8.1 Introduction
8.2 Hypophysation
8.3 Existence of a steroidogenic relay in gonadotropin-induced maturation
8.4 Source of maturation-inducing steroids: The ovary versus the interrenal
8.5 Hypotheses
8.6 Mechanism of steroid-induced oocyte maturation
8.7 Ovulation
8.8 Ovulating agents other than gonadotropins and steroids
Maturation is accomplished in the fish oocyte by means of two successive meiotic divisions during the course of which the oocyte gives out two polar bodies. During this process, the enlarged nucleus of the primary oocyte, which is arrested at the dictyotene stage, moves towards a more peripheral position, its membrane breaks down and with the extrusion of a polar body the first meiotic division is completed. The second meiotic division starts immediately thereafter but progresses only up to the metaphase stage (Goswami and Sundararaj, 1971a, b; Sundararaj and Goswami, 1977a, b; Masui and Clarke, 1979). During these events, in some species, a distinct animal pole differentiates from the vegetal pole. The yolk also undergoes some sort of maturation as well as hydration and becomes less opaque (Stevens, 1966; Goswami and Sundararaj, 1971a; Jalabert et al., 1973); the oil droplets, when present, coalesce to form one or more larger globules (Stevens, 1966; Fostier, Jalabert and Terqui, 1973). Soon after, the mature (secondary) oocyte is ovulated out of its follicular envelope and acquires a jelly coat (Goswami and Sundararaj, 1971a; Sundararaj and Goswami, 1977a., b). The ovulated eggs are then spawned in water and almost immediately fertilized by the male.
Environmental conditions required for initiation of oocyte maturation, ovulation and spawning are much more strict than those for gametogenesis. In the majority of fishes, maturational processes are initiated only when thermal conditions are optimum. In others, a precise combination of daylength and temperature is required. In still others, radically different environmental stimuli such as rainfall and inundation precipitate maturational events.
8.2.1 Hypophysation by homoplastic and heteroplastic pituitary extracts
8.2.2 Hypophysation by partially purified and purified gonadotropins
8.2.3 Hypophysation by steroids
The precise combination of environmental factors required for maturation, ovulation and spawning when available, brings about an accelerated release of gonadotropin from the pituitary into the blood stream. As a result, gonadotropin, which was hitherto being secreted only at a tonic level for the maintenance of yolky oocytes, suddenly appears as a surge. The elevated titres of gonadotropin bring about maturational changes culminating in the act of spawning. However, quite often, under farm conditions, the requisite environmental factors are either not available or do not persist for sufficient length of time for spontaneous maturation to occur. Thus, one of the greatest constraints to fish culture has been the problem of obtaining fry of commercially important fishes in sufficient numbers and of good quality for stocking culture ponds. The pioneering discovery of Houssay (1931) and von Ihering (1935, 1937) that fishes can be induced to spawn by injecting pituitary homogenates has somewhat mitigated the problem. The principal advantage of this technique, referred to in aquacultural parlance as hypophysation, is that by simulating the natural gonadotropin surge, it bypasses, to some extent, the environmental variables of photoperiod, temperature, rainfall, etc. The technique, therefore, permits a rather accurate prediction of the time of spawning and the aquaculturist can plan his work well in advance. Also, the nurseries can be stocked with seed of uniform age, size and quality. Furthermore, hybrids can be produced by hand stripping and fertilization, whenever possible or feasible.
