The enlargement of the oocyte that takes place during vitellogenesis is due largely to an accumulation of ex-ogenously derived yolk protein precursors (Begovac and Wallace, 1988). This protein is, in general, called female-specific serum protein (Fujita, Takemura, and Takano, 1998). In teleosts, the relative contribution of protein yolk deposition to oocyte growth is obscured to some extent by the extensive elaboration of cortical alveoli that was initiated prior to vitellogenesis and by the hydration that follows vitellogenesis (Wallace and Selman, 1981). Most proteins appear to be synthesized outside the oocyte (heterosynthetic) although autosynthetic origin of yolk proteins may also occur (Wallace, 1978). The female specific protein, vitellogenin, is a large lipoglycophosphoprotein which is synthesized by the liver, released into the blood, and transported to the ovary. Indeed, the association between liver and ovary is indicated by simultaneous increases in the hepatosomatic and gonadosomatic indexes during vitellogenesis in the catfish Hetero-pneustes fossilis (Srivastava and Saxena, 1996). This phase is dependent on pituitary gonadotropin (Khoo, 1979) which stimulates oestrogen production by the ovary (Wallace, 1985). Oestrogen, especially oestradi-ol-17p, transported in the blood to the liver, regulates synthesis and secretion of vitellogenin (Nagahama, 1984). Also in response to gonadotropin, vitellogenin is selectively sequestered from the bloodstream by growing oocytes. Vitellogenin itself is not incorporated into teleost egg yolk but rather is proteolytically cleaved within the oocyte into yolk proteins of which lipovitellin and phosvitin are the most familiar (Wallace, 1985; Greeley, Calder, and Wallace, 1986). By now, all types of non-mammalian vertebrates, from hagfish (Yu et al„ 1981), sharks (Craik, 1978a), and other fish (Wiegand, 1982) to turtles and birds, have been shown to synthesize and secrete vitellogenin as a response to oestrogen.
Macromolecules destined for the production of yolk follow an intercellular route from the maternal circulation to the surface of the oocyte, penetrating between the endothelial cells of the perifollicular capillaries to enter the connective tissue surrounding the follicle (Selman and Wallace, 1982b). On reaching the follicular epithelium they pass between, rather than through, adjacent follicular cells en route to the oocyte. Occasional evidence has been found for the internalization, by micropinocytosis, of electron-dense tracers within the follicular cells but most macromolecules appear to pass through extracellular channels of varying sizes between the follicular cells (Selman and Wallace, 1982a). The molecules then penetrate the patent pore canals of the zona pellucida. Follicular cells do not appear to contribute to yolk formation in teleosts (Selman and Wallace, 1986).
Protein yolk precursors are incorporated into the oocyte by intense micropinocytosis of the oolemma between the bases of the microvilli (Figures 2.30 and 2.31) (Droller and Roth, 1966; Anderson, 1968; Ulrich, 1969; Shackley and King, 1977; Selman and Wallace, 1982a, 1983; Abraham etal., 1984; Brusle, 1985; Kjesbu, Kryvi, and Norberg, 1996) and translocated to yolk spheres in less than 20 min (Selman and Wallace, 1982b). Coated pits are numerous along the oolemma between the microvilli and micropinocytotic vesicles as well as smooth-surfaced tubules lie together in the cortical ooplasm. Micropinocytosis of external materials by the oocyte is the most characteristic cellular event of vitellogenesis (Selman and Wallace, 1982a).
During vitellogenesis, the zona pellucida thickens and the oocyte continues to form cortical alveoli (Selman and Wallace, 1982a, 1986). The most obvious event marking the initiation of vitellogenesis, however, is the appearance of membrane-bound, fluid-filled yolk spheres lying between the cortical alveoli in the peripheral ooplasm (Figure 2.32).
