Initiation of sperm motility

Of the species so far described it is now known that sperm become motile in response to one or more of the following:

a) Dilution

The sperm of sea urchins, salmonid fishes and amphibians become motile upon dilution in the external medium which occurs following release from the body cavity. In the case of sea urchins, a sodium dependent release of protons'occurs upon their dilution in sea water and results in an alkalinisation of the cell (see

Trimmer & Vacquier, 1986). This alkalinisation is o^ the order of 0.4 pH units and motility is stimulated by a change in intracellular pH to that which is within the optimal range for the pH sensitive flagellar dyenin ATPase. The high levels of potassium in salmonid fishes suppress speim motility in the testes (see Morisawa,

1987) and motility is stimulated due to the lowering of potassium ion concentration upon dilution. It has been shown in trout spermatozoa that this serves to hyperpolarize the cell which in turn stimulates motility (Omoto, pers, comm.). In marine teleosts, it is the osmolarity of the seminal plasma which inhibits sperm motility in the testis, and exposure to the hypertonicity of sea water upon the release of spermatozoa which appears to initiate their motility (Morisawa and Suzuki, 1980). Conversely, in freshwater cyprinid fishes and amphibians, it is the decrease in external osmolarity which is à trigger for the initiation of motility, although, the isosmolarity of the seminal fluid remains as a suppressor of sperm motility in the body cavity.

b) Interaction with egg derived substances

In the horseshoe crab, sperm undergo only a brief flurry of motility when they are released and they remain immotile until they come into contact with a sperm motility initiating factor (SMI) derived from the egg (Clapper & Brown, 1980). The only other example of sperm activation of this kind is observed in the herring (Yangimanchi, 1957). The interaction of sperm with factors that effect sperm motility has been described in over twenty species (see Garbers etal, 1986; Gabers & Kopf, 1980). Unlike the situation in the horseshoe crab or the herring however, these effect the motility pattern of already motile spermatozoa. In sea urchins for example, five classes of egg derived, species specific peptides, that stimulate the respiration and motility of spermatozoa have been isolated and sequenced (see Domino & Garbers, 1990). The two most studied of these peptides (termed

‘speract’ and Tesact’), act by stimulating a net proton efflux and a transient elevation of cAMP & cGMP concentrations in a receptor-mediated response which lead to modification of sperni behaviour and ultimately fertilisation (see Garbers et aL,

1986).

c) Male body fluids

The study of mammalian sperm motility acquisition is hindered by the complex system of cells and fluids in which the process takes place (Mitchell eta].,

1976) but it seems clear that sperm leaving the testes are non-functional, immotile and are not capable of fertilisation. Mammalian sperm develop their capacity for forward motility progressively. During transit through the epididymis, binding of a specific component, forward motility protein (FMP) takes place (Brandt et ah,

1978). Forward motility protein activity has also been observed in seminal fluid (Acott et al, 1979). It is only when sperm become mixed with fluids from the accessory glands at ejaculation however, that they become motile. This is due either to their release form immobilizing factors present in the epididymis (see section 1.3.1), or due to the mixing of sperm with seminal fluid during ejaculation. Seminal fluid is rich in sodium bicarbonate and this has been demonstrated in the pig, to be a specific stimulator of the sperm adenylate cyclase system; a mechanism now thought to be common to all mammals (Okamura eta], 1987).

In the lugworm Arenicola marina, the subject of this investigation, sperm become motile in the body cavity prior to spawning in response to a ‘Sperm Maturation Factor’ (SMF) released from the prostomium. This will be discussed in greater detail in section 1.5.

From this summary of sperm activation mechanisms, several parameters can be seen to be important at the biochemical level. Intracellular pH (pHi) and cAMP are important in many systems as well as various intracellular and extracellular ionic conditions.

