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Experimental
MITOCHONDRIAL IN DIFFERENT URCHIN
Cell Research I1 1 (1978) 205-209
PROFILE
DENSITIES
DEVELOPMENTAL
AND
STAGES
SPHAERECHINUS
AREAS
OF THE
SEA
GRANULARIS
HORST BRESCH Abteilung
fir
experimentelle
Pathologie, Medizinische Hochschule and Stazione Zoologica, Naples,
Hannover, Italy
D-3000
Hannover,
Germany
SUMMARY Mitochondrial profde densities in electronmicrographs were counted in the swimming blastula, mesenchyme blastula, gastrula and prism stages of the sea urchin embryos Sphaerechinus granularis. No numerical changes were statistically apparent. When profde areas were investigated, the mean values of the swimming blastula, the gastrula and the prism stage showed no statistical differences. However, increased areas were measured in the mesenchyme blastula stage. This increase might be related to an increase of the embryonic volumina in the mesenchyme blastula stage. In contrast to earlier reported data, the results indicate that the mitochondrial density in S. granularis embryos does not alter during development in these stages.
After fertilization of the sea urchin egg, cell division proceeds quickly. Within 24 h, the embryo contains several hundred ceils. It remains unclear whether intracellular differentiation occurs during the time of rapid cell cleavage. With respect to this question it is of interest to trace the distribution of mitochondria to daughter cells. Mitochondria are of particular interest for the study of internal cell differentiation, since structural components are encoded in the nuclear, as well as in the mitochondrial genome [ 1, 21. It also remains to be explained whether replication of certain structural components of mitochondria takes place during the early cleavage period in sea urchin development, or whether the particles are only passively distributed to daughter cells and if so up to which stage. To-date, published data do not permit a
detailed description of what happens to and in mitochondria up to the pluteus stage. Apart from lack of basic knowledge, previous results are incomplete, since most studies were conducted using different species in different developmental stages. Indeed, several publications have reported investigations of only one stage. Moreover, accepted opinions are based on earlier studies, which were carefully done, but with techniques that have now been superseded by more sophisticated methods. The investigation presented here describes mitochondrial densities in different stages. If mitochondria are only distributed passively after fertilization from egg to daughter cells a lower threshold level must be reached somewhere during development and new particles must occur. A severalfold increase in the number of mitochondria Exp Cell
Res 111 (1978)
206
H. Bresch
from late blastula to gastrula stage has been reported in Psammechinus miliaris embryos [3] and in Lytechinus pictus and Strongylocentrotus purpuratus embryos during the same stages [4]. In the presented investigation, the fluctuations of mitochondrial densities in Sphaerechinus granularis embryos were measured in four stages from swimming blastula up to prism stage. It was intended to observe whether a similar increase in mitochondrial number occurs as in other species. A further aim of this investigation was to correlate the different biochemical parameters [5] with fluctuations in mitochondrial densities. MATERIAL
AND
METHODS
Gametes from S. granularis were obtained by the KC1 method. After several washings in artificial sea water, eggs were fertilized and development proceeded in Erlenmeyer flasks in a shaking water bath at 20°C. Embryos of only one female were taken. A minimum of %% of them developed synchronously to at least the four cell stage; further development resulted in normal plutei. After the distinct stages had been reached, samples were taken and the embryos sedimented at 500 g. Fixation was done in a 2% glutaraldehyde-sea water solution pH 7.8 for 30 min at 20°C and post-fixation in 2% osmium tetroxide for 1 h. In the epon blocks a minimum of six pl es, each 10 pm apart were sectioned with a LKB ul“framicrotome for each stage, a lead citrate staining was used. The optimal section from each plane was scanned in the Zeiss EM 10 microscope and serial photographs were taken at a magnification of x 7 900. In order to calculate the exact magnification factor, a replica of a diffraction grid was also photographed. For measurement of the cytoplasmic areas, and for counting the mitochondrial profiles, the 6x9 cm negatives were 2.4-fold enlarged and printed on Agfa P90 paper. The areas of the mitochondrial profiles were measured by enlarging the negatives 4&fold, projecting them on paper and outlining the profiles by pencil. All areas were determined electronically with the MOP-AM-01 apparatus from Kontron/Eching, W. Germany. At least 60 photographs were taken for each stage. The data were statistically analysed using a Students t-test and a Komogorov-Smimov-test for suitability [6]. These tests are independent of each other.
