It is now accepted that sperm trigger deuterostome egg activation by causing an increase in egg cytoplasmic Ca2+ levels (Jaffe, 1983; Whitaker & Swann, 1993). This increase generally takes the form of a single wave of Ca2+ release that crosses the egg from the point of sperm-egg interaction (Jaffe, 1983)eggs Ca2+ waves occur repetitively and soon turn into synchronous pulses, or homogeneous oscillations, that last for hours after sperm-egg fusion (Miyazaki et al., 1993b; Homa et al., 1993). Despite their extensive characterisation it is still not established how sperm trigger these Ca2+ changes in eggs. The signal transduction mechanism is missing. There is a proliferation of overlapping and complex schemes for how the sperm may initiate Ca2+ release (Miyazaki et al., 1993b;Whitaker & Swann, 1993). Here, my aim is to present one simple scheme in its generic form. The brevity of this essay restricts citations and necessitates using reviews to reference original work.

Egg activation at fertilisation is composed of a complex, choreographed series of events, the initiation of which still is not understood. Two major hypotheses have emerged as explanations for the mechanism of egg activation (reviewed by Nuccitelli, 1991; Whitaker & Swann, 1993). The first holds that the sperm delivers an ‘activating factor’ (e.g. inositol trisphosphate, calcium, or a protein) that diffuses into the egg cytoplasm after gamete fusion. The second hypothesis holds that sperm bind to receptors spanning the egg plasma membrane which then transduce a signal to second messenger enzymes. We present here the evidence for receptor-mediated signal transduction in egg activation at fertilisation. By ‘receptor’ we mean only molecules that bind an extra-membrane ligand and which transduce a signal to molecules residing on the cytoplasmic side of the egg membrane. It is critical to be aware that several alternative activating mechanisms are not mutually exclusive and that species differences may exist. In fact, as more has been learnt about the molecules involved in gamete recognition and binding and of the signalling pathways in the egg, it seems likely that multiple pathways exist to trigger complete egg activation.

Approximately 50% of rat 8-cell embryos obtained from the oviduct on day 4 of pregnancy developed to the balstocyst stage after 18 h of incubation in vitro. The embroys were compared with in vivo blastocysts derived from the uterus on day 5 of pregnancy as regards their response to vitrification treatment. Before vitrification, both types of embryos were exposed to vitrification solution, and subsequent embryonic development was inhibited with increasing time of exposure. A greater suppression of development after exposure was observed in the embryos cultured in vitro compared with in vivo blastocysts. However, development was gradually restored as in vitro incubation was continued. The response of embryos to vitrification and warming treatments was shown to be almost the same for both in vitro and in vivo blastocysts, though the former were significantly (p < 0.05) less tolerant of exposure to vitrification solution. These results suggest that in vitro blastocysts were much more susceptible to vitrification solution than in vivo blastocysts. The degree of damage caused by vitrification and thawing treatments, and the inhibition of in vitro embryonic development, were significantly (p < 0.05) more serious for in vitro blastocysts.

The fluroscence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage (GV) ovarian oocytes exhibited a bright, amorphous, mesh-like staining pattern (immature type). In contrast, mature polar body stage (PB) oocytes, either ovarian or oviductal, displayed faint, spotty fluorescence labelling of the ZP (mature type). The perivitelline space (PVS) of mature ovarian oocytes (12 h post-hCG) failed to label, whereas approximately 50% of oviductal oocytes showed PVS labelling. The incidence of PVS staining increased with postovulatory age, possibly as a result of the accumulation of materials secreted by the oviduct. Following in vivo or in vitro fertilisation of oocytes, a characteristic pattern of plasma membrane (PM) labelling was observed. Similar patterns of PM labelling were seen in oocytes parthenogenetically activated with ethanol or ionophore (A23187) but not in control oocytes. The pattern of PM labelling observed with FITC-protein conjugates was strikingly similar to that observed with FITC-labelled lectins, which are thought to interact with glycoconjugates released from cortical granules. Immature type of ZP staining also occurred when GV oocytes were treated with FITC alone or with a variety of FITC-protein conjugates. Thus, protein may not be required for labelling of the ZP by FITC-protein conjugates as previously thought. FITCconjugated proteins including casein, bovine serum albumin, peroxidase and non-immune immunoglobulin G (IgG), all labelled the PM of activated oocytes; however, FITC-IgG failed to label the PVS. Results demonstrate for the first time that various components of viable mouse oocytes exhibit and undergo characteristic structural and functional changes during periovulatory differentiation as evidenced by their interaction with one or more FITC-protein conjugates and/or FITC. On the basis of these results the intrafollicular and oviductal mechanisms mediating these changes are discussed as is the possibility that the fluorescent molecule attached to conjugates may play a role in oocyte labelling.

