Female Genital Systems Of Fish

1.1 Introduction

There is an amazing variation in methods of reproduction in fishes1. Some species, at large outlays of energy, release enormous numbers of moderately yolky, mesolecithal eggs that are subjected to subsequent parental neglect. A single ocean sunfish, Mola mola, for example, by a prodigious feat of productivity, is said to spawn over 300,000,000 eggs (Nelson, 1984). Other fish may produce relatively small numbers of much yolkier, telolecithal eggs, often improving their chances of survival by providing parental support. Elaborate nest-building activities may precede sexual behaviour and parental care may continue for some time after hatching. The young may be sheltered, for varying periods, within the body of the female and even the male. Some viviparous2 teleosts produce only one to four well-developed young at a time (Thibault and Schultz, 1978). An amazing array of structural adaptations has evolved for the protection and nourishment of these developing young.

A remarkable reproductive strategy is seen in bitterlings (Cyprinidae, Acheilognathinae) where parents transfer responsibility for the care of their young to a freshwater unionid mussel (Aldridge, 1998). The female bitterling extends her long ovipositor into the mantle cavity of the mussel and deposits her eggs between the gill filaments. The male then ejects his sperm into the mussel's inhalent water current and fertilization takes place within the gills of the host. Early developmental stages are protected from the risk of predation within the body of the mussel; subsequently the larvae swim away from the host to continue life on their own.

Although external fertilization is usual among fishes, copulation may occur in some species. Sometimes

1 Detailed in an exhaustive chart, see Breder and Rosen, 1966.

2 Producing living young instead of eggs.

this merely ensures that spermatozoa are shed near the eggs but more often it involves insemination of the females. Internal fertilization may be followed immediately by the laying of newly fertilized eggs or some time later by the release of embryos at various stages of development.

Viviparity is highly specialized in some groups of elasmobranchs and teleosts and the developing young may be housed within the ovary or in the genital ducts. Viviparous species of elasmobranchs range from internal incubators that simply retain large, yolked eggs to other species in which the complexity of placentation and egg yolk retention approaches the eutherian condition (Callard et al., 1989). The cartilaginous fishes are of particular interest to reproductive biologists because of their structural and functional similarities in patterns of reproduction and development with those of amniotes (Wourms, 1977). In these highly advanced animals, several processes either made their appearance for the first time among vertebrates or became well established: internal fertilization, viviparity, placental mechanisms for foetal maintenance, patterns of genital tract development and sex differentiation, and the vertebrate type of reproductive endocrinology.

Although most fish are dioecious or gonochoristic, hermaphroditism does occur where ovarian and testicular tissues appear in the same individual (Atz, 1964; Greenwood, 1975; Chan and Yeung, 1983; Grandi and Colombo, 1997). It is found especially among cyclostomes and teleosts and, in some species, is the normal way of life. Male and female sex cells ripen at the same time in synchronous hermaphrodites. In consecutive hermaphrodites, however, there is sex reversal: protogynous hermaphrodites function first as females, then as males while protandrous hermaphrodites transform from males into females.

Reproduction in most fishes is cyclic, although the length of the cycle is extremely variable (see reviews by Greenwood, 1975; Wourms, 1977; Billard and Breton, 1978; Dodd and Sumpter, 1984; Hamlett and

Koob, 1999). Some, like the lampreys and certain sal-monid and eel species, spawn only once and then die; others may breed every two or three years, but most breed once or several times a year. Some hagfishes, some teleosts, and some species of oviparous skates appear to breed throughout the year.

The appearance of the ovary of cyclical breeders varies greatly at different times of the cycle and three ovarian types have been recognized on the basis of the pattern of oocyte development (Wallace and Sel-man, 1981; Nagahama, 1983). Fish that spawn once and then die, as lampreys, anadromous salmon species, Oncorhynchus spp., or catadromous eels, Anguilla spp., have synchronous ovaries in which all oocytes are at the same stage of development. Species that generally spawn once per year during a short breeding season display group-synchronous ovaries with at least two populations of oocytes at different developmental stages; this is the commonest situation in teleosts. Ovulation of group synchronous ovaries may occur at intervals over the breeding season so that the oocytes are released in "batches". The yellowtail flounder Pleuronectes ferrugineus of the continental shelf of the western North Atlantic is such a batch spawner (Manning and Crim, 1998). An asynchronous ovary contains oocytes at all stages of development and occurs in species that spawn many times during a long breeding season: e.g., medaka Oryzias spp., kil-lifish, Fundulus heteroclitus, and goldfish, Carassius auratus.

