Fertilisation has been the most investigated point in the history of research, and the sea urchin has been the most investigated model in this respect: authors such as Epel (Epel 1975; Kuo et al. 2000) and Jaffe,both Laurinda and

Lionel (Jaffe 1976, 1980, 1983; Jaffe and Robinson 1978; Turner et al. 1984), investigated the electrophysiological events; Vacquier (1969, 1981; Vacquier and Moy 1977) and Lennarz (Schmell et al. 1977; Ohlendieck et al. 1994; Ohlendieck and Lennarz 1996; Just and Lennarz 1997) investigated sperm-egg reception and adhesion; Shen (Rakow and Shen 1990; Shen 1995) and Chambers (Chambers and De Armendi 1979; Longo et al. 1986; McCulloh et al. 1987; Crossley et al. 1988; Ivonnet and Chambers 1997) investigated calcium dynamics in egg responses; Schuel (e.g. Schuel et al. 1991) investigated sperm dynamics; and Whitaker (Whitaker and Irvine 1984; Crossley et al. 1988; Wilding et al. 1996; Harrison et al. 2002) investigated the mechanisms of calcium release from inner stores. Each author's approach included morphological, biochemical and electrophysiological methods, sometimes with different hypotheses and interpretations.

Essentially, fertilisation occurs in two phases: the first phase is characterised by sperm activation events; the second, by the egg activation events.

First Phase: Sperm Activation

Sperms are immobilised in male gonads and genital ducts, as the membrane in these sites is stabilised by cholesterol and glycoproteins (Gilbert 1994) and by the sperm fluid, which has a low pH and contains calmodulins (Gilbert 1994).

During the first phase of fertilisation, the sperm motility is activated, the sperm are able to swim towards the egg, and are capacitated to the acrosome reaction, during which the acrosome vesicle releases the enzymes it contains into the water surrounding the egg envelope. During these phases, electrical events caused by inward-outward ion fluxes take place.

Actually, in each of these events, calcium is made available for the sperm since it is abundant in seawater as well as in the peri-ovular jelly coat; the latter probably also plays a role in modifying the sperm membrane, and also in activating the acrosome reaction (Darszon et al. 1987; Garcia-Soto et al. 1987; Lievano et al. 1990). Moreover, K+ and Ca2+ channels are activated by speract, the attractant molecule released by the egg, thus leading the sperm to find the egg of the same species (Babcock et al. 1992).

At the same time, Na+, abundant in the marine environment, causes an increase in the intracellular pH level, which in turn activates flagellum dynein, maintained low by the low pH of the sperm fluid. This event seems to be guided by the activation of nicotinic receptors, present in the membrane of the flagellum, as shown by experiments with a-bungarotoxin, a type of snake venom which is specific for these receptors (Nelson 1976; Falugi et al. 1993b), and by histochemical staining of agonist and antagonist drug binding (Bac-cetti et al. 1995). In each case, ionic changes play a role in the phase in which swimming is activated as well as in the phase in which the egg membrane is

"attacked"; these events take place thanks to: (1) an enhanced swimming activity and (2) exocytosis of the acrosome granule which, releasing lytic enzymes, exposes the egg membrane, free from accessory envelopes (jelly coat), and perforates the primary envelope (vitelline membrane). Simple neurotransmitter signal molecules play a crucial role in these processes as well.

