FERTILIZATION IN THE SEA-URCHIN AS A FUNCTION OF SPERM-TO-EGG RATIO

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1 FERTILIZATION IN THE SEA-URCHIN AS A FUNCTION OF SPERM-TO-EGG RATIO H. TIMOURIAN, C. E. HUBERT and R. N. STUART Bio-Medical Division, Lawrence Livermore Laboratory, University of California, Livermore, California 94550, U.S.A. (Received 17th April 1971, accepted 25th October 1971) Summary. Fertilization of the sea-urchin egg has been determined for low sperm concentrations in various sperm-to-egg ratios. It was found that only about 2 % of the spermatozoa were successful in fertilizing the eggs. There was no way of distinguishing between the possibility that only 2 % of the spermatozoa were capable of fertilization, or that only 2 % of the surface of the egg was receptive to the spermatozoa. The information obtained by measuring fertilization as a function of sperm-to-egg ratio was discussed as a possible method for the study of factors affecting the capacity of either the spermatozoa or egg to participate in fertilization. INTRODUCTION Rates of fertilization are measured after various treatments of the eggs and spermatozoa (Runnström, Hagstroom & Perlman, 1959) in order to study the factors that affect their ability to perform fertilization. For studies of the kinetics of fertilization, sea-urchin spermatozoa and eggs have been used because fertilization can be accomplished in vitro. However, when using seaurchin gametes, several problems have been encountered in measuring the rate of fertilization. A basic problem explored by Presley & Baker (1970) was how to determine the percentage of eggs fertilized during very short exposures to spermatozoa. This has been done by adding a spermicide seconds after the spermatozoa have been added to the eggs. The spermicide must be capable of inactivating the spermatozoa while allowing the eggs to develop until fertilization can be assessed by visual inspection. Furthermore, the spermicide must be one that neither influences the rate of fertilization nor kills the eggs, and does not allow poly- was that spermy. Another problem encountered by Presley & Baker (1970) different spermicides inactivated the spermatozoa at different stages during the process of fertilization and thus gave different rate curves in the kinetic studies. In this preliminary report, we explore the possibility of using a sperm-to-egg ratio as a model standard for later studies of various treatments and conditions affecting fertilization. The method is based on measurement of the fraction of eggs fertilized in low concentrations of spermatozoa and at various sperm-toegg ratios after the gametes have remained together for a long period of time. * Permanent address: Biology Department, Clark College, Atlanta, Georgia. 381

2 382 H. Timourian et al. MATERIALS AND METHODS The spermatozoa and eggs were obtained from the sea-urchin, Lytechinus pictus, (obtained from Pacific Bio Marine Company, Venice, California) and were kept in artificial sea-water at 12 to 15 C in aquaria as described by Tyler & Tyler (1966). The gametes were obtained from excised gonads. The sperm suspensions were kept undiluted until the time they were used. The eggs were washed once with artificial sea-water at ph 5-0 to remove the jelly coat and then several times in artificial sea-water. To accomplish fertilization, sea-water (1 ml) containing approximately 10,000 eggs was placed in a test tube, and a previously diluted sperm suspension (0-1 ml) was added. To obtain a range of sperm-to-egg ratios, initial sperm dilutions were made to an estimated ratio of 200 spermatozoa/egg, and two subsequent 1 : 10 dilutions were made from each. The spermatozoa in the diluted suspensions were counted to obtain more accurate sperm-to-egg ratios. The fertilization reaction was allowed to proceed for 5 min and then stopped by the addition of 1 ml 10% formaldehyde in sea-water. The test tubes were given random numbers and decoded only after the percentage of eggs fertilized had been determined. To determine the percentage fertilization, 100 random eggs were counted. Membrane elevation was the criterion used to determine fertilization. The motility indices of the sperm suspensions were measured using the spectrophotometric technique published previously by Timourian & Watch maker (1970). The method depends on orientating the spermatozoa by making them flow and then measuring their capacity to return to randomness when the orientating force is stopped. Due to the optical anisotropy of spermatozoa (Walton, 1952), their orientation can be followed with a spectrophotometer (Beckman DB) equipped with a flow cell. RESULTS One of the problems considered was the possibility that the fertilizing capacity of the spermatozoa would decrease during the 5-min incubation period. It has been shown that spermatozoa rapidly decrease in motility when diluted with sea-water (Timourian & Watchmaker, 1970). When fertilizing capacity and sperm motility were plotted as a function of time, the initial rapid drop in motility was not accompanied by a drop in the fertilizing capacity (Text-fig. 1 ). From our previous work (Timourian & Watchmaker, 1970), it could not be determined whether the initial rapid drop in motility reflected immobility of a fraction of the spermatozoa or a general slowing down of all spermatozoa. From the data presented here, it appears more likely that all the spermatozoa are slowed down during this period of declining activity. The important point, however, is that during the period of decreasing activity due to dilution of the spermatozoa, their fertilizing capacity remains rather stable. When the percentage of eggs fertilized is plotted as a function of the spermto-egg ratio (Text-fig. 2), the data are fitted by a sigmoidal curve that can be expressed as:

