ABSTRACT
In eukaryotic cells, variations in shape, active movement, and mechanics of cell division depend on the cytoskeleton, a sophisticated array of protein fi laments that extends throughout the cytoplasm. The cytoskeleton also directs and provides the machinery for the intracellular transport of organelles. These functions are assured by three types of fi laments, namely microtubules, actin fi laments, and intermediate fi laments. Each fi lament, formed from a different protein monomer, can be assembled in different structures whose functions, such as coordinated polymerization and depolymerization in time and space or interaction to one another and other cell components, are fi nely regulated by a wide range of associated proteins. In the context of oocyte physiology, the microtubules and actin fi laments assist the cytoplasmic choreography of the meiotic process, and, in the course of oogenesis, contributes to an endowment of maternal-derived molecules and spatial clues that are essential for the development of the embryo (1,2). An example of the importance of the cytoskeleton for the function of the oocyte is represented by the involvement of microtubules-in the form of the metaphase I (MI) and metaphase II (MII) spindles-and actin fi laments in the sequential segregation of homologous chromosomes and sister chromatids through two successive and highly asymmetric cell divisions that coincide with the emission of the fi rst (PBI) and second (PBII) polar body. This process ensures that the zygote receives a haploid set of maternal chromosomes. The organization and dynamics of microtubules and actin fi laments are known be affected by a variety of intrinsic and extrinsic factors (3-5). In fact, it is paradoxical that much of current understanding of microtubule dynamics during cell division in somatic and germ cells is a consequence of studies on the cold sensitivity of these structures. Damage to the MII spindle has emerged from studies that have tested the effects of physical-chemical conditions which are imposed, often unknowingly, during the return journey to and from physiological temperatures to cryogenic storage in liquid nitrogen (–196°C). This has generated the credence that oocytes cannot be safely cryopreserved. In reality, the question as to whether the oocyte cytoskeleton is subjected to damage after cryostorage does not imply a simple answer. Many factors, such as specifi c conditions imposed by the diverse cryopreservation protocols or nature of the biological material (differences in species, maturation stage, and intrinsic quality), can act independently or through complex interactions, infl uencing the response of the cytoskeleton to cryopreservation conditions, in a fashion that is not always understood or even recognized. For example, relatively minor differences in the degree of dehydration of the cytoplasm generated by exposure to cryoprotective agents (CPAs) before controlled rate slow cooling (CRSC) cryopreservation can infl uence the proportion of frozen-thawed oocyte with a normal MII spindle (6). Current evidence, which has expanded considerably over the last few years, collectively does not appear to be entirely consistent, probably as a result of differences in the protocols subjected to scrutiny and/or criteria and methods of analysis. It is perceived, however, that specifi c sets of cryopreservation conditions may generate rather unique infl uences that may be not necessarily reproduced under other conditions, making arduous the attempt to draw general conclusions. In this chapter, evidence on the effects of cryopreservation on the actin and microtubular structures of the fully grown human oocytes will be discussed, without taking in consideration the countless number of regulatory factors on which virtually no information is presently available.
