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.

The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction.PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes.

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.