Ultrastructure and distributional arrangement of mitochondria in prepubertal and adult sheep oocytes

"\"The objective of this study was to compare the in vitro developmental competence of prepubertal (one-month-old) and adult sheep oocytes in terms of the 1) ultrastructural morphology and 2) mitochondrial distribution. Cumulus-oocyte complexes undergo IVM in standard condition for 24 h. Following decumulation by pipetting, half of the mature metaphase II (MII) stage oocytes were fixed in 2.5% glutaraldehyde and processed for light and transmission electron microscopy (LM and TEM) observations. The other half were subjected to immunostaining with MitoTracker Red (MT-Red, to label mitochondria with functionally active membrane potential) and Hoechst 33342 and analyzed by confocal microscopy (CM). Immature germinal vesicle (GV) stage oocytes, retrieved at 0 h IVM, were used as controls.. By LM and TEM all the oocytes were regularly rounded, covered by microvilli and surrounded by an intact zona pellucida. Numerous rounded, oval or hooded mitochondria appeared either isolated or arranged in clusters in the ooplasm. At 0 h IVM the GV was rounded in prepubertal oocytes, whereas in the adult it appeared often flattened against the oolemma, with a crescent-shaped outline interpretable as an early sign of resumption of meiosis. Scattered cortical granules (CGs) were rarely found in the ooplasm of both prepubertal and adult oocytes. After 24 h of IVM, CGs became abundant and distributed in a single row under the oolemma, particularly in adult oocytes. By CM analysis it was found a homogeneous fine-to-granular mitochondrial distribution in prepubertal GV and MII oocytes. Conversely, in adult GV oocytes the mitochondrial pattern appeared granular to become arranged in clusters in MII oocytes. . In conclusion, both prepubertal and adult oocytes completed maturation after 24 h in culture and showed an overall good preservation following IVM. However, the diverse distribution of mitochondria in prepubertal oocytes could explain, at least in part, their scarce developmental competence. . \""