Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences.

摘要

Oocytes were matured in vitro for 22 h and cryopreserved by vitrification. After warming and additional 2 h of culture, the oocytes, at the metaphase II (MII) stage, were inseminated in vitro. Oocytes were fixed for transmission electron microscopyimmediately after warming, at 4 h after warming (i.e., 2 h post insemination [hpi]), at 26 h after warming (24 hpi), and at 74 h after warming (72 hpi). Control (non-vitrified) oocytes were processed at 22 h after in vitro maturation and at 2, 22, and 72hpi. Compared to the controls, the vitrified oocytes fixed immediately after warming presented an additional category of small membrane-bound vesicles and lacked the typical compartment of solitary cortical granules aligned along the oolemma. Instead, they presented clusters of cortical granules that displayed varying degrees of degeneration. In vitrified oocytes fixed at 2 hpi, the small vesicles were less abundant, and more advanced degeneration of the cortical granule clusters was noted. In vitrified oocytes fixed at 24 hpi, the small vesicles were practically absent, and polyspermic penetration was observed as were vacuoles containing degraded cortical granule content. In vitrified oocytes fixed at 72 hpi, lack of cleavage as well as vacuolizationand degeneration of blastomeres were noted. Moreover, the nucleolar ultrastructure signalled aberrant activation of the ribosomal RNA genes. In conclusion, vitrification of bovine oocytes at the MII stage resulted in cell biological alterations in the oocyte after warming that apparently were reflected in the subsequent fertilization and embryonic development.