South American, Russian and Asian aquaculturists have employed the hypophysation method for large-scale spawning of economically important fishes (see Pickford and Atz, 1957; Atz and Pickford, 1959; Chaudhuri, 1968, 1969, 1976; Clemens, 1968; Singh, 1969; Donaldson, 1973; Shehadeh, 1973, 1975; Yamazaki, 1976; Fontaine, M., 1976; Chaudhuri and Tripathi, 1979; Harvey and Hoar, 1979). Maturation and ovulation have been induced in a variety of fishes including many cultivated fishes such as Prochilodus sp., Curimatus elegans (see Pickford and Atz, 1957), sturgeon, Acipenser güldenstädti and Acipenser stellatus (Gerbil'skii, 1938, 1950), catfish, Heteropneustes fossilis (Ramaswami and Sundararaj, 1956; Ramaswami, 1962; Sundararaj and Goswami, 1969a), catfish, Clarias batrachus (Ramaswami and Sundararaj, 1957; Devaraj, Varghese and Rao, 1972; Thakur, 1976), catfish, Clarias macrocephalus (Sidthimunka, Sanglert and Pawapootanon, 1968; Carreon, Estocapio and Enderez, 1976), the Brazilian catfish, Rhamdia hilarii (Machado and Castagnolli, 1979), channel catfish, Ictalurus punctatus (Sneed and Clemens, 1960; Clemens and Sneed, 1962), catfish, Pangasius sutchi (Potaros and Sitasit, 1976); murrel, Channa punctatus (Banerji, 1974), Indian major carps, Labeo rohita, Cirrhina mrigala, and Catla catla (Chaudhuri and Alikunhi, 1957; Chaudhuri, 1963, 1968, 1969, 1976; Bhowmick and Kowtal, 1973; Varghese et al., 1975; Varghese and Rao, 1976), grass carp, Ctenopharyngodon idella (Lin, 1965; Chaudhuri, Singh and Sukumaran, 1966; Boonbrahm, Tarnchalanukit and Chuaposhuk, 1970; Chen, Chow and Sin, 1969), silver carp. Hypophthalmichthys molitrix (Lin, 1965; Chaudhuri, Singh and Sukumaran, 1966; Boonbrahm, Tarnchalanukit and Chuaposhuk, 1970; Chen, Chow and Sin, 1969; Pruginin and Cirlin, 1976; Varghese and Rao, 1976), bighead carp, Aristichthys nobilis (Boonbrahm, Tarnchalanukit and Chuaposhuk, 1970; Chen, Chow and Sin, 1969), common carp, Cyprinus carpio (Pickford and Atz, 1957; Clemens and Sneed, 1962; Woynarovich, 1969), rainbow trout, Salmo gairdneri (Hasler, Meyer and Field, 1939; Pickford and Atz, 1957), grey mullets, Mugil cephalus (Tang, 1964; Yashouv, 1969; Shehadeh, 1973), and Mugil macrolepis (Sebastian and Nair, 1975), milkfish, Chanos chanos (Vanstone et al., 1976, 1977; Harvey and Hoar, 1979) and other marine fishes(Shelbourne, 1964) by injecting homogenates of homoplastic (same donor and recipient species) or heteroplastic (different donor and recipient species) pituitary glands.
The technique of hypophysation is now an established method for inducing spawning in cultivated fishes that mature under confined conditions (Pickford and Atz, 1957). Nevertheless, the collection of pituitary glands is not only time-consuming but involves the sacrifice of a potential broodfish or the collection of glands from fish to be marketed, which often results in considerable devaluation of the product. The alternatives of using pituitary material from trash fish or marine fishes (see Varghese et al., 1975; Varghese and Rao, 1976) or ampouling of the defatted pituitary extracts with known potency and shelflife (see Ibrahim, 1969a, b) have not found wide acceptance.
A serious drawback of the conventional hypophysation technique is the lack of standardization of the pituitary material, the demand for which generally exceeds supply. The pituitary extract is a mixture of several hypophysial hormones some of which are not even concerned with reproduction. The gonadotropic potency of the injected material depends on the stage of sexual maturity and physiological state of the donor, as well as on the method of pituitary collection and preservation (see Pickford and Atz, 1957; Clemens and Sneed, 1962; Clemens, 1968; Shehadeh, 1973; Yamazaki, 1976), while its effect in the recipient depends on its sexual maturity and on donor-recipient phylogenetic relationships (see Pickford and Atz, 1957; Fontaine, Y.A., 1980). Table II summarizes a number of procedures available for bioassay of gonadotropin potency of fish pituitary extracts or preparations. The radioimmunoassay techniques for precise measurements of plasma and pituitary levels of gonadotropic hormones of fish have been standardized (see Breton et al., 1971b; 1972b; Crim, Meyer and Donaldson, 1973; Crim, Watts and Evans, 1975; Tan and Dodd, 1978; Burzawa-Gérard and Kerdelhué, 1978).