RESULTS The complete investigation consisted of two measurements for obtaining quantitaExp Cdl
RPS 111 (1978)
tive data regarding the variability of mitochondrial populations in the four stages. In the first measurement, the mitochondrial profile densities were determined. In the second, mitochondrial profile areas of the four stages were analysed. In the investigation presented here, no trial studies were conducted to select special cells, such as mesenchyme cells, owing to the fact that such cells are too scarce within the sections available. Therefore, the data represent mean values for the total embryo. In addition, according to Berg et al. [7] mitochondrial profile densities were not different in the animal, equatorial, vegetal and mesenthyme cells of early gastrulae in four species. Mitochondrial profile densities
Mitochondrial profiles in the electronmicrographs were counted and the number of profiles per 100 pm2 calculated. The number of evaluated electronmicrographs, each containing from l-4 cell fractions, are indicated in table 1, as are the total cytoplasmic areas, the total number of mitochondria and the mean values of profile densities. To detect whether the differences in the mean values were randomly caused, the samples were statistically analysed and data from the prism stage taken as a reference. Since the null hypothesis could not be disproved by both statistical tests (P~0.05) the differences in mean values are interpreted as being randomly caused. Hence, according to these tests, the embryos of the investigated stages contain the same number of mitochondrial profiles. From these data and from the equation Nv=Na/D [8] the mitochondrial densities were calculated. NV, signifies number of mitochondrialunit volume; Na, number of mitochondria/unit area; D, average “tangent diameter” of the mitochondria, determined as described by
Mitochondrial
profile
densities
and areas in S. granularis
207
embryos
Table 1. Mitochondrial
profile
Calculations of mitochondrial in [8] and [9]
densities are based on mean values of profile densities and on procedures described
Stage Swimming blastula Mesenchyme blastula Gastrula Prism stage a r-test. b Kolmogorov-Smimov-test
densities
in four different
No. of micrographs
Area of cytoplasm km*)
No. of mitochondria
Mean (mit/ 100 pm*)
78 75
3 032 2 525 952 1 420
1 030 782 352 568
34f 1.88 31k2.11 37f2.10 40f3.88
54:
embryos
Mit/ 100 pm3
Significance P”>O.3 P >0.05 P >0.6
Pb>0.2 P b-o.2 ,P >0.2
14 12 19 18
for homogenicity.
Bach [9]. The values are listed in table 1. The mitochondrial densities measured in these stages are in the same range as has been reported for rat liver [lo].
Mitochondrial
stages of S. granularis
plotted on probability paper, as seen in fig. 1. Most probably, mainly two reasons could be responsible for this difference: (1) Altered metabolic activities during the mesenchyme blastula stage, as indicated by the increased synthetic rates of cardiolipin [5], might cause a swelling of the mitochondria in this stage. (2) Such an increase might result from a general expansion of the cytoplasm in the mesenchyme blastula stage. In order to clarify this situation the total cytoplasmic areas in the micrographs of a stage were added together, as well as all mitochondrial areas, and the ratios of the totals were determined. The ratios are listed in table 2. The fact that the ratio of the mesenchyme blastula stage is the same as that of the swimming blastula stage sug-
profile areas
The number of measured profile areas and the mean values of the samples are demonstrated in table 2. As can be seen the statistical tests did not reveal differences between swimming blastula or gastrula and prism stage. However, a statistically significant difference resulted when these data were compared with the profile areas of the mesenchyme blastula stage. Its enlarged values are also obvious if cumulative frequency distributions of profile areas are
Table 2. Mitochondrial profile areas in four different stages of S. granularis embryos In addition the ratios of cytoplasmic and mitochondrial
Stage Swimming blastula Mesenchyme blastula Gastrula Prism stage
areas are indicated. For further explanation see text
No. of mit. measured
Area of mit. mean &m”)
Significance
390
0.210+0.004
PQO.2
P90.2
14.0
E 401
0.229f0.004 0.203f0.004 0.214+0.004
P 0.05
P co.01 P >0.2
14.1 13.4 11.7
area of cytoplasm (table 1) no. of mit. (table 1) X area of mit. mean
(1 t-test.
b Kolmogorov-Smimov-test 13t-771807
for homogenicity. Exp
Cd
Rr.5 I I I (1978)
208
H. Bresch
I
I
?
50 OOSL
I
,
100 0128
Fig. 1. Abscissa:
200 0255
area; ordinate:
300 038L
560 Gnm4 06LO
[Id]
cumulative frequency
(%I. Cumulative frequency distributions of mitochondrial profile areas of four developmental stages of S. granufaris embryos. The almost straight lines indicate that raw-data have to be logarithmically transformed before entering the r-test. In addition the F-test allowed the evaluation of data by a t-test. Median value 0, +, A, 0,
Blastula Mes. blastula Gastnda Prism stage
(mm?
