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The first cleavage furrow in sea urchin eggs does not pass through the sperm entrance point
Y. Endo much less. Paralysis may be caused by a deficiency in ATPase, but interpretation of the result is difficult because it is not known whether all the ATPase activity of normal flagella is concerned with movement. REFERENCES BROKAW, C. J., Submitted for publication (1960). GIBBS, S. P., LEWIN, R. A. and PHILPOTT, D. E., Ezptl. 3. LEWIN, R. A., .J. Gen. Microbial. 6, 233 (1952). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and 1. 2.
5.
MINTZ,
265 (1951). R. H. and
THE
R. A.,
LEWIN,
Canad.
J. Mierobiol.
Cell Research RANDALL,
R.
15, 619 (1959). J.,
.I. Biol.
Chem.
193,
1, 65 (1954).
FIRST CLEAVAGE FURROW IN SEA URCHIN NOT PASS THROUGH THE SPERM ENTRANCE
EGGS DOES POINT
Y. END0 Biological
Laboratory,
Keio Received
A~-rmwH
University, January
Yokohama-Hiyoshi.
Japan
20, 1960
the question whether the first cleavage furrow of sea urchin eggs passes through the entrance point of the spermatozoon or not is certainly not new, it yet has not been definitely settled (for references, see [2]). According to one opinion there is a coincidence between the entrance point of thk spermatozoon and the first cleavage furrow; this accordance is, however, also vigorously denied by others. This disagreement between research workers may be due to the fact that the method for determining the entrance point of the spermatozoon under normal conditions is not sufficiently conclusive. It is well known that the “entrance” or “fertilization cone” is formed at the attachment point of the spermatozoon soon after the fertilization membrane begins to be elevated. In most species of sea urchins, this entrance cone, with the lapse of time, gradually changes in form and finally becomes indistinguishable. In Pseudocentrotus depressus, however, the entrance cone remains for a long time and is possible to detect at the time of the first cleavage. Since the entrance cone is the most exact indication of the entrance point of the spermatozoon, Pseudocentrotus egg was used to test the relation between the cone and the first cleavage furrow. Immediately after membrane elevation in some of these eggs, the entrance cone can be seen in the optical section of the egg surface. Location of the cone is facilitated by the fact that cortical granule substance in the form of rods [l, 31 is distributed in the previtelline space following a gradient which is highest at the sperm entrance point (unpublished). At the time of cleavage, the entrance cone appears as a hyaline protuberance on the surface of the hyaline layer (Fig. 1). Experimental
Cell Research
19
Sperm entrance and cleavage furrow
433
In practice, the positions of entrance cones on the greatest optical sections were recorded with the camera lucida; in those eggs which were divided with the cleavage furrow perpendicular to the optical section, the angle between the entrance cone and the furrow was measured, using the egg center as apex.
Fig. 1. Fig. 2. Fig. l.-Disaccordance between entrance point of spermatozoon and the first cleavage furrow. f, cleavage furrow, c, fertilization cone. Fig. 2.-Distribution of fertilization cones with respect to the first cleavage plane. A-B, position of the first cleavage plane, U, O1 - - - - 30°, b, 30” - - - - 60°, c, 60” - - - - 90”.
From 53 such examples (Fig. 2), 19 were found in which the furrow formed an angle of less than 30” with the cone; in 17 casesthe angle was between 30 and 60”, and in 17 casesthe angle was between 60 and 90”. On the basisof these data, it must be concluded that the direction of the first cleavage furrow is not affected by the location of the sperm entrance point. In other speciesof seaurchins, the fertilization cone cannot be detected at the time of the first cleavage. As stated before, however, the sideat which the spermatozoon entered can be roughly determined from the distribution of the rods in the perino invariable coincidence was obvitelline space. In Hemicenfrofus pulcherrimus, served of the first furrow with the side of sperm entry. The work Department
was partly supported of Education.
by the Scientific
Research
Expenditure
Experimental
Cell
of the
Research
19
M.
434
Yoshida
REFERENCES 1. ENDO, Y., Expll. Cell Research 3, 406 (1952). 2. H~RSTADILJS, S., Bid. Rev. 104, 132 (1939). 3. RUNNSTR~M, J., Arkiu Zool. 40, Nr. 1 (1948).