Cholinergic system molecules were discovered in sperm cells of different animal species (Bishop et al. 1977; Sastry et al. 1979; Baccetti et al. 1995). These findings led researchers to investigate the possible role played by neurotransmitter system molecules in these cells. In particular, the presence in sperm of cholinergic molecules has been reported and studied by Nelson (1973,1978, 1990) and Nelson et al. (1970), who supplied evidence for the implication of the cholinergic system in sperm propulsion. The activity of the enzyme acetylcholinesterase (AChE, E.C. was evaluated by biochemical methods in the sperm flagellum of the sea urchin P. lividus (Cariello et al. 1986). AChE is the lytic enzyme of the cholinergic system, and its function is to cleave ACh into choline and acetate, thus restoring ACh receptor excitability. In sperm of the same sea urchin species, AChE was also localised by histo-chemical methods on the head membrane (Falugi et al. 1991). ACh receptors were also found and localised in the sperm cell structures: muscarinic receptors mainly in the acrosome, nicotinic receptors both in the acrosome and in the flagellum membrane (Baccetti et al. 1995). The two different families of receptors seem to play different roles in the different moments of fertilisation: the nicotinic ones (nAChRs, see Stroud et al. 1990), gating Na+ channels on activation by their specific ligands (ACh and cholinomimetic agonist drugs), may result in a change of the Na+ influx and consequently H+ efflux, increasing the internal pH and activating the dynein of the flagellum. The muscarinic ones, known in five different molecular forms, associated with G-protein in the intracellular domain, trigger a second messenger transduction cascade (Birdsall et al. 1978; Watson and Arkinstall 1994), activating intracellular dynamics related to fertilisation (Falugi et al. 1993a).

In addition, the effects of cholinomimetic drugs on P. lividus sperm motil-ity and fertilisation ability suggested an involvement of the nAChRs and mAChRs in these phenomena (Falugi et al. 1993b). This was also clear from experiments with potentiometric dyes, which helped to evidence the pattern of membrane depolarisation and fluidity during swimming and acrosome reaction caused by the presence of either eggs, calcium ionophore A 23187 or nicotine (Falugi et al. 1993a).

The localisation of the studied molecules in the different cell districts is useful in order to understand their function. The presence of molecules immunologically related to AChRs was found to be constant in gametes of different organisms, and their localisation seems to be related to the different mode of interaction between sperm and egg in the different species. In particular, P. lividus sperm presented molecules immunologically correlated to muscarinic receptors mainly at the acrosome and nicotinic receptors at the acrosome and along the flagellum (Fig. 3), thus confirming previous

Sea Urchin Sperm Png
Fig. 3. Comparison of the localisation of muscarinic and nicotinic receptors in human and sea urchin sperm (sp). Acr Acrosome; eq equatorial ring; p.acr post-acrosomal region; midp midpiece; flag flagellum

results (Dwivedi and Long 1989; Baccetti et al. 1995). This localisation suggests that the nicotinic receptors play a role in sperm propulsion, as previously suggested by in vivo pharmacological experiments, with agonist and antagonist drugs (Nelson 1976; Dwivedi and Long 1989; Falugi et al. 1993b). As to the receptors present in the acrosome, their function may be related to sperm-egg interaction, including acrosome reaction (AR) and membrane fusion. Similarly, Bray et al. (2002), recently investigating whether the nAChR may have a role in the acrosome reaction (AR) of human sperm, have obtained strong evidence that ACh is capable of initiating the AR by activating an a7 subunit-containing nAChR and that this receptor is essential for the AR initiated by purified recombinant human zona pellucida (ZP) protein (rhZP3), thus playing a potential role in the initiation of the AR by intact ZP in vivo. In the sea urchin, this reaction could also be enhanced by molecules immunologically related to choline acetyltransferase (ChAT, E.C., the biosynthetic enzyme of ACh (Mautner 1986) present on the surface of mature eggs (Angelini et al. 2004), as this enzyme is capable of autonomously synthesising ACh, active on the ACh receptors located in the acro-some (Fig. 4).

The function of muscarinic AChRs localised in the acrosome of P. lividus sperm might be related to the fusion of the membrane between the gametes. This assumption arises from the comparison between their localisation in sea urchin sperm and that in mammalian sperm, respectively: to be precise, in sea urchin sperm, the first fusion between the sperm and the egg membrane takes place at the tip of the acrosome, where these ACh receptors are also localised, while mammalian sperm present the receptors at the acrosome equatorial ring (Baccetti et al. 1995), where the first contact and fusion with the egg takes place (see Longo 1987 for comparison among the different models).

