Reviews in Fish Biology and Fisheries 12: 33–58, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 33 Gamete physiology, fertilization and egg activation in teleost fish K. Coward1 , N.R. Bromage2 , O. Hibbitt1 & J. Parrington1 1 Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK (Phone: +44 (0) 1865 271850; Fax: +44 (0) 1865 271853; E-mail: kevin.coward@pharm.ox.ac.uk); 2 Institute of Aquaculture, University of Stirling, Scotland, FK9 4LA Received 7 April 2002; accepted 17 October 2002 Contents Abstract Introduction Modes of reproduction in teleost fist The teleost gonad Ovarian physiology Anatomy of the ovary Ovarian growth and regulation Testicular physiology Testicular morphology and spermatogenesis Morphology, physiology and biochemistry Activation of sperm motility Sperm quality and cryopreservation Fertilization and activation of teleost oocytes Exogenous and endogenous factors affecting fertilization Problems associated with fertilization in commercial species Physiology of fertilization The mechanism of egg activation The three models of egg activation Post-fertilization signalling events Summary and future studies Acknowledgements References page 33 34 34 35 36 38 42 51 51 51 Abstract The fertilization and activation of fish oocytes are vital, but unfortunately overlooked, processes in fisheries research. This paper sets out to review our present understanding of these important events in teleost fish and, drawing comparisons with mammalian research, to highlight areas in which research effort is urgently required. Presently, the commercial culture of many important freshwater, but especially marine, teleosts is beset by problems associated with fertilization, hatching and early embryonic development. These problems have been particularly acute in certain species leading to the application of spawning induction technologies in an effort to optimize production. Increased knowledge of the processes of egg activation and fertilization in these groups of fish is likely to make significant contribution to commercial aquaculture. Studies of a wide variety of animal and plant species has demonstrated that development at fertilization is triggered by an increase in intracellular Ca2+ concentration within the egg that occurs as either a single transient or a series of distinctive oscillations depending upon the species under investigation. This increase in intracellular Ca2+ activates the egg and also appears to play an important role in later embryonic development. Teleost reproductive strategies and more importantly, teleost oocytes and spermatozoa, exhibit a remarkable variety of adaptations. Currently, studies of egg activation in teleosts are confined to laboratory species such as medaka Oryzias latipes and zebrafish Brachydanio rerio. Nevertheless, even between these two species, although an increase in intracellular Ca2+ appears to be the trigger 34 in both cases, the mechanism of Ca2+ release may be quite different. Activation in medaka is initiated only through direct contact with conspecific sperm, suggesting the involvement of a sperm-specific factor, while zebrafish eggs appear to require only contact with the external spawning medium. In view of the highly variable fertility rates evident in many commercially cultured teleosts, it could be very rewarding to investigate the mechanism of egg activation in representative teleost groups using the findings and theories emerging from other animal groups as a starting point. In order to successfully conduct such an investigation, it will be necessary to employ a combination of physiological, molecular and recombinant approaches. Introduction The processes of fertilization and egg activation are highly important issues in fish reproductive biology, particularly in those species intensively cultured. Marine and freshwater aquatic environments support over 24,000 species of teleost (bony) fish. Fish, like all other vertebrates, reproduce sexually. In the vast majority of fish species, eggs and spermatozoa are produced in separate individuals and gametes expelled into the external aquatic environment, whereupon fertilization and egg activation take place. The array of reproductive strategies evident amongst teleosts is however, extraordinarily diverse and will be discussed briefly later. Much of our knowledge concerning reproductive physiology in fish originates from commercially-important species including salmonids such as the Pacific salmon and rainbow trout (Oncorhynchus spp.), the Atlantic salmon Salmo salar (L.), tilapia (Oreochromis and Tilapia spp.), carp (Cyprinus spp.) and catfish (Clarias spp). However, our present knowledge concerning the key intracellular and molecular events accompanying fertilization and egg activation in fish is limited solely to small laboratory species that have no real commercial importance but represent useful laboratory models for developmental biology; these species include zebrafish Brachydanio rerio (Hamilton), medaka Oryzias latipes (Temminck & Schlegel) and bitterling Rhodeus ocellatus ocellatus (Kner). It is well known that certain commercially important fish species, notably marine species, suffer from low fertilization and hatching rates; examples include Atlantic halibut Hippoglossus hippoglossus (L.) (Norberg et al., 1991; Holmefjord et al., 1993; Bromage et al., 1994), sole Solea solea (L.) (Houghton et al., 1985), turbot Scophthalmus maximus (L.) (Bromley et al., 1986), gilthead seabream Sparus auratus (L.) (Carrillo et al., 1989) and some salmonids (Bromage et al., 1992). Furthermore, similar problems often arise when fish are held captive in artificial environmentally controlled conditions. These problems have serious ramifications for successful and profitable culture. There are numerous possible explanations for these observations; which will be discussed later. However, there is a distinct paucity of focused research in this vital area of teleost reproductive biology. In contrast, decades of research in other animal species has revealed much about the chemical messengers and mechanisms involved in fertilization and egg activation (Swann and Parrington, 1999). Most recently, the focus has been on uncovering the molecular identity of the signalling proteins involved (Parrington, 2001). The findings should form an important starting point for investigation of the molecular mechanisms of reproduction in commercially-relevant fish species if we are to successfully address fertilization and hatching problems in the future. This paper sets out to review our present knowledge of gamete physiology, fertilization and egg activation in fish. The review focuses only upon the bony (teleost) fishes and does not include sharks, rays and skates (Chondrichtyes). Reproduction in the Chondrichtyes is reviewed at length by Dodd (1983). Here, we look briefly at the different modes of reproduction adopted amongst teleost species along with overviews of oocyte and sperm physiology. This is followed by an overview of our present understanding of fertilization and oocyte activation in fish and by comparison with other animal species, we make recommendations for future research. Modes of reproduction in teleost fish The array of reproductive strategies evident amongst the teleostei is extraordinarily diverse (see Hoar, 1969; Jobling, 1995). Some species, such as the live-bearing Amazon molly Poecilia formosa (Girard), are represented solely by the female sex; females court and mate with males of a related species. In these species, gametes do not fuse, the sperm serves only to initiate development of the egg. Other fish undergo natural 35 sex change, a process indicating sexual plasticity not evident in other vertebrate groups. Hermaphroditism is common in coral reef and deep sea species. Those species that exhibit maturation of both parts of the ovo-testis at the same time are known as synchronous hermaphrodites and are considered both male and female from a functional viewpoint. Species that undergo sex reversal are known as successive hermaphrodites; these either begin life as male and then become female (protandrous) or vice versa (protogynous). For detailed descriptions of teleost hermaphroditism, see Hoar (1969) and Dodd (1977). In the majority of teleosts, eggs and sperm develop in separate female and male sexes, although a wide array of reproductive patterns is evident. Some teleosts establish close pair-bonding strategies, others adopt group spawning mechanisms (for review see Jobling, 1995). Most teleosts are oviparous and release yolky eggs into the external aquatic environment where they are subsequently fertilised (e.g. salmonids, cyprinids and tilapiines). Egg laying (or oviparity) involves fertilization and subsequent embryonic development outside of the maternal body cavity; juveniles hatch when the egg envelope is broken. The egg yolk provides the appropriate nutrients for developing embryos. Oviparous fish can be sub-classified into ovuliparous and zygoparous species. Ovuliparous fish are those in which mature oocytes are released from the female and fertilized/activated in the external environment. Zygoparous fish, on the other hand, retain fertilized ova within the body of the female for short periods before being released into the external environment. Zygoparous fish, although egg laying, therefore undergo internal fertilization and include skates, some sharks and a small number of teleosts; see Hoar (1969) for review. External fertilization (where