THE
DESOXYRIBOSE NUCLEUS
NUCLEIC OF THE M.
Institute
of Biology, Received
ACID CONTENT HAPLOID FROG
IN THE LARVA’
RETINAL
YOSHIDAB Oita
University,
February
Oita,
Japan
9, 1960
A
RELATIVELY proportional relationship between the nuclear volume and the set of chromosomes in retinal cells has been proved to be existent among diploid, triploid and pentaploid amphibian embryos [3]. Recently, Moore [4] reported that an approximate ratio of the nuclear desoxyribose nucleic acid (DNA) content was shown as two to one between diploid and haploid frog embryos, and that the wide range of the nuclear DNA content was correlated with the differentiation of embryonic tissues. As is well known, in the adult frog retina the color intensities of the DNA-Feulgen dye on nuclei, which are approximately identical in nuclei within the same nuclear layer of the retina, are remarkable in the nuclei of the inner nuclear layer among three nuclear layers, while this is not so marked in the embryonic retina. Therefore, the experiment was performed to ascertain whether or not the constancy concept of the nuclear DNA content, which was first proposed by Boivin, Vendrely and Vendrely [l], applied in the retinal cells between diploid and gynogenetic haploid frog larvae. Materials and Methods.-Both diploid and haploid frog larvae developed from eggs were derived from a single female frog, Rana nigromaculata nigromaculata, by the usual pituitary injection method. Eggs were, in part, inseminated with sperms treated with dye, toluidine blue (10-O M, pH 8.8 in tris aminomethane buffer), to produce gynogenetic haploid embryos [2]. Both the diploid and haploid frog larvae 21 days after their fertilization were simultaneously fixed with Carnoy’s acetic acidalcohol (3:l) for two hours. Materials embedded in paraffin were cut as thin as 15 micra. Sections of both diploid and haploid retinal tissueswere attached on the same thin cover glass. Feulgen reaction for histochemical demonstration of DNA was applied,
where
hydrolysis
of sections
with
N
hydrochloric
acid
lasted
for
a period
of 20 min. at 60 C. The extinctions of the DNA-Feulgen dye on the nucleus were 1 This work (July, 1957). * Research Tissue Culture
was reported
at the Thirtieth
fellow of the National Society for the Laboratory, The University of Texas,
Experimental
Cell Research
19
Annual
Meeting
of the Japanese
Biochemical
Society
Prevention of Blindness. Present address: Medical Branch, Galveston, Texas, U.S.A.
5.
MINTZ,
265 (1951). R. H. and
THE
R. A.,
LEWIN,
Canad.
J. Mierobiol.
Cell Research RANDALL,
R.
15, 619 (1959). J.,
.I. Biol.
Chem.
193,
1, 65 (1954).
FIRST CLEAVAGE FURROW IN SEA URCHIN NOT PASS THROUGH THE SPERM ENTRANCE
EGGS DOES POINT
Y. END0 Biological
Laboratory,
Keio Received
A~-rmwH
University, January
Yokohama-Hiyoshi.
Japan
20, 1960
the question whether the first cleavage furrow of sea urchin eggs passes through the entrance point of the spermatozoon or not is certainly not new, it yet has not been definitely settled (for references, see [2]). According to one opinion there is a coincidence between the entrance point of thk spermatozoon and the first cleavage furrow; this accordance is, however, also vigorously denied by others. This disagreement between research workers may be due to the fact that the method for determining the entrance point of the spermatozoon under normal conditions is not sufficiently conclusive. It is well known that the “entrance” or “fertilization cone” is formed at the attachment point of the spermatozoon soon after the fertilization membrane begins to be elevated. In most species of sea urchins, this entrance cone, with the lapse of time, gradually changes in form and finally becomes indistinguishable. In Pseudocentrotus depressus, however, the entrance cone remains for a long time and is possible to detect at the time of the first cleavage. Since the entrance cone is the most exact indication of the entrance point of the spermatozoon, Pseudocentrotus egg was used to test the relation between the cone and the first cleavage furrow. Immediately after membrane elevation in some of these eggs, the entrance cone can be seen in the optical section of the egg surface. Location of the cone is facilitated by the fact that cortical granule substance in the form of rods [l, 31 is distributed in the previtelline space following a gradient which is highest at the sperm entrance point (unpublished). At the time of cleavage, the entrance cone appears as a hyaline protuberance on the surface of the hyaline layer (Fig. 1). Experimental
Cell Research
19
Sperm entrance and cleavage furrow
433
In practice, the positions of entrance cones on the greatest optical sections were recorded with the camera lucida; in those eggs which were divided with the cleavage furrow perpendicular to the optical section, the angle between the entrance cone and the furrow was measured, using the egg center as apex.
