Many molecules and biochemical cascades that mediate sperm–egg interactions and egg activation have been discovered, but much still remains to be understood. Studies in the past were often focused on the identification of specific molecules such as egg and sperm receptors and egg activation and transduction pathways, but relatively few studies dealt with more general factors such as charge and pH that may affect these processes.

Histochemical studies on cells and tissues usually involve the use of dyes and fixed non-living material, but we have developed other histochemical approaches that identify cellular properties using derivatized agarose beads and various compounds to probe the biochemical components of fertilization and egg activation processes in living systems. The present study is the third presented in this journal that examines fertilization and egg activation in a model living system using free sugars and amino acids as probes instead of derivatized beads or dyes.

In previous studies, we used derivatized agarose beads as a model non-living system to show that charge–charge bonding may be involved in controlling cellular adhesive interactions. We followed up these studies examining sperm–egg interactions in the sea urchin model to show that negatively charged phosphorylated amino acids and sugars block sea urchin fertilization, whereas uncharged molecules had no inhibitory effect. These results were intriguing, suggesting that charge–charge interactions may be involved in sperm–egg binding.

Here we expand these studies by examining the effects of positively charged, negatively charged and uncharged molecules on fertilization and egg activation. In the early experiments on negatively charged molecules, no attempt was made to determine whether charged molecules affect sperm, eggs or both, whether their effect was due to a change in sea water pH, or whether the effects are due to chelation of positive ions such as Ca²⁺ or Zn²⁺.

A resolution of these questions should help to determine how charged molecules are involved. Answers to these questions may also lead to a better understanding of sperm–egg interactions and egg activation. In addition, this information may lead to a new approach in the development of contraceptives. In the present studies, we explore these questions by:

(1)examining the effects of positively charged, negatively charged and uncharged molecules on fertilization and egg activation;

(2)performing rinse-out experiments that remove charged molecules from eggs before the addition of sperm or addition of an artificial egg activator;

(3)determine whether the pH of sea water is affected by the added molecules;

(4)addition of Ca²⁺ or Zn²⁺ to examine the chelation issue, that is also examined in (2) above.

The results show that sperm and eggs are exquisitely sensitive to charged molecules. In at least one case, it is shown that a charged molecule with the same structural formula as another, but that is a ketone instead of an aldehyde, may reveal new information on species-specific components involved in the fertilization process.

Lytechinus pictus sea urchins were purchased from Marinus Inc. (Long Beach, CA, USA). Gametes were obtained and treated as previously described. The fertilization was performed in some experiments after the addition of specific sugars or amino acids as described below as. All reagents were obtained from Sigma (St. Louis, MO, USA). Experiments were run in triplicate by at least two investigators and the results were recorded as percentages of eggs displaying fertilization membranes 10 min after the addition of sperm or ionophores with standard error of the mean or standard deviation (pH readings were recorded as means±standard deviation). In some cases, fertilization and/or activation was monitored by establishing whether eggs were cleaved at 1.5–2 h after fertilization.

For pH-adjustment experiments, the pH of each solution consisting of artificial sea water (ASW) was adjusted with Trizma base (Sigma) or HCl to be as close to pH 8.0 as possible. The pH was checked at the start and end of all experiments. Suspensions of eggs in 100 ?l of pH-unadjusted solutions, pH-adjusted solutions or ASW controls were placed in three separate droplets on a microscope slide. Approx 1 ?l of undiluted sperm was added to each drop with the blunt end of a flat toothpick.

Calcium ionophore A23187 and other reagents were obtained from Sigma. The calcium ionophore (5 mg) was dissolved in 9.55 ml dimethylsulfoxide (DMSO). This stock solution of 10 mM was stored in 100 ?l aliquots in small foil-wrapped tubes and these aliquots were further diluted with 50 ml DMSO and stored in 2 ml aliquots (2 ?M ionophore concentration) in foil-wrapped tubes at ?20°C. Final stock solutions (40 ?l) were used in experiments and this concentration of DMSO alone had no effect on egg activation. For the non-rinse-out experiments, 465 ?l of egg suspension and 500 ?l of either ASW or test compound in ASW and 40 ?l of ionophore were mixed. For the rinse-out experiments, 495 ?l of egg suspension and 500 ?l of either ASW or test compound in ASW were mixed. Eggs were allowed to settle and rinsed 3 times with ASW, pH 8, followed by addition of 40 ?l ionophore.

For the rinse-out fertilization experiments, eggs were washed 3 times in ASW, pH 8.0, and then incubated in 1 ml aliquots of solutions to be tested for 10 min. Eggs were allowed to settle and the supernatant was removed and replaced with ASW, pH 8.0. Eggs were allowed to settle and the supernatant was removed and replaced with ASW once again. Diluted sperm (5 ?l of 10-times diluted sperm in ASW) was added to each sample. Sperm motility was observed and fertilization was evaluated by the presence of fertilization membranes and egg cleavage at 1.5–2.0 h afterwards. The non-rinse-out samples were handled as above except that the original solutions were never rinsed out.

Fig. 1 shows that the positively charged amino acid -arginine (50 mM) inhibited fertilization whether or not it was rinsed out from eggs prior to the addition of sperm. Similar results were obtained with negatively charged o-phospho- -serine, o-phospho- -threonine, -glucosamine-6-phosphate, and -ribose-1-phosphate, whereas other negatively charged molecules ( -mannose-1-phosphate, -glucose-6-phosphate, -glucose-1-phosphate, -glucose-6-sulfate and -maltose-1-phosphate) were only inhibitory when not rinsed out before the addition of sperm. Fructose-1-phosphate, however, was never inhibitory even in the non-rinsed-out samples. Uncharged molecules ( -threonine, -serine, -valine, -mannose, -glucose, -maltose, -fructose, and -ribose) had no effect. -glucosamine and -glucosamine-6-sulfate inhibition was partially restored when rinsed out.

Most of the molecules that were inhibitory at 50 mM were not inhibitory at 5 mM, except for o-phospho- -threonine and o-phospho- -serine that were still inhibitory at 5 mM whether or not these negatively charged molecules were rinsed out prior to the addition of sperm. -arginine and -ribose-1-phosphate inhibition at 5 mM was partially restored when rinsed out prior to the addition of sperm.

Table 1 shows that sperm cells were motile in sugar-phosphate samples whether or not the solutions were rinsed out prior to the addition of sperm, except for -glucosamine-6-phosphate. In the presence of 50 mM o-phospho- -serine and o-phospho- -threonine, sperm cells were not motile in the rinsed-out or non-rinsed-out samples, whereas in the presence of 5 mM, sperm cells were motile in the rinsed-out samples but not in the non-rinsed-out samples. -arginine affected sperm motility only occasionally.