Memoirs of the College of Education Akita University (Natural Science) 41, 63-67, (1990)
The Process of Fertilization in the Eggs of the Sea Urchin,Hemicentrotuspulcherrimus
Masahiko YAMAMOTO
(Received August 31. 1989)
Summary
The process of fertilization was studied in the eggs of the sea urchin, Hemicentrotus pulche- rnmus.
As reported previously, the unfertilized eggs were first immersed in hypotonic sea water, then centrifugalized by an ordinary driven electric centrifuger. By the centrifugation, the egg was remarkably stratified into definite three layers, oil droplets in the centripetal side, clear hyaloplasmic and dark layers in the centrifugal side, respectively.
In the case of the sperm attachment at the site of the hyaloplasmic layers, the behavior of the spermatozoon in the process of invasion, and the course of penetration through the egg cortex were precisely reported below.
Introduction
Significance of fertilization involves a new combination of genetic materials, through a synkaryon of the male and the female pronuclei between different gamates. As the results of the fertilization, the egg acquires the potency to survive an unlimitted series of generations.
Thus, the fertilization is essential to all of the living organisms. Fertilization consists of three different processes involving first, the attachiment of spermatozoon onto the egg surface. This can be accomplished by an acrosomal reaction, induced by the contact of the spermatozoon to the jelly coat. Second, sperm invasion into the egg interior, the process of the sperm invasion and the penetration. In this report, the 'penetration' deals with the spermatozoon, passing througn the superficial cytoplasm of the fertilized egg (egg cortex) . Third, the process of the synkaryon of the male and the female pronuclei within the inner part of the fertilized egg. In the living sea urchin egg, the process of the sperm invasion is hardly observable, because of the minimum size of the spermatozoon with an opacity of the superficial region of the egg itself as a background of the view.
In the previous paper, the writer obtained the clearly stratified layer, consisting of egg cytoplasm, when the eggs of the sea urchin, Hemicentrotus pulcherrimus were first treated with the hypotonic sea water, then centrifugalized for a short time (Yamamoto, 1964, 1988 and 1989). The cortical granules were immovable during the centrifugation, and they were clearly recognizable arround the periphery of the egg, embedding in the original position in the cortex of the unfertilized egg. By using the above treated eggs, the course of the sperm
invasion was observed in the living condition, and then photographed into high magnification.
In this manner, the behavior of the spermatozoon at the time of fertilization, was ascertained in detail.
Material and Method
The present experiments were carried out by using the eggs of the sea urchin, Hemicentrotus pulcherrimus, collected in the vicinity of Asamushi Marine Biological Station, Tohoku University.
Unfertilized eggs and spermatozoa were supplied during the spawning season at March to April, by cutting out the ovary and the test into the ordinary sea water. Eggs taken from the ovary were washed repeatedly with the sea water, then inseminated. In the present experi- ment, the unfertilized eggs were pretreated with 58 per cent hypotonic sea water for five minutes, then centrifugalized by an electrically driven centrifuger for five to ten minutes, at the speed of 3,000 revolutions per minute. The centrifugal force calculated, was about 1,300 times gravity. The eggs just after centrifugation were put back to the ordinary sea water, through the exposure to 80 per cent hypotonic sea water for five minutes.
Antecedently to microscopic observation, small amount of white vaseline was mounted on the corners of a cover slip, then the treated unfertilized eggs were put on a slide. For the microscopical observation, the eggs were slightly flattend by pressing the cover slip toward the slide glass, then a drop of sperm suspension was laid down at one edge of the cover slip.
By using high magnification, the moment of sperm invasion pushing the acrosomal region of the sperm head into the egg surface, could clearly be observed.
Result and Discussion
Just at the completion of the centrifugation, the unfertilized sea urchin eggs were remark- ably stratified into three layers, as compared to the eggs without pretreatment. Since the ordinary unfertilized eggs without pretreatment, can never stratified. The reasons for the stratification seems to be attributable to a lowering of the viscosity of the cytoplasm, caused by the pretreatment of the hypotonic sea water. The stratification was distinct, and the female pronucleus was visible in the clearly stratified hyaloplasmic layer, as a transparent small sphere. The cortical granules were seen around the periphery of the egg cortex.
The most conspicuous phenomenon in the fertilization, is the breakdown of the cortical gra- nules and the elevation of the vitelline membrane, for the formation of the fertilization mem- brane. These granules were first described by Motomura (1936) as the sort of granules, scattered in the egg cortex of the unfertilized Hemicentrotus egg. He named them as the
"Janus Green Granule", because they were stained intensely with a solution of Janus Green B in sea water in the living state. Up to the present, the role of the cortical granules for the formation of the fertilization membrane, has been elucidated by many investigators by using electron microscopy (Takashima 1960 and others). Unfortunately, these granules are hardly seen in the living condition, because of the homogeneity of the refractive index, to the un- transparent particles in the background view residing in the egg interior.
