MÉTODO DE ENSAYO PARA DETERMINAR EL CONTENIDO DE HUMEDAD

2.1 Introduction

2.1.1 The Visualisation of Oocyte Maturation using

Chromosomal Characteristics and the Development of an

In

vitro Assay for Maturation

The investigation of the hormonal control of oocyte maturation in Arenicola

marina requires a reliable assay for maturation. Oocytes of Arenicola sp. undergo a number of morphological changes as they mature; a general change in shape, retraction of the microvilli, cortical granule changes and the breakdown of the germinal vesicle (GVBD) including chromosome migration (Howie, 1961a; Meijer,

1979a). The usual method for scoring the maturation of A. marina oocytes has been

to use bright field microscopy to examine the breakdown of the germinal vesicle. This however, is not a reliable method of determining if maturation has occurred because the high levels of yolk within oocytes can lead to considerable errors in the estimation of the extent of GVBD. The use of the fluorescent compounds known as Hoechst dyes provides a much faster and more reliable means of assaying maturation.

The assay used here, relies on the use of the bisbenzimide compounds known as Hoechst 33342 and 33258. These dyes bind to the minor groove of DNA, preferentially to contiguous AT base pairs (Müller and Gautier, 1975; Latt and Stetten,

1976; Pjura et al, 1987), and are excited at 345 nm and emit at 460 nm (Latt and

Stetten, 1976). The dyes are cell permeant, with Hoechst 33342 being slightly more cell permeant than 33258 (Amdt-Jovin and Jovin, 1977). Both are relatively non toxic (Arndt-Jovin and Jovin, 1977). Upon binding to the chromosomes the dyes fluoresce bright blue in comparison with a pale blue of the oocyte cytoplasm. This peimits their relative positions to be easily identified so that mature and immature eggs can be distinguished by the differences in chromosome position and form. The permeant nature of the dyes and low toxicity allows living as well as fixed oocytes to be stained with the dyes.

The characterisation of maturation by the examination of chromosome characteristics allows numbers of mature versus immature eggs to be counted quickly

and accurately. The assay permits the assessment in vitro of the presence of

maturation inducing activity of coelomic fluid, and the ability of other test solutions to induce oocyte maturation to be evaluated.

2.1.2 Visualisation of Oocyte Maturation using Microtubule

Characteristics

The labelling of microtubule structures with fluorescent antibodies in conjunction with the use of Hoechst dyes allows the visualisation of changes in microtubular structures duiing maturation. Specifically, it can enable the appearance of the meiotic spindle and a time series of events occurring during maturation to be recorded.

The internal framework of the eukaryotic cell, termed the cytoskeleton, is composed of three types of proteinaceous structure: actin filaments, intermediate filaments and microtubules. These elements contribute to the shape of the cell, movement of cell organelles and structures, and cell locomotion (Bray, 1992).

The Composition of Microtubules

Microtubules are made of the globular, slightly acidic protein, tubulin. It has a molecular weight of 110 kDa with a subunit molecular weight of 55 kDa, confirming that tubulin is a dimer. Each dimer was found to consist of one a and one B tubulin (the two isoforms of tubulin). Each heterodimer of tubulin in solution is known to contain two molecules of guanine trisphosphate (GTP), one of which is hydrolysed during the course of polymerisation (Bray, 1992).

The Structure of Microtubules

Microtubules aie polar structures due to the asymmetric stiucture of each tubulin molecule. Thirteen linear protofilaments, each of 5 nm diameter, are formed along, the axis of the microtubule with a total diameter of 25 nm with a central lumen of 7 nm. There are other proteins involved in the structuie and these aie termed microtubule associated proteins (MAPs). Their functions are in a stabilising or regulatory role of the microtubule structure (Bray, 1992).

Assembly of Microtubules

Microtubules are very dynamic structures constantly growing and depolymerising. Rapidly growing microtubules are more stable than slow growing ones and this is termed dynamic instability. Polarity is also found in the kinetic behaviour of microtubules; in most polymerising conditions, tubulin binds faster onto one end, termed the plus end than at the minus end (Bray, 1992). Tubulin dimers are added by the hydrolysis of the GTP molecule after the dimer has added to the microtubule. At rapidly growing ends, dimers attach faster than the GTP can be hydrolysed producing a GTP cap facilitating further attachment. A negative feedback mechanism at the level of protein synthesis in which ftee tubulin reduces the hall-life of tubulin mRNA also controls microtubule levels within a cell (Bray, 1992).

Heterogeneity in Microtubules

Variation in microtubule use suggests that microtubules may not all be

identical. Most eukaryotes have small multigene families encoding both a and |3

tubulin which can lead to different isoforms (Cleveland, 1987). Further heterogeneity can be introduced by various post-translational modifications of tubulin. These include addition of an acetyl group to, or the removal of a terminal tyrosine residue

Microtubule Function

The function of microtubules within cells is varied. They can be found within three main types of structure; mitotic and meiotic spindles, cytoplasm, and ciliaiy axoneme of which their function during mitosis and meiosis is concentrated, on in this study.

Mitosis like meiosis is divided into a number of stages according to the behaviour and position of the chromosomes. Whereas meiosis results in the reduction by half in the chromosome content and chromosome mixing in the daughter c^lls, mitosis is essentially replication; with daughter cells maintaining the chromosome number with no genetic changes. The function of microtubules wUI be discussed in terms of mitosis involving the stages prophase, metaphase, anaphase and telophase and these will be discussed in turn in relation to cytoskeletal mechanisms. Similar arrays of microtubUles to those found in mitosis (discussed below) are used in meiosis to segregate chromosomes into germ-line cells and during fertilization to facilitate the fusion of male and female pronuclei (Bray, 1992).

Prophase

Just before the cell enters prophase, chromosomes condense and centrosomes outside the nuclear envelope duplicate producing two centres that move to opposite sides of the nucleus. These act as focal points for the microtubule anay known as the mitotic spindle. The mitotic spindle forms by microtubule growth on the centrosomes. At the end of prophase the nuclear membrane breaks down and the microtubules enter the space previously occupied by the nucleus.

Metaphase

Three types of microtubule structure (shown in Fig. 2.1) are produced as the cell enters the pro-metaphase stage: Polar microtubules grow together from opposite

spindle pole