Two types of yolky inclusions are formed during vitellogenesis: lipid yolk droplets and masses of pro tein yolk (Yamamoto, 1964; Shackley and King, 1977, 1979; Wallace and Selman, 1981; Mayer, Shackley, and Ryland, 1988; Selman and Wallace, 1989; Selman et al., 1993). The first type of yolky inclusion to accumulate in most species is lipid yolk in the form of distinct lipid droplets; in the bass these make their appearance in the outer cortex but in most teleosts they first appear in the perinuclear cytoplasm giving rise to suggestions that they are probably formed endog-enously (Figure 2.5). Protein yolk accumulation is exogenous in origin and occurs after, and concomitant to, lipid yolk accumulation. In most teleosts, yolk proteins accumulate in fluid-filled yolk spheres that may either maintain their integrity throughout oocyte growth or fuse centripetally, eventually forming a continuous mass of fluid yolk, a process which confers on many teleost eggs their characteristic transparency (Figure 2.33). This coalescence can occur relatively soon after the initial formation of yolk spheres, as in sticklebacks and the pipefish, during the later stages of vitellogenesis, as in the sheepshead minnow, or during maturation, as in those marine teleosts that produce pelagic eggs. Creation of a central mass of fluid yolk displaces the ooplasm, with its cortical alveoli and lipid droplets, to the periphery of the oocyte (Figure 2.5). A few yolk spheres always remain in the peripheral ooplasm, but appear to be reduced in number as the central mass increases (Figure 2.32) (Shackley and King, 1977).
In some species, protein yolk is sequestered in the form of discrete granules that stain intensely for proteins but not for lipids or carbohydrates (Figure 2.34) (Selman and Wallace, 1989; Selman et al., 1993). The surface of the oocyte displays considerable endocyto-tic activity during this stage and electron-dense material is visible in some endocytotic structures. The yolk platelets of the zebrafish Brachydanio rerio are variable in size and some may attain diameters of 40 pm. The contents of most of these yolk bodies are not homogeneous and generally display one to several rectilinear masses embedded in a homogeneous matrix. In appropriately oriented sections, these masses display an orthorhombic crystalline structure with parallel dense bands approximately 10 nm apart (Figure 2.35). This granular yolk becomes the predominant yolk inclusion and densely packs the mid and outer cortex. Unlike the fluid yolk droplets that coalesce as they migrate centripetally, protein yolk granules maintain their structural integrity until maturation, after which they actively coalesce.
Yolk granules are characteristic of freshwater te-leosts (Lange et al., 1983) although they have been noted in eggs of the cod Gadus morhua (Kjesbu and Kryvi, 1989). As described in a wide range of fishes they consist of one or more crystalline main bodies within an amorphous superficial layer enclosed by a membrane (Figure 2.36) (Yamamoto and Oota, 1967; Wallace, 1978). Elegant crystals have been described within the main body of yolk platelets from a wide variety of fishes (cyclostomes: Karasaki, 1967; Ulrich, 1969; Patzner, 1975; Lange and Richter, 1981; Lange, 1982; teleosts: Lange et al., 1983; and the ancient fishes Polypterus bichir, Amia calva, and Lepisosteus osseus: Lange, Grodzinski, and Kilarski, 1982). The crystals of cyclostomes appear to be a monoclinic lattice of possibly symmetric lipovitellin-phosvitin di-mers whereas those of teleosts and the ancient fishes are an orthorhombic array of lipovitellin-phosvitin complexes consisting of two subunits that are likely not identical. This crystalline structure is rare in marine species, suggesting that the crystals may store essential nutrients, unavailable in fresh water, providing regularly packed microchambers that sequester low molecular weight particles or ions (Lange, Grodzinski, and Kilarski, 1982). X-ray analysis of cryosections indicates the presence of chloride and a variety of cations within the platelets (Lange, 1981, 1982; Lange et al., 1983).