In the writer's present experiment, the collapse of the cortical granules was easily recog-
Photomicrograph showing the process of sperm entry. The process of sperm invasion and the penetration into the eggs of the sea urchin,Hemicentrotus pulcherrimus.The unfertilized eggs were pretreated with hypotonic sea water, and then centrifugalized. The eggs thus treated was insemi- nated, and the process of fertilzation was photographed.1.Spermatozoon just after sprem invasion.
2. Breakdown of cortical granules. The fertilization membrane is elevating. 3. Spermatozoon, passing through the egg cortex. 4. Formation of thin transparent layer just beneath the egg surface. Broad fertilization cone is forming just over the invading spermatozoon.
C; clear cytoplasmic layer. CG; cortical granules. FM; fertilization membrane. N; Female pronucleus. aD;oil droplets. T; transparent hyaloplasm. Y; yolk granules.
nized at the point of sperm invasion, spreading out all the directions abruptly. The time required for the breakdown of these granules took about ten to twenty seconds. At the end of the collapsion, a feeble transparent layer was found in a original position, where the corti- cal granules were previously seen. The layer thus formed, vanished soon in accordance with the several processes, occuring in the superficial regions caused by sperm invasion. Simultane- ously, a tiny cone-like protrusion appeared at the point of sperm invasion, and transformed finally into a transparent columnar form as shown in the figure. This is the fertilization cone, designated by Runnstrom. According to Hagstrom (1950), the fertilization cone last more than 30 minutes, the period of its endurance coincides with the writer's observation. In the meantime, the head of the spermatozoon (containing the nucleus and the middle piece) was swallowed through the egg surface, remaining its tail out of the fertilization cone. Later, the tail was engulfed into the egg and disappered from the outward observation. Consequently, it becomes clear that in the case of the sea urchin, the entire spermatozoon was incorporated into the egg 1.5 to 3 minutes after the fertilization. This has been ascertained by J. C. Dan (1950), as a sperm induced protrusions in sea urchin oocyte. In the writer's cases, the fertili- zation cone was conspicuous as compared to the control eggs. J. C. Dan also described the formation of abnormal fertilization cone production, when the unfertilized eggs were subjected to heat, cold and several drugs such as nicotine and others. She showed the endurance of the cones, until the period of the first cleavage. Therefore, the writer's present case will be possi- bly based upon the pretreatment with the hypotonic sea water.
According to the writer's present experiment, the spermatozoon for passing through the egg cortex, took comparably long time about two and three minutes. This will be due to the rigi- dity of the cortex, residing just beneath the egg surface layer. During the biginning of the sperm penetration, the cortex at the point of sperm invasion, caved slightly down forming the concavity of the egg cortex, as shown in the figure. In the previous paper (Yamamoto 1989), the writer reported the thickness of the cortex about less than 4 f.l.m or so by using centrifu- gation experiment, after the treatment of the unfertilized eggs with hypotonic sea water.
Hiramoto (1957), by using the micrurugical technique, mesured the thickness of the cortical cytoplasm (egg cortex), that is about 3 f.l.m thickness, get thicker after the fertilization, reaching 4 f.l.m shortly before the cleavage. The writer's mesurement was nearly the same as Hiramoto's description. Because of the rigidity of the egg cortex, this has been supposed that the cortex has a nature of the gel. Consequently, it can be understood, that the spermatozoon take such a long time for the penetration, passing through the gelated superficial cytoplasmic layer. The spermatozoon which accomplished the penetration, was able to migrate in the fluid hyaloplasmic layer without any restriction. From this period of time, the spermatozoon grad- ually became impossible to distingwish owing to the various endoplasmic granules scattering as the background. The sperm head turns to 1800 ,and its end faces to the egg interior (J. C.
Dan 1950). Unfortunately, the writer could not recognize the behavior of the spermatozoon, dur- ing the course to meet the female pronucleus. Although the writer recognized sperm pronucleus elswhere in the egg interior as a small transparent sphere, he could not find the way, known as the copulation path. The mechanism of synkaryon was discussed in the writer's previous report, which was based upon the analysis of the astral behaviors, in the fertilized eggs of the
sea urchin treated with phenylurethan (Yamamoto 1967). Recently, the analysis of the astral changes from the initiation of fertilization to cleavage, was made by using indirect immuno- fluorescence for tubulin-containing structures, and copulation precess of the both nuclei was elucidated (Obson and Weber, 1980).Generally speaking, these results coincide with the writer's conclusion. In the amphibian egg, the invasion path of the spermatozoon is straight in most cases, but it does not lead the spermatozoon directly toward the final position of the female pronucleus. Therefore, the course of the spermatozoon is diverted in the midway. The new curved line was designated as the copulation path in the text book (Rugh,1951).In the present experiment, the writer could not distingwish these courses. It will be based upon the lack of pigments in the eggs of the sea urchin as compared to the amphibian egg.
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