eggs and sperm are simultaneously released into the external environment) is by far the most common in teleosts, though an estimated 2–3% of teleosts, along with >50% of cartilaginous fish, adopt internal fertilization (Jobling, 1995). Fertilised eggs in those species that adopt internal fertilization develop within the maternal reproductive system and hatching either precedes or coincides with parturition resulting in the female giving birth to a free living juvenile. In teleosts, developing eggs and embryos are retained within the ovarian follicle or lumen. In ovoviparous species, eggs usually develop within a modified section of the oviduct of the female. Following internal fertilization, eggs are retained until hatching. Developing embryos obtain nutrients from the egg yolk and oxygen from the female via highly vascularised oviduct walls. Viviparous teleosts produce eggs that develop either in the ovary or uterus. Nutrient requirements are fulfilled by both egg yolk and additional maternal secretions. In some species, a primitive “placenta” may develop, allowing more efficient nutrient transfer. For reviews on viviparous species, see Hoar (1957), Amoroso (1960), Hogarth (1976) and Wourms (1981). Irrespective of the mode adopted, teleost reproduction occurs in cycles that involve gamete development, maturation and spawning. Fish in equatorial regions such as Nile tilapia Oreochromis niloticus (L.) will breed all year round (Lowe-McConnell, 1958) whilst other more temperate species such as the rainbow trout Oncorhynchus mykiss (Walbaum) exhibit distinct spawning seasons confined to a discrete time period (Bromage et al., 1992). Generally speaking, spawning cycles of teleost fish are adjusted by an interaction of a series of environmental factors including photoperiod, temperature, rainfall and salinity (de Vlaming, 1974). In teleosts, ovarian and testicular development and the ultimate production of mature oocytes and sperm are highly complex processes modulated by various exogenous and endogenous pathways such that juveniles are produced only at times when the chances of fry survival are optimal, i.e. usually when food availability is highest. The teleost gonad As in all other vertebrates, the gonads of teleost fish originally arise as paired structures in the dorsal lining of the peritoneal cavity. Although gonads develop as paired structures, some adult fish species possess only a single gonad (Jobling, 1995). This can be the result of fusion during developmental phases, or the failure of one gonad to develop fully. In other species, although two gonads are present, one appears small, under-developed, and is non-functional. Partial fusion, involving the posterior gonadal regions or the gonadal ducts, may also arise. For reviews on gonadal development and functional morphology in teleosts see Dodd (1977), Grier (1981), Wallace and Selman (1981), Billard et al. (1982), de Vlaming (1982), Nagahama (1983), Billard (1995), Tyler and Sumpter (1996) and Brooks et al. (1997). 36 Ovarian physiology Anatomy of the ovary Ovaries are paired structures although in some species one of the ovaries is reduced in size and remains non-functional. The reproductive system of female teleosts tends to show marked variation, reflecting the diverse nature of reproductive strategies adopted. As in other vertebrates, the teleost ovary develops as an attachment to the dorso-lateral lining of the peritoneal cavity. Developing ovaries are paired, elongate hollow organs positioned immediately ventral to the swim bladder and consist of numerous partial transverse septa projecting into the ovarian lumen (van den Hurk and Peute, 1979). It is upon these septa that developing oocytes grow. Two forms of ovarian structure have been recognized: cystovarian (closed) and gymnovarian (naked) (Hoar, 1969). The cystovarian ovary (e.g. the tilapia ovary) remains fully enclosed by the peritoneum (mesovarium) and following maturation, oocytes are released into a central ovarian lumen leading to the posterior oviduct (Mullerian duct). The gymnovarian ovary, however, is only partially covered by the peritoneum and releases mature oocytes directly into the body cavity (e.g. salmon, trout). Oocytes are subsequently released into the external environment via the oviduct, a posterior continuation of the peritoneum opening into a urogenital sinus (van den Hurk and Peute, 1979). Oviducts are formed from posterior outgrowths of the tissues that encircle the ovary (the ovarian tunic). As a result, in many teleosts, oocytes pass straight from the ovary to the oviduct without entering the body cavity. In some teleosts (e.g. salmonids, anguillids, cyprinids), partial or total degeneration of the oviducts occur such that oocytes are released from the ovary into the body cavity, before being released into the external environment. A major structural difference between mammalian oocytes and teleost oocytes is the presence of the micropyle at the animal pole of the teleost oocyte. Teleost spermatozoa do not possess an acrosome and therefore penetrate the oocyte via a small opening in the oocyte membrane, the micropyle (for review, see Riehl and Götting, 1974). The size of the micropyle is species-dependent but is usually only wide enough to allow one sperm through at a time. This adaption is crucial in the prevention of polyspermy. Very little is known of the processes forming the micropyle, although it first appears during early stages of ovarian development before exogenous yolk incorporation (vitellogenesis) begins. Riehl (1977) proposed that the micropyle is formed from highly specialized follicle cells during formation of the oocyte membrane. The micropyle is occupied by highly specialized micropylar cells until ovulation; studies suggest that these cells provide a plug for the micropyle as well as having a secretory role (Suzuki, 1958). The fine structure of the micropyle was recently described in the hagfish (Morisawa, 1999). In zebrafish, there is a tuft of plasma-membrane derived microvillii at the micropyle to which a sperm head binds (Hart and Yu, 1980); the tuft is thought to increase the surface area of the plasma membrane coming into contact with the external spawning medium. Ovarian growth and regulation To date, all teleosts studied exhibit the same basic pattern of oocyte growth (reviewed by Tyler and Sumpter, 1996). Ovarian development is sub-divided into distinct developmental stages according to physiological, biochemical, morphological and histological criteria (Nagahama, 1983, 1994; Selman et al., 1986; Bromage and Cumaranatunga, 1988; Tyler and Sumpter, 1996; Coward and Bromage, 1998). Although classification schemes tend to vary, the generally accepted system is stage 1 (chromatin nucleolar), stage 2 (early perinucleolar), stage 3 (late perinucleolar), stage 4 (cortical alveolar), stage 5 (early vitellogenesis), stage 6 (maturing/late vitellogenesis) and stage 7 (germinal vesicle migration and breakdown) (see Figure 1). Major developmental phases are classified into six main phases: oogonial proliferation, oogenesis, primary growth and folliculogenesis, cortical alveolus stage, vitellogenesis, maturation and ovulation. For a detailed description of these phases see Bromage and Cumaranatunga (1988) and Tyler and Sumpter (1996). For detailed discussion of the ultrastructural and molecular events associated with oocyte development see Selman et al. (1993). Teleosts possessing well defined breeding cycles exhibit periods of oogonial proliferation immediately before, during or after the main spawning period (Franchi et al., 1962; Khoo, 1979) although pools of oogonia are available for recruitment throughout (Bromage and Cumaranatunga, 1988). Folliculogenesis begins during the meiotic transformation of oogonia and results in primary oocytes being drawn away from oogonial nests to become closely related with pre-follicle cells (Moser, 1967). Following meiotic prophase, oocytes enter a growth phase 37 Figure 1. Transverse histological thin section of a tilapiine ovary stained with haematoxylin and eosin showing a stage 6 (vitellogenic) oocyte with centrally-positioned germinal vesicle. N = Nucleus (germinal vesicle), S2 = stage 2 oocyte, S6 = stage 6 oocyte. involving amplification of ribosomal genes and the transport of RNA and mRNA from the nucleus to the periphery to form “Balbiani bodies”. As primary oocytes grow, “Balbiani bodies” disperse and a follicular layer consisting of thecal and granulosa cell layers begins to develop. Oocytes then enter the cortical alveolar stage in which numerous vesicles/ alveoli appear at the oocyte periphery. The temporal distribution and size of the cortical alveoli varies quite widely amongst teleosts (see Gilkey et al., 1978; Hart and Donovan, 1983). The contents of these alveoli has been the source of much speculation; suggestions include glycosaminoglycans (Yammamoto, 1956), polysaccharides (Khoo, 1975), endogenously derived glycoprotein (Korfsmeier, 1966; teHeesen and Engels, 1973; teHeesen, 1977) and lectins with sugar binding properties (Krajhanzl et al., 1984a, b; Nosek, 1984). Selman et al. (1986) further detected a large glycoconjugated protein (>200 kDa) within cortical alveoli. Towards the end of this growth phase, but depending upon species, the cortical alveoli can almost entirely fill the oocyte cytoplasm. As oocyte growth proceeds, the cortical alveoli gradually become displaced to the periphery of the oocyte and at fertilization they release their