(rm”)
156 172 150 158
0.199 0.219 0.191 0.201
gests that the second hypothesis is correct. This is further supported by a study of Mtiller et al. [l l] according to which the S. granularis embryo demonstrates its largest volume in the mesenchyme blastula stage during development from fertilization to prism stage. The increased volume could be due to cytoplasmic expansion. DISCUSSION According to the presented results, no increase in mitochondrial number was registered in the S. granularis embryo from the swimming blastula up to the prism stage. Exp
Cell
Res
111 (1978)
These results do not support the pronounced increase in mitochondria observed in other species during these stages [3, 41. However, the results presented here do not necessarily contradict the earlier studies as different species might react differently. The data presented here nevertheless indicate that earlier studies should be repeated in the light of new technical methodology. Since mitochondrial activity is dependent not only on the number of the particles and their volumes, but also on other factors including the surface area of the inner membrane, tie attempted to count the number of cristae from the micrographs. The results consist of preliminary data, since for a precise counting a higher magnification is needed. The average number of cristae was found not to vary in the investigated stages. In conclusion, no significant mitochondrial changes were registered by morphological methods, with the exception of the increase in mitochondrial volume in the mesenthyme blastula stage. In these investigations the embryos of only one female were studied and it might be argued that in embryos of other females fluctuations in mitochondrial densities might occur. However, it this is the case such differences are probably not important for development at least up to the pluteus stage. The lack of mt (mitochondrial) DNA replication in S. granularis embryos [5] is in direct #accordance to the morphological data. As reported earlier, incorporation rates of glycerol into mtphosphatidylethanolamine and phosphatidylcholine were constant from blastula to prism stage, as was the incorporation rate of leucine into mitochondrial membranes [5]. These results are also in accordance with the interpretation of morphological data if it is assumed that the rates indicate a basic and constant turnover of the corresponding
Mitochondrial
profile
densities
macromolecules. Apart from this hypothesis, these biochemical data might also be related to intramitochondrial differentiation processes not manifested by the morphological measurements, The increased synthetic rates of cardiolipin and mtphosphatidylinositol measured in the mesenchyme blastula [5] is obviously not related to an increase in mitochondrial number. Therefore, the meaning of this requires further analysis in separate experiments. Results obtained from other sea urchin species demonstrated that synthetic rates of other mitochondrial components did not change in early development. Thus, as in S. granularis embryos, no mtDNA synthesis was detected in L. pictus and in Arbacia punctulata embryos [12, 131 and in S. purpuratus and L. pictus embryos polyA-containing mtRNA synthesis proceeded at constant rates [14]. In the latter study, measurement was started soon after fertilization, but unfortunately was only extended up to the 21 h embryo. Piko & Chase have investigated the morphology of mitochondria during the early development of the mouse embryo [15], another deuterostome, the egg of which also cleaves equally. In contrast to the sea urchin embryo, the mitochondrial volume increases continuously from the two cell stage and intramitochondrial differentiation has also been observed. It would be interesting to investigate the cause of this difference between the two species and to examine whether it might be linked to various metabolic activities in the early embryos. In order to complete such investigations, data regarding the matrix compounds would also be needed. Such in-
and areas in S. granularis
embryos
209
formation would also be necessary to find a correlation between mitochondrial activity and the S-shaped rates of oxygen uptake measured in embryos of different sea urchin species between fertilization and pluteus stage [ 161. I am greatly indebted to Professor R. Martin and his staff, the section of electronmicroscopy, University Ulm, for technical help, and to Drs W. Drommer and U. Veltmann, Tiermedizinische Hochschule, Hannover, who kindly allowed the use of the MOP-AM-01. I thank U. Arendt, K. Hoffmann and D. Kracke for skilful assistance, P. Schneider for advice in statistics, and Miss C. Murphy for correcting the translation of the manuscript. I am also indebted to Professor U. Mohr who sponsored this work. This research was carried out under contract 02174-1 ENVD of the European Communities Environmental Research Program.