A perusal of Table II shows that there are no easy methods for determining the gonadotropic potency of the pituitary extracts and those used for assessing intraovarian oocyte development without sacrificing the fish are, at best, unsatisfactory (see Harvey and Hoar, 1979).
As a matter of fact, the fish farmer even now reckons dosages as mg wet or dry weight. of pituitary per kg body weight of the recipient. A reliable gonadotropin assay that can be performed under field conditions without the aid of sophisticated instruments is a desideratum if hypophysation is to be a success.
Limitations of the technique notwithstanding, hypophysation is still the only method available to the fish farmer for use under field conditions. Fish endocrinologists are now looking for an ovulating agent that is inexpensive, potent, with known activity and long shelflife. The only preparation now available is the acetone-dried salmon pituitary powder (see Harvey and Hoar, 1979).
The mammalian gonadotropic hormones, the luteinizing hormone (LH) and the human chorionic gonadotropin (hCG), are effective in inducing maturation and ovulation in fishes (see Pickford and Atz, 1957; Ramaswami, 1962; Atz and Pickford, 1964; Sundararaj and Goswami, 1973, 1977a, b; Fontaine, M., 1976; Chaudhuri, 1976; Hirose, 1976). Partially purified or purified piscine pituitary gonadotropins isolated from carp and salmon have been extensively used for inducing ovulation and spawning in a number of fishes. Maturation, ovulation and spawning have been induced in the catfish, Heteropneustes fossilis, with hCG, LH, partially purified salmon gonadotropin (SG-G100), purified carp and catfish (Heteropneustes fossilis) gonadotropin (Sundararaj and Goswami, 1966a, 1973, 1977a, b; Sundararaj and Samy, 197A; Sundararaj et al., 1976), and in the ayu, Plecoglossus altivelis, with hCG and SG-G100 (Ishida, Hirose and Donaldson, 1972; Hirose, 1976). However, in the Indian major carps, hCG or Synahorin (a mixture of chorionic gonadotropin and mammalian pituitary extract) alone does not induce successful spawning but is effective when injected in combination with carp pituitary extract (Chaudhuri, 1976). Comparable results have been obtained in the grass carp (Lin, 1965; Chen, Chow and Sin, 1969). However, Bhowmick (1979) has recently reported on the use of crude hCG prepared in the laboratory for inducing spawning in Labeo rohita. In the grey mullet, hCG (Kuo, Shehadeh and Nash, 1973) or SG-G100 (Shehadeh, Kuo and Milisen, 1973; Kuo, Nash and Shehadeh, 1974a) alone or pituitary homogenates from grey mullet or Pacific salmon along with Synahorin (Liao, 1969, 1975; Shehadeh and Ellis, 1970), or SG-G100 along with hCG or even deoxycorticosterone (Kuo and Nash, 1975) are effective in inducing spawning. Ovulation has been reported by injecting SG-G100 in the captured milkfish females (Nash and Kuo, 1976; Vanstone, Villaluz and Tiro, 1976) and coho salmon (Jalabert et al., 1978). Ovulation and spawning have been induced with hCG in Dicentrarchus labrax (Barnabé, 1976) and Sparus aurata (Arias, 1976; Gordin and Zohar, 1978). Nevertheless, extracts of whole fish pituitary glands in glycoprotein solvents such as trichloroacetic acid (see Ramaswami and Lakshman, 1960; Bhowmick and Chaudhuri, 1968), and partially purified or purified piscine gonadotropins are generally more potent in inducing maturation and ovulation in fishes than mammalian gonadotropins (Sundararaj and Anand, 1972; Ishida, Hirose and Donaldson, 1972; Chaudhuri, 1976; Fontaine, M., 1976). Ng and Idler (1978a, b; 1979) and Idler and Ng (1979) have recently reported the isolation of a separate maturational hormone from the pituitary glands of common carp, chum salmon, winter flounder and plaice (see section 5 on Gonadotropic hormones).