contents into the perivitelline space (Yamamoto, 1961). In rainbow trout, the 200 kDa glycoproteins undergo depolymerization into much smaller 9 kDa fragments (Inoue and Inoue, 1986). The contents of the cortical alveoli serve to harden the vitelline envelope after ovulation and prevent polyspermy. There are also reports that polyspermy is prevented in sturgeon and trout eggs by the discharge of cortical alveoli into the micropylar region (Ginsburg, 1972). Cortical alveoli and their role in fertilization have been extensively studied in teleosts such as the medaka (Iwamatsu et al., 1988; Iwamatsu, 1998) and the rose bitterling (Ohta et al., 1990). In the rose bitterling, cortical alveoli of various sizes exist in multiple layers within the cortical cytoplasm of each egg. Two types of alveolus are distinguishable, one possessing homogeneous contents, the other possessing heterogeneous contents (Ohta et al., 1990). At fertilization, cortical alveoli breakdown is associated with a large increase in intracellular free Ca2+ ions (Ohta et al., 1990). Undoubtedly, the most critical period of oocyte growth is vitellogenesis (a process common to all oviparous animals). Vitellogenesis involves the uptake of a hepatically-derived glycolipophosphoprotein (vitellogenin, VTG) by receptor-mediated endocytosis involving a specific receptor bound to the oocyte membrane (Prat et al., 1998; Perazzolo et al., 1999). VTG is sequestered from the blood by growing oocytes to form an embryonic nutrient source. In teleosts such as the rainbow trout, sequestration and packaging of VTG into its yolk derivatives (lipovitellins and phosvitins) by proteases such as cathepsin D (Brooks et al., 1997; Kwon et al., 2001), accounts for over 95% of final egg size (Tyler, 1991). There then follows a period of maturation during which VTG sequestration continues such that yolk globules fill the entire ooplasm. The nucleus (germinal vesicle) later migrates to the oocyte periphery to adopt a position at the animal pole immediately beneath the micropyle. Hydration during the final phases of development can account for up to 88% of final egg size in some teleosts 38 but remains negligible in others (Wallace and Selman, 1981). Hydration is important in species that produce pelagic (or buoyant) eggs and is seen typically in marine teleosts. The acquisition of buoyancy in such species is a key event in reproduction and affects both fertility and survival of spawned eggs (Carnevali et al., 1999). The process of ovarian development and the ultimate release of mature oocytes is synchronized by an interrelated series of internal (endogenous) and external (exogenous) stimuli (for reviews, see Lam, 1983; Peter, 1983; Idler and Ng, 1983; Nagahama et al., 1995). Exogenous factors such as temperature, photoperiod, food availability, water quality and a variety of social factors (e.g. visual, chemical or tactile contact with conspecifics) are perceived by the brain and translated into neural impulses that stimulate the endocrine pathways of the hypothalamopituitary-gonadal axis to respond in appropriate fashion. Key endogenous hormones involved in this process include hypothalamic gonadotropin releasing hormone (GnRH), pituitary gonadotropins GTH I and GTH II (analogous to mammalian FSH and LH respectively, Prat et al., 1996), sex steroids, progestagens, and prostaglandins. Evidence also exists to suggest that, as in mammals, a variety of growth factors and paracrine factors also play an important role in teleost ovarian development (Tyler and Sumpter, 1996). Ovulation (i.e. the release of a mature oocyte from its follicle) involves the neuroendocrine system (Peter et al., 1978) and numerous other regulators: proteases, protease inhibitors, eicosanoids, catecholamines, vasoactive peptides and especially progestational steroids and prostaglandins (see Berndtson and Goetz, 1986; Hsu and Goetz, 1992; Goetz, 1993; Goetz and Bradley, 1994). Testicular physiology Testicular morphology and spermatogenesis Testis size is highly variable amongst teleosts and varies from 0.2 to 10% of total body weight (Billard, 1969). Testes arise as paired structures within the dorsal aspect of the body cavity. In most teleosts, testes are elongate, white/cream coloured lobulate organs attached to the dorsal wall of the body cavity. Teleost testicular structure is variable but essentially two distinct types, lobular and tubular, are evident (Billard et al., 1982). Lobular testis are far more common amongst teleosts and consist of numerous lobules separated from each other by thin layers of connective tissue extending from the testicular capsule (Roosen-Runge, 1977). The interstitium between lobules consists of interstitial cells, fibroblasts and blood and lymph vessels. The lobular component contains two cell types: germ cells and distinct somatic cells lining the periphery of the lobule. The terminology used to describe these somatic cells has been the source of much debate. Originally referred to as lobule boundary cells (Marshall and Lofts, 1956), the somatic cells were considered homolous to mammalian Leydig cells (Marshall and Lofts, 1956; O’Halloran and Idler, 1970). The term lobule boundary cell is considered acceptable, anatomically speaking, but functional homology to mammalian Leydig cells is doubtful (see Nagahama, 1983). In some species, the lobule boundary cells are considered more likely to be homologous to Sertoli cells as cells are found in close proximity to spermatids/developing sperm and possess various structures that indicate a phagocytic role (Billard, 1970; Billard et al., 1972; Grier, 1975). For more information on the terminology of testicular components, refer to Grier (1981). A sperm duct (vas deferens) originates from the posterior region of each testis and leads to the urinary papilla located in between the rectum and urinary ducts. The teleost sperm duct, unlike those of most other vertebrates, are not part of the nephric or Wolffian duct. Van den Hurk et al. (1974) report that epithelial cells lining the sperm duct possess microvillii with numerous mitochondria and alkaline phosphatase activity within the apical region; these observations imply an involvement in the regulation of ionic composition and osmotic pressure of the seminal fluid. It is thought that epithelial cells lining the efferent duct are derived from Sertoli cells during spermiation (Pandey, 1969; van den Hurk et al., 1974; Gardiner, 1978; Grier et al., 1978; Grier, 1981). Specific glandular structures, often referred to as seminal vesicles, are also commonly found in teleosts. Protrusions along the medial ventral wall of the goldfish sperm duct have been reported and suggested to be homologous to the mammalian seminal vesicle, serving essentially as a sperm reservoir (Takahashi and Takano, 1972). Accessory structures to the male reproductive system are known to exist in several teleost families, but their role in sperm production and fertilization remains unclear. In gobies (gobidae spp.), the struc- 39 ture of seminal vesicles exhibits great variability (Scaggiante et al., 1999); larger males have smaller testes and larger seminal vesicles compared to those of smaller males. The main role of seminal vesicles in large male gobies is mucin production, but sperm storage, is important in smaller fish (Scaggiante et al., 1999). In brief, sperm (spermatozoa) are formed from germ cells via a series of developmental changes known collectively as spermatogenesis, the final part of the process being spermiogenesis. Spermatogenesis involves initial proliferation of spermatogonia through repeated mitotic division then a growth period to form primary spermatocytes. These then undergo reduction division (meiosis) to form secondary spermatocytes; leading to the spermatid phase, and following metamorphosis, into the motile spermatozoa. In teleosts with lobular testis, primary spermatogonia undergo meiosis resulting in the formation of numerous cysts surrounded by Sertoli cells. Within each cyst, germ cells divide synchronously. However, spermatogenesis tends to proceed asynchronously in the lobules, and therefore, provides batches of germ cells at different stages of differentiation. As spermatogenesis proceeds, cysts grow in size and eventually rupture to release spermatozoa into the lobular lumen (although sometimes, spermatids may be released instead). As more cysts burst, more spermatozoa collect in the lumen. Following a species-dependent period within the lobular lumen, sperm are released into the sperm duct (spermiation). A very different mechanism is found in those few teleosts (e.g. the guppy, Poecilia reticulata Peters) possessing tubular testis (Billard et al., 1982). In these species, primary spermatogonia are only found in cysts located at the blind end of the tubules. As spermatogenesis proceeds, cysts gradually migrate towards the sperm duct; there is no structure analogous to the lumen found in lobular testis (Roosen-Runge, 1977; Pilsworth and Setchell, 1981; Billard et al., 1982). Morphology, physiology and biochemistry Morphology of the teleost spermatozoon has been studied in many species (for reviews, see Jamieson, 1991; Billard, 1995); see Figure 2a and 2b). Fine structure of spermatozoa in two marine teleosts, the red mullet Mullus barbatus (L.) and the white bream Diplodus sargus (L.) are discussed by Lahnsteiner and Patzner (1995). Spermatozoa are subdivided into head, neck, neck-piece, mid-piece and tail. The head piece lacks the