Fig. 4A-C. ChAT ultrastructural immunogold reaction in Paracentrotus lividus sperm. A Control; B, C reacted sperm (x12,000)

A matter of debate over recent years has been the fact that the binding sites for pharmacological agents may differ between in vitro and in vivo experiments, as suggested from experiments on mouse sperm (Florman and Storey 1982). However, experiments on sea urchin fertilisation are normally carried out in seawater, the natural medium in which this event takes place, so as to eliminate any possible difference between in vivo and in vitro experiments.

Second Phase: Egg Activation

It is well known that sea urchin egg activation and fertilisation are led and followed by electrical phenomena, especially as far as ion dynamics are concerned. Following the contact of the egg's membrane with that of the sperm, an early depolarisation event of the egg membrane takes place, caused by an influx of Na+ ions (see Epel 1975,1980), which makes the membrane permissive to the fusion (McCulloh et al. 1987).

This spike is immediately followed by an explosive increase in intracellular [Ca2+], released from intracellular stores (Giudice 1973; Longo 1987). This event evokes a strong membrane depolarisation (about 90 mV) and activates the egg, causing the so-called fast block to polyspermy as well as the cortical reaction. The egg, thus activated, begins to "breathe" and starts its metabolic activities, including DNA synthesis and the trigger for the first cell cycles (Giudice 1973; Longo 1987).

As far as the mode of regulation and unfolding of these ion dynamics is concerned, a great deal of research has been carried out since the beginning of the last century and, according to Ohlendieck and Lennarz (1996), the number of investigations on the subject has greatly exceeded that of the works concerning the philosopher's stone over the past centuries! However, recently some progress has been made towards the comprehension of these phenomena.

First Step: Na+ Influx

This event was described by Epel (1980): as soon as the sperm touches the egg membrane, a minor influx of Na+ takes place throughout the egg membrane; at the same time, sperm-egg membrane fusion takes place, immediately followed by calcium release from intracellular stores. The meaning of this first Na+ influx was apparently forgotten by researchers in the following years, who rather chose to focus on the calcium wave responsible for the block to polyspermy.

Just recently, this event has been re-examined,by considering old papers on the effects of nicotine exposure during sea urchin egg fertilisation and by observing that the ionic dynamics (first Na+ influx, immediately followed by explosive Ca2+ release) are very similar to what happens in the neuromuscu-lar synapses. Nicotine had been reported by Jaffe (1980) to cause polyspermy; thus it is very likely that nicotinic receptors may be present just as in the neu-romuscular post-synaptic membrane. To be precise, nicotinic receptors were histochemically revealed by curare-prevented a-bungarotoxin binding (Falugi and Prestipino 1988; Falugi et al. 1989) at the egg surface. More recently, by exposing sea urchin unfertilised eggs to acetylcholine together with eserine (Physostigmine, Sigma), we have observed, after the addition of sperm, a membrane depolarisation of about 40 mV (starting from a rest potential of -70 mV,to the spike of -30 mV), while the normal final depolarisation in control eggs is +0 mV, with a depolarisation ranging between 70 and 90 mV. Moreover, the exposure to acetylcholine prior to fertilisation significantly increased the percentage of polyspermic eggs as compared to controls (Harrison et al. 2002; Angelini et al. 2004). The pharmacological properties of these receptors were verified with electrophysiological methods by Chambers and co-workers, who confirmed the presence of nicotinic receptors in the unfertilised sea urchin egg, and characterised them as being identical to the ones present in the neuromuscular synapses (Ivonnet and Chambers 1997). According to these authors, the presence of nAChR channels at the surface of unfertilised eggs can account for the capacity of nicotine to impair the block to polyspermy, by lowering the positive shift of the egg's membrane potential caused by sperm.