Up until now steroids have seldom been used for large-scale breeding of cultivated species. This is surprising since evidence obtained in several laboratories has shown that progestational and adrenocortical steroids are nearly as effective as the protein hormones in inducing oocyte maturation in intact or hypophysectomized fishes; estrogenic and androgenic steroids are rarely, if ever, effective (see Sundararaj and Goswami, 1966a, 1977a, b; Sundararaj, Goswami and Lamba, 1979; Jalabert et al., 1977, 1978; Jalabert, Breton and Fostier, 1978; de Montalembert, Jalabert and Bry, 1978; Hogendoorn, 1979).
Even though LH and LH-like gonadotropins could act directly on fish oocytes to induce ovulation, the possibility of mediation by other endocrine organs cannot be ruled out. In this context, it is relevant to mention that pretreatment of hypophysectomized gravid catfish, Heteropneustes fossilis, with metopiron (SU-4885: Ciba), an inhibitor of 11ß-hydroxylase, greatly reduces the ovulatory response to gonadotropin (Sundararaj and Goswami, 1966b). This is a clear indication that the ovulation-inducing ability of gonadotropin is in some manner mediated through a steroidogenic tissue. This is further confirmed by studies in which maturation-inducing ability of gonadotropic preparations has been tested on oocytes (with or without follicular layers) cultured in a variety of media to isolate them from other endocrine influences (Goswami and Sundararaj, 1971a, 1974; Jalabert, 1976; Sundararaj and Goswami, 1977a, b). The results of such in vitro experiments indicate that the action of gonadotropin is invariably mediated through a steroidal substance whose source may be the oocyte follicle itself or another steroidogenic organ such as the interrenal.
Ovarian origin of maturation inducing steroids (MIS) is supported by the observation that in vitro maturation of oocytes of medaka (Hirose, 1971, 1972a, b, 1973; Iwamatsu, 1978), Misgurnus anguillicaudatus (Iwamatsu and Katoh, 1978) rainbow trout (Jalabert, Breton and Billard, 1974, Breton, Jalabert and Reinaud, 1976), brook trout and walleye (Goetz and Bergman, 1978a, b) as well as killifish (Wallace and Selman, 1978) can be induced by mammalian and piscine gonadotropins. Further, in medaka, metopiron, a specific inhibitor of 11(3-hydroxylase at low dose levels, prevents gonadotropin-induced maturation and ovulation but does not affect maturation and ovulation induced by cortisol (Hirose, 1973). These observations suggest that the gonadotropin acts first on the follicular tissue to produce an effective steroidal substance which in turn induces maturation and ovulation (Hirose, 1972b; Hirose et al., 1975; Jalabert, 1976; Iwamatsu, 1978). The follicular envelope of the oocyte is, therefore, necessary for the gonadotropin to induce maturation (Jalabert, Breton and Bry, 1972).
On the other hand, equally impressive evidence can be cited to prove that MIS are not synthesized in the ovary but more peripherally, possibly in the interrenal. In this category, are fishes such as Misgurnus fossilis (Kirshenblatt, 1959), catfish (Goswami and Sundararaj, 1971a; Sundararaj, Goswami and Donaldson, 1972; Sundararaj, Goswami and Lamba, 1979), goldfish (Jalabert et al., 1973), and yellow perch (Goetz and Bergman, 1978a, b) in which mammalian or piscine gonadotropins are marginally effective in inducing oocyte maturation under in vitro conditions. Also, the demonstration that oocyte maturation in catfish is accomplished 2 h earlier following systemic administration of deoxycorticosterone acetate than following LH injection (Goswami and Sundararaj, 1971b) possibly shows that the 2-h time lag represents the duration necessary for LH to act on the interrenal to build up sufficient titres of corticosteroids to induce oocyte maturation.