Many fish, in both temperate and tropical regions, exhibit an annual rhythm of reproduction that is correlated with photoperiod and temperature variations; in the tropics the increased productivity of the rainy season is also an influence (Billard and Breton, 1978). The breeding seasons of marine teleosts of the northern hemisphere are characteristically shorter in northern (3 to 4 months) than in southern forms (5 to 6 months) (Qasim, 1955). Oocytes in the northern species develop synchronously and are probably released in a single spawning; there is a wide range in sizes of maturing oocytes in asynchronous ovaries of southern species indicating that several spawnings occur in a single season. The appearance of continuous breeding, however, may be deceptive in that individuals or populations may exhibit synchronized annual reproductive cycles that are simply out of phase with one another (Scott, 1974).

1.2 Origin of the Genital Systems

Ovaries and testes develop from paired masses of mesodermal tissue on either side of the dorsal mesentery in the dorsolateral lining of the peritoneal cavity (Figure 1.1). These indifferent genital ridges bulge into the developing coelom and are later invaded by primordial germ cells that will eventually give rise to oogonia or spermatogonia (Nieuwkoop and Sutas-urya, 1979; Nakamura et al., 1998). Covering the surface of the ovary is the so-called germinal epithelium which is continuous with the peritoneum lining the coelom (Figure 1.2). During embryonic development the germinal epithelium is the proliferative layer that gives rise to much of the somatic tissue of the rudimentary gonad. In the common snook Centropomus undecimalis, oogonia are distributed among the stratified squamous cells of this epithelium; they proliferate by mitosis and sink into the stroma of the ovary where further development takes place (Figure 1.3) (Grier, 2000). Presumably the germ cells have invaded the epithelium from an external source. Since it is not known at this time whether this epithelium is the source of germ cells in all fish, some authors consider it misleading to refer to it as a "germinal epithelium", preferring the term "gonadal epithelium" for this proliferative layer (Nieuwkoop and Sutasurya, 1979).

In most vertebrates, the somatic tissue of each gonad has a double origin, developing from two distinct but closely associated mesenchymal sources: the cortex is derived from proliferation of the coelomic epithelium and is destined to become an ovary; the medulla comes from a more medial cellular proliferation and produces the testis (Wourms, 1977; Wolff, 1991)3. Usually one of these portions grows rapidly while the other fails to develop and the sex of the individual is determined early. This pattern of gonadal differentiation from two components is characteristic of elasmobranchs and all tetrapods (Chieffi, 1967). In contrast, both male and female gonads of cyclo-stomes and teleosts develop directly from coelomic epithelium that corresponds to the cortex of other vertebrates (Wolff, 1991) and there appears to be no contribution from medullary tissue (Dodd and Sumpter, 1984). It has been suggested that these differences may account for the widespread occurrence

3 The phenomenon of sexual differentiation has been discussed at length by Lepori (1980).

of intersexuality among cyclostomes and teleosts (Hoar, 1969).

Primordial germ cells arise early in development within embryonic endoderm or mesoderm, long before the rudiments of the gonads are formed and at a distance from the site they will eventually occupy (Figure 1.4). Their site of origin is extragonadal and, in some fishes, may be extraembryonic. They migrate into the rudimentary gonad and establish residence in the outer margin of the stroma,just beneath the gonadal epithelium (Figure 1.5) (Wourms, 1977; Nieuwkoop and Sutasurya, 1979; Hamaguchi, 1982; Timmermans and Taverne, 1989; Gevers et al., 1992; van Winkoop et al., 1992; Grandi and Colombo, 1997). Later, they will be ensheathed by cells derived from the gonadal epithelium (Figure 1.6). In contrast to other species, where primordial germ cells migrate to previously formed germinal ridges, the primordial germ cells of the carp Cyprinus carpio are present at the site of the gonadal ridges before a gonadal anlage appears (Par-mentier and Timmermans, 1985).