acrosome seen in all other vertebrate groups, this being a functional adaptation to the presence of a micropyle on the teleost oocyte. However, acrosomes are found in agnathans (hagfish and lampreys), paddlefish and sturgeons (Acipenseriformes) (Ciereszko et al., 2000). Within the teleosts, temporary acrosomelike structures have been reported, for example, in Lepadogaster lepadogaster (Bonnaterre) (Mattei and Mattei, 1978), brown trout (Billard, 1983) and several others (Jamieson, 1991). The sperm head is generally spherical or sickle cell shaped, although crescent-shaped heads occur in eels (Colak and Yamamoto, 1974; Todd, 1976). The shape of the nucleus is highly variable and appears to be related to the complexity of spermatogenesis and spermiogenesis (Grier, 1981; Billard et al., 1982; Billard, 1986; Billard, 1990). The mid-piece consists of a central flagellum surrounded by a mitochondrial sheath; the number of mitochondria present is highly species dependent (Jamieson, 1991). Studies in black porgy Acanthopagrus schlegeli (Bleeker), black grouper Epinephelus malabaricus (Bloch and Schneider) and Atlantic croaker Micropogonias undulatus (L.) (Gwo, 1995) suggest that mitochondria within the midpiece form the energy source for motility. The morphology of teleost sperm appears to reflect the mode of fertilization adopted (Nagahama, 1983). Species exhibiting external fertilization strategies produce primitive, round-headed spermatozoa (Grier, 1981), whereas various morphological modifications exist in those species with internal fertilization (Billard, 1970; Grier, 1981); these modifications usually involve elongation of the sperm nucleus and enhanced development of the mid-piece. For a comparison of sperm morphology between teleosts adopting internal and external fertilization, see Pavlov et al. (1997). The biochemistry of sperm is reviewed by Billard and Cosson (1990), Linhart et al. (1991) and Billard (1995). Organic composition and enzymic content are discussed by Belova (1982), Billard and Cosson (1990), King et al. (1990) and Jamieson (1991). Seminal plasma of teleosts are known to contain several proteinase inhibitors. Anti-proteinase activity has been reported in rainbow trout, whitefish Coregonus clupeaformis (Mitchill) and yellow perch Perca flavescens (Mitchill) (Dabrowski and Ciereszko, 1994), thereby raising the question of whether these proteins play a regulatory role in anacrosomal spermatozoa. The presence of uric acid (Ciereszko 40 Figure 2. Electron micrographs of fish spermatozoa. Left, guppy Poecilia reticulata × 25000; right, Mozzambique tilapia Oreochromis mossambicus × 40000. Key: C, centrioles; CC, cytoplasmic canal; MP, mid-piece; H, head. Kindly provided by Dr. Roland Billard (Ifremer, France). 41 et al., 1999) and ascorbic acid (Ciereszko and Dabrowski, 2000) in teleost appear to protect the spermatozoa against oxidative damage. Interestingly, common biochemical parameters for fish and mammal sperm provide evidence for the use of fish sperm as a model in biomedical research (Cieresko and Dabrowski, 1994). Production of sperm has been quantified in several species (Billard, 1995). In the guppy, a species with continual spermatogenesis, the number of sperm produced is 150 × 106 sperm per gram of testis per day (Billard, 1969). Generally, spermatozoa concentrations in teleosts range from 2 × 106 to 5.3 × 1010 spermatozoa per ml (Leung and Jamieson, 1991). Spermiation in teleosts involves the thinning or hydration of sperm by a hormonally-regulated process (Billard et al., 1972) and usually occurs just prior to spawning. The precise physiological significance of hydration is unclear but is thought to increase interlobular pressure thereby aiding transport of the sperm (Billard et al., 1972). Activation of sperm motility Generally, teleost spermatozoa (particularly in marine species) do not become fully motile until they have been exposed to the external aquatic environment or ovarian fluid, although changes in sperm duct pH during final maturation may trigger a certain degree of motility. Ionic composition (Schlenk and Kahmann, 1938; Cosson et al., 1989; Morisawa and Morisawa, 1990), pH (Hines and Yashow, 1971; Billard et al., 1993), but particularly osmotic pressure (see Stoss, 1983; Billard, 1995), appear to be the most important factors in the determination of teleost sperm activation. In freshwater salmonid species, initial immobility of sperm is due to high concentrations of K+ (Schlenk and Kahmann, 1938); subsequent dilution in the surrounding water column initiates sperm motility. In some marine species, exposure to a hyperosmotic environment is insufficient to induce a marked motility increase. In these species, sperm need to be exposed to unfertilized eggs for them to demonstrate maximal motility. It was initially proposed that unfertilized teleost eggs might release a substance, proteinaceous in nature, which activates sperm (see Griffin et al., 1996). Contact with this substance is known to increase the number of motile sperm and swim duration. It has also been demonstrated that the teleost sperm-activating substance acts as an attractant, guiding the sperm to the micropylar region in some cyprinid and salmonid species. This factor is now known as the sperm motility initiation factor (SMIF). In Pacific herring, antibodies to SMIF blocked fertilization and sperm motility inhibited; SMIF was localized to the region of the egg that encircles the micropyle (Griffin et al., 1996). Other mechanisms of sperm activation in a variety of animals adopting external fertilization are discussed by Tosti (1994). Calcium, potassium, and possibly sodium and chloride ions, are known to play a key role in the motility of Atlantic croaker sperm motility (Detweiler and Thomas, 1998); membrane-bound ion channels were also found to be heavily involved. Further evidence for the key involvement of potassium ions are discussed by Takai and Morisawa (1995); in brief, changes in intracellular potassium ion concentration caused by an external osmolality change appear to regulate sperm motility in both marine and freshwater teleosts. Some studies report that acquisition of sperm motility also involves various hormonal regulators. In masu salmon, for example, gonadotropin-induced testicular production of 17α-20-β-dihydroxy-4pregnen-3-one appears to be responsible for the induction of sperm motility (Miura et al., 1992) and was mediated through an increase in sperm duct pH. Neither testosterone nor 11-ketotestosterone, the two major androgens in teleosts, appeared to affect motility. It is already established that milt volume can be increased in the goldfish Carrassius auratus (L.) by spawning interactions involving females injected with prostaglandin F2-α and 17α-20-β-dihydroxy-4-pregnen-3-one (Zheng and Stacey, 1996). Gonadotropin releasing hormoneanalogue treatment also increased sperm motility and production in the yellowtail flounder Pleuronectes ferrugineus (Storer) (Clearwater and Crim, 1998). In most teleosts adopting an external fertilization strategy, sperm activity is brief and tends to decline during the period of motion. Duration of motility tends to be very short, for example 20 to 25 seconds in rainbow trout, and sperm motility declines rapidly thereafter. Mechanisms involved in the initiation and control of motility are discussed at length in Billard (1995). Those species exhibiting internal fertilization however, exhibit much longer windows of motility; the viviparous guppy, for example, displays motility in seminal fluid for periods up to 48 h (Billard, 1978). In some species, for example, the Nile tilapia, there is a high molecular weight glycoprotein in seminal plasma that acts as a sperm immobilizing 42 factor (Mochida et al., 1999); sperm that have acquired potential for motility are kept immobilized within the plasma. Studies showed that this immobilizing factor was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells. It should also be noted that it is possible to induce spermiation in some teleosts by application of exogenous hormones. For example, maturation and spermiation can be induced very effectively in the male European eel Anguilla anguilla (L.), by use of weekly injections of human chorionic gonadotropin (Pérez et al., 2000). Sperm quality and cryopreservation The evaluation of sperm quality in teleosts concerns motility and fertilizing capacity and, to a lesser extent, sperm concentration (tested by spectrophotometry or haemocytometry). Sperm motility is the most commonly used parameter used to assess sperm quality, although fertilization capacity remains the most conclusive. Sperm tend to be immotile in the genital tract and are only activated upon contact with external media. Motility is tested under a microscope using standardized criteria (Goryczko and Tomasik, 1975; Sanchez-Rodriguez and Billard, 1977; Baynes et al., 1981; Cosson et al., 1985; Billard et al., 1987; Billard and Cosson, 1989, 1992; Cosson et al., 1991) and more recently using computerized sperm tracking systems. Tests of fertilizing capacity tend to be confounded by variations in egg quality and are thus, far more prone to error. Percentage fertilization is the usual parameter determined and refers to the percentage of fertilised eggs hatching or successfully developing to a characteristic stage, such as eyeing or first-feeding. Some quarters however, consider that percentage of developing larvae to first feeding may be a better index of gamete