First Cell Cycle

Therefore, cholinergic molecules do not seem to be involved in the block to polyspermy and cortical reaction. Nevertheless, several authors have found a complete set of cholinergic system molecules in the eggs both before and after fertilisation. Among these, our group has localised nicotinic AChR molecules in the sea urchin P. lividus by use of curare-prevented FITC-a-bungarotoxin binding at the surface of unfertilised and fertilised eggs of P. lividus (Falugi et al. 1989), alongside muscarinic AChR molecules (Piomboni et al. 2001), as well as other cholinergic molecules, such as acetylcholinesterase (Piomboni et al. 2001) and maternal choline acetyltransferase (ChAT) molecules, assembled on the oolemma during egg maturation and fertilisation processes (Angelini et al. 2004). Actually, muscarinic receptor molecules are present in the egg starting from the first maturation events, but are assembled on the egg membrane only after fertilisation. This was demonstrated by both ultrastructural localisation of anti-mAChR immunoreactivity and Western blot of cytoplasm homogenates compared with membrane extracts of unfertilised and fertilised eggs (Piomboni et al. 2001). A similar distribution pattern was found for ChAT (Angelini et al. 2004). Moreover, the cortical granules display a content of active molecules of AChE, identified by the enzyme reaction product as suggested by Karnovsky and Roots (1964). These molecules are synthesised within the endoplasmic reticulum, processed in the Golgi vesicles and from there exposed at the membrane surface (Piomboni et al. 2001). During the cortical reaction, these molecules are extruded into the perivitelline space (Fig. 6).

Such a coordinated distribution of signal molecules suggests a function for the cholinergic system, displayed after fertilisation. The key to the understanding of this possible function lies in the role played by muscarinic AChRs, which consists of triggering intracellular signal transduction (Watson and Arkinstall 1994). This appears to be the case in all the systems in which they have been identified. It has been recently reported that mAChRs are functional only when assembled on the cell surface, while inactive subunits are contained in ER-derived vesicles (Mei and Xiong 2003). This is true for ChAT as well, as it is known that ChAT activity is regulated by its binding to the cholinergic membrane (Dobransky and Rylett 2003) and by interaction with other cellular proteins related to this membrane (Gabrielle et al. 2003). In other words, the enzyme is not active when it is inside the cytoplasm, while it is active when it is exposed on the cholinergic membrane.

Furthermore, by FURA-2 experiments, it was shown that ACh and car-bamylcholine, the natural signal and the cholinomimetic agonist of the mAChR, respectively, are capable of evoking a [Ca2+] spike. This occurs when the exposure is performed at a time preceding the nuclear envelope breakdown (NEB) occurring at the first cell cleavage, i.e. approximately 15 min for P. lividus and 40 min for L. pictus after fertilisation. In contrast, atropine (antagonist of mAChRs, non-selective among the different molecular forms of the receptors) prevented further Ca2+ spikes caused by agonist molecules. The ability to respond to them was acquired again after rinsing the atropine present in the egg-containing water (Harrison et al. 2002); for this reason, we hypothesised that mAChRs could be involved in the regulation of intracellu-lar ionic dynamics related to the cleavage of the first cell cycles (Harrison et al. 2002). All our findings seem to suggest a model of autocrine regulation of these dynamics. To be precise, those ChAT molecules that after fertilisation are exposed at the egg surface may autonomously synthesise ACh. The egg cannot receive ACh from elsewhere, because after fertilisation the zygote is enclosed in the fertilisation membrane, which is in turn completely surrounded by seawater, and transported by waves and currents. The ACh pro-

Fig. 6. A Schematic drawings of the relative localisation of AChE activity, ChAT immunoreactivity and muscarinic receptors during maturation phases of sea urchin eggs. lc Lampbrush chromosomes; nu nucleus. B AChE histochemical localisation in the cortical granules of Paracentrotus lividus mature egg. C Cortical granules extrusion (arrows) during the cortical reaction. D AChE activity in the elongated microvilli of the fertilisation cone. E Muscarinic receptors included in cortical vesicles in unfertilised eggs. F Muscarinic receptor immunoreactivity at the membrane surface of fertilised egg. G ChAT immunoreactivity in immature ovarian egg. H ChAT immunoreactivity in fertilised eggs. Bars 50 |m. The electron microscopy figures were taken at instrument magnification x20,000