REFERENCES 1. Borst, P, Ann rev biochem 41 (1972) 333 2. Bucher, T et al. (ed), Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/NorthHolland Biomedical Press, Amsterdam (1976). 3. Gustafson, T & Lenicque, P, Exp cell res 8 (1955) 114. 4. Shaver. J R. EXD cell res 11 (1956) 548. 5. Bresch; H, Genetics and biogenesis of chloroplasts and mitochondria (ed T Bticher et al.) p. 83 1. Elsevier/North-Holland Biomedical Press,.Amsterdam (1976). 6. Massey jr, F J, J Am stat assoc 46 (1951) 68. 7. Berg, W E, Taylor, D A & Humphreys, W J, Dev bio14 (1%2) 165. 8. DeHoff, R T & Rhines, F N, Trans Am inst min metal1 pet eng 221 (l%l) 975. Bach, G, Z angew Math Phys 16 (1%5) 224. 1:: Loud, A V, J cell bio137 (1968) 27. 11. Miiller, W E G, Forster, W, Zahn, G & Zahn, R K, Wilhelm Roux arch Entwicklungsmech Org 167 (1971) 99. 12. Piko, L, Am zoo1 9 (1%9) 1118. 13. Matsumoto, L & Piko, L, Biol bull 141 (1971) 397. 14. Delvin, R, Dev biol50 (1976) 443. 15. Piko, L & Chase, D G, J cell biol58 (1973) 357. 16. Yanagisawa, T, The sea urchin embryo (ed G Czihak) p. 510. Springer Verlag Berlin, Heidelberg, New York (1975). Received June 14, 1977 Accepted July 13, 1977
Exp
Cd
Res
I I1 (1978)
Experimental
MITOCHONDRIAL IN DIFFERENT URCHIN
Cell Research I1 1 (1978) 205-209
PROFILE
DENSITIES
DEVELOPMENTAL
AND
STAGES
SPHAERECHINUS
AREAS
OF THE
SEA
GRANULARIS
HORST BRESCH Abteilung
fir
experimentelle
Pathologie, Medizinische Hochschule and Stazione Zoologica, Naples,
Hannover, Italy
D-3000
Hannover,
Germany
SUMMARY Mitochondrial profde densities in electronmicrographs were counted in the swimming blastula, mesenchyme blastula, gastrula and prism stages of the sea urchin embryos Sphaerechinus granularis. No numerical changes were statistically apparent. When profde areas were investigated, the mean values of the swimming blastula, the gastrula and the prism stage showed no statistical differences. However, increased areas were measured in the mesenchyme blastula stage. This increase might be related to an increase of the embryonic volumina in the mesenchyme blastula stage. In contrast to earlier reported data, the results indicate that the mitochondrial density in S. granularis embryos does not alter during development in these stages.
After fertilization of the sea urchin egg, cell division proceeds quickly. Within 24 h, the embryo contains several hundred ceils. It remains unclear whether intracellular differentiation occurs during the time of rapid cell cleavage. With respect to this question it is of interest to trace the distribution of mitochondria to daughter cells. Mitochondria are of particular interest for the study of internal cell differentiation, since structural components are encoded in the nuclear, as well as in the mitochondrial genome [ 1, 21. It also remains to be explained whether replication of certain structural components of mitochondria takes place during the early cleavage period in sea urchin development, or whether the particles are only passively distributed to daughter cells and if so up to which stage. To-date, published data do not permit a
detailed description of what happens to and in mitochondria up to the pluteus stage. Apart from lack of basic knowledge, previous results are incomplete, since most studies were conducted using different species in different developmental stages. Indeed, several publications have reported investigations of only one stage. Moreover, accepted opinions are based on earlier studies, which were carefully done, but with techniques that have now been superseded by more sophisticated methods. The investigation presented here describes mitochondrial densities in different stages. If mitochondria are only distributed passively after fertilization from egg to daughter cells a lower threshold level must be reached somewhere during development and new particles must occur. A severalfold increase in the number of mitochondria Exp Cell
Res 111 (1978)
206
H. Bresch
from late blastula to gastrula stage has been reported in Psammechinus miliaris embryos [3] and in Lytechinus pictus and Strongylocentrotus purpuratus embryos during the same stages [4]. In the presented investigation, the fluctuations of mitochondrial densities in Sphaerechinus granularis embryos were measured in four stages from swimming blastula up to prism stage. It was intended to observe whether a similar increase in mitochondrial number occurs as in other species. A further aim of this investigation was to correlate the different biochemical parameters [5] with fluctuations in mitochondrial densities. MATERIAL
AND
METHODS
Gametes from S. granularis were obtained by the KC1 method. After several washings in artificial sea water, eggs were fertilized and development proceeded in Erlenmeyer flasks in a shaking water bath at 20°C. Embryos of only one female were taken. A minimum of %% of them developed synchronously to at least the four cell stage; further development resulted in normal plutei. After the distinct stages had been reached, samples were taken and the embryos sedimented at 500 g. Fixation was done in a 2% glutaraldehyde-sea water solution pH 7.8 for 30 min at 20°C and post-fixation in 2% osmium tetroxide for 1 h. In the epon blocks a minimum of six pl es, each 10 pm apart were sectioned with a LKB ul“framicrotome for each stage, a lead citrate staining was used. The optimal section from each plane was scanned in the Zeiss EM 10 microscope and serial photographs were taken at a magnification of x 7 900. In order to calculate the exact magnification factor, a replica of a diffraction grid was also photographed. For measurement of the cytoplasmic areas, and for counting the mitochondrial profiles, the 6x9 cm negatives were 2.4-fold enlarged and printed on Agfa P90 paper. The areas of the mitochondrial profiles were measured by enlarging the negatives 4&fold, projecting them on paper and outlining the profiles by pencil. All areas were determined electronically with the MOP-AM-01 apparatus from Kontron/Eching, W. Germany. At least 60 photographs were taken for each stage. The data were statistically analysed using a Students t-test and a Komogorov-Smimov-test for suitability [6]. These tests are independent of each other.
