PROLONGATION OF LIFE-SPAN OF SEA URCHIN SPERMATOZOA, AND IMPROVEMENT OF THE FERTILIZATION-REACTION, BY TREATMENT OF SPERMATOZOA AND EGGS WITH METAL-CHELATING AGENTS (AMINO ACIDS, VERSENE, DEDTC, OXINE, CUPRON)

Abstract

The duration of motility and of fertilizing capacity of sea urchin sperm diluted in sea water can be considerably extended by the addition of any one of certain metal-chelating agents. These include ethylenediaminetetraacetate (Versene), diethyldithiocarbamate, 8-hydroxyquinoline, alpha-benzoinoxime and various amino acids and peptides (glycine, alanine, valine, leucine, lysine, glutamic acid, histidine, phenylalanine, tryptophane, cysteine, glycylglycine, glutathione). The relative increase in life-span, in the presence of these agents, is greater the more dilute the sperm suspension, while the absolute increase is generally greater for the more concentrated suspensions. Over 100-fold extension of life-span of dilute suspensions has been obtained by use of these agents. An artificial sea water of low heavy metal content also enables the sperm to survive longer than in ordinary sea water. This effect is obtained with artificial solns. in which the Ca concn. ranges from 8 times to 1/100 that of sea water. The results support the previously suggested view that the increased survival of the sperm in presence of amino acids, proteins, etc. is due to the ability of these agents to bind heavy metals present in the diltuion medium. It is suggested that Cu++ and Zn++ are among the metals involved. The "Dilution Effect" (decreasing life-span with increasing dilution of suspension) is largely explained on the basis of similar action of the seminal fluid proteins which are present in higher concentration in the denser suspensions prepared by direct dilution of the semen. Treatment of the sperm with glycine or with Versene improves the fertilization-reaction (membrane-elevation) induced upon insemination of eggs in sea water with a given amount and age of sperm suspension. This effect persists along with the increased survival of the sperm in these agents. The results demonstrate that the type of response given by the egg can be determined by the condition of the inseminating sperm. The spermatozoon evidently does not simply act in "all-or-none" manner, in the sense of operating a trigger mechanism in the egg. Fertilization is also improved when the eggs are inseminated in the presence of Versene of glycine, but the effect is largely eliminated if the eggs are washed and inseminated in sea water. Swelling of the jelly-coat that occurs in these solutions is not an important factor in the results. Eggs that have been deprived of their jelly-coat behave similarly, showing improved fertilization in the solutions and no persistent pretreatment effect. Such eggs also show the improved response to treated sperm. It is concluded that if there is any effect of these agents on the egg in the direction of improving fertilizability such effect is readily reversed upon transfer to sea water and does not constitute a "maturating" effect. Eggs of L. pictus and S. purpuratus develop normally to the pluteus stage in 10-3 M Versene but not 10-3 [image] glycine.