This could also explain our results described above (Angelini et al. 2004) Actually, the same effect of maintaining the fertilisation potential of the egg at low levels could be exerted by an increased quantity of ACh, which in this case could maintain the membrane fusibility for a longer period of time than under physiological conditions. This relationship between the first step of depolarisation due to Na+ influx and membrane fusibility seems to be reliable. In fact, as previously demonstrated (Longo 1987; McCulloh et al. 1987; McCulloh and Chambers 1992), a low membrane depolarisation allows sperm entry and membrane fusion in the sea urchin Lytechinus pictus and Strongy-locentrotus purpuratus.

What is the source of ACh able to activate the nicotinic receptors present on the egg surface? The answer to this question could be the discovery of molecules related to choline-acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholinesterase) in the acrosome and head membrane of P. lividus sperm, reported by immunogold methods and electron microscopy (Piom-boni et al. 2001). To be precise, we found that ChAT immunoreactive molecules, revealed by immunogold particles, were mainly localised in the sperm head of P. lividus. In immotile sperm ("fixed-dry sperm"), gold particles appeared to be particularly concentrated in the acrosome vesicle. However, when sperm began to swim actively as a result of their suspension in seawater containing eggs, the gold particles were observed in the membrane all along the head region. This may suggest that the sperm autonomously synthesises ACh, and that this function is active as far as the first sperm-egg interaction takes place (Angelini et al. 2004). The cholinergic molecules present in the sperm acrosome might play a role in sperm-egg interaction, as hypothesised by Ibanez et al. (1991) for mammalian sperm, where ChAT molecules and their mRNAs were found in the equatorial ring, which is where the first contact with the egg takes place.

Therefore, our hypothesis is that ACh released by the sperm surface (where ChAT is exposed) may, at the first contact, excite the nicotinic AChRs present in the egg membrane, thus evoking the first event of Na+-induced depolarisation, responsible for membrane fusion (Angelini et al. 2004; Fig. 5).

Exposure ACh synthesis

Exposure ACh synthesis

(close channel)

Fig. 5. Schematic drawing of the possible function of ChAT and nicotinic receptors during fertilisation

(close channel)

Fig. 5. Schematic drawing of the possible function of ChAT and nicotinic receptors during fertilisation

Calcium Dynamics

Although the egg membrane is effectively an excitable membrane, its similarity with the synapses is only limited to the nicotinic receptors, because the influx of Na+ ions caused by acetylcholine in the sea urchin egg is not sufficient to evoke the release of calcium ions and the rapid block to polyspermy, as we demonstrated in cooperation with the laboratory of Prof. Whitaker (Harrison et al. 2002), thanks to the use of cholinomimetic agonists and antagonists on P. lividus and Lytechinus pictus eggs and zygotes. We did not find any evidence of an involvement of cholinergic molecules in this process, as nicotinic AChR agonists were not capable of evoking any [Ca2+] variation in unfertilised eggs by themselves, nor were the antagonists capable of preventing the calcium spike provoked by sperm. Also, muscarinic agonists and antagonists were completely inactive in the course of these events.

Thus, in the fast block to polyspermy a different kind of signal and receptor molecule must be active for the Ca2+ ion release. The latter was elicited by a signal transduction cascade, since Whitaker and Irvine (1984) had shown that an inositol-triphosphate (IP3) injection in the cytoplasm was sufficient to activate the sea urchin unfertilised egg and to cause the cortical reaction.

Actually, in August 2000, a paper by Kuo et al. was published, reporting that IP3 release was due to an atypical neurotransmitter, nitric oxide (NO). These authors showed that nitric oxide synthase (NOs) is present in the post-acroso-mal area of the sperm, and that it is activated by contact with the jelly coat, to synthesise NO, which is subsequently carried inside the egg together with the sperm material. This would be the signal that triggers the transduction cascade, including the release of IP3; these events result in an explosive release of calcium from the inner stores, identified as cortical vesicles of the endoplas-mic reticulum (see Giudice 1986; Shen 1995 for extensive reviews) and, recently, also vacuolar-like structures (Churchill et al. 2002).

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