Various mechanisms have been proposed to explain the migration of primordial germ cells in fish: amoeboid movements of the germ cells themselves, a passive deliverance produced by morphogenetic movements of the surrounding tissues, and transport in the bloodstream. Migration is said to be powered by amoeboid movement in largemouth black bass Micropterus salmoides salmoides (Johnston, 1951), and the carp Cyprinus carpio (Nedelea and Steopoe, 1970), while in the medaka Oryzias latipes (Hamaguchi, 1982) and barbel Barbus conchonius (Timmermans and Taverne, 1989), morphogenetic movements of the surrounding tissues are thought to provide the motive force. Suggestions of a vascular route of migration have received no recent substantiation.

Primordial germ cells are mitotically active as they become segregated from somatic cells but mitosis ceases during the period of migration to the gonadal primordia (Hamaguchi, 1982; Parmentier and Timmermans, 1985; Timmermans and Taverne, 1989; Grandi and Colombo, 1997). In some species a second proliferative phase occurs soon after gonadal colonization but in others there may be a period of weeks or months of intragonadal mitotic rest: intragonadal primordial germ cells of the carp Cyprinus carpio, for example, remain "mitotically silent" for an extended period making them especially suitable for studies before the onset of proliferation (van Winkoop, et al., 1992).

Primordial germ cells are larger than surrounding cells, are rounded, oval, or pear-shaped, and show a distinct cell boundary (Figure 1.7) (Nedelea and Steopoe, 1970; Hogan, 1978; Nieuwkoop and Sutasurya, 1979; Hamaguchi, 1982; Parmentier and Timmermans, 1985; Timmermans and Taverne, 1989; van Winkoop et al., 1992; Grandi and Colombo, 1997). The large nucleus has a distinct membrane and is often eccentric; it contains one or two prominent nucleoli. The cytoplasm is often homogeneous but, depending upon the stage of development and the species being studied, it may contain yolk granules, oil droplets, or pigment granules. Granular endoplasmic reticulum and abundant ribosomes have been described in the cytoplasm of some species and an active Golgi complex is found in some cells, often near clumps of mitochondria (Figure 1.8). Coated vesicles, suggestive of metabolic exchanges with the gonadal environment, were described in the cytoplasm of primordial germ cells of the carp Cyprinus carpio, often near the surface membrane (Figure 1.9) (van Winkoop et al., 1992).

Two cytoplasmic markers of uncertain significance are said to distinguish primoridal germ cells of Oryzias latipes from somatic cells: sheets of agranular endoplasmic reticulum and electron-dense mitochon-drial-associated granular material (Figure 1.10) (Hogan, 1978). The fenestrated sheets of agranular endoplasmic reticulum, perhaps produced by bleb-bing of the nuclear envelope, follow the contours of the nuclei, sometimes almost encircling them. In the germ cells of many species, mitochondrial associated granular material forms one or two compact masses that have been designated variously as balbiani bodies, perinuclear nuages, perinuclear dense bodies, and germinal dense bodies (Timmermans and Taverne, 1989; van Winkoop et al., 1992; Grandi and Colombo, 1997). They are loosely-woven and strandlike in premigratory germinal cells of Oryzias latipes; this appearance gradually changes as the cells begin their migration and, in the gonadal anlagen, these bodies are amorphous, of various sizes and shapes, and composed of electron-dense, fine fibrils (Figure 1.11) (Hamaguchi, 1985). In the carp Cyprinus carpio they increase in size with development and part of the material becomes more electron dense; these dark masses adhere to mitochondria and may be centres for mitochondrial multiplication (Figure 1.12) (van Winkoop et al., 1992).

1.3 Anatomy of the Female Genital System

The female genital system consists of the ovaries that produce eggs or ova and, in most species, a duct system communicating with the exterior. In addition to its cytocrine function of producing fertilizable gametes, the ovary shares with the testis the complementary endocrine function of secreting a variety of steroid hormones that regulate development of the germ cells. Oviducts may be simple passageways for the eggs but often their lining is glandular, and forms protective coverings for the eggs. In some viviparous species, young may develop within the ovary and, in others, within the oviducts.