3 Fertilization as a function of sperm-to-egg ratio 383 % Fertilization 96-6 [l-e-(f-s/e)] In this expression, E is the number of eggs, S is the number of spermatozoa, and f is a numerical efficiency factor. Text-figure 2 shows computed curves f0-025 and Minutes after dilution Text-pig. 1. Fraction of eggs fertilized with sperm-to-egg ratio of 200 (a) and of 20 ( ) and sperm motility (#) as a function oftime after dilution of the spermatozoa in sea-water. I Or cj-^l I A SS I IO 20 S perm/eggs V j_ Text-fig. 2. Points are the measured fraction of eggs fertilized as a function of the spermto-egg ratio.-, calculated curve for F [l-e-(f S/B)], when f 0-025; -, whenf DISCUSSION In order to determine the significance of the data in Text-fig. 2, several possible models for the interaction of spermatozoa and eggs have been considered. The first model was based on the assumption that only some fraction of the sperm atozoa is capable of fertilization. If this is the case, then the average number of

4 384 H. Timourian et al. spermatozoa available to fertilize one egg is f S/E. If A f S/E, and the probability that one spermatozoon will attach to one egg is P where Pi e-aip (1) If W represents the probability that one of these effective spermatozoa will succeed in fertilizing an egg, the fraction F of fertilized eggs is F P,W, oo»i '?,^ (2, When we attempted to fit the data of Text-fig. 2 with an expression of the form of Equation (2), we found that the W values were all approximately equal to 1. That, is if one or more of the 'effective' spermatozoa interacted with an egg, it was always fertilized. Setting W equal to 1, Equation (2) becomes -a y A e"a e-a[ea-l] Fl-e"A (3) From Text-fig. 2, it is clear that not all of the eggs can be fertilized. In the present study, this fraction was taken as 96-6%, and Equation (3) should therefore be replaced by F0-966(l-e-f-s/E) The value offwhich best fitted the data was about 0-02 ; that is, only 2 % of the spermatozoa are 'effective'. The second model that was considered was based on the assumption that all the spermatozoa were capable of fertilization, but only some small fraction of the egg's surface was sensitive to fertilization. There is no way to distinguish between the first and second possibilities from the data. If a spermatozoon can fertilize only by entrance through a particular area on the egg surface, we have found that area to be equal to the numerical efficiency factor f, or approximately 2 % of the surface of the egg. A third possible model is that some large number of spermatozoa must attach to the egg before one spermatozoon can fertilize. Such a model predicts a very sudden rise in F at some sperm-to-egg ratio. It is clear from the data that this is not the case. We have, therefore, ruled out this model. Besides the practical problems encountered when the rate of fertilization is measured, theoretical treatment of the data is also beset with problems. For example, Rothschild & Swann (1951) measured the rate of fertilization with

5 Fertilization as a function of sperm-to-egg ratio 385 different sperm concentrations and estimated the number of egg-sperm collis ions necessary for fertilization. They found that the more spermatozoa present, the more collisions were necessary for fertilization to occur. They assumed that these unexpected results were due to sperm-sperm collisions, but the addition of excess foreign spermatozoa to the egg-sperm suspensions did not alter the fertilization rate by homologous spermatozoa (Runnström et al., 1959). The importance of measuring precise rates of fertilization remains; the practical and theoretical problems encountered, however, appear to justify attempts to describe fertilization in a simpler way, considering only the ratio of spermatozoa to eggs during a long period of time. The characteristics of the curve in Text-fig. 2 are susceptible to predictable changes. These changes reflect whether various treatments and conditions affect the capacity ofeither the spermatozoon or egg to participate in fertilization. In our system, any treatment that affected only the spermatozoa would be reflected a by shift of the whole curve to the right. On the other hand, any effect on the eggs would lower the maximum fraction of eggs fertilized at the high sperm-to-egg ratios. ACKNOWLEDGMENTS The technical assistance of Mr G. Watchmaker is greatly appreciated. This work was performed under the auspices of the U.S. Atomic Energy Commission. Reference to a company or product name does not imply approval or recom mendation of the product by the U.S. Atomic Energy Commission or the University of California to the exclusion of others that may be suitable. REFERENCES Presley, R. & Baker, P. F. (1970) Kinetics of fertilization in the sea urchin: a comparison of methods. J. exp. Biol. 52, 445. Rothschild, Lord & Swann, M. M. (1951) The fertilization reaction in the sea-urchin. The probability of successful sperm-egg collision. J. exp. Biol. 28, 403. Runnström, J., Hagstroom, E. E. & Perlman, P. (1959) Fertilization. In: The Cell. Eds. J. Brächet and A. E. Mirsky. Academic Press, New York and London. Timourian, H. & Watchmaker, G. (1970) Determination of spermatozoan motility. Devi Biol. 21, 62. Tyler, A. & Tyler, B. S. (1966) The gametes: some properties andproceduresan: Physiology ofechinodermata, pp Ed. R. A. Boolootian. Interscience Publishers, New York. Walton, A. (1952) Flow orientation as possible explanation of wave-motion and rheotaxis of sperm atozoa. J. exp. Biol. 24, 520.