Mammalian Oocyte in Vitro Maturation: The Endomembrane System Dynamics

Abstract

The process of oocyte maturation encompasses a serie 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), while PP2A phosphatase dephosphorylates the residue during telophase. Since mammalian oocytes contain large stockpiles of cyclin B1, cyclin B2 and cdc2, 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. Prospective and descriptive basic study performed in an IVF private clinic affiliated to University of Buenos Aires. Confocal Scanning visualization of bovine and human immature and mature oocytes was performed after in vivo and in vitro maturation (220 bovine and 16 human oocytes). Human in vitro oocyte maturation was performed in human tubal fluid supplemented with 15% of synthetic serum in 5% CO2 ,100% humidity at 37°C for 24hs. Bovine in vitro oocyte maturation was performed in M199 media; 5% fetal calf serum and FSH in 5% CO2 , 100% humidity at 39°C for 24hs. Fixation and permeabilization were done with formaldehyde and Triton X-100 respectively. Golgi and ER were identified using GM-130 and Calreticulin antibodies respectively. DNA was labeled using TOTO-3 and samples were imaged using a confocal microscope. Golgi and ER dynamics was also studied after microtubule stabilization and depolymerization (taxol and nocodazole respectively). Our 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. 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.