Following vitellogenesis, oocytes undergo maturation with the resumption of the meiotic or "maturation" division that has been arrested in prophase I since their transformation from oogonia. At this time the follicles appear to attain a critical size, becoming competent to respond to maturation-inducing hormones (MIH) by initiating maturation8. Diameters of 0.5 to 0.6 mm have been recorded in seabream Sparus aurata (Gothilf et al., 1997), 0.69 to 0.73 mm in zebrafish Brachydanio rerio (Selman et al., 1993), 0.8 to 1.2 mm in medaka Oryzias latipes (Iwamatsu et al., 1988), 1.5 to 1.9 mm in killifish Fundulus heteroclitus (Selman and Wallace, 1986), and 1.1 to 1.3 mm in the pipefish Syngna-thus scovelli (Begovac and Wallace, 1988). A further increase in size occurs by hydration during maturation in many teleosts, especially marine forms. Maturation division is followed by condensation of the chromosomes and expulsion of the first polar body.
At the beginning of maturation, the oocyte has a large nucleus or germinal vesicle located centrally or halfway between the centre and periphery (Wallace and Selman, 1978, 1981; Nagahama, 1983; Kjesbu, Kryvi, and Norberg, 1996; Gothilf et al., 1997); it is inconspicuous in whole oocytes of many teleosts (Figure 2.43). The nucleus is in meiotic prophase and contains lampbrush chromosomes. The first visible event associated with final oocyte maturation is the migration of the germinal vesicle to the animal pole at which time it becomes visible under the dissecting microscope. The envelope of the germinal vesicle breaks down (germinal vesicle breakdown: GVBD) and the nuclear contents blend with the surrounding cytoplasm. This provides a morphologically identifi able event that may conveniently be used to divide maturation into two physiologically distinct phases: early maturation (pre-GVBD) and late maturation (post-GVBD) (Selman and Wallace, 1986). Germinal vesicle breakdown generally occurs in Fundulus heteroclitus when the follicle reaches a diameter of 1.5 to 1.7 mm (Wallace and Selman, 1978).
Early maturation is characterized by a rapid increase in follicular volume, primarily due to hydration but the continued accumulation of heterosynthetic macromol-ecules by endocytosis accounts for about 16% of this increase (Wallace and Selman, 1985). Protein incorporation appears to stop abruptly at the time of germinal vesicle breakdown. This is consistent with the observation that oocytes in early maturation continue to display endocytotic activity at their surface, whereas those in late maturation do not. During hydration, yolk polypeptides present in early maturational oocytes undergo proteolysis. In late maturation, after germinal vesicle breakdown, the follicle continues to enlarge by hydration. Within the oocyte, lipid droplets continue to coalesce, lose their peripheral attachments, and are able to move freely through the oocyte (Figure 2.44). Immediately prior to ovulation they collect at the upper surface of the oocyte and will float to the opposite pole if the follicle is inverted. During maturation, the yolk bodies of the zebrafish Brachydanio rerio lose their crystalline masses and develop a homogeneous interior (Figure 2.45) (Selman et al., 1993).
Follicular maturation is controlled by pituitary gonadotropin which induces a shift in follicular steroidogenesis away from the production of oestradiol-17p, necessary for vitellogenesis and oocyte growth, to the production of the maturation-inducing hormone (MIH) required for the resumption of meiosis (Patino and Thomas, 1990; Thomas and Patino, 1991; Nagahama et al., 1994). Oocytes must be primed with gonadotropin before maturation can be induced by MIH. This occurs before coalescence of the lipid droplets and includes synthesis of receptors for the MIH. Considerable evidence has accumulated, especially in Salmoniformes (Jalabert, 1976; Duffey and Goetz, 1980; Nagahama, 1983,1984; Nagahama and Adachi, 1985; Kanamori, Adachi, and Nagahama, 1988), but in other bony fish as well (Jalabert, 1976; Goetz and Theofan, 1979; Greeley et al., 1986; Levavi-Zermon-sky and Yaron, 1986; Asahina, Taguchi, and Hibiya, 1987; Kobayashi et al., 1988; Schoonen et al., 1989; Suzuki, Tan, and Tamaoki, 1989; Haider, 1990; Less-man, 1991; Petrino et al., 1993; Fukada et al., 1994;
8 The extensive literature on maturation-inducing hormones in teleosts has been reviewed by Goetz (1983), Scott and Canario (1987), Nagahama et al., (1994), and Peter and Yu (1997).
