B.E. Hagström

A method of determining the fertilization rate in sea urchins

Abstract A method for estimating the rate of fertilization has been described. The fertilization rate is increased by pretreating the eggs with albumin, glycine or sodium periodate. These substances facilitate fertilization in underripe sea-urchin eggs.

Interruption of the cortical reaction by heat

Abstract The fertilization impulse and associated structural changes accompanying the cortical response to sperm attachment in the sea urchin egg were interrupted within 20 seconds after sperm attachment by a 20 second exposure to warm sea water containing small amounts of the detergent sodium laurylsulfate (to prevent entry of a second sperm). Substantial numbers of partially fertilized eggs were isolated and observed for surface structure, nuclear movements, cleavage details and rate, and later development. Partially fertilized eggs showed a fertilized zone , an unfertilized zone and an intermediate zone . The unfertilized zone remained freely penetrable to refertilizing spermatozoa, showing that no irreversible block to polyspermy had preceeded the cortical reaction. The intermediate zone exhibited a gradient of hyaline layer thickness inversely related to a gradient of cortical granule number. From this and other evidence, it was concluded that the increase in hyaline layer thickness depended upon prior breakdown of the cortical granules. It was suggested that the hyaline layer might play and important role in the final establishment of the block to polyspermy, because thin hyaline layers could be penetrated by refertilizing spermatozoa. The presence of unfertilized cortex in partially fertilized eggs was found to retard or arrest some of the processes associated with early and late development. Various degrees of inhibition of nuclear movements were correlated with corresponding degrees of partial fertilization. Eggs which exhibited nuclear fusion usually cleaved, and in such cases the cleavage furrow began to form on the side nearest the point of sperm entry. Increasing amounts of unfertilized cortex were found to cause more delay in cell division. The development of most partially fertilized eggs led to a formation of “cell plates” because of the failure of hyaline layer growth and cement formation. The embryos the cells of which held together usually formed animalized larvae, for eggs fertilized on the animal hemisphere had a better chance to undergo nuclear fusion.

The mechanism of nicotine-induced polyspermy

Abstract The effect of nicotine on sea urchin eggs with and without jelly coats was examined. Sperm adhere irreversibly to the surface of nicotine-pretreated eggs, thus perhaps partly accounting for the increased probability of a successful spermegg interaction. The height of membrane elevation was considerably reduced by nicotine-pretreatment. This effect is probably of more importance as a symptom of the injurious effect of nicotine on the egg cortex than as indicating any direct influence on the block to polyspermy on the part of the membrane. Nicotine-pretreatment of sea urchin eggs often predetermines the failure of the cortical reaction on subsequent fertilization. Many eggs containing one or more sperm nuclei were found to be susceptible to polyspermy, largely by virtue of the partially fertilized character of their surfaces. Nicotine-pretreated eggs admit supernumerary sperm long after the block to polyspermy would have been complete in the controls. This was correlated with the facts that (1) the hyaline layer development was extremely poor even in completely fertilized nicotine-pretreated eggs, and (2) the hyaline layer has been suggested to play an important role in the establishment of the block to polyspermy.

Studies on the fertilization of jelly-free sea urchin eggs.

Abstract Removal of the jelly coat from the sea urchin egg at pH 5.8 improves the fertilization rate. Addition to the egg suspension of the jelly solution obtained at pH 5. 8 causes a decrease in the fertilization rate. Removal of the jelly coat at a lower pH results in an injury of the egg.

The effect of decreased and increased temperatures on fertilization

Abstract The influence of decreased and increased temperatures on fertilization in sea urchins was studied using the fertilization rate method. It was found that temperatures lower than that of the sea inhibited fertilization whereas fertilization was promoted by a slight increase in temperature. At temperatures above 32 °C the fertilization rate was retarded. The gametes were also subjected to a short treatment at decreased or increased temperatures before insemination. In both types of experiments there was a considerable increase in the fertilization rate.

Further experiments on jelly-free sea urchin eggs

Abstract The jelly coat of the sea urchin egg was found to reduce the fertilizing capacity of the spermatozoa. Measurements of the fertilization rate indicate that about 80–90 per cent of the reactive spermatozoa, after piercing the jelly coat, become uncapable of fertilizing the egg.

The influence of the jelly coat in situ and in solution on cross fertilization in sea urchins.

Abstract The jelly coat represents in Psammechinus microtuberculatus and Echinocardium cordatum a strong barrier to heterologous fertilization. The inhibiting action on fertilization exerted by jelly solutions is not species-specific. Very dilute jelly solutions (= egg water) enhance the fertilization rate of jelly-free eggs inseminated with heterologous sperm. This effect of jelly solution does not seem to be species-specific. The addition of dilute jelly solution to eggs with intact jelly coats and inseminated with homologous or heterologous sperm does not markedly influence the fertilization rate. This is probably explained by the fact that the eggs are already surrounded by “egg water” (= the jelly coat). Heparin was found to exert an action similar to that of jelly solution. Heparin and jelly solution are only effective when present at the time of insemination and they both increase the motility of the spermatozoa.

The variability in the fertilization rate

Abstract The rate of fertilization in a mixture of eggs and spermatozoa can be characterized by the time FT 50 at which 50 per cent of the eggs are fertilized and by the standard deviation of the distribution about the 50 per cent fertilization time. The logarithms of the times, at which the individual eggs are fertilized, are—at least approximately—normally distributed about the logarithm of the 50 per cent fertilization time. The calculation of the 50 per cent fertilization time and of the standard deviation is easily made from a graph in which the probits of the percentage of fertilized eggs are plotted versus the logarithms of the times; cf. dosage-mortality curves from toxicity experiments. There are four kinds of fertilization rate variability for the eggs of Paracentrotus lividus : the occurrence of several groups of eggs with different 50 per cent fertilization times in one single female, the distribution of the eggs in one group about their 50 per cent fertilization time, the difference between females collected at the same time and place and the difference between females collected at different times and places. The rate of the development of the fertilized eggs is different for different groups of eggs. The variability in fertilizability and in fertilization rate is remarkable even in selected material of apparently the best quality available. For the various groups of eggs from 12 high quality females FT 50 varied between 7 and 135 seconds. The standard deviation for the logarithm of the fertilization time for the eggs in the various groups differed between 0,1 and 0,4. The differences between the females collected at the same time and place were not noticeably less than the differences between females collected at different times and places. The variability is thus so great that it is indispensible to check the fertilization rate of eggs intended to be used for investigations in which material of a high uniformity is desirable.

Refertilization of partially fertilized sea urchin eggs.

Abstract The mechanism preventing polyspermy in sea urchins was studied in experiments with partially fertilized eggs. The rate at which these eggs became polyspermic upon reinsemination was determined and was found to be high. The development of the block to polyspermy was found to be correlated with the visible cortical changes. The results did not point to the existence of any rapid propagated block preceding the visible cortical changes.

The influence of jelly coat solution on sea urchin spermatozoa

Abstract The influence on the fertility of sea urchin spermatozoa of homologous and heterologous jelly substance was investigated. It was found that the jelly substance diminishes the fertilizing capacity of the sperm. This also diminishes the incidence of polyspermy. These effects of the jelly substance are in keeping with previous results which showed that the jelly coat in situ or in solution acts as an inhibitor of fertilization.