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Notes: We have considered how a transient rise in calcium can occur and how it can result in the activation of the egg. Our evidence with the “artificial sperm”—the ionophore-coated glass rod—suggests that even if sperm release calcium locally, this may be insufficient to completely propagate around the egg. Something else is needed and this something else can be mimicked by placing the eggs in higher than normal concentrations of potassium ion. A possibility is that the potassium effect results from inducing a membrane depolarization similar to the one that normally follows sperm-egg binding. If correct, then the membrane depolarization might not only function in the block to polyspermy10 but could also be involved in “relaxing” or overcoming the normal calcium-sequestering ability of the cell. A possible mechanism is a voltage-dependent Ca++ -induced Ca++ release.The other question we have addressed is how this transient rise in calcium might act to trigger development of the egg. An obvious candidate is calmodulin and as noted this is indeed present in eggs in fairly high concentrations. Irrespective, one must then ask, “how does calmodulin act?” We propose that one site of calcium action, perhaps through calmodulin, is to initiate a brief bout of Na+– H+ exchange with a resultant rise in pHi. Our data indicates that the sodium influx per se is not critical but that the resultant hydrogen efflux and the pHi change is essential.This further suggests that during the normal formation of gametes the synthetic activity of the oocyte is turned off by a lowering of pHi at the end of oogenesis and that at fertilization the pHi is returned to a normal level through the brief episode of Na+– H+ exchange. Correlative evidence exists that the rise in pHi is related to the well known increase in protein synthesis and that there may be a pH-dependent unmasking of mRNA. The pHi rise does not seem to be related to a 2.5-fold increase in ribosomal transit time.The last question I wish to consider is whether these two ionic triggers, a transient rise in intracellular calcium and a permanent increase in intracellular pH, are used as triggers only once in the lifetime of an organism—i.e., only at fertilization—or whether these are general regulatory mechanisms used by many cells throughout the life history of the individual. The modulation of activity in adult cells by regulating Ca+2 levels appears general; the role of calcium in controlling contraction of muscle and the permeability of nerves is well known and the presence of calmodulin in many cells further suggests that calcium is used as a general regulatory cation in these cells. Also, as detailed at this symposium, levels of calcium ion may be critical for regulating mitosis.The question of whether pHi can be used as a regulatory element for cell activity is less clear. One problem, similar to that encountered in measuring calcium levels in cells, is the difficulty of in situ measurements. The two methods that are probably best suited are direct measurements with microelectrodes47 and measurements based on phoning of weak acids, such as the DMO method.48 Both of these procedures are almost in their infancy and still used in relatively few laboratories.In sea urchin eggs, the rise in pHi is signaled by the concomitant efflux of hydrogen ions from the cell. Can this efflux be used as a simple indicator of whether there are similar intracellular pH changes accompanying other cell activities? Looking at fertilization, the preliminary indications would be that raises in pHi are spotty. Acid release is seen in eggs of Urechis49 (an echiuroid related to the annelids) and in Spisula (a mollusk).50 However, acid release is not seen in other mollusks such as Mytilus and Acmaea49 nor is it seen in the starfish and tunicate eggs.51 Yet, acid release is associated with activation of both starfish and tunicate sperm.15,52These scanty results would suggest that although the potential for utilizing pH as a regulatory element is widespread it is not necessarily used as a regulator at fertilization. A probable scenario is that the rise in calcium is the universal response to fertilization but that the mechanisms by which calcium acts to activate metabolism vary. In the sea urchin egg, it appears to be through pHi; in other eggs alternate regulatory factors, such as calcium-dependent protein phosphorylation, may be more important. It remains to be seen whether pHi is also utilized for regulation after fertilization, as during later development and in the cells of the adult organism.

Notes: Sperm motility in Limulus is initiated by a sperm motility initiating factor (SMI) that emanates from Limulus eggs. This report describes the partial purification of SMI (greater than 230-fold purification with respect to protein content) with 40% recovery. SMI appears to be a hydrophobic peptide of 500-2,000 MW. Although probably not purified to homogeneity, SMI is estimated to be active at a concentration of less than 0.2 μM.

Notes: Upon dilution into sea water, Limulus spermatozoa undergo a brief flurry of motility (duration 〈 60 sec), after which they are nonmotile until encountering a sperm motility initiating peptide (SMI) that emanates from eggs. Utilizing highly purified SMI extracts and simplified seawater formulations (from which individual ions have been deleted), we found that no specific extracellular ion is required for either dilution-initiated or SMI-initiated motility. Indeed, deletion of one ion (Na+) produced dilution-initiated motility of very long duration (several hours). When motility is initiated by SMI (in normal seawater) there is an increase in intracellular pH (pHi), as indicated by the fluorescent probe, 9-amino acridine; however, this pH, change is not a trigger for motility. As a more general method examining ion movements, the fluorescent probe diS-C3-(5) was used to qualitatively measure changes in the membrane potential of spermatozoa. Although crude SMI extracts caused membrane depolarization, further purification resulted in an almost complete separation of this activity from SMI, thus showing that SMI activation is apparently an electroneutral event. (The membrane-depolarizing factor has a molecular weight 〉 30,000 and does not initiate acrosome reactions.) Experiments utilizing the ionophore A23187 and Ca+2-blocking agents (verapamil and TMB-8) provided tentative evidence that mobilization of intracellular Ca+2 may be required for motility initiation. These results show that neither changes in pHi nor the influx of specific extracellular ions are direct mediators of SMI-initiated motility; however, experiments with pharmacologic agents indicate a possible role for intracellular Ca+2.