Fig. 1. Fig. 2. Fig. l.-Disaccordance between entrance point of spermatozoon and the first cleavage furrow. f, cleavage furrow, c, fertilization cone. Fig. 2.-Distribution of fertilization cones with respect to the first cleavage plane. A-B, position of the first cleavage plane, U, O1 - - - - 30°, b, 30” - - - - 60°, c, 60” - - - - 90”.
From 53 such examples (Fig. 2), 19 were found in which the furrow formed an angle of less than 30” with the cone; in 17 casesthe angle was between 30 and 60”, and in 17 casesthe angle was between 60 and 90”. On the basisof these data, it must be concluded that the direction of the first cleavage furrow is not affected by the location of the sperm entrance point. In other speciesof seaurchins, the fertilization cone cannot be detected at the time of the first cleavage. As stated before, however, the sideat which the spermatozoon entered can be roughly determined from the distribution of the rods in the perino invariable coincidence was obvitelline space. In Hemicenfrofus pulcherrimus, served of the first furrow with the side of sperm entry. The work Department
was partly supported of Education.
by the Scientific
Research
Expenditure
Experimental
Cell
of the
Research
19
M.
434
Yoshida
REFERENCES 1. ENDO, Y., Expll. Cell Research 3, 406 (1952). 2. H~RSTADILJS, S., Bid. Rev. 104, 132 (1939). 3. RUNNSTR~M, J., Arkiu Zool. 40, Nr. 1 (1948).
THE
DESOXYRIBOSE NUCLEUS
NUCLEIC OF THE M.
Institute
of Biology, Received
ACID CONTENT HAPLOID FROG
IN THE LARVA’
RETINAL
YOSHIDAB Oita
University,
February
Oita,
Japan
9, 1960
A
RELATIVELY proportional relationship between the nuclear volume and the set of chromosomes in retinal cells has been proved to be existent among diploid, triploid and pentaploid amphibian embryos [3]. Recently, Moore [4] reported that an approximate ratio of the nuclear desoxyribose nucleic acid (DNA) content was shown as two to one between diploid and haploid frog embryos, and that the wide range of the nuclear DNA content was correlated with the differentiation of embryonic tissues. As is well known, in the adult frog retina the color intensities of the DNA-Feulgen dye on nuclei, which are approximately identical in nuclei within the same nuclear layer of the retina, are remarkable in the nuclei of the inner nuclear layer among three nuclear layers, while this is not so marked in the embryonic retina. Therefore, the experiment was performed to ascertain whether or not the constancy concept of the nuclear DNA content, which was first proposed by Boivin, Vendrely and Vendrely [l], applied in the retinal cells between diploid and gynogenetic haploid frog larvae. Materials and Methods.-Both diploid and haploid frog larvae developed from eggs were derived from a single female frog, Rana nigromaculata nigromaculata, by the usual pituitary injection method. Eggs were, in part, inseminated with sperms treated with dye, toluidine blue (10-O M, pH 8.8 in tris aminomethane buffer), to produce gynogenetic haploid embryos [2]. Both the diploid and haploid frog larvae 21 days after their fertilization were simultaneously fixed with Carnoy’s acetic acidalcohol (3:l) for two hours. Materials embedded in paraffin were cut as thin as 15 micra. Sections of both diploid and haploid retinal tissueswere attached on the same thin cover glass. Feulgen reaction for histochemical demonstration of DNA was applied,
where
hydrolysis
of sections
with
N
hydrochloric
acid
lasted
for
a period
of 20 min. at 60 C. The extinctions of the DNA-Feulgen dye on the nucleus were 1 This work (July, 1957). * Research Tissue Culture
was reported
at the Thirtieth
fellow of the National Society for the Laboratory, The University of Texas,
Experimental
Cell Research
19
Annual
Meeting
of the Japanese
Biochemical
Society
Prevention of Blindness. Present address: Medical Branch, Galveston, Texas, U.S.A.