Mammalian oocyte in vitro maturation: the endomembrane system 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:  The process of oocyte maturation encompasses a complex series of molecular and structural events, culminating in the arrest of the oocyte chromosomes on the metaphase II plate (MII). Contrary to bovines, maturation in vitro can be accomplished in humans, but it is associated with a loss of developmental and functional competence. This loss can be associated with a poorly understood absence or abnormal distribution of specific proteins and organelles during attempts of germinal vesicles (GV) culture to MII in vitro. Cyclin B-Cdc2 kinase phosphorylates Golgi apparatus (GM130) on serine 25, while PP2A phosphatase dephosphorylates the residue during telophase. Since mammalian oocytes contain large stockpiles of cyclin B1, cyclin B2, cdc2 and PP2A mRNA and protein, it is likely that these molecules act on Golgi matrix proteins during meiosis to partition the organelle. In this report we focus on the dynamics of the Golgi matrix and Endoplasmic Reticulum (ER) in bovine and human oocytes during meiosis. Differences between these two animal models and between in vivo and in vitro human oocyte maturation were considered for Golgi and ER distribution. Material and methods: Confocal Scanning visualization of bovine and human immature and mature oocytes was performed (bovine GV:…., bovine MII: ….., human GV: 9 and human MII: 7). Fixation and permeabilization were done with 2% formaldehyde and 0.1% Triton X-100 respectively. Golgi and ER were identified using GM-130 and Calreticulin antibodies respectively. Block of unspecific epitopes was done before incubating with primary and secondary antibodies. DNA was labeled using TOTO-3 and samples were imaged using an Olympus spectral confocal microscope. Golgi and ER dynamics was also studied after microtubule stabilization and depolymerization (taxol and nocodazole respectively). Results: Our very preliminary results show bovine and human immature oocytes (GVs,) with independent Golgi structures that vesiculate following Germinal Vesicle Breakdown (GVBD). The matrix fraction of the Golgi fragments after GVBD and disperses in bovine and human MII oocytes. In preliminary observations, human oocyte fragmentation of the Golgi matrix during in vitro maturation redistributes punctate to clusters of ER in a similar way to bovines. No apparent differences were found in terms of the endomembranes distribution between in vivo and in vitro human oocyte maturation. In vitro disruption of microtubules during oocyte maturation affects Golgi and ER distribution and the maturation rate, suggesting that a microtubule-dependent movement of these organelles is essential during in vitro oocyte maturation. Conclusion: Although differences in terms of bovine and human oocyte competence during in vitro maturation have been described, these two models are comparable in terms of Golgi and ER distribution until MII arrest. Enlargement of the sample and more research is needed to better describe the presence of checkpoints and the role of translation and protein uptake on completing oocyte maturation in vitro and in vivo. These investigations will enhance our understanding of mammalian oocyte maturation with special emphasis on clinically valuable applications in humans.