those matured totally in vivo. Such small follicles constitute the vast majority of the surface visible follicle population and removal of the oocyte may shorten the final phase of folliculogenesis. This necessitates a culture system that reproduces the ovarian follicular environment, in which nuclear maturation is prevented. The aims of this study were to assess the effect of various GVBD-inhibiting substances on meiotic resumption of bovine oocytes and then to examine the developmental potential of such blocked oocytes following removal of the inhibitory conditions and reinstatement of conditions conducive to normal IVM. Concentrations of inhibitors were chosen on the basis of preliminary dose response trials. Immature cumulus oocyte complexes (COCs) were cultured for 24 h in (a) Ml99 alone, or supplemented with (b) 10% FCS, (c) ll.tg/ml cycloheximide (CX), or (d) 2mM 6dimethylaminopurine (6-DMAP). Following 24h, groups (a) and (b) were inseminated with frozen-thawed sperm and subsequently cultured, while groups (c) and (d) were washed in PBS and cultured for a second 24 h period in M 199+FCS, following which they were inseminated and cultured. At all time points a representative sample of oocytes were fixed and stained with orcein to observe the nuclear status. To test whether maintenance of meiotic arrest affected oocyte protein neosynthesis, COCs were labeled with [35S]-methionine at (a) 0 h, (b) following 21 h maturation in M199, (c) following 21 h culture in the presence of 2mM 6DMAP or ll.tg/ml CX, (d) following 24 h culture in the presence of 6-DMAP or CX and a second period of culture in M199+FCS. Data were analyzed by chi-square analysis. Incubation with 6-DMAP or CX completely blocked GVBD with most oocytes remaining at GV after 24 h culture (64/72, 89% and 54/54, lOO%, for 6-DMAP and CX, respectively). The inhibitory effect of both was fully reversible. Following a second period of incubation GVBD occured in almost all cases (6-DMAP: 104/105,99%; CX: 60/60, 100%). The majority of these oocytes reached metaphase II (6-DMAP: 89/105, 85%; CX: 50/60,83%). Cleavage rates and day 8 blastocyst yields were, respectively : (a) M199,72% (71198) and 34% (33/98); (b) +FCS, 80% (74/93) and 45% (42/93); (c) +CX, 81% (SWlO5) and 19% (20/105, PcO.05); (d) +6-DMAP, 77% (80/104) andl4% (15/104, PcO.05). In general, the pattern of protein neosynthesis observed was linked to the nuclear stage of the oocytes rather than to conditions of culture (although the latter dictated the former). 6DMAP did not modify methionine incorporation. However, CX completely blocked protein synthesis when present during the period of labeling. When CX was omitted from the labelling medium, blocked oocytes resumed protein synthesis in a manner similar to oocytes at the GV stage immediately after removal from the follicle. In conclusion, our results show that it is possible to reversibly inhibit meiotic resumption in bovine oocytes using CX or 6-DMAP, with over 80% of blocked oocytes reaching metaphase II after removal of the inhibitory conditions and about lo-20% developing to the blastocyst stage. These results give an indication of the feasability of in vitro meiotic inhibition as a tool in the study of the mechanisms involved in competence aquisition. Improved knowledge of the mechanisms involved in vivo in the follicle will allow the establishment of techniques which better preserve the viability of the oocyte.