In catfish, Heteropneustes fossilis, the only potent MIS are the 11-deoxycorticosteroids. Incubation of homogenates of gravid ovary of catfish during the spawning season with pregnenolone-7-3H clearly shows that the catfish ovary does not have the ability to synthesize 11-deoxycorticosteroids unlike in some other fishes (see Colombo et al., 1973) but yields only two metabolites, 3a -hydroxy-5ß-pregnan-20-one (epipregnanolone) and 5ß-pregnan-3a -20a -diol (pregnanediol) (Ungar et al., 1977) the former has slight and the latter has no maturation-inducing effect.
That the ovary of the catfish does not contribute significantly towards the synthesis of MIS is clear from the above discussion. On the other hand, there is good evidence to suggest that the MIS are elaborated principally by the interrenal. Sundararaj and Goswami (1966c, 1969b) have demonstrated that in vitro synthesis of the two catfish corticosteroids (hydro-cortisone and deoxycorticosterone) can be increased several-fold by gonadotropic stimulation. Truscott et al. (1978) have recently shown that the concentration of plasma cortisol increases approximately four-fold after injection of either ovine LH or SG-G100 in the sexually regressed as well as gravid catfish. Further, ovariectomy in the sexually regressed catfish does not prevent the LH-induced rise in plasma cortisol levels. Katz and Eckstein (1974), who studied changes in steroid concentration in blood of female Sarotherodon (Tilapia) aurea, reported a 38-fold increase of deoxycorticosterone (DOC) during the initiation of spawning. The source of DOC was, however, not ascertained. Nonetheless, the fact that a large increase in DOC concentration occurs in the peripheral blood during the initiation of spawning indicates its possible importance in the ovulation of this fish.
Even in the goldfish the participation of an extraovarian relay (probably interrenal) in gonadotropin-induced oocyte maturation is evident since in vitro oocyte maturation is induced by 17a -20ß-dihydroprogesterone but only marginally by carp pituitary extract, carp gonadotropin and SG-G100 (Jalabert et al., 1973; Jalabert, 1976). The recent report that the ovulatory GtH surge (Stacey, Cook and Peter, 1979) precedes the preovulatory rise in cortisol (Cook, Stacey and Peter, 1980) indicates that cortisol, presumably of interrenal origin, may be involved in GtH-stimulated ovulation in goldfish.
Further evidence in support of the interrenal origin of MIS comes from studies on the ovary-interrenal coculture where the greatly attenuated in vitro oocyte maturational ability of gonadotropins (LH or SG-G100) is restored to a great extent by inducting the interrenal tissue into the culture medium. It is clear, therefore, that not only is the interrenal the principal source of MIS but that the synthesis of MIS can be greatly stimulated by gonadotropins (Sundararaj and Goswami, 1974; Goswami, Sundararaj and Donaldson, 1974). These findings strongly suggest the existence of a pituitary-interrenal-ovarian axis in the maturation of catfish oocytes.
Jalabert (1975, 1976) has shown that some steroids such as testosterone, cortisol and cortisone, increase the maturation-inducing efficiency of gonadotropin or gonadotropic extracts. Dettlaff and Davydova (1974, 1979) have reported that triiodothyronine increases the sensitivity of follicles isolated from cold-stored sturgeon eggs to gonadotropins. These results suggest that some steroids and even thyroid hormones can sensitize the oocytes to gonadotropic action.
In vitro culture of oocytes of catfish, Heteropneustes fossilis, with cortisol acetate or LH alone and in various combinations has shown that not only is cortisol acetate a much more potent maturation-inducing agent than LH but that the two hormones act synergistically over a wide range of dosages. Prior exposure of oocytes to LH or epipregnanolone for 90 min enhances subsequent cortisol action on maturation, thereby indicating that possibly LH sensitizes oocytes through the formation of epipregnanolone. Addition of testosterone or estradiol to the culture medium simultaneously with cortisol, LH or epipregnanolone markedly inhibits oocyte maturation (Sundararaj, Goswami and Lamba, 1979).
