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In vitro maturation of sheep ovarian oocytes.

An in vitro system has been developed which induces full meiotic maturation in 98% ovarian sheep oocytes isolated from follicles 2-6 mm in diameter. 45.7% of these were fertilized, determined by the presence of two pronuclei, extrusion of the second polar body and the presence of the sperm flagellum. This culture system was used to describe the morphological changes during meiotic maturation, examining the nucleus, the cytoplasm and cumulus (corona)-oocyte relationship. 24 h are required for maturation of sheep oocytes. The culture medium must contain FSH, LH (10 micrograms/ml of each), estradiol-17 beta (1 micrograms/ml) and coculture of 10(6) mural granulosa cells in suspension (Crozet et al., 1987). Nuclear changes were the first evident transformations, showing that chromatin condensation leads to nuclear deformation, to germinal vesicle breakdown and to formation of the first and second meiotic metaphases. The axis of both spindles are oriented perpendicularly to the egg membrane. At each pole a bent disc composed of filamentous material represents the microtubule organizing centers (MTOC). The key event may be the initiation and control of chromosome condensation. Cytoplasmic changes include the development of a cortical layer of 1-4 microns thickness poor in cell organelles. Golgi complexes are localized in three distinct areas with possibly different functions: (1) around the germinal vesicle; (2) in the oocyte cortex, of regular distance; (3) in the central part of the oocyte. Cortical granules (CG) of different maturation stages (condensation) form clusters near the peripheral Golgi complexes while at Meta I they form a nearly continuous single layer. At Meta II the CGs are apparently anchored to the cell membrane by means of small spokes. The cumulus (corona) cells are attached by junctional complexes to each other and to the oocyte. Foot processes cross the zona and indent the oocyte. The termini are gradually exteriorized and contacts must be broken to isolate the oocyte. The sum of all the above changes represent meiotic maturation.