Gonads of vertebrates always originate from bilateral primordia, but many species possess only one gonad as adults (Franchi, Mandl, and Zuckerman, 1962; Harder, 1975). In hagfish, the left gonad degenerates and only the right develops; in lampreys, the two primordia fuse during development forming a single midline gonad. Ovaries of elasmobranchs develop as paired structures along the dorsal wall of the coelom, on each side of the midline (Wourms, 1977; Dodd, 1983; Dodd and Sumpter, 1984; Callard et al„ 1989; Hamlett and Koob, 1999). In several species, either the right or left ovary fails to mature. The functional ovary may be the right (as in several viviparous sharks) or the left (some viviparous rays); in some oviparous skates, however, both ovaries are functional. Atrophy of the oviduct may occur on the same side as the nonfunctional ovary. There is a range in teleost fishes from complete fusion to partial fusion, involving only the posterior part of the gonad orjust the gonoducts (Franchi, Mandl, and Zuckerman, 1962; Harder, 1975). Sometimes one of the gonads is rudimentary or merely smaller but still present.

The ovaries are suspended in the posterior body cavity, dorsal to the gut, by mesovaria that run their entire length (Figure 1.13) (Harder, 1975). The ovaries may be displaced toward the intestine, coming to lie with the intestine within the mesentery (Figure 1.14). Beneath the visceral peritoneum that envelops each ovary is a capsule of connective tissue and smooth muscle, the tunica albugínea. The ovarian artery arrives by way of the mesovarium and branches repeatedly into the tunica albuginea.

The ovaries of cyclostomes, most elasmobranchs, and some teleosts are solid and ova are shed directly into the body cavity. No duct system is present in cyclostomes and the eggs are expelled from the body through genital pores that develop in the body wall shortly before spawning and lead into the urinary sinus or urinary duct and thence to the cloaca or urinogeni-tal papilla. In elasmobranchs, a duct system typical of higher vertebrates develops from the mesonephric or Mullerian ducts, gathering up shed eggs or embryos in a funnel-shaped ostium and passing them through an oviduct to the outside, often adding a shell or providing refuge for developing embryos along the way (Figure 1.15) (Hoar, 1969; Dodd, 1983). Two ovaries and two oviducts are present in the chimaera Hydrola-gus colliei. In general, the reproductive system of this holocephalan resembles that of elasmobranchs.

Although the ovaries of most elasmobranchs are solid, the visceral peritoneal covering of the ovary of lamnid sharks invaginates to create a hollow organ (Matthews, 1950; Pratt, 1988). The hollow is a direct extension of the peritoneal cavity and forms a branching invagination from a pocket on the right side of the outer surface (Figure 1.16). The channels penetrate all parts of the ovary and provide a means of exit for ripe oocytes (Figure 1.17).

Teleosts display two types of ovaries, gymnovar-ian and cystovarian (Dodd and Sumpter, 1984; Koya, Takano, and Takahashi, 1995). In the simpler gymn-ovarian condition, the ovaries are suspended in the coelom and ovulation occurs directly into the coelomic cavity. In cystovarian ovaries, adhesions occur which isolate a part of the coelom, thereby forming a closed sac, the ovarian cavity or lumen, into which ovulation occurs directly (Figure 1.18). This ovarian cavity communicates only with its duct and is lined with me-sothelium derived from the coelomic peritoneum. Following ovulation, eggs from both types of ovaries are bathed in a fluid. In gymnovarian ovaries, this fluid is coelomic fluid and is synthesized and secreted by the epithelium of the dorsolateral coelomic wall and mesovarium. In the enclosed cavity of cystovarian ovaries, the fluid is designated as ovarian fluid and originates in part from the epithelial lining. Since teleosts display a wide spectrum of reproductive methods, these fluids have manifold functions.