Intraperitoneal injections of extracts of Spartium junceum in adult male rats were shown to reduce the rate of fertility and acrosin enzyme activity. For the latter the drug effects could be divided into two phases: in the first stage a significant increase in proteolytic activity was observed in all experimental animals examined; and in the second stage, there was a general decrease of up to 50% in enzyme hydrolytic properties. There is a notable difficulty in correlating the acrosin activity and the data obtained from mating tests. The effect of drugs on the activity of another acrosomal protease, benzamidine-resistant protease, was assayed. A significant decrease in this enyme was noted though the modality was different from that of acrosin. Extrapolating the data of the two co-extracted acrosomal proteases, the ratio of acrosin-like protease [E] and benzamidine-resistant protease [E]x, it was possible to correlate the value expressed to the rate of pregnancy. The implications of this finding is discussed although the exact molecular basis of the phenomenon is uncertain.

High- and low-molecular weight spawning-female-specific (H-SF and L-SF) substances are liver-derived putative precursors of the inner layer of egg envelope of the medaka Oryzias latipes, and are also found in the ascites which are accumulated in the male fish on prolonged treatment with oestrogen. In the present study, the H-SF substances purified from the ascites of the oestrogen-treated male medaka were found to consist of three proteins. Their molecular weights ranged from 74 000 to 76 000, which corresponds to those of ZI-1 and ZI-2, the major components of medaka egg envelope. The amino acid composition of the H-SF substances was characterised by high contents of proline (17.58 mol%) and glutamic acid/glutamine (13.34 mol%). The characteristics were almost identical to those of ZI-1 and ZI-2 but differed from those of L-SF substance, a precursor of ZI-3, another major component of the egg envelope. These results confirm our view that H-SF substances are the precursors of ZI-1 and ZI-2. The average amino acid compositions of H-SF substances and L-SF subsatance coincided with the amino acid composition of whole egg envelope. This result strongly suggests that an equal amount of H-SF substances and L-SF substance would construct the inner layer of egg envelope after a little modification.

Sand dollar eggs were microinjected with botulinum C3 exoenzyme, an ADP-ribosyltransferase from Clostridium botulinum that specifically ADP-ribosylates and inactivates rho proteins. C3 exoenzyme microinjected during nuclear division interfered with subsequent cleavage furrow formation. No actin filaments were detected in the equatorial cortical layer of these eggs by rhodamine-phalloidin staining. When microinjected into furrowing eggs, C3 exoenzyme rapidly disrupted the contractile ring actin filaments and caused regression of the clevage furrows. C3 exoenzyme had no apparent effect on nuclear division, however, and multinucleated embryos developed from the microinjected eggs. By contrast, C3 exoenzyme did not affect the organisation of cortical actin filaments immediately after fertilisation. Only one protein (molecular weight 22000) was ADP-ribosylated by C3 exoenzyme in the isolated cleavage furrow. This protein co-migrated with ADP-ribosylated rhoA derived from human paltelets when analysed by two-dimensional gel electrophoresis. These results strongly suggest that a rho-like, small GTP-binding protein is selectively in the organisation and maintenance of the contractile ring.

We have examined immunocytochemically the subcellular distribution of the cell adhesion molecule uvomorulin in cleavage stage mouse embryos using conventional and confocal microscopy, under a range of detergent extraction and fixation regimes. Only traces of uvomorulin were detectable on the surface of unfertilised oocytes, whereas between 6 and 11 h after activation detergent-resistant surface expression was evident. This shift correlates with previously demonstrated changes in the pattern of synthesis and accumulation of uvomorulin from precursor state in unfertilised oocytes to mature protein after fertilisation. Embryos at subsequent stages up to the 8-cell stage exhibited a uniform distribution of uvomorulin on free surfaces and its concentration in regions of contact between blastomeres. At the 8-cell stage, during compaction, there was increased intercellular adhesion with concomitant accumulation of uvomorulin at intercellular contacts, whilst free surface uvomorulin was reduced and became relatively more susceptible to detergent extraction. When compact 8-cell embryos were decompacted in calcium-free medium, uvomorulin at contacts decreased while free surface and cytoplasmic staining increased. Blastomeres disaggregated from 4- and 8-cell embryos showed traces or ‘footprints’ of anti-uvomorulin staining in regions previously in apposition. These footprints disappeared over 45–60 min, during which time uvomorulin distribution became uniform. Possible mechanisms underlying the rearrangements which take place both at fertilisation and during compaction and experimental decompaction are discussed.

The zona pellucida of mammalian oocytes stored in highly concentrated solutions of neutral salts is known to retain its biological and biochemical properties. However, the zona may become resistant to sperm penetration as the storage period is increased. In cattle and hamsters, the penetrability of zonae of salt-stored oocytes was restored or increased by treating the oocytes with moderate heat without altering the gross morphology of the zona. Although this technique did not work for salt-stored human ova, this may have been due to the use of so-called inseminated- unfertilised ova which (1) may have been fertilised but failed to activate, or (2) were not fertilised because of functionally defective zonae.