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thawed bovine in vitro matured oocytes
Theriogenology
39:304,
1993
ULTBASTRUCl-URE OF FROZEN/THAWED BOVINE IN VITRO MATURED ODCYTES M. Schmidt, P.Hyttel’, T.Greve and B.Avery Dep,artment of Clinical Studies, Reproduction and Department of Anatomy and Physiology. Royal Veterinary and Agricultural University, Biilowsvej 13, 1870 Frederiksberg C, Copenhagen, Denmark
Most studies have shown that freezing/thawing of mature bovine oocytes is detrimental for subsequent fertilization and development in vitro. The precise nature of the damages upon the oocytes caused by the freezing and thawing process is by and large unknown and it was the purpose of the present ultrastructural study to investigate possible subcellular damages inflicted by the cryopreservation. In vitro oocyte maturation (IVM) followed our standard procedure. Following IVM the oocytes were frozen/ thawed as follows: They were equilibrated for 15 min in PBS containing 10% fetal calf serum (FCS) and 10% glycerol and then loaded in 0.25 ml french straws. The straws were placed in an alcohol freezer directly at 5°C and seeded 10 min later. At that stage, 5 oocytes were thawed and rehydrated (see later) and processed for transmission electron microscopy (TEM). The remaining oocytes continued at a freezing rate at -0.4”C/min, and at -35°C the straws were plunged directly into liquid nitrogen. Thawing occured in a water bath at 37°C and the glycerol was removed in 1.O M sucrose in PBS containing 10% FCS for 8 min, after which another 5 oocytes were processed for TEM. Unfrozen IVM oocytes (n = 5) from the same batch were processed for TEM, as controls. In the unfrozen oocytes an abundance of small membrane bound vesicles were scattered throughout the ooplasma except peripherally, where a vesicle free zone was observed. In the seeded/thawed oocytes the vesicles had migrated peripherally in the ooplasm, had to some degree become confluent and displayed large variation in size. The frozen/thawed oocytes exhibited a high degree of heterogeneity in vesicle size and even more vesicles had become confluent. Again, no peripheral vesicle free zone was observed, and in three of the five oocytes the vesicle acculmulation peripherally in the ooplasm had resulted in rupture of the oolemma. In conclusion frozen/thawed oocytes display ultrastructural damages especially during the latter part of the freezing process, which is incompatible with normal fertilization and embryonic development. Supported
304
by the Animal
Biotechnology
Research
Centre.
Copyright (8 1993 Butterworth-Heinemann
39:304,
1993
ULTBASTRUCl-URE OF FROZEN/THAWED BOVINE IN VITRO MATURED ODCYTES M. Schmidt, P.Hyttel’, T.Greve and B.Avery Dep,artment of Clinical Studies, Reproduction and Department of Anatomy and Physiology. Royal Veterinary and Agricultural University, Biilowsvej 13, 1870 Frederiksberg C, Copenhagen, Denmark
Most studies have shown that freezing/thawing of mature bovine oocytes is detrimental for subsequent fertilization and development in vitro. The precise nature of the damages upon the oocytes caused by the freezing and thawing process is by and large unknown and it was the purpose of the present ultrastructural study to investigate possible subcellular damages inflicted by the cryopreservation. In vitro oocyte maturation (IVM) followed our standard procedure. Following IVM the oocytes were frozen/ thawed as follows: They were equilibrated for 15 min in PBS containing 10% fetal calf serum (FCS) and 10% glycerol and then loaded in 0.25 ml french straws. The straws were placed in an alcohol freezer directly at 5°C and seeded 10 min later. At that stage, 5 oocytes were thawed and rehydrated (see later) and processed for transmission electron microscopy (TEM). The remaining oocytes continued at a freezing rate at -0.4”C/min, and at -35°C the straws were plunged directly into liquid nitrogen. Thawing occured in a water bath at 37°C and the glycerol was removed in 1.O M sucrose in PBS containing 10% FCS for 8 min, after which another 5 oocytes were processed for TEM. Unfrozen IVM oocytes (n = 5) from the same batch were processed for TEM, as controls. In the unfrozen oocytes an abundance of small membrane bound vesicles were scattered throughout the ooplasma except peripherally, where a vesicle free zone was observed. In the seeded/thawed oocytes the vesicles had migrated peripherally in the ooplasm, had to some degree become confluent and displayed large variation in size. The frozen/thawed oocytes exhibited a high degree of heterogeneity in vesicle size and even more vesicles had become confluent. Again, no peripheral vesicle free zone was observed, and in three of the five oocytes the vesicle acculmulation peripherally in the ooplasm had resulted in rupture of the oolemma. In conclusion frozen/thawed oocytes display ultrastructural damages especially during the latter part of the freezing process, which is incompatible with normal fertilization and embryonic development. Supported
304
by the Animal
Biotechnology
Research
Centre.
Copyright (8 1993 Butterworth-Heinemann