An increase in cAMP levels has been demonstrated to be a stimulator of speiTO motility in mammals (Babcock etal., 1983; Babcock & Pfeiffer, 1987), in sea urchins (see Trimmer & Vacquier, 1986; see also Garbers eta/., 1986), in the horseshoe crab, (Tubb et al, 1979) and in rainbow trout, Salmo gairdneri (Morisawa & Okuno, 1982). A change in pHi is noted during sperm activation of sea urchins (see Trimmer & Vacquier, 1986), and in mammals (Babcock et al, 1983; Babcock & Pfeiffer, 1987). Although an increase in pHi is observed during sperm activation in the horseshoe crab Limulus, this is thought to be a side product of the activation sequence rather than a control mechanism (Clapper & Epel, 1982).

Intracellular pH serves to trigger sperm activation by, at least in the case of sea urchins, altering the intracellular pH to that which is optimal for the flagellar dyenin ATPase (see Trimmer & Vacquier, 1986). In rat spermatozoa a sodium dependent control of pHi similar to that of sea urchins has been proposed as a regulator of motility acquisition (Wong et a l, 1981). The cellular function of cyclic AMP is known to involve the phosphorylation of protein via the activation of a cAMP dependent protein kinase and the role of this in the activation of spermatozoa has recently been reviewed (see Brockaw, 1984; Brokaw, 1987; Morisawa & Morisawa, 1990; Tash, 1990). Work on mammals has identified a heat stable 56 KDa protein in sperm of the dog which is a significant substrate for cAMP phosphorylation. Termed ‘axokinin’, this protein is able to fully reactivate motility even in the presence of inhibitors to protein kinase (Tash et al, 1986). In the bovine spermatozoa, the same single protein is also thought to be present (Noland et al, 1987). The presence of a protein with axokinin properties has also been

identified in sea urchins, trout and man (see Brokaw, 1987). It has been demonstrated that phosphorylated proteins are localised along the length of the mammalian flagellum and there is evidence that they may play a pivotal role in the second messenger regulatory mechanisms of flagellar movement (see Tash, 1990).

Although each of these variables is singularly important in the stimulation of motility, in some systems factors such as pHi, cAMP levels, various ions, and ATP levels all interact with one another in the regulatory process (see Hoskins & Vijayaraghavan, 1990). In mammalian spermatozoa for example, it is thought that intracellular pH is a permissive event for motility development in that if cAMP levels are elevated without raising pHi no motility is observed until the pHi is raised. Thus, the increase in pHi ‘permits’ all preceding biochemical events to manifest themselves.

Upon sperm activation, motility results from the transient interaction of the dyenin arm of the A tubule with the adjacent B tubule, which induces a sliding of microtubules which is resisted and coordinated by the other structures of the axoneme to generate the flagellar waveform (see Gibbons, 1981). The dyenin heavy chains, which constitute the globular heads of the dyenin arm bouquet (see section 1.2.4), contain sites of ATP binding and hydrolysis. The binding of ATP induces conformational changes in the dyenin arm. In its absence the dyenin arm is attached to the adjacent subfibre, but in the presence of ATP the arm adopts a relaxed configuration in which it is detached and tilted toward the base of the axoneme. Therefore, the cycle of ATP binding and hydrolysis models suggest that the dyenin arms ‘push’ the adjacent doublet toward the tip or plus end of the axoneme as they ‘walk’ toward the minus end (see Porter & Johnston, 1989).

The ATP utilised in this process is thought to be directed from the mitochondria to the flagellum by a phosphorylcreatine (PCr) shuttle (Tombes &

Shapiro, 1985). If ATP was available to the axoneme only by diffusion, then the normal production of flagellar waves would be disrupted at some point along its length due to the preferential hydrolysis of ATP by the more proximal dyenin ATPase. That this occurs in sea urchin spermatozoa in the presence of a specific inhibitor of creatine kinase helps to support the existence of this system in sea urchin spermatozoa. Such a shuttle ensures high concentrations of ADP are present at the mitochondria to permit maximal respiration and that sufficient ATP is present at all points of the axoneme. This is achieved because the diffusing metabolite is not directly accessible to dyenin ATPase molecules along the sperm tail and the energy is available only once transphosphorylation of PCr with ADP has occurred.