The cavity of cystovarian ovaries may be formed in two ways. An endovarian cavity is derived from internalized invaginations of the genital ridges where the ribbonlike ovaries roll up in a lateral and dorsal direction so that the side at which ovulation occurs is turned inward surrounding the cavity (Figure 1.18) (Goodrich, 1930). Alternatively, a parovarian cavity arises from a space enclosed by adhesions between the gential ridge and the parietal peritoneum. A range is seen from a simple ovary, where ova are shed into the ovarian cavity and soon expelled, to the complex, hollow organ of viviparous teleosts that produces eggs, stores spermatozoa, serves as a site for fertilization, and may provide nourishment for developing young for a prolonged period.

Steroid-producing cells are described in the ovary of tilapia Sarotherodon niloticus during ovarian differentiation (Nakamura and Nagahama, 1985). As the ovarian cavity is forming, these cells appear near the blood vessels of the stroma on the side facing the mesentery (Figure 1.19). They are presumed to originate from stomal elements and, as the ovary develops, their numbers increase surrounding the blood vessels. These cells display the tubular mitochondrial cristae and tubular agranular endoplasmic reticulum which are characteristic of steroid production (Figure 1.20). It is suggested that the steroids produced by these cells play a role in the formation of the ovarian lumen (Figure 1.21) and in the differentiation of germ cells.

In many teleosts, the visceral peritoneum covering the ovary and the peritoneum lining its cavity often continue posteriorly, beyond the mass of ovarian tissue, to form the unique oviduct (Hoar, 1969; Harder, 1975) (Figure 1.22). This extension of the ovarian cavity continues posteriorly to connect with the genital pore leading to the exterior. In some teleosts, including Osteoglossidae, Hiodontidae, Notopteridae, Anguilli-dae, the loach Misgurnus (Cobitididae), Galaxiidae, and Salmonidae, the oviducts degenerate in whole or in part so that the ova pass into the body cavity and make their exit by way of funnels or pores, depending upon the degree of degeneration of the oviducts (Hoar, 1969; van Tienhoven, 1983).

1.4 Ovary

The vertebrate ovary is an aggregation of developing follicles enmeshed in a vascular stroma of loose connective tissue and enclosed within an envelope of gonadal epithelium (Figure 1.23). The stroma consists of collagenous, elastic, and reticular fibres and becomes greatly distended as the follicles enlarge. Only in a spent ovary, when the stroma is collapsed, is it easily seen. Beneath the ovarian envelope, a condensation of stromal tissue forms the tunica albugínea, a capsule of connective tissue and, sometimes, smooth muscle (Figure 1.24). Collagenous and elastic fibres are most abundant in the peritoneum, less so in the tunica albuginea, and less again in the stroma itself. Reticular fibres are numerous in both the capsule and the stroma, appearing as fine, anastomosing argyro-philic threads surrounding each egg within a basket of fibres (Figure 1.25).

Early follicles consist of primordial germ cells, newly arrived by migration, enclosed within a cluster of follicular cells derived from divisions of the gonadal epithelial cells. Primordial germ cells divide by mitosis to produce diploid oogonia (Figure 1.26). By mitotic division these oogonia form diploid primary oocytes. Meiotic division of a primary oocyte results in a secondary oocyte and first polar body, both diploid (Figure 1.27) (Grandi and Colombo, 1997). The follicular epithelium eventually completely surrounds the secondary oocyte and the follicle increases greatly in size, first by the elaboration of more cytoplasm and later by the accumulation of yolk during the process of vitellogenesis. Finally the secondary oocyte undergoes maturation where a second meiotic division yields an ovum and another polar body, both haploid. The development and maturation of diploid oogonia to produce haploid ova is oogenesis. Polar bodies are small, functionless haploid cells resulting from unequal division during oogenesis. They allow most of the cytoplasm to pass to the ovum while providing for disposal of excess chromatin; polar bodies subsequently degenerate.