The above discussion points to a dichotomy in the hormonal regulation of oocyte maturation in teleost fishes. In one group of fishes exemplified by the rainbow trout, the medaka and others, the ovary seems to be the source of MIS, whereas in the other group exemplified by the catfish, and possibly the goldfish, the interrenal seems to be the major source of MIS. This kind of diversity in reproductive adaptations is only to be expected in a group as diverse and specialized as the teleosts. We are only beginning to get an insight into this vast diversity and not unless representatives from a large number of teleostean families are investigated shall we be in a position to understand the magnitude of species variations within the teleost taxon.
While numerous investigators have tested the effectiveness of various hormones on fish oocyte maturation, few attempts have been made to understand the mechanism of hormone-induced oocyte maturation in fishes. However, some information is available in the case of two species, catfish (Goswami and Sundararaj, 1973), and rainbow trout (Jalabert, 1976). In the-former, puromycin and cycloheximide, inhibitors of protein synthesis, can completely block deoxycorticosterone-induced in vitro oocyte maturation when added to the culture medium along with or within three hours of hormonal stimulation, whereas actinomycin D and mitomycin C are totally ineffective. These findings suggest that maturation of catfish oocytes involves a de novo synthesis of protein(s) possibly through translation of preformed stable messenger RNAs; and that the process of protein synthesis is completed within three hours of hormonal stimulation.
In the case of Salmo gairdneri, where in vitro oocyte maturation can be induced by gonadotropin as well as by steroids, actinomycin D, mitomycin C or puromycin can inhibit gonadotropin-induced maturation but have no effect on maturation induced by steroids. Cycloheximide, on the other hand, completely blocks maturation-inducing action of all hormones (Jalabert, 1976). Thus, the situation in rainbow trout is somewhat different from that in catfish where both cycloheximide as well as puromycin block steroid-induced in vitro oocyte maturation.
In catfish (Goswami and Sundararaj, 1971a, b; Sundararaj and Goswami, 1977a, b) and yellow perch (Goetz, 1979; Goetz and Theofan, 1979), maturation in vitro is often followed by ovulation, whereas in rainbow trout maturation and ovulation are distinct phases, the steroid responsible for stimulating maturation may also initiate a process to bring about detachment of oocyte from the follicle (Jalabert, 1976). Expulsion of oocytes might involve prostaglandins since under in vitro conditions these agents induce ovulation of oocytes of trout (Jalabert and Szöllösi, 1975) and yellow perch (Goetz, 1979; Goetz and Theofan, 1979) and restore ovulation in indomethacin-inhibited goldfish (Stacey and Pandey, 1975). Stimulation of synthesis and release of prostaglandins possibly requires an extra-ovarian relay since ovulation does not normally follow steroid-induced in vitro maturation (Jalabert and Szöllösi, 1975).
The role of hypothalamic hormones in stimulating the pituitary gland to secrete gonadotropin has been discussed in section 3 on the. hypothalamus.
In addition to steroids, a synthetic nonsteroidal antiestrogen - clomiphene citrate - has been used to induce ovulation in a number of fishes (Harvey and Hoar, 1979). It induces maturation and ovulation in intact (Pandey and Hoar, 1972) but not in hypophysectomized goldfish (Pandey, Stacey and Hoar, 1973). Kapur and Toor (1979) have induced spawning in the common carp following treatment with clomiphene, whereas Bhowmick et al. (1979) failed to induce ovulation in Labeo rohita with clomiphene citrate. Breton, Jalabert and Fostier (1975) reported that treatment with cis-clomiphene induces gonadotropin surge in the common carp at low but not at high dosages. Abraham (1975) has suggested that clomiphene can be used to overcome arrested ovarian development in grey mullet reared in freshwater. Clomiphene possibly acts by interfering with the steroid feedback at the level of the hypothalamo-hypophysial system by competing with estrogen (see Pandey and Hoar, 1972) and thereby counteracts its negative feedback effect (see also section 11 on Feedback action of gonadal steroids).