quality (Billard, 1995). The cryopreservation of fish sperm has received much research attention. For reviews, see Stoss (1983), Leung and Jamieson (1991), Billard (1992, 1995) and Rana (1995). Although, sperm from over 200 fish species can now be cryopreserved, few practical applications have been achieved (Billard, 1995), although some operative sperm banks have now been developed for certain teleosts (see Lubzens et al., 1997). Attempts to cryopreserve fish oocytes and embryos have, so far, been ineffective (see Stoss, 1983; Rana, 1995). Fertilization and activation of teleost oocytes Exogenous and endogenous factors affecting fertilization There have been many studies concerning the effects of various factors upon fertilization in teleosts, athough we still know very little about the intracellular and molecular mechanisms involved. In the medaka, shortened photoperiod regimes are known to influence fecundity, spawning interval and fertility (Koger et al., 1999). Diet, particularly dietary ascorbate (Cieresko and Dabrowski, 1995, 2000) and lipid levels (Pustowka et al., 2000) are also known to influence fertility in rainbow trout. Water temperature also plays a key role in the fertility of rainbow trout (Pankhurst et al., 1996); elevated autumn holding temperatures had a deleterious effect upon ovulation, egg production and fertility. The effects of water temperature upon sperm motility and fertility in Atlantic salmon and brown trout Salmo trutta (L.) are discussed by Vladic and Jarvi (1997). In brief, a reduction in water temperature had little effect upon fertility in the two species, although brown trout eggs exhibited greater resistance to higher temperatures. Temperature had a highly significant effect upon the duration of sperm motility in both species, but was more pronounced in salmon (Vladic and Jarvi, 1997). The potential effects of salinity on fertilization in the marine teleost black bream Acanthopagrus butcheri (Munro) are discussed by Haddy and Pankhurst (2000); high levels of deformity, poor hatching rates and increased mortality were observed when salinity was lower than that normally experienced. Diet composition heavily influences the fatty acid (phosphatidylcholine, phosphatidylethanolamine, phosphatidyserine and phosphatidylinositol) composition of sea bass Dicentrachus labrax (L.) sperm (Bell et al., 1996) and is thereby likely to influence sperm viability and egg activation. In a further study of sea bass, Asturiano et al. (2001) investigated the effect of feed formulation on various aspects of sperm physiology. Fish fed upon a diet enriched with polyunsaturated fatty acids (PUFAs) exhibited increases in spermiation period, milt volume and spermatozoa density compared to fish fed a basic wet diet. Although no differences in sperm quality or motility were detected, fish fed supplemented diets exhibited higher rates of embryonic and larval survival (Asturiano et al., 2001). Ascorbic acid is known to protect fertility in male teleosts (Dabrowski and Ciereszko, 1996). The 43 high concentration of ascorbic acid in semen plays a key role in maintaining the genetic integrity of spermatozoa by preventing oxidative damage to sperm DNA. In the rainbow trout, dietary ascorbate level directly affected sperm quality and influenced male fertility (Dabrowski and Cierszko, 1996) and dietary supplementation aids the maintenance of high sperm motility and fertilizing capacity (Ciereszko and Dabrowski, 2000). The steroid hormone 17α-20-β-dihydroxy-4pregnen-3-one has been found to play a key role in fertility and paternity in goldfish (Zheng et al., 1997). This hormone functions as a pheromone in this particular species and also serves to increase sperm volume and male serum gonadotropin II (GtH II, analagous to mammalian LH). Sperm from goldfish males exposed to 17α-20-β-dihydroxy-4-pregnen3-one fertilized more eggs than sperm from control males (Zheng et al., 1997). Some biochemical constituents are also known to affect fertility in teleosts. For example, highly significant relationships were found in sperm and seminal plasma, between aspartate aminotrasferase activity and fertility in both rainbow trout and whitefish (Ciereszko and Dabrowski, 1994); in rainbow trout, highly significant correlations were detected between sperm concentration, motility and fertilization rate. Problems associated with fertilization in commercial species Temporal variations in quality of spermatozoa and oocytes have serious effects upon the success of fertilization. There are numerous factors governing egg quality in teleosts. These include, amongst others, seasonal variations, diet, physiological, endocrinological status of broodstock and genetic influences. For a comprehensive review see Brooks et al. (1997). Studies on the composition of teleost spermatozoa indicate large intra- and inter-specific variations in sperm concentration and seminal plasma concentration (Rana, 1995). Variations have been attributed to genetic variability, intra-testicular ageing of spermatozoa, seasonality, breeding state and reproduction strategy (Billard et al., 1977; Scott and Baynes, 1980; Kruger et al., 1984; Munkittrick and Moccia, 1987; McAndrew et al., 1993). Changes in sperm quality, spermatocrit, motility and seminal plasma composition have also been reported during a spawning season, particularly in sea bass and salmonid species (Billard et al., 1977; Sanchez-Rodrigues et al., 1978; Buyukhatipoglu and Holtz, 1984; Muntkittrick and Moccia, 1987). These variations mostly involve a gradual decline as the breeding season progresses, although spermatocrit in the rainbow trout has been observed to increase over the spawning season (Sanchez-Rodriguez et al., 1978; Piironen and Hyvarinen, 1983). Sperm quality is also known to vary significantly between virgin- and repeat-spawning cod (Trippel and Neilson, 1992); hatching success was more variable in virgin spawners. There is also strong evidence to suggest that certain plasma characteristics such as protein concentration and anti-proteinase activity show variability amongst individual rainbow trout males (Glogowski et al., 2000). There are growing concerns over the “capacity to fertilize” of certain species of cultured fish held in captivity, notably marine species (Houghton et al., 1985; Bromley et al., 1986; Carrillo et al., 1989; Norberg et al., 1991; Holmefjord et al., 1993; Bromage et al., 1994). In captivity, fertilization and hatching success rates are often much lower than that witnessed in the wild, particularly if eggs and sperm are stripped from broodstock and fertilized manually. For example, in the channel catfish Ictalurus punctuatus (Rafinesque), mean fertility rate varied between 25% and 67% with a mean of just 53% (Bart and Dunham, 1996). In cases where spawning is induced by application of exogenous hormones, mean fertilization rate can be as low as 34% (Coward et al., 2000). Cod Gadus morhua (L.) and Atlantic halibut are important species in the North Atlantic aquaculture industry (Kjørsvik and Holmeford, 1995). Cod spawn naturally in captivity and fertilization rates are normally reasonably high; stressed fish however, exhibit irregular spawning intervals, low fertilization rates and increased occurrence of abnormal embryos (Kjesbu, 1989). Halibut culture is presently based upon manual stripping of eggs and sperm from viable broodstock. Stripping time in relation to ovulation time is a crucial factor in obtaining optimal egg yield and fertilization rates (Holmefjord, 1991, Norberg et al., 1991; Bromage et al., 1992; Holmefjord et al., 1993), as with other batch spawning species (McEvoy and McEvoy, 1991). However, even considering ovulatory rhythms for individual broodstock, fertilization rates and egg viability remain highly variable in stripped halibut (Norberg et al., 1991; Bromage et al., 1994; Holmefjord et al., 1993); hatching rate can be as low as just 1% (Norberg et al., 1991). Similar variability is evident for captive sole and turbot, where 44 up to 50% of annual egg production may be unfertilized (Houghton et al., 1985; Bromley et al., 1986). In sea bass and gilthead seabream, hatching rates are often just 10–15% of the total number of eggs spawned (Carrillo et al., 1989). In salmonids, losses of up to 50% are common (Bromage et al., 1992). However, it is important to remember that wild stocks of fish also demonstrate pronounced variability in fertilization success; Baltic herring Clupea harengus membras (L.), for example, exhibit variation from one year to the next possibly due to the variability of key nutritional and environmental events (Laine and Rajasilta, 1999). There are also age-related effects upon fertility and sperm quality in some teleosts. For example, in the striped bass Morone saxatalis (Walbaum), 3 year old fish exhibited far superior sperm quality than 1 or 2 yr old fish, both in terms of higher sperm production and increased sperm longevity (Vuthiphandchai and Zohar, 1999). Manipulative techniques are often used to induce spawning in many teleost species, particularly those that exhibit reduced spawning activity in captivity, or demonstrate pronounced asynchronous spawning periodicity. In some species, the induction of spawning is becoming a vital part of their management. Spawning induction techniques involve temperature or photoperiod manipulation, injection or implantation of exogenous hormones. These techniques, although useful, tend to have detrimental effects upon subsequent fertilization, for