A detailed description of folliculogenesis in teleosts has been provided for adult females of the common snook Centropomus undecimalis (Grier, 2000). Oogonia, presumably having arisen from some external source, are dispersed among the somatic cells of the gonadal (germinal) epithelium that lines the ovarian lumen (Figure 1.3). The oogonia divide by mitosis, often forming nests of cells. At the outset of folliculogenesis, some oogonia begin to divide by meiosis to produce oocytes. These oocytes descend into the deeper, basal regions of the epithelium. Some epithelial cells, whose processes envelop meiotic oocytes, transform into prefollicular cells which become follicular cells at the completion of folliculogenesis (Figure 1.28). The oocyte, surrounded by its simple layer of prefollicular cells, presses on the basement membrane of the gonadal epithelium, evaginating it and eventually causing the pinching off of a discrete follicle consisting of the oocyte surrounded by its layer of folliclar cells and enclosed in a basement membrane. Cells of the stroma aggregate around the basement membrane of the follicle to form the vas-

cular theca interna and avascular theca externa. Fol-liculogenesis is considered to be concluded when the basement membrane completely encloses the follicular epithelium; the thecal layers may be fragmentary at this time. Ensconced within the ovarian stroma, the follicle continues its development.

In the past it has been proposed that oogonia and early meiotic stages are not present in the ovaries of adult cartilaginous fishes. It has been shown that oogenesis occurs early in the life of the spotted ray Torpedo marmorata, an aplacental, viviparous elas-mobranch with a reproductive cycle of three years (Prisco, Ricchiari, and Andreuccetti, 2001). The site of origin and migratory route of the primordial germ cells was not determined. Clusters of oogonia and early meiotic oocytes were observed in the gonadal anlage of embryos; germ cells in the clusters were connected by intercellular bridges bordered by an electron-dense membrane about 30 nm thick (Figure 1.29). In ovaries of subadult and adult females, however, all the germ cells were organized into follicles and no clusters of oogonia or early meiotic cells were found with a single exception. In one adult female, the persistence of germ cells not organized into follicles may support the concept of "reproductive plasticity" and represent the variation that is targeted by natural selection, thereby accounting for the different reproductive strategies adopted by cartilaginous fishes.

In lampreys, primary germ cells divide slowly in the gonadal ridge beneath the peritoneum (Lewis and McMillan, 1965; Hardisty, 1971). This is followed by a period of more rapid division when the germ cells form germ cell nests or cysts. Meiotic prophase begins in both isolated germ cells and in the cell nests. Cells from the peritoneal epithelium give rise to follicular elements. Germ cells may begin cytoplasmic growth or undergo degeneration (atresia) in early stages of meiotic prophase. Oogenesis occurs to some extent in most or all lamprey gonads. In gonads destined to become ovaries, oogenesis is more synchronous than in testes and, eventually, only oocytes are present. In male lampreys, oocytes that have proceeded to the cytoplasmic growth phase are eliminated by atresia so that the testis becomes smaller during differentiation and contains only small numbers of residual germ cells; at metamorphosis these develop, by mitosis, into nests of primary spermatogonia.

Division of developing germ cells in the ovaries of lampreys causes evaginations of the ovarian wall to expand ventrally, subdividing the ovary into lobes and lobules that contain a stroma of vascular connective tissue (Figures 1.30A to C) (Lewis and McMillan, 1965). These flat, leaf-like extensions of ovarian tissue, usually two follicles in thickness, are the oviger-ous folds (Figure 1.30D).

The ovary of the hagfish Myxine glutinosa is a dorsal, narrow ribbon suspended in the midline from the mesovarium (Walvig, 1963; Patzner, 1974, 1975). It consists of "series" of follicles that develop from oogonia contained within a band of germinal cells that runs along the free edge of the ovary (Figure 1.31A). When the oocytes enter the vascular connective tissue of the inner part of the ovary, undifferentiated epithelial cells from the mesothelium covering the ovary become attached to them, grow, and stretch to enclose them within a follicular epithelium. "Generations" of oocytes develop from oogonia in the germinal ridge; they grow to a diameter of 1 to 2 mm whereupon they enter a resting period, awaiting ovulation of the previous generation of oocytes (Patzner, 1974). The primary follicle consists of an oocyte surrounded by its follicular epithelium and is contained within a connective tissue sheath, the theca (Figure 1.31E). Two layers can be discerned in this connective tissue: the theca interna and theca externa. The largest and oldest of the immature eggs are found dangling in individual peritoneal slings, the follicular ligaments, in a single row at the ventral boundary of the ovary (Figure 1.31F). Some of these eggs mature, are ovulated, and a new row of follicles develops to take their places (Figure 1.31G). Empty postovulatory follicles remain for a time, hanging from the free surface of the ovary; later they withdraw into the stroma and undergo regression.

