Mitochondrial Distribution and Adenosine Triphosphate Content of Bovine Oocytes Before and After In Vitro Maturation: Correlation with Morphological Criteria and Developmental Capacity After In Vitro Fertilization and Culture1

Abstract

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

“…In our study, we did not find differences in mitochondrial distribution between BCB oocyte groups, while Egerszegi et al (2010) concluded that, in pigs, BCBK oocytes showed more heterogeneous and non-aggregated mitochondrial distribution than BCBC oocytes. Different authors have reported differences in mitochondrial distribution patterns among species, during IVM and in vivo maturation (Stojkovic et al 2001, Sun et al 2001, Torner et al 2004, Velilla et al 2006, Dell'Aquila et al 2009, Egerszegi et al 2010. Previous studies showed that GV-oocytes do not present MPF activity (Dedieu et al 1996) but, in our study, we detected MPF activity before IVM; this may be due to the BCB staining time.…”

“…This would indicate a positive relationship between mitochondrial activity at MII stage and embryo development. Stojkovic et al (2001) showed that mitochondrial reorganisation was different between morphologically good and poor quality oocytes. In our study, mitochondria migrated throughout the IVM process.…”

“…Mitochondria are maternally inherited organelles that use oxidative phosphorylation to supply energy (ATP) to the cell (Stojkovic et al 2001). The distribution of mitochondria changes during oocyte maturation and fertilisation with the aim of bringing mitochondria to the region of the cell where a higher level of ATP (Van Blerkom & Runner 1984) or calcium (Sousa et al 1997) is required.…”

“…It has been demonstrated that mitochondrial function and the cytoplasmic ATP level can affect fertilisation, resulting in a significant increase in blastocyst rates or their total failure after IVF (Van Blerkom et al 1995, Liu et al 2000. Mitochondrial distribution and activity are modified during oocyte in vitro maturation (IVM) and this differs among species such as cattle (Stojkovic et al 2001, Tarazona et al 2006, dogs (Valentini et al 2010), goats (Velilla et al 2006), horses , humans (Van Blerkom et al 1995, Dell'Aquila et al 2009), mice (Calarco 1995) and pigs (Torner et al 2004, Brevini et al 2005. Using the fluorescence probe MitoTracker Green, Sun et al (2001) concluded that in vitro matured pig oocytes present changes in the distribution of mitochondria causing the incomplete movement of mitochondria into the inner cytoplasm affecting the cytoplasmic maturation.…”

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity

“…This could easily be explained by the fact that an embryo fully depends on maternal cytoplasm until the maternal embryonic genome transition takes place from the 8-to 16-cell stage, since no (or very few) mRNAs are formed until then (Rodriguez & Farin 2004). Fully mature cytoplasm of the oocyte provides the embryo with a battery of maternal mRNAs, proteins, and mitochondria, which are related to oocyte development competence and which may contribute to apoptotic pathways but also to defense mechanisms against apoptosis (Fair et al 1995, Otoi et al 1997, Stojkovic et al 2001. Microinjection of healthy cytoplasm protected oxidative-stressed zygotes from apoptotic death in mice (Liu & Keefe 2000) and improved fertility in older patients with compromised embryos in humans (Cohen et al 1998, Barritt et al 2001.…”