Primary Growth

Studies with many teleosts indicate that primary growth is independent of pituitary control (Khoo, 1979). During the primary growth stage, oocytes of most teleosts increase in volume about 1000 fold, growing in diameter from about 10-20 pm at lepto-tene to 100-200 pm; this is accompanied by a noticeable decrease in nuclear/cytoplasmic ratio (Selman and Wallace, 1989). As the oocyte grows, the definitive follicle forms. Oocytes undergo the early stages of prophase and become arrested in diplotene of the first meiotic division. The primary growth stage may be divided into two phases based on nucleolar morphology: the chromatin-nucleolus phase and the perinucleolus phase. In the zebrafish Brachydanio rerio, the follicular cells do not completely isolate individual oogonia and surround nests of early germ cells (Figure 2.11) (Selman et al., 1993). During the primary growth phase, the meiotic oocytes become enclosed by follicular cells thereby forming definitive follicles.

Chromatin-nucleolus stage

Oogonia entering the early stages of meiotic prophase within the germinal ridge of syngnathids transform into oocytes during the chromatin-nucleolus phase (Figure 2.12). DNA replication (pre-leptotene) occurs at this time. Homologous chromosomes pair and begin to condense (leptotene, zygotene); these pairs subsequently shorten and thicken to form synaptonemal complexes (pachytene); finally the chromosomes take on the "lampbrush" configuration (diplotene). The nuclei of pipefish oocytes become more spherical than those of oogonia (Begovac and Wallace, 1988). Nuclei of leptotene oocytes contain dense-cored granules and the chromatin remains electron-dense. Nuclei of oocytes in zygotene and pachytene stages of prophase I are less electron-dense and can be distinguished by the presence of synaptonemal complexes between homologous chromosomes within the nucleus (Figure 2.7B). By the pachytene stage, the synaptonemal complexes are long and the nucleoli have become dispersed and fragmented. Dense-cored granules are still present in the nuclei. Oocytes in zygotene and pachytene grow slightly with an increase in organelles, usually aggregated at one side of the nucleus: mitochondria, Golgi complex, ribosomes, and endoplasmic reticulum. Nuage continues to be present.

Newly formed meiotic oocytes in syngnathids initially are in contact with other oocytes. As the oocytes grow within the germinal ridge, they become separated from adjacent oocytes by processes of prefollicu-

lar cells (Figure 2.4) (Selman, Wallace, and Player, 1991). Soon after the oocytes enter the diplotene, pre-follicular cells proliferate, flatten, and eventually form the follicular epithelium that completely envelops the growing oocytes (Brusle, 1980; Begovac and Wallace, 1988). Each oocyte extends numerous microvilli toward the follicular cells (Figure 2.13E) and later the follicular cells reciprocate by extending microvilli toward the oocyte. The squamous follicular cells have large nuclei with cytoplasm containing mitochondria, granular endoplasmic reticulum, and scant Golgi complexes. The follicular epithelium is enclosed by its basal lamina.

Chromatin-nucleolus oocytes become arrested in late diplotene of meiotic prophase I as the synaptone-mal complexes dissociate within the oocyte nucleus (Selman and Wallace, 1989). During this phase, the oocytes begin an extensive period of growth. The ooplasmic matrix increases as well as the organelles which tend to remain juxtanuclear. Nucleoli reappear adjacent to the inner layer of the nuclear envelope. The appearance of multiple nucleoli seems to coincide with the formation of the definitive follicle as it separates from the germinal ridge. Each nucleolus consists of two parts: a cortical region of tightly packed ribo-somes, and a central area composed largely of fine filaments and scant amounts of the granular component (Figure 2.14) (Anderson, 1968). The definitive follicle now consists of an oocyte surrounded by a single layer of flattened follicular cells with the entire complex enclosed by a basement lamina. Outside the basal lamina there is a sparse thecal layer of vascular connective tissue and a thin surface epithelium consisting of mesothelial cells. The nucleus of the oocyte is generally spherical with multiple nucleoli in a perinuclear position adjacent to the inner layer of the nuclear envelope (Figure 2.13). Dense-cored granules can still be observed in the nucleus. The nucleus grows and nucleoli increase in number.

Perinucleolusphase

As the oocyte grows, the nucleus enlarges to form the germinal vesicle5; the nuclear membrane becomes undulating and, with further growth of the oocyte, it forms large folds and evaginations and becomes lobed

(Guraya, 1986). Soon after the arrest in prophase, ri-bosomal genes are amplified and several nucleoli appear in the oocytes of most teleosts, generally arranged at the periphery of the germinal vesicle although later they may lie randomly within it (Figure 2.15) (Selman and Wallace, 1989). This is the perinucleolus stage of primary oocyte growth. These nucleoli are present throughout oocyte growth.

At this stage, the "lampbrush" appearance of the chromosomes becomes more apparent (Figure 2.16). These chromosomes are festooned with loops of DNA that presumably represent unwindings of a single chromosome and are sites of active transcription of heterogeneous RNA in addition to the ribosomal RNA provided by the multiple nucleoli.

Unlike the situation in most teleost oocytes, where large numbers of nucleoli are produced during the chromatin-nucleolus period of primary growth, only one or two nucleoli are present at this time in the viviparous teleost Xiphophorus helleri (Azevedo and Coimbra, 1980). These nucleoli display a compact fibrillar core surrounded by a looser, granular periphery (Figure 2.17). Following the diplotene stage, when meiotic activity is suspended and lampbrush chromosomes are present in the nucleus, these nucleoli enlarge from about 6 to 18 pm and become increasingly vacuolated, indicating increased ribosomal production as the cells prepare for vitellogenesis (Figure 2.18). The nucleoli suddenly decrease in size during vitellogenesis; they disappear when the first polar body is emitted and the oocyte II is ready for fertilization.