Gothilf et al., 1997), indicating that the major naturally occurring MIH is 17a,20p-dihydroxy-4-preg-nen-3-one (17a,20p-DP). This steroid is synthesized collaboratively by both the thecal and follicular layers: gonadotropin stimulates thecal cells to produce 17a-hydroxyprogesterone which is converted within the follicular cells to 17a,20p-dihydroxy-4-pregnene-3-one (Figure 2.46) (Nagahama and Adachi, 1985; Nagahama, 1987; Nagahama et al., 1994). Other C21 steroids have been shown to be equally potent in inducing maturation in a number of species, at least in vitro, and all have a 17- and 20p-hydroxyl group and are structurally similar to 17a,20p-dihydroxy-4-preg-nene-3-one (Scott and Canario, 1987). In two species of sciaenid fish (Perciformes), the predominant steroid produced during final oocyte maturation and the natural MIH is 17a,20p,21-trihydroxy-4-pregnene-3-one (20P-S) (Trant, Thomas, and Shackleton, 1986; Thomas and Trant, 1989; Trant and Thomas, 1989a,b). Both hormones (17a,20p-DP and 20P-S) may play a role in inducing maturation in the striped bass Morone saxatilis and white perch M. americana (Perciformes) (King et al., 1994; King, Thomas, and Sullivan, 1994; King, Berlinsky, and Sullivan, 1995). Maturation of oocytes of the winter flounder Pleuronectes america-nus (Pleuronectiformes) appears to be induced by the synergism of several steroids distinct from the progesterones which predominate in other fish (Truscott et al., 1992). It is of interest to note that 17a,20p-dihy-droxy-4-pregnene-3-one has been detected in the serum of the sea lamprey Petromyzon marinus during the early spawning migration, suggesting a role in maturation and ovulation of this cyclostome (Weisbart et al., 1980).
Proteasomes appear to play a role in the maturation of fish oocytes (see review by Tokumoto, 1999). These proteolytic complexes are found in eukaryotic cells and are responsible for the degradation of most cellular proteins, including structural proteins, enzymes, and proteins that regulate cell cycles (Coux, Tanaka, and Goldberg, 1996). Proteasomes help to regulate the amount of a particular protein present in a cell at a given time. There are two types of proteasomes: 20S (700 kDa) and 26S (2000 kDa). The 20S proteasome forms the catalytic core of the 26S which consists of a central cylinder formed from proteases whose active sites are thought to face an inner chamber. Each end of the cylinder is capped by a large protein complex; these complexes are thought to bind proteins destined for digestion and then feed them into the inner cham ber. Activity of proteasome 26S increased in oocytes of the goldfish Carassius auratus within one hour after exposure to MIH and then declined; it rose again after completion of GVBD (Tokumoto et al., 1997). It was concluded that proteasome 26S is involved in two steps in the meiotic cycle of maturation: the early stage, following stimulation by MIH, before migration of the germinal vesicle, and later at the transition from metaphase I to anaphase I. The nature of the proteins being degraded at these times is still uncertain but proteins critical to the regulation of the cell cycle have been suggested.