example in brown trout (Mylonas et al., 1992), sea bass (Carrillo et al., 1995), Japanese eel Anguilla japonica (Temminck and Schlegel) (Ohta et al., 1996) and the red bellied tilapia Tilapia zillii (Gervais) (Coward et al., 2000). In brown trout injected with gonadotropin releasing hormone analogue (GnRHa) for example, premature ovulation resulted in significant reduction of fertility thought to be due to disruption of final oocyte maturation and ovulation (Mylonas et al., 1992). Physiology of fertilization Fertilization is the process whereby male and female gametes interact to form a diploid zygote. Studies in a wide variety of animal and plants groups have demonstrated that sperm make two essential contributions to this process. Firstly, the addition of paternal chromosomes and secondly, the initiation of key intracellular signalling processes within the oocyte that will result in egg activation and development. Without both contributions, fertilization would be unable to proceed (Whitaker and Swann, 1993). Many of the processes occurring during teleost fertilization are similar to those seen in marine invertebrates and mammalian eggs (Iwamatsu and Ohta, 1978). Much of our existing knowledge of the mechanisms involved in teleost fertilization are based upon few data from small model species such as zebrafish, medaka and bitterling which have no real commercial value. Research is urgently required to address mechanisms involved in fertilization in species of great commercial value such as the cyprinidae, salmonidae, cichlidae, pleuronectidae and gadidae. Early research into fertilization in fish was reviewed by Yamamoto (1961), most of the more recent work only concerns medaka, zebrafish and bitterling. The unfertilized teleost egg is bounded by a series of membranes. The outer membrane, the chorion, is relatively thick and tough and possesses a small funnel-shaped micropyle through which sperm enter to fertilise the egg. In contrast to mammals, teleost sperm do not possess an acrosome. Fertilisation is usually monospermatic; the micropyle is only wide enough to allow the passage of one sperm at any given time. The chorion and ooplasm separate as the egg is activated and a plug forms in the micropyle, to prevent entry by further sperm. Polyspermy does occur in some elasmobranches, but only one sperm ultimately fuses with the egg; remaining sperm are probably reabsorbed for nutritive puposes. Fertilization of the egg requires the presence of small quantities of divalent ions (Ca2+ and Mg2+ ) and once the egg has been activated, the micropyle is plugged. Fertilization initiates the second meiotic division in the egg, which at spawning contains two sets of maternal chromosomes. Following the second meiotic division, however, one of these chromosome sets is lost as a polar body and is expelled leaving one maternal and one paternal set. The mechanisms associated with fertilization in the freshwater teleost rose bitterling are discussed by Ohta and Nashirozawa, 1996). In this species, as with other teleosts, sperm penetrate eggs via a defined sperm entry site, a region of plasma membrane just beneath the micropyle. In rose bitterling, the sperm entry site transforms from a tuft of microvillii into a swollen mass that continues to plug the micropyle after sperm penetration. Data suggest that sperm penetration was not necessary for swollen mass formation in rose bitterling and actin microfilaments did not participate in the swollen mass formation (Ohta and Nashirozawa, 1996). 45 In medaka, chorionic proteins in the diluted mucous area of glycoprotein on the outermost layer of the chorion, demonstrate an affinity for spermatozoa and are therefore thought to play a key role in guiding sperm to the micropyle (Iwamatsu, 1997). Scanning and transmission electron microscopic observations of sperm entry into rose bitterling eggs are discussed by Ohta and Iwamatsu (1983). A spermatozoon that has penetrated a medaka egg and reached the vitelline layer is quickly enclosed by ooplasmic protrusions (the fertilization cone). After plasma membrane fusion, the sperm nuclear envelope disappears, vesicles appear in the apical head region, and the sperm chromatin nuclear envelope is reformed by fusion of elongate or flattened vesicles (Iwamatsu and Ohta, 1978); the mature male pronucleus has a large nucleolus with a wrinkled envelope. Interestingly, medaka eggs containing low histone H1 kinase activity fertilized normally but the pronuclear membrane did not break down and chromosomes did not condense (Iwamatsu et al., 1999a). Data also suggested that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy. Further interactions between nuclear behaviour and histone H1 kinase activity in the medaka egg are reported by Iwamatsu et al. (1999b). Following fertilization, the teleost egg absorbs water leading to various changes in ultrastructure and cytochemistry. The chorion separates from the vitelline membrane and creates the perivitelline space. Enzymes located in the hardened chorion serve to protect the developing embryo from bacterial and fungal attack. Hardening of the chorion is thought to be due to alveolar colloid, Ca2+ ions, phospholipids and enzymes in the inner chorion layer of glycoprotein or enzymes present in the perivitelline space (Zotin, 1958). The chorion is only permeable to water and small molecules; larger molecules of a colloidal nature are retained in the perivitelline space and maintain osmotically-based tension. It is thought that these colloids are derived from the cortical alveoli (Blaxter, 1969). Recent research in the medaka has lead to the identification and cloning of alveolin, an extracellular metalloproteinase that induces chorionhardening (Shibata et al., 2000). This protein has a deduced molecular mass of 21.5 kDa and is the first example of a molecular candidate for a chorion hardening-inducing factor from any teleost cortical alveoli. The chorion of an unfertilized medaka egg consists of two major (73–77 kDa) and one minor (150 kDA) protein (Iwamatsu et al., 1995); upon fertilization, these proteins are polymerized to insoluble high molecular weight proteins thereby increasing chorion toughness. Current data suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space (Iwamatsu et al., 1995). Fertilization is followed by a breakdown of the cortical alveoli, which, in the rose bitterling, occurs in a wave that propagates from the vegetal pole to the animal pole of the egg. This process, in medaka eggs, occurs within 30 sec of the onset of activation (Lee et al., 1999). Cortical alveoli breakdown occurs concurrent with a large increase in intracellular free Ca2+ ions (Ohta et al., 1990). Calcium signalling is a key event in fertilization in all animals studied thus far and is discussed more fully later in this review. Salmonid eggs, and the eggs of many other species, are activated by water when eggs are released into hypotonic solutions, the vitelline membrane becomes opaque and changes in permeability occur. If sperm are not immediately available, there is significant reduction in rates of fertilization. Fish eggs, particularly those produced by most freshwater species, can lose their ability to fertilize very quickly (Yamamoto, 1961), presumably due to water hardening of the chorion. Marine species retain their capacity for fertilization much longer. In common with several other animals, eggs produced by some teleosts such as medaka and sticklebacks Gasterosteus aculeatus (L.) can be activated by pricking (Blaxter, 1969) thereby inducing artificial parthenogenesis. The mechanism of egg activation At fertilization, in practically all species studied, egg activation and the initiation of development are always triggered by an increase in intracellular Ca2+ concentration within the egg (Whitaker and Swann, 1993; Miyazaki et al., 1993; Stricker, 1999). In certain species such as sea urchin, frog and some fish eggs, a single transient of Ca2+ is triggered during egg activation at fertilization (Stricker, 1999). In other animals however, including mammals, ascidians and nemertean worms, a distinctive series of intracellular Ca2+ oscillations are observed (Miyazaki et al., 1993; Swann and Ozil, 1994; Stricker, 1999; see Table 1). 