Oocytes increase in size with the continued elaboration of cytoplasm, mitochondria, Golgi complexes, ribosomes, endoplasmic reticulum, and multivesicular bodies (Begovac and Wallace, 1988). This growth is accommodated by mitotic division of the follicular cells (Figure 2.19). During the primary growth phase most teleost oocytes accumulate extensive aggregations of basophilic and electron-dense material in the perinuclear cytoplasm. This loosely defined structure is the juxtanuclear complex of organelles (BAL-

biani's vitelline body)6 (Figure 2.20) (Clérot, 1976; Bruslé, 1980; Mayer, Shackley, and Ryland, 1988; Selman and Wallace, 1989); there is one report of the juxtanuclear complex in an elasmobranch, the dogfish Scoliodon sorrakowah (Guraya, 1979). A conspicuous

5 The greatly enlarged nucleus of an oocyte during the prophase of the first meiotic division is designated a germinal vesicle.

6 The references by Guraya (1979, 1986) discuss Balbiani's vitelline body at length.

"vitelline body" described at the end of the primary growth period in oocytes of the brook lamprey Ento-sphenus wilderi is probably the same structure (Ok-kelberg, 1921).

The juxtanuclear complex is composed of particles of ribonucleoprotein associated with a heterogeneous population of cytoplasmic organelles: granular endoplasmic reticulum, Golgi elements, mitochondria, lysosomes, multivesicular bodies, and lipid droplets (Figures 2.13C,D). Annulate lamellae, of unknown function, have been often described within the juxtanuclear complex (Iwamatsu, 1988). They consist of several parallel smooth cisternae, each containing regularly arranged fenestrations corresponding to the nuclear pores (Figures 2.21 and 2.22). It has been suggested that the cisternae arise from the nuclear envelope and are a special type of endoplasmic reticulum7.

Depending upon the species of fish, thejuxtanucle-ar complex shows different combinations of various organelles and inclusions. It may be a consequence of this variability that an extensive array of confusing terms has arisen in association with it. These terms include yolk nucleus, which is sometimes used synonymously with the term juxtanuclear complex; nuage or cement, which appears to be a material rich in ribonucleoprotein occupying the ooplasm between the aggregated organelles; and pallial substance, a deeply basophilic cloak enclosing less basophilic material (Beams and Kessel, 1973). The presence of nuage has been used as a criterion for identifying primordial germ cells in the cyprinid Barbus conchionus (Gevers et al., 1992) and the centropomid Centropo-mus undecimalis (Grier, 2000).

The substance of the juxtanuclear complex originates as a crescent or cap adjacent to the nuclear membrane and increases in size as the oocyte grows (Figure

2.23) (Guraya, 1963). The complex disappears at the beginning of vitellogenesis. The circumnuclear ring, seen with the light microscope in oocytes of the cod Gadus morhua and the centropomid Centropomus unidecimalis, is thought to be homologous to thejux-tanuclear complex and is considered to be the first sign that the fish will spawn in the coming season (Figure

The juxtanuclear complex may assume various shapes in different teleosts. In oocytes of some fish,

7 The topic of annulate lamellae is reviewed extensively by Kessel (1992).

a single discrete structure develops near the nucleus (e.g., Channa marulis, Dicentrarchus labrax) (Guraya, 1963; Mayer, Shackley, and Ryland, 1988); it may expand to form a perinuclear ring (e.g., Tilapia nilotica, Syngnathus scovelli) (Begovac and Wallace, 1988); and in some oocytes a single crescent that later becomes ovoid and migrates to the periphery of the oocyte (eight Indian species, but especially Het-eropneustes fossilis) (Figure 2.25) (Nayyar, 1964). The complex may appear double in oocytes of the Atlantic mackerel Scomber scombrus (Coello and Grimm,1990). Nuage of the common snook Centropomus undecimalis is a polymorphic organelle associated with mitochondria (Grier, 2000). It occurs as dense cytoplasmic inclusions, generally perinuclear in position (Figure 1.28B).

In the medaka Oryzias latipes, the yolk nucleus is an electron-dense, thread-like structure inhabiting the vegetal pole side of the oocyte (Figure 2.26) (Iwamatsu and Nakashima, 1996). It is suggested that the orientation of the vegetal pole may primarily be determined by the random positioning of the yolk nucleus within the oocyte and established by reciprocal interactions between the cytoplasm containing the yolk nucleus and the surrounding follicular cells.

The function of the juxtanuclear complex is enigmatic. It has been suggested that it acts as a centre for metabolic activities related to the formation, multiplication, and accumulation of other organelles and inclusions that are finally distributed in the outer ooplasm before the deposition of yolk, representing a mobilization of the forces needed for laying down yolk (Clerot, 1976; Brusle, 1980; Selman and Wallace, 1989).

Oocyte volume increases a few hundred fold during primary growth, the nuclear/cytoplasmic ratio decreases, and many membranous organelles are elaborated within the ooplasm (Figures 2.5C,D). At the end of this phase, the oocyte and follicular cells have established an intimate relationship. Patches of translucent material, a precursor of the zona pellucida, accumulate around microvilli that have formed on the surface of the oocyte; follicular cells display fewer microvilli. During oocyte growth, both thecal and follicular cells thicken and multiply.

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