Fig. 6. A Schematic drawings of the relative localisation of AChE activity, ChAT immunoreactivity and muscarinic receptors during maturation phases of sea urchin eggs. lc Lampbrush chromosomes; nu nucleus. B AChE histochemical localisation in the cortical granules of Paracentrotus lividus mature egg. C Cortical granules extrusion (arrows) during the cortical reaction. D AChE activity in the elongated microvilli of the fertilisation cone. E Muscarinic receptors included in cortical vesicles in unfertilised eggs. F Muscarinic receptor immunoreactivity at the membrane surface of fertilised egg. G ChAT immunoreactivity in immature ovarian egg. H ChAT immunoreactivity in fertilised eggs. Bars 50 |m. The electron microscopy figures were taken at instrument magnification x20,000

duced by the ChAT assembled on the egg membrane is released into the peri-vitelline space and reaches the mAChRs that at this stage are also exposed at the surface (Piomboni et al. 2001). As a result, muscarinic activation evokes a [Ca2+] response, which further enhances development. The hypothesis that calcium may be involved in the regulation of the first cell cycle may be also supported by the findings of Steinhardt (1990a, 1990b), who proposed that intracellular [Ca2+] changes might be possible signals for the regulation of different mitosis stages, in both animal and plant cells. In particular, together with coworkers, he forwarded the hypothesis that calcium might be responsible for the regulation of the NEB at the moment of cleavage. The above-cited authors showed that a calcium transient immediately precedes the NEB, which may also be prevented by subtracting calcium from the cell (Baitinger et al. 1990).

According to these discoveries, we proposed a model that could help to explain the function of inter- and intracellular signals mediated by choliner-gic molecules during fertilisation and the first cell cycles - both gametes possess cholinergic molecules: ChAT,which is necessary in order to produce ACh,

Fig. 7. Schematic drawing of the hypothesis about "autocrine"behaviour of acetylcholine in fertilised eggs: ChAT synthesises ACh, which activates the muscarinic receptors exposed at the surface after fertilisation, triggering the transduction cascade causing Ca2+ release from inner stores. Musc rec Muscarinic receptors; G-prot G-protein

Fig. 7. Schematic drawing of the hypothesis about "autocrine"behaviour of acetylcholine in fertilised eggs: ChAT synthesises ACh, which activates the muscarinic receptors exposed at the surface after fertilisation, triggering the transduction cascade causing Ca2+ release from inner stores. Musc rec Muscarinic receptors; G-prot G-protein

AChE, which cleaves ACh bound to the receptors, and ACh nicotinic and mus-carinic receptors (Figs. 6,7). Moreover, their activities are displayed at different times: during the first phase, the nicotinic receptors in the sperm tail are involved in promoting swimming [a-bungaro toxin blocks their movement (see Nelson 1976; Falugi et al. 1993b); then the receptors present in the acro-some (both nAChRs and mAChRs) may be excited by the ACh synthesised by the ChAT present on the egg surface.

During the second phase, when sperm activate the eggs, ACh, autonomously synthesised by ChAT on the sperm head, activates the nicotinic receptors present in the egg membrane (characterised by Ivonnet and Chambers 1997) and the first step of depolarisation caused by Na+ influx mediated by the nicotine receptors takes place. This makes the egg membrane permissive to fusion with the sperm membrane.

Successively, the cholinergic molecules are not involved in the calcium wave, which is released by intracellular stores at fertilisation through IP3 release by membrane phospholipases; this, in turn, is caused by the NO, inserted into the egg intracellular domain by the sperm ingression (Kuo et al. 2000). However, the cortical reaction following the calcium wave causes the exposure of muscarinic receptors at the egg surface, and AChE is released into the perivitelline space together with the other cortical granule components. After this exposure, mAChRs become active and excitable, evoking further calcium spikes (as demonstrated by Harrison et al. 2002) when bound by the ACh, autonomously synthesised by ChAT exposed on the egg membrane. This event might be related to the calcium spike that was reported to take place during the nuclear envelope breakdown (Steinhardt 1990a, b; Wilding et al. 1996).

A Disquistion On The Evils Of Using Tobacco

A Disquistion On The Evils Of Using Tobacco

Among the evils which a vitiated appetite has fastened upon mankind, those that arise from the use of Tobacco hold a prominent place, and call loudly for reform. We pity the poor Chinese, who stupifies body and mind with opium, and the wretched Hindoo, who is under a similar slavery to his favorite plant, the Betel but we present the humiliating spectacle of an enlightened and christian nation, wasting annually more than twenty-five millions of dollars, and destroying the health and the lives of thousands, by a practice not at all less degrading than that of the Chinese or Hindoo.

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