Mammalian oocyte maturation and microtubule-associated proteins dynamics

RAWE VANESA; ESPAÑOL ALEJANDRO; NODAR FLORENCIA; BRUGO OLMEDO SANTIAGO

Montreal, Quebec, Canadá

Workshop; 61 Annual Meeting of the American Society for Reproductive Medicine; 2005

Objective: Although the fertilization rate of in vivo and in vitro human matured oocytes is similar after ICSI, in vitro oocyte maturation in humans is associated with a loss of developmental competence and therefore smaller pregnancy rates. After in vitro culture, previous works have reported an inaccurate segregation of oocytes chromosomes and consequently an elevated rate of aneuploidy in embryos. In this sense, the maintenance of the microtubule structure in culture and the role of microtubule-associated molecular motors like HSET, Eg5 and the mitotic antigen NuMA for the correct formation of the mitotic spindle have gained attention in the last years. In the present project, we aim to explore the presence and distribution of HSET, Eg5 and NuMA during bovine and human oocyte in vitro maturation. Proteins distribution and their association with microtubules after its stabilization and depolymerization have been also studied. Material and methods: A total of 32 human oocytes (16 immature and 16 mature) and 184 bovine oocytes (98 immature and 86 mature) have been analyzed. Human in vitro oocyte maturation was performed in human tubal fluid supplemented with 15% of synthetic serum in 5% CO2 and 100% humidity at 37ºC for 24hs. Bovine in vitro oocyte maturation was performed in M199 media; 5% fetal calf serum; 2 mg/ml FSH in 5% CO2 and 100% humidity at 39ºC for 24hs. For immunofluorescence, bovine and human immature and mature oocytes were fixed with a glycerol-based microtubule stabilizing buffer previously described. To study microtubule-associated molecular motors, antibodies to kinesin antagonistic molecular motor proteins HSET, Eg5 and the spindle pole matrix protein NuMA were used. Incubation with primary and secondary antibodies was done over night at room temperature and for one hour at 37oC respectively. DNA was labeled using TOTO-3 and samples were imaged using a spectral confocal microscope. Microtubule stabilization and depolymerization were carried out using taxol and nocodazole respectively and the distribution of the mentioned proteins analyzed.  Results: Our very preliminary observations show the presence of HSET, Eg5 and NuMA in human and bovine oocytes. An expected distribution of these proteins on the meiotic spindles of MII oocytes obtained from ovaries (in vivo maturation) was visualized. Contrary to that, after in vitro maturation, absence or ectopic localization of NuMA and HSET has been observed in the majority of human oocytes. Disruption of microtubules during maturation in bovines induces the formation of aberrant spindles as well as an anomalous distribution of the proteins of interest. Conclusion: The identification of HSET, Eg5 and NuMA in mammalian in vivo and in vitro matured oocytes suggests that their presence may be necessary for controlled microtubule dynamics during oocyte maturation. Bipolar spindle assembly in bovine and human eggs depends on microtubules and motor proteins. Disruption of microtubules during oocyte maturation severely affects the microtubule-associated motor proteins distribution. Further investigation of these proteins should enhance our understanding of their contribution during oocyte maturation.