The visceral epithelium lining the hollow, cystovar-ian ovaries of many teleosts is thrown into a complex series of ovigerous folds (Figure 1.32) (Hoar, 1969). In the stickleback Eucalia inconstans, the folds project into the centre of the cavity, usually extending the entire length of the ovary, crowding the cavity into a ventrolateral position where no folds originate (Braekevelt and McMillan, 1967). Early germ cells are first found on the edge of the folds bordering the ovarian cavity. As the follicles mature and increase in size, they are pushed deeper into the stroma. When the follicles are mature, they burst through the visceral peritoneum and are ovulated into the ovarian cavity where they remain until spawning. The cavity, which becomes largely obliterated as the eggs reach maturity, continues posteriorly as the lumen of the oviduct.

The ovary of teleosts is supplied with blood from several arteries that branch off the dorsal aorta, entering the dorsal side of the ovary through the mesovari-um. Each follicle in the ovary of Fundulus is supplied by a primary arteriole that reaches it through a stalk of stromal elements (Figures 1.33A,B) (Brummett, Dumont, and Larkin, 1982). Branches of the arteriole penetrate deeply into the investing layers of the follicle. The superficially located vessels in the apical or luminal hemisphere of the follicle are more readily apparent and are presumed to be venules that collect blood from the intervening capillary bed and transport it to a vessel that forms a loop on the luminal side of the follicle (Figures 1.33C and A). These vessels terminate in a single collecting vessel that courses toward the outer surface of the ovary, merging with others into two large veins that unite in the dorsal, anterior region of the ovary to form the single ovarian vein. Thick bundles of non-myelinated nerves, presumably autonomic, accompany the ovarian artery and vein in the ovary of tilapia Oreochromis niloticus (Figure 1.34) (Nakamura, Specker, and Nagahama, 1996). Groups of a few axons ramify from these nerves to terminate among the cells surrounding the follicles.

In the killifish Fundulus heteroclitus, the posterior portion of the ovary is a thin-walled nongerminal ovisac that is continuous with the short oviduct and apparently serves as a receptacle for ovulated eggs prior to their release to the external environment (Brummett, Dumont, and Larkin, 1982). The luminal epithelium of this thin-walled region displays a localized population of unusual cells with long cytoplasmic extensions bearing short microvilli (Figure 1.35). These cells may continue to line at least part of the oviduct and may function in the transport of ovulated eggs or they may secrete a jelly that forms a surface coat for the extruded eggs.

The microscopic structure of the ovarian luminal wall has been described in a few oviparous teleosts. (The ovarian wall of viviparous teleosts is described in the section on Viviparity.) In the medaka Oryzias latipes, its epithelium displays cyclic changes corresponding to changes in the ovary (Yamamoto, 1963). As the ova mature, the luminal wall thickens and its simple cuboidal epithelium becomes simple columnar as secretory activity increases (Figure 1.36). The underlying tunica albuginea thickens as vascularization increases and smooth muscle develops. It is presumed that the fluids produced by the epithelial cells facilitate extrusion of the eggs at spawning as well as playing some role during external fertilization. After spawning, the epithelium and tunica albuginea undergo involution.

