Tubulin posttranslational modifications in in vitro matured ovine oocytes

Microtubules (MTs), polymers of alpha/beta-tubulin heterodimers, are involved in several cellular functions including shape maintaining, mitotic/meiotic spindle organization and intracellular trafficking. MTs physiology can be influenced by a variety of posttranslational modifications (PTMs), including tyrosination, detyrosination, delta 2 modification, acetylation, polyglutamylation, polyglycylation, phoshorilation and palmitoylation. In the mammalian oocyte, MTs are essential for first and second meiosis, regulating the correct formation of meiotic spindle, crucial step for chromosomes movements. In the last decades, several works investigated the role of few tubulin PTMs (acetylated and tyrosinated alpha-tubulin), especially in mouse and human oocytes. Considering that tubulin PTMs expression and function have not been investigated in ovine oocytes, this study aimed at extending our knowledge on tubulin PTMs expression in in vitro matured ovine oocytes. Cumulus–oocytes complex (COCs) were obtained from adult Sarda ewes regularly slaughtered at the local abattoir. COCs were in vitro matured in TCM 199 supplemented with 10% heat-treated oestrus sheep serum, 10 μg /mL of FSH/LH, 100 μM cysteamine, 8 mg/mL of pyruvate for 24 h in 5% CO2 in air at 39°C. Metaphase II oocytes were fixed in PBS/2.5% paraformaldehyde solution and processed for indirect immunofluorescence and confocal microscopy analyses. Primary antibodies against the most representative tubulin PTMs were used (tyrosinated n=20, detyrosinated n=20, acetylated n=20, polyglutamylated n=20, delta 2 n=20, total alpha-tubulin n=20, total oocytes n=120). Through the fluorescence emission quantification, we found that acetylated and tyrosinated alpha-tubulin signal was well detectable in both meiotic spindle and polar body. In particular, tyrosinated tubulin was significantly higher than acetylated in meiotic spindle (p=0.0414), whereas no differences were found on the polar body (p=0.5166). In contrast, no immunopositivity were appreciable for detyrosinated, polyglutamylated and delta 2 tubulin in both meiotic spindle and polar body. Our results provide first information about the diverse tubulin PTMs involved in oocytes spindle organization of ovine species. This data could be useful in order to better understand the role played by each tubulin PTMs in the acquisition of oocyte competence.