308 MITOCHONDRIA RELOCATION, MICROTUBULE ASSEMBLING AND PARTHENOGENETIC DEVELOPMENTAL COMPETENCE OF PIG OOCYTES

Abstract

Developmental competence of in vitro-produced porcine embryos appears to be limited by specific maternally inherited cytoplasmic factors. We previously reported a relationship between mitochondria distribution during IVM, energy status, and oocyte developmental ability after parthenogenetic activation. The aim of the present study was to investigate the timing of mitochondria relocation during meiosis and the possible relationship with cytoskeleton organization in high and low competence oocytes. To this purpose, homogeneous groups of oocytes were matured in vitro (IVM) with 25% or 0% porcine follicular fluid (pff) to obtain different cytoplasmic competence (high and low, respectively) but similar nuclear maturation (Brevini et al., 2003, Theriogenology, 59, 440). After maturation, oocytes were parthenogenetically activated and cultured as previously described by Grupen et al., 2002 (Mol. Reprod. Dev., 62, 387–396). Active mitochondria were stained with MitoTracker® Orange CMTM Ros (Molecular Probes, Leiden, The Netherlands) at GV, MI and MII meiotic stages. At the same time microtubule organization was determined by immuno-cytochemistry using an antibody raised against α-tubulin (Sigma, St. Louis, MO, USA). Meiotic stages were assessed with DAPI. Specimens were examined with a Leica TCS-NT confocal microscope through an equatorial optical section. Nuclear maturation rate was comparable in the two groups at the end of IVM (46 h). Mitochondria relocation from the periphery to the center of the oocyte was evident as early as 20 h IVM in the 25% pff group (high competence), while 0% pff oocytes (low competence) did not show any mitochondria relocation at this time point. In agreement with the literature, α-tubulin was not detectable in GV oocytes, while at the end of IVM, α-tubulin was associated with the DNA, forming the meiotic spindle both in high and low competence oocytes. However, oocytes in the 25% pff group displayed a cytoplasmic microtubular organization that co-localized with mitochondria at 20 to 28 h IVM. Conversely, α-tubulin was not detected in the cytoplasm of 0% pff oocytes at the same time points and 71% of these oocytes did not undergo any mitochondria relocation at all by the end of IVM. Altogether the present results show that mitochondria relocation takes place at a well-defined time during IVM and is temporally associated with the formation of the microtubule mesh in the oocyte cytoplasm. Low-developmental-competence oocytes display an altered mitochondria distribution and microtubule arrangement or seem to lack the temporal coupling of the two phenomena. We speculate that a tightly linked timing of mitochondria relocation and cytoskeleton microtubule formation in the cytoplasm of the oocyte during IVM may represent a key cytoplasmic factor regulating pig embryo parthenogenetic development.