A simple columnar epithelium of microvillous cells lines the ovarian cavity of the bleak Alburnus alburnus (Lahnsteiner, Weismann, and Patzner, 1997). Around the time of ovulation, these cells bulge api-cally into the lumen and a few small clusters are also found in the oviduct (Figure 1.37). These cells have diverse functions: they are secretory, maintaining the ionic gradient of the ovarian fluid by the secretion of glucose, proteins, and enzymes (acid phosphatase, protease, and |3 D-glucuronidase); they synthesize glucuronide steroid (which may act as a pheromone that attracts males and increases the volume of milt); and they are phagocytic. Protein production by microvillous cells is indicated by the prominent nucleolus and abundant tubular granular endoplasmic reticulum that forms concentric layers around the basal nucleus (Figure 1.38). Coated vesicles pinched off from the endoplasmic reticulum are associated with the ubiquitous Golgi complexes; secretory vesicles are released from the Golgi cisterns. The involvement of the microvillous cells in ionic regulation of the ovarian fluid is demonstrated by the presence of apical junctional complexes sealing the epithelium, the abundance of apical mitochondria, and positive tests for apical adenosine triphosphatase activity. Maintenance of the inorganic composition of the ovarian fluid is essential to prevent egg activation: closure of the micropyle, swelling of the eggs due to water uptake, and shell hardening. Table 1.1 records analyses of the composition of the ovarian fluid in the bleak4. The microvillous cells of the bleak contain heterophagic vacuoles and lamellar bodies and are active in the phagocytosis of débris from the lumen. Autophagy occurs in regions containing secretory vesicles. The cytoplasm also contains lipid vacuoles and accumulations of free ribo-somes. Agranular endoplasmic reticulum is rare.

Ovaries of the syngnathids, the pipefish Syngnathus scovelli, and seahorse Hippocampus erectus, are unusual in that they lack ovigerous lamellae and present a sequential array of developing follicles arranged within a single sheet in order of their developmental maturity (Begovac and Wallace, 1987, 1988; Sel-

4 Similar values for four species of salmonids are recorded in an earlier paper by these authors (Lahnsteiner, Weismann, and Patzner, 1995).

man, Wallace, and Player, 1991). This feature is especially useful for studies of follicular development. The ovaries are asynchronous and, because these fish are reproductively active throughout the year, a heterogeneous population of follicles is available in all developmental stages.

The ovaries of both species of syngnathids consist of a sheet of follicles, with their supporting vascular stroma, in the form of a tubular scroll extending lengthwise (Figure 1.39) (Begovac and Wallace, 1987; Selman, Wallace, and Player, 1991). A single germinal ridge of the pipefish ovary forms the inner margin of the scroll and, extending from this, is a sequential array of follicles arranged according to their developmental age, sandwiched between the outer ovarian wall and the inner luminal epithelium. Mature oocytes are ovulated from the opposite margin of the scroll, the mature edge, into the ovarian lumen (Begovac and Wallace, 1987, 1988). In the seahorse, there are two germinal ridges that run the length of the tubular ovary on opposite edges of the ovarian sheet (Selman, Wallace, and Player, 1991). Developing follicles arise from each germinal ridge and grow toward a shared mature region near the middle of the sheet. These sequences may be seen in any cross section of sygnathid ovaries (Figure 1.40). In contrast to the random organization of ovaries of other fish, this temporal and spatial correlation of oocyte development, as seen in these two species, facilitates the study of specific events in follicular development. In addition, the earliest events of oogonial proliferation and early follicle formation, because of their specific location within the ovary, are accessible to experimental manipulation.

These syngnathid ovaries are covered on the outside by visceral mesothelium of squamous to low cu-boidal cells that is continuous with the mesovarium; these cells have irregular apical surfaces and numerous pits and vesicles on both their apical and basal surfaces (Figure 1.41A). They contain abundant cytoplasmic filaments and have junctional complexes at their apico-lateral surfaces. This epithelium is subtended by variable amounts of collagenous connective tissue connecting it to several layers of smooth muscle (Figure 1.41B). Blood and lymph vessels are visible in this connective tissue, with the larger vessels apparent at the connection with the mesovarium. Smooth mus

TABLE 1.1. Composition of the ovarian fluid of the bleak Alburnus alburnus (n = 10). (From Lahnsteiner, Weismann, and Patzner, 1997)

Component

Value

pH

8.61 ±0.10

Osmolality (mosmol kg1)

237.0 ±27.12

Na+ (mmol l1)

171.58 ±25.83

K+ (mmol l1)

2.93 ± 0.57

Ca++ (mmol l1)

0.63 ±0.11

Protein (mg x 100 ml

158.48 ± 37.27

Glucose (pmol 1~')

2064.76 ± 529.23

Fructose (pmol 1~')

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