NIVEL DE SEVERIDAD ALTO (H):

Sexually mature specimens of Arenicola marinahave been induced previously to spawn following the injection of macerated extracts or ‘saponifiable lipid’ extracts of whole body homogenates (Howie, 1961b). The saponifiable lij^id fraction includes the fatty acids, and therefore, that the fatty acid 8,11,14 - eicosatrienoic acid can induce Spawning, supports these observations.

Following the injection of active tissue extracts, Howie ( 195 lb) noted that worms underwent violent contractions of the body wall which led him to suggest that a muscle stimulant must be present in such tissue extracts. The utilisation of muscular contractions during spawning has been observed in other polychaete species. For example; during spawning in Nephtys caeca and Nephtys hombergi,

violent muscular contractions result in the emission of gametes from the anus

(Bentley et a/., 1984). To discharge gametes in this way is not common within poychaetes, and to date, has been described only in these species. More commonly, polychaetes not forming epitokes, undergo garhete discharge through a coelomoduct system with most ducts leading into the nephridial canal rather than directly t6 the exterior (Schroeder and Hermans, 1975).

The number of gametes observed being released in these experiments is large, and is comparable with studies in other broadcast spawrtèrs. For example,

Asterias rubens and Strongylocentrotus purpuratus have been shown to liberate 2x108 and 8x10^° sperm respectively during a breeding season (Giese & Kanatani,

Ç .

1987). In Arenicola there is little variation, both between individuals and between treatment groups, in the number of spermatozoa released, although, over lower doses of 8,11,14 -eicosatrienoic acid which are capable of stimulating spawning, the

number of sperm released begins to decline. Above threshold doses however, spawning generally appears to be an ‘all or nothing’ response and the variability of gamete release that has been observed in other polychaete species (Olive et a l,

1981a ; Olive etal,, 1981b) does not occur.

Chapter 4 of this study presented evidence which suggested that the chemical characteristics of prostomial SMF and 8,11,14- eicosatrienoic acid were identical. Data presented in Chapter 5 however, suggested that the responses of spennatozoa

in vitro, to incubation with pure 8,11,14 -eicosatrienoic acid and prostomial homogenate, were slightly different. Principally, there was a difference in the time taken for sperm to become independently motile and the corresponding oxygen consumption rate to rise above basal levels. During incubation with 8,11,14- eicosatrienoic acid, this time was significantly shorter than during incubation with prostomial SMF. During spawning experiments however, no such time differences are observed and this suggests that the repsonse may be modulated in vivo. That this might occur due to carrier proteins has already been suggested (see section 5.4). In fact, in every respect, the spawning response of male Arenicolato either 8,11,14 -eicosatrienoic acid or SMF is identical and this adds further support to the evidence derived from chromatographic, enzymic and respirometric experiments, that SMF is 8,11,14 -eicosatrienoic acid. That the fatty acid isomer 11,14,17 -eicosatrienoic acid is unable to induce spawning (and is unable to induce sperm activation in vitro - see section 4.3.2), is probably a reflection of its stereochemistry. Whilst 8,11,14- eicosatrienoic acid is the parent molecule of a wide variety of biologically active substances, due to the differences in the position of the conjugated double bonds, the 11,14,17- isomer is not (see section 4.4). Quinacrine is unable to induce

spawning, but it has been demonstrated dearly that its mode of action differs from that of the natural activator (see sections 4.3.5; 5.3.1 & 5.3.3).

Howie (1961c) described that free spermatozoa, when injected into the coelomic cavities of male Arenicola, were quickly released to the exterior, presumably as a result of the spermatozoa being accepted automatically by the ciliated funnels of the nephromixia. A similar series of events have been suggested for the spawning mechanism of Pectinaria gou/d/r (Tweedell, 1980), and an

endocrine factor activates the admission of fully developed gametes into the

nephromixia where they then undergo their final maturation. In the terebellid Lanice conchilega however, there is no evidence for selective uptake of spermatozoa and both ripe and unripe gametes are observed in the nephromixia of post spawning animals (Smith, 1989). In Arenicola, only free swimming spermatozoa or morulae in which the sperm are swimming (Howie 1961c) and oocytes which have undergone germinal vesicle breakdown, are observed being spawned (Howie, 1961b), so clearly some selection of gametes is occUring. The observations of muscular contractions and periods of definite ejaculation described in this chapter, suggest that an active spawning mechanism is involved.

Either during passage through the nephromixia or during sperm activation within the coelomic fluid of Arenicola, the spermatozoa undergo changes in which their motile life and viability are prolonged over and above that observed during in vitro activation (section 5.3.1). Presumably these enhance the success of interaction with oocytes and subsequent fertilisation. Additional cytochemical changes often occur following sperm motility acquisition in many animals and these prepare the spermatozoa for the acrosome reaction and fertilisation (reviewed in section 1.3.3). Such events, known as capacitation, usually occur in the female genital tract, but in invertebrates, which undergo ‘external fertilisation' and no sperm transfer to females, it has been suggested that capacitation and the acquisition of

motility may occur concurrently (Longo, 1987). The precise moment of sperm motility acquisition is subject to significant intraspecies variation and is reviewed in section 1.3.2. Sperm motility acquisition in Arenicola occurs in the coelomic cavity, but is not a simple process, and the dissociation of sperm morulae and the activation of spermatozoa is clearly quite separate from the changes which result in the increase in sperm viability.

That 8,11,14- eicosatrienoic acid is capable of stimulating spawning in males, but not in females, supports the observations made by Howie (1961a & b). He described that the lipid fraction of whole worm homogenates, which can induce spawning in males, cannot do so in females. Spawning in females can be induced by a maturation factor present within prostomial homogenates but this clearly is not 8,11,14-eicosatrienoic acid. Meijer (1979a; 1979b; 1980) investigated an oocyte maturation factor in the prostomia of sexually mature females. He concentrated however on describing its biochemical and morphological effects on oocytes, rather than on its chemical identity and these are reviewed in section 1.5.2. There are, therefore, clear sex specific differences between the endocrine signalling mechanisms which surrounds gamete maturation and spawning in this animal.