The ultrastructure of oocytes approaching the resumption of meiosis has been described in vitro and in vivo in oocytes of the medaka Oryzias latipes (Iwamat-su et al., 1976, 1988). Immediately before germinal vesicle breakdown, tetrad chromosomes may be detected in the central part of the large germinal vesicle. Before maturation begins, there are many mitochondria, annulate lamellae, granular endoplasmic reticulum, and Golgi complexes in the cortical cytoplasm (Figure 2.22). Germinal vesicle breakdown leads to the formation of the spindle of meiosis and, within three hours, meiosis progresses through prometaphase I to metaphase I. Following a short time in anaphase I, the first maturation division is complete by four hours. The oocytes reach metaphase II about five hours after germinal vesicle breakdown and the first polar body is extruded at the animal pole. All cytoplasmic projections of both the oocyte and follicular cells have withdrawn from the zona pellucida by the time of ovulation. Ovulation takes place at the end of this stage. A few Golgi complexes and no annulate lamellae are noted in the ovulated eggs of the medaka (Iwamatsu et al., 1988). (The meiotic process, leading to the expulsion of the second polar body, is resumed at the time of fertilization and sperm penetration.)
Reorganization of the microtubular cytoskeleton during maturation in oocytes of the goldfish Carassius auratus has been recorded by confocal immunofluorescence microscopy using an anti-tubulin antibody (Jiang et al., 1996). In fully grown, immature oocytes, the yolk granules appear as dark masses suspended within the glowing meshes of a microtubular network that extends throughout the ooplasm except for the region containing the germinal vesicle. A condensed band of microtubules surrounds the germinal vesicle (Figure 2.47); these microtubules play a role in stabilizing the germinal vesicle at the centre of the oocyte. When the germinal vesicle begins its migration, the microtubule network disappears, permitting movement toward the animal pole. Cytoplasmic microtubules gradually concentrate in the animal hemisphere. As the germinal vesicle migrates, it trails a glowing "perinuclear tail" of condensed microtubules from its vegetal surface; it appears that the perinuclear tail permits the accumulation of cytoplasmic materials that are necessary for the subsequent maturation (Figure 2.48). It is presumed that cytoplasmic microfilaments contribute the motive power for migration of the germinal vesicle. Coincident with the breakdown of the germinal vesicle (GVBD), numerous microtubules penetrate the germinal vesicle from its vegetal surface and a disc-shaped ring of condensed microtubules is seen at the animal pole region, suggesting a role in spindle formation or the organization of meiotic chromosomes (Figures 2.49 and 2.50).
The marked hydration of oocytes during maturation is especially pronounced in marine teleosts with pelagic or floating eggs, although it occurs to a lesser extent during maturation in many brackish water and marine species with non-floating or demersal eggs (Craik and Harvey, 1984, 1986; Greeley, Calder, and Wallace, 1986; Thorsen and Fyhn, 1996). The floating eggs of many marine teleost species contain about 92% water (Craik and Harvey, 1984,1986). They owe their buoyancy to this high water content rather than, for example, to low density lipids. Such eggs contain a more dilute aqueous solution than seawater itself and are consequently less dense. In these species, there is a massive and rapid uptake of water during maturation, increasing the volume of the oocyte by a factor of four or five; the resulting mature, buoyant egg is ovulated shortly thereafter. This water uptake is clearly an adaptation leading to wide dispersal of the floating eggs by water currents and is thus related to the high fecundity of these species (Fulton, 1898, cited by Wallace, 1978).
There is evidence of secondary proteolysis of yolk proteins during maturation, being most pronounced in marine species with pelagic eggs where its extent is well correlated with the extent of oocyte hydration (Greeley, Calder, and Wallace, 1986). A large pool of free amino acids that is present in pelagic marine fish eggs originates mainly from the hydrolysis of yolk proteins during final oocyte maturation (Thorsen and Fyhn, 1996). This may assist hydration by increasing osmotic potential within the ooplasm or by supplying energy for hydration to occur, perhaps by breaking high-energy phosphorus bonds.
Three factors have contributed to the enlargement of oocytes: extensive elaboration of cortical alveoli before vitellogenesis, deposition of protein yolk, and hydration following vitellogenesis; the relative contributions of each are difficult to ascertain. Follicular growth in Fundulus heteroclitus, from a diameter of 0.55 ± 0.5 up to 1.35 ± 0.5 mm, is due to vitellogenin uptake by the growing oocyte while final enlargement, from 1.35 ± 0.05 up to 1.85 ± 0.05 mm during maturation, is the result of hydration (Wallace and Selman, 1985).