46 Table 1. Brief overview of calcium dynamics at fertilization in a variety of organisms. Modified from Stricker (1999) Organism investigated Egg diameter Calcium transients (single or multiple)∗ Wave of calcium traversing egg/oocyte at fertilization Examples of relevant literature Cnidaria 185 µm Single Unclear Freeman and Ridgway (1991, 1993) Nemertea 120 µm Multiple Debated Stricker (1996, 1997) Mollusca Bivalve 60 µm Multiple Debated Abdelmajid et al. (1993) Deguchi and Osanai (1994) Deguchi and Morisawa (1997) Annelidae 105 µm Multiple Yes Eckberg et al. (1993) Eckberg and Miller (1995) Echinodermata Sea urchin 110 µm Single Yes Starfish 160 µm Single Yes Stricker et al. (1992) Swann and Whitaker (1986) Stricker (1996) Eisen and Reynolds (1996) Chordata Ascidian 140 µm Multiple Yes Fish Frog 1.1 mm 1 mm Single Single Yes Yes Mammal 80 µm Multiple or single Yes Brownlee and Dale (1990) McDougall and Sardet (1995) Gilkey et al. (1978) Busa and Nuccitelli (1985) Fontanilla and Nuccitelli (1998) Miyazaki et al. (1986) Jones et al. (1995) Deng et al. (1998) ∗ Within 30 minutes post-fertilization. Gilkey et al. (1978) were the first to describe a free calcium wave traversing the activating teleost egg (medaka); aequorin-injected eggs exhibited an explosive rise in free calcium during fertilization followed by a slow return to resting level, similar to that seen in echinoderm eggs. This wave began at the animal pole, close to the site of germinal vesicle breakdown, and vanished at the antipode some minutes later. Fertilization was thus thought to be propagated by calcium-stimulated calcium release, primarily from some internal sources other than cortical alveoli. Similar observations were later made in sand dollar Peronella japonica (Yoshimoto et al., 1986) and zebrafish eggs (Lee et al., 1999). The cytoplasmic release of Ca2+ ions at fertilization in the medaka are discussed by Iwamatsu et al. (1988); microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli that was initiated at the injection site. The dynamics of Ca2+ release in medaka eggs is discussed at length by Iwamatsu et al. (1988) and was assessed by the microinjection of inositol 1,4,5triphosphate (IP3 ), calcium ionophore A23187, Ca2+ , Sr2+ , Ba2+ and cyclic guanosine 5′ monophosphate (cGMP). Micro-inection of IP3 or calcium ionophore induced Ca2+ release without time lag, whilst injection of Ca2+ or cGMP required a time lag of 5 to 30 seconds for Ca2+ release to be detectable. IP3 injection resulted in Ca2+ release in a cytoplasmic region close to the egg surface suggesting that cytoplasmic Ca2+ induces Ca2+ release from cytoplasmic stores indirectly, probably via a membrane factor such as IP3 (Iwamatsu et al., 1988). Zebrafish eggs are thought to be activated by contact with the spawning medium (Lee et al., 1999); there then follows a short time window (5 to 30 seconds) within which fertilization can occur. This strategy serves to ensure that only the successful pairbonded male is able to fertilize a particular female’s eggs. This mechanism, however, is a very different 47 activation/fertilization mechanism than that previously described in medaka (Gilkey et al., 1978; Yoshimoto et al., 1986) in which sperm appear to activate the egg and not the external media. A search of the literature however, reveals that the zebrafish is not alone in not appearing to require sperm for egg activation. Other species exhibiting a similar strategy include goldfish and pond smelt Hypomesus olidus (Pallus) (Yamamoto, 1954), some invertebrates such as marine shrimp Sicyonia ingentis (Burkenroad) (Lindsay et al., 1992), and prawn Palaemon serratus (Pennant) (Goudeau and Goudeau, 1996). Exposure to Mg2+ ions in the external marine environment is thought to activate eggs in these two invertebrates. Irrespective of the mode of egg activation (i.e. whether activated by sperm or external media), the induced calcium wave always begins in the micropylar region (Gilkey et al., 1978). It is thought that the extensive network of endoplasmic reticulum beneath the plasma membrane in this region (Hart, 1990), plays a key role in calcium activation waves and may, in fact, be the source of calcium (Jaffe, 1991). Other possible sources of calcium in egg activation are reviewed in depth by Jaffe (1983). The calcium activation wave is propagated only through the peripheral cytoplasm of the egg in medaka and through the cortex in the zebrafish (Gilkey et al., 1978; Lee et al., 1999); these differences are thought to be due to differences in egg morphology (see Beams et al., 1985). For instance, these two species exhibit noticeable differences in the temporal distribution and size of cortical alveoli in the egg (Gilkey et al., 1978; Hart and Donovan, 1983). The duration of the cytosolic calcium activation wave in zebrafish correlates well with the time required to complete the cortical reaction (Hart and Yu, 1980). The mechanism seen in zebrafish is unusual; the spawning medium activates the calcium activation wave without the aid of sperm-derived factors as appears to be the case for the vast majority of species. For a comprehensive review of the comparative biology of intracellular signalling during activation and fertilization across a range of animal groups, see Stricker (1999). The three models of egg activation In other animal groups besides teleosts, the mechanism whereby the sperm triggers intracellular Ca2+ release within the egg remains a major unsolved question. There are currently three main models to explain this phenomenon (Whitaker and Swann, 1993; Schultz and Kopf, 1995; Evans and Kopf, 1998; Swann and Parrington, 1999; Parrington, 2001; see Figure 3). Firstly, there is the ‘Ca2+ bomb’ or ‘Ca2+ conduit’ model, in which Ca2+ is introduced by, or channeled through, the sperm (Jaffe, 1983, 1991; Creton and Jaffe, 1995). Consistent with this model is the finding that sperm-egg fusion in the sea urchin and mouse always precedes egg Ca2+ release (McCulloch and Chambers, 1992; Lawrence et al., 1997) and that sperm from many species take up Ca2+ prior to fusion (Jaffe, 1983, 1991). However, since injection of Ca2+ into sea urchin, ascidian and mouse eggs fails to cause Ca2+ release (Whitaker and Swann, 1993; Swann and Ozil, 1994), Ca2+ imaging studies in mice failed to show an elevation in the local cytoplasmic Ca2+ concentration and removal of extracellular Ca2+ failed to block Ca2+ release in the egg (Jones et al., 1998), there are now doubts about the validity of this model. The second hypothesis for egg activation is the ‘membrane receptor’ model. In this case, a sperm surface ligand binds to an egg membrane receptor and activates a signaling cascade, leading to generation of the second messenger inositol trisphosphate (InsP3 ). This then causes Ca2+ release through the IP3 receptor via the activation of an egg phospholipase C (PLC) (Jaffe, 1990; Schultz and Kopf, 1995; Evans and Kopf, 1998). Receptor theories have centred upon G-proteins, tyrosine kinase and the “adhesion model” (see Table 2). The fact that a similar set of events occurs in many examples of signalling pathways in somatic cells (Berridge, 1993) made this a very popular model. Studies in echinoderms have provided evidence that an egg PLC may play a central role in egg activation. Injection of recombinant proteins containing the SH2 domains of bovine PLCγ block Ca2+ release in both starfish (Carroll et al., 1997) and sea urchin (Carroll et al., 1999; Shearer et al., 1999) eggs at fertilization. It is thought that the SH2 domains interfere with an upstream component of the signalling cascade operarating at fertilization. Despite these promising findings, there still remain some unresolved problems with the model and doubts about its general validity. Firstly, there is little evidence for involvement of an egg PLC in vertebrates. Thus, injection of PLCγ SH2 domain containing proteins, similar to those described above, have failed to show any inhibitory effect on Ca2+ release at fertilization in frogs and mice (Mehlmann et al., 1998; Runft et al., 1999). Secondly, it still remains to be shown that a surface mediated interaction between a 48 Figure 3. The three models of egg activation: (a) The calcium bomb model – spermatozoon acts a conduit to transport Ca2+ into the egg where it is taken up and then released from intracellular stores. (b) The surface receptor model – spermatozoon binds to membrane receptors which initiate transmembrane signaling and lead to the generation of InsP3. (c) The sperm factor model – sperm-derived PLC activity is introduced into the egg and acts upon PIP2 (phosphatidylinositol-4,5-bisphosphate) stores in the egg to generate InsP3 (inositol 1,4,5-triphosphate), leading to Ca2+ release from internal stores via the InsP3 receptor. sperm ligand and an egg receptor does indeed trigger activation of a PLC in the egg. This is despite the fact that many of the proteins involved in spermegg binding have now been identified (see Parrington, 2001). It thus remains a possibility that even in species where an egg PLC has been implicated in egg activation, it may be via another mechanism than a surface mediated interaction between sperm and egg. The third hypothesis for egg activation is the ‘soluble sperm factor’ model, in which a soluble sperm Ca2+ releasing factor is introduced into the egg at sperm-egg fusion (Whitaker and Swann, 1993; Swann and Ozil, 1994; Swann and Lai, 1997; Fissore et al., 1998; Parrington et al., 1998; Swann and Parrington, 1999). Although this model attracted much initial controversy, over the last few years, it is becoming increasingly accepted as the mechanism likely to be operating in mammals (Fissore et al., 1998; Swann and Parrington, 1999), and probably in other species such as ascidians and nemertean worms (Stricker, 1997; Kyozuka et al., 1998; see Table 3). Like the calcium ‘bomb’ or ‘conduit’ model, the soluble sperm factor hypothesis requires that gamete fusion precedes egg Ca2+ release and activation, which has now been demonstrated in sea urchins and mice (McCulloch and Chamber, 1992; Lawrence et al., 1997). Indirect evidence for the existence of an intracellular sperm factor comes from the clinical procedure intracytoplasmic sperm injection (ICSI), in which the sperm is injected directly into the egg. Besides leading to egg activation, during ICSI a series of Ca2+ oscillations are observed in human (Tesarik 49 Table 2. Evidence for the “receptor theory” for egg activation at fertilization Receptor theory Evidence for receptor theory Species Evidence against receptor theory G-proteins Over-expression of exogenous Gprotein linked receptors Mouse (Moore et al., 1994) Xenopus (Kline et al., 1991) Starfish (Shilling et al., 