Early vitellogenesis in the pipefish is characterized by the appearance of individual small, membrane-bound yolk spheres in the oocyte periphery and interior (Figures 2.37A to C) (Begovac and Wallace, 1988). As vitellogenesis progresses, the yolk spheres increase in size while the cortical alveoli and lipid are displaced to the peripheral ooplasm. Eventually the yolk spheres coalesce to form a central fluid mass; the cortical alveoli and lipid remain in the peripheral part of the yolk mass. Yolk spheres are moderately electron dense and contain highly electron-dense inclusions that occasionally have a finely granular appearance and provide a specific, reliable marker for following yolk formation (Figures 2.37D,E). There are three types of small yolk spheres: primary, transitional, and mature. Primary yolk spheres appear to contain proteinaceous material interspersed among multivesicular elements. Intermediate, transient yolk spheres contain proteinaceous material, variable vesicular elements, and the yolk-specific, electron-dense inclusion. Mature yolk spheres contain homogeneous, condensed yolk and a heterogeneous size and distribution of the yolk-spe cific marker (Figures 2.37D,F). It is suggested that the multivesicular bodies represent a modified lysosomal compartment involved in yolk sphere formation (Begovac and Wallace, 1988). Secondary lysosomes or multivesicular bodies are seen adjacent to cortical alveoli in oocytes of the seahorse and pipefish at about the time of the onset of vitellogenesis (Figure 2.38) and may be required for the proteolytic processing into mature yolk proteins of the vitellogenin taken up by endocytosis (Figure 2.37G) (Selman, Wallace, and Player, 1991). Multivesicular bodies seem to disappear as vitellogenesis progresses.
The involvement of lysosomes or multivesicular bodies in vitellogenesis has been demonstrated in oocytes of the trout Salmo truttafario (Busson-Mabillot, 1984). At an early stage of development, lysosomes arise in the cortical ooplasm and migrate centripetally, almost reaching the nucleus; eventually they constitute the most voluminous compartment of the oocyte (Figure 2.39). They are membrane-bound and contain small vesicles and clumps of finely granular material (Figure 2.40A). Continuities are abundant between the agranular endoplasmic reticulum and the membrane enclosing the lysosomes (Figure 2.40B). At the beginning of vitellogenesis, endocytotic vesicles containing vitellogenin fuse with one another to form larger vesicles and these then fuse with lysosomes to form yolk bodies that may attain diameters up to 10 pm (Figure 2.41). Granular material gradually fills the lysosomes at the onset of vitellogenesis and, over several weeks, this transforms into homogeneous yolk (Figure 2.42).
Other changes are apparent within the follicle during vitellogenesis (Selman and Wallace, 1986; Begovac and Wallace, 1988; Kjesbu, Kryvi, and Norberg, 1996). The germinal vesicle is progressively displaced toward the animal pole as yolk accumulates centripetally within the oocyte. Some small lipid droplets remain associated with the germinal vesicle while others begin to fuse, reaching diameters of 40 to 50 pm, and reside in the peripheral ooplasm. In Fundulus, the zona pellucida continues to enlarge, acquiring an elaborate structure whereas, in Syngnathus, it undergoes condensation. There is a close structural relationship between bundles of microfilaments and annulate lamellae in vitellogenic oocytes (Kessel, Beams, and Tung, 1984). It is suggested that there is a coparticipa-tion of pores of the annulate lamellae and polyribosomes in the development of microfilament bundles.
Vitellogenesis in the elasmobranch Scyliorhinus canicula does not appear to differ fundamentally from other vertebrates either in the mechanism of vitello-genesis or in its immediate endocrine control (Craik, 1978a,b,c,d,e). Vitellogenin is present in the plasma throughout the year and its uptake by growing ovarian follicles seems to be stimulated by a hormone from the cranial pars distalis of the pituitary.
An unusual form of yolk uptake is described in the lamprey Petromyzon marinus where both the oocyte and certain enlarged follicular cells, the "nurse cells", simultaneously form yolk platelets (Lewis and McMillan, 1965). These cells eventually fuse and the yolk-rich cytoplasm from the nurse cells is incorporated into the oocyte. The nucleus of the nurse cell disappears.
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