During maturation, the oocyte becomes more translucent as lipid and protein yolk droplets coalesce. Complete fusion of yolk droplets does not occur in all species and it has been noted that the degree of coalescence of the lipid droplets follows a phylogenetic pattern, forming one major droplet in higher teleosts (such as yellow perch Perca flavescens, walleye Sti-zostedion vitreum, striped bass Roccus saxatilis, and paradisefish Macropodus opercularis) but showing less coalescence in lower teleosts (brook trout Salvelinus fontinalis and rainbow trout Salmo gairdneri) where ovulated oocytes contain a large number of lipid droplets (Goetz, 1983). In some intermediate teleosts (sticklebacks Gasterosteus aculeatus and Apeltes quadracus and the killifish Fundulus heteroclitus) the degree of coalescence is intermediate.
The oocytes soon ovulate into the ovarian lumen and become mature eggs. The vitelline envelope is highly compacted and possesses pore canals prior to maturation (Begovac and Wallace, 1988). At some undefined time during maturation, and before ovulation, the pore canals disappear as the microvilli are retracted or lost from the oocyte and follicular cell. The mature egg is surrounded by the tough chorion that is derived from remnants of the zona pellucida. The chorion may be decorated with a secondary envelope that appears to have been synthesized by the follicular cells during oocyte growth (Wourms and Sheldon, 1976).
Marine fishes with pelagic eggs differ from other oviparous vertebrates in that a characteristic component of the yolk, protein phosphate, is synthesized and laid down in the ovary in the typical vertebrate manner but does not nourish the young animal (Craik and Harvey, 1984). Instead, it is apparently utilized at a much earlier stage in the life cycle, before the eggs leave the ovary, to provide the energy necessary for the considerable transport of ions by the potassium/sodium pump (K+ in, Na+ out). In species such as herring Clupea harengus, with demersal eggs, ripening is accompanied by a smaller uptake of water and a signifi cant but correspondingly smaller decrease in protein phosphate (Craik and Harvey, 1986).
In captive females of the striped bass Morone saxatilis, a certain vulnerability following vitellogen-esis presents a problem for the acquaculture industry (Mylonas et al., 1997; Mylonas, Woods, and Zohar, 1997). Final oocyte maturation fails to occur due to an absence of a surge in plasma gonadotropin II so that oocytes become atretic and no ovulation or spawning occurs. Although captive broodstocks would provide more reliable egg production, wild caught females undergoing final oocyte maturation must be used for artificial spawning.
Development of the follicular envelopes has been described for several species of bony fish, both oviparous and viviparous, and is essentially the same for all (Hurley and Fisher, 1966; Flugel, 1967a,b; Anderson, 1967; Gotting, 1967; Ulrich, 1969; Busson-Mabillot, 1973, 1977; Azevedo, 1974; Wourms, 1976; Caporic-cio and Connes, 1977; Tesoriero, 1977a; Dumont and Brummett, 1980; Stehr and Hawkes, 1983; Hosokawa, 1983, 1985; Brusle, 1985; Kessel etal., 1985; Schme-hl and Graham, 1987; Iwamatsu et al., 1988; Mat-suyama, Nagahama, and Matsuura, 1991; Giulianini and Ferrero, 2000). Oocytes of all species produce a refractile, acellular primary envelope around themselves (Wourms, 1976). Several names have been applied to this layer, including "zona pellucida", "zona radiata", "chorion", "vitelline membrane", and "vitelline envelope". The term most commonly used for the homologous structure in mammals is "zona pellucida" and this will be used in this text.9 Around the zona pellucida, the follicular cells may form a secondary envelope, often highly ornamented. Tertiary envelopes, such asjelly coats or leathery egg cases, may be formed by glands in the oviduct, or elsewhere, as the ovum makes its way to the outside world.
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