1994) Inhibitory monoclonal antibodies to G-proteins do not inhibit fertilisation in the mouse (Williams et al., 1992) and frog (Runft et al., 1999) Injection of G-protein agonist inhibits fertilisation Mouse (Moore et al., 1994) Hamster (Miyazaki et al., 1993) Injection of G-protein antagonist stimulates fertilisation response Mouse (Miyazaki, 1988; Fissore and Robl 1994) Over-expression of exogenous tyrosine kinase linked receptors Starfish (Shilling et al., 1994) Xenopus (Yim et al., 1994) Injection of dominant negative SH2 domains Starfish (Carrol et al., 1997) Sea Urchin (Carrol et al., 1999; Shearer et al., 1999) Inhibition of endogenous tyrosine kinase activity Starfish (Glahn et al., 1999) Frogs (Dupont et al., 1996) Mice (Glahn et al., 1999) Disintegrin peptides stimulate fertilisation response Frog (Shilling et al., 1997, 1998) Marine worm Urechis Caupo (Gould and Stephano, 1991) Mice lacking fertilin B are fertile and egg activation is unaffected (Cho et al., 1998) RGD peptides, ADAM peptides Xenopus (Iwao and Fujimura, 1996; Shilling et al., 1996) Disintegrin domains do not activate mammalian eggs (Evans and Kopf, 1998) Tyrosine kinase Adhesion model and Sousa, 1994; Tesarik et al., 1994), nemertean worm (Stricker, 1996) and mouse (Nakano et al., 1997) eggs, similar to those seen at fertilization. Most importantly, there is direct evidence for the existence of a soluble sperm factor. Injection of soluble mammalian sperm extracts from boars, hamsters, or humans can trigger Ca2+ oscillations similar to those seen at fertilization in mouse, hamster, human and cow eggs (Swann, 1990, 1992, 1994; Homa and Swann, 1994; Palermo et al., 1997; Wu et al., 1998; Fissore et al., 1998). Sperm extracts also cause Ca2+ oscillations in eggs from non-mammals such as marine worms (Stricker, 1997), ascidians (Kyozuka et al., 1998), and newts (Yamamoto et al., 2001), while sperm extracts from frogs, chickens and plants trigger Ca2+ oscillations when injected into mouse eggs (Dong et al., 2000). In each case, injection of sperm extract closely mimics the particular SH2 domains do not block fertilisation in mouse (Mehlmann et al., 1998) and frog (Runft et al., 1999) eggs characteristics of the pattern of Ca2+ release seen at fertilization (Swann and Ozil, 1994; Kyozuka et al., 1998). Calculations suggest that the amount of sperm factor in a single sperm is within the range that would be expected if it is the physiological agent of egg activation (Rice et al., 2000). Undoubtedly, major unresolved questions of the soluble sperm factor model of egg activation is the identity of the factor, and indeed, whether the sperm factor is conserved across species as different as nemertean worms, ascidians and humans and other mammals, or whether the factor is different in each case. In mammals, two main candidates were initially proposed: a 33 kDa hamster sperm protein (Parrington et al., 1996) and a truncated form of the c-kit receptor termed tr-kit, which is present in mouse sperm (Albanesi et al., 1996). However, subsequent findings have tended to rule out a role for either of 50 Table 3. Species in which evidence for an active sperm factor has been documented Species with active sperm factor Researcher Human Tesarik et al., 1994; Homa and Swann, 1994; Dozortsev et al., 1995; Palermo et al., 1997; Kim et al., 1999 Monkey (Cynomolgus) Ogonuki et al., 2001 Porcine Wu et al., 1998; Witton et al., 1999; Kim et al., 1999; Machaty et al., 2000 Bovine Kim et al., 1999; Okitsu et al., 2001 Hamster Swann 1990; Homa and Swann, 1994 Mouse Kim et al., 1999 Rabbitt Stice et al., 1990 Chicken Dong et al., 2000 Frog Dong et al., 2000 Newt Yamamoto et al., 2001 Ascidian Wilding and Dale, 1998; Kyozuka et al., 1998; McDougall et al., 2000 Marine worm Stricker, 1997 Sea urchin Osawa, 1994 Flowering plant Li et al., 2001 (Brassica campestris) these proteins in egg activation (Parrington, 2001; Swann et al., 2001). Another reason for doubting these initial sperm factor candidates is that there are now several pieces of evidence indicating that the soluble sperm factor in mammals is a sperm derived PLC with distinctive properties (Parrington, 2001; Swann et al., 2001). Studies combining intact mouse eggs with cell free sea urchin egg homogenates have demonstrated, firstly that the sperm factor mediates Ca2+ release through the InsP3 receptor and that it generates InsP3 in the homogenate, indicating involvement of a PLC (Jones et al., 1998, 2000); secondly, that the sperm factor itself appears to be, or contains, a sperm-derived PLC, rather than it being another type of protein that activates a PLC in the egg (Jones et al., 2000); thirdly, that this sperm PLC has some distinctive features compared to PLCs in other tissues (Rice et al., 2000). A major question now is to identify whether the sperm factor is a known PLC, or a sperm-specific novel isoform (Parrington, 2001). The identity of the sperm factor in non-mammals is less clear. There is currently no clear evidence that the sperm factor activities identified in non-mammalian species are due to a sperm PLC. In ascidians, egg activation may be triggered by a sperm protein that activates an egg PLC, as both the Ca2+ oscillations triggered at fertilization and those induced by ascidian sperm extract are inhibited by SH2 domain constructs (Runft and Jaffe, 2000). Thus, in both mammalian and non-mammalian species egg activation may be triggered by a sperm factor that induces Ca2+ release through a common mechanism, the PI signaling pathway, but which enters the pathway at a different point, depending on the species. Other studies in sea urchins have suggested a role for a sperm nitric oxide synthetase in egg activation (Kuo et al., 2000). In contrast to these findings, crossspecies injection studies indicate that a sperm factor with the qualities of the mammalian sperm factor may be conserved across very different species (Parrington et al., 2001). This suggests that, although the pattern of Ca2+ release seen at fertilization may vary in these different species, the trigger of release may involve the same protein factor, possibly a sperm-derived PLC. It is also possible that in some species a sperm factor is the initial trigger of Ca2+ release, but that the Ca2+ wave that crosses the egg is then carried by a different Ca2+ releasing agent (Whitaker and Swann, 1993). Research into the mechanism of egg activation in teleosts will need to address which of these possible scenarios is taking place, as well as differences between teleost species. Such a research initiative will be aided by the findings and hypotheses emerging from studies in the other animal groups outlined. Although in its embryonic stages, recent research has already demonstrated the presence of a calcium-release inducer (cyclic ADP ribose, cADPR) in sea bream (Polsonetti et al., 2002). Post-fertilization signalling events Apart from egg activation, intracellular calcium signalling appears to play an important role in early embryogenesis. Fluck et al. (1991) described slow calcium waves accompanying cytokinesis in medaka eggs. Animal cells are cleaved by the formation and contraction of a thin actomysin band. In medaka, contractile band growth was studied by injection of aequorin; results revealed two successive waves of faint luminescence moving along each of the first three cleavage furrows. The first waves are thought to aid furrow growth whilst the second waves act to induce exocytosis which provides new furrow membrane (Fluck et al., 1991). Further research showed that localized domains of elevated free cytosolic calcium 51 are essential for ooplasmic segregation in zebrafish (Leung et al., 1998). More recently, multicellular large-scale rhythmic calcium waves were described, using imaging techniques, during zebrafish gastrulation (Gilland et al., 1999). Summary and future studies It is of great concern that, aside from small noncommercially important, laboratory model species such as medaka, zebrafish, and rose bitterling, we know little about the precise intracellular and molecular events accompanying egg activation and fertilization in teleost fish. Presently, the culture for food of many important freshwater, and especially marine teleosts is beset by problems associated with successful fertilization, hatching and early embryonic development. Increased knowledge of the processes of egg activation and fertilization in these groups of fish is likely to make a significant contribution to aquaculture. Comparison of existing data for medaka and zebrafish, although very limited, clearly highlight the fact that two very different activation/fertilization mechanisms are in operation. Teleost reproductive strategies and more importantly, teleost oocytes, exhibit a remarkable variety of adaptations as might be expected from a group which has more species than the rest of the vertebrate kingdom put together. It is therefore crucial that we address the intracellular and molecular mechanisms involved in teleost fertilization in a wide variety of species. Over the last ten years or so, our knowledge of mammalian activation and fertilization has grown enormously. In view of the highly variable fertility rates evident in many commercially cultured teleosts, it is essential that some of the lessons learnt from this work are swiftly applied to representative teleost groups using a combination of physiological, molecular and recombinant technology. 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