RESULTS The complete investigation consisted of two measurements for obtaining quantitaExp Cdl
RPS 111 (1978)
tive data regarding the variability of mitochondrial populations in the four stages. In the first measurement, the mitochondrial profile densities were determined. In the second, mitochondrial profile areas of the four stages were analysed. In the investigation presented here, no trial studies were conducted to select special cells, such as mesenchyme cells, owing to the fact that such cells are too scarce within the sections available. Therefore, the data represent mean values for the total embryo. In addition, according to Berg et al. [7] mitochondrial profile densities were not different in the animal, equatorial, vegetal and mesenthyme cells of early gastrulae in four species. Mitochondrial profile densities
Mitochondrial profiles in the electronmicrographs were counted and the number of profiles per 100 pm2 calculated. The number of evaluated electronmicrographs, each containing from l-4 cell fractions, are indicated in table 1, as are the total cytoplasmic areas, the total number of mitochondria and the mean values of profile densities. To detect whether the differences in the mean values were randomly caused, the samples were statistically analysed and data from the prism stage taken as a reference. Since the null hypothesis could not be disproved by both statistical tests (P~0.05) the differences in mean values are interpreted as being randomly caused. Hence, according to these tests, the embryos of the investigated stages contain the same number of mitochondrial profiles. From these data and from the equation Nv=Na/D [8] the mitochondrial densities were calculated. NV, signifies number of mitochondrialunit volume; Na, number of mitochondria/unit area; D, average “tangent diameter” of the mitochondria, determined as described by
Mitochondrial
profile
densities
and areas in S. granularis
207
embryos
Table 1. Mitochondrial
profile
Calculations of mitochondrial in [8] and [9]
densities are based on mean values of profile densities and on procedures described
Stage Swimming blastula Mesenchyme blastula Gastrula Prism stage a r-test. b Kolmogorov-Smimov-test
densities
in four different
No. of micrographs
Area of cytoplasm km*)
No. of mitochondria
Mean (mit/ 100 pm*)
78 75
3 032 2 525 952 1 420
1 030 782 352 568
34f 1.88 31k2.11 37f2.10 40f3.88
54:
embryos
Mit/ 100 pm3
Significance P”>O.3 P >0.05 P >0.6
Pb>0.2 P b-o.2 ,P >0.2
14 12 19 18
for homogenicity.
Bach [9]. The values are listed in table 1. The mitochondrial densities measured in these stages are in the same range as has been reported for rat liver [lo].
Mitochondrial
stages of S. granularis
plotted on probability paper, as seen in fig. 1. Most probably, mainly two reasons could be responsible for this difference: (1) Altered metabolic activities during the mesenchyme blastula stage, as indicated by the increased synthetic rates of cardiolipin [5], might cause a swelling of the mitochondria in this stage. (2) Such an increase might result from a general expansion of the cytoplasm in the mesenchyme blastula stage. In order to clarify this situation the total cytoplasmic areas in the micrographs of a stage were added together, as well as all mitochondrial areas, and the ratios of the totals were determined. The ratios are listed in table 2. The fact that the ratio of the mesenchyme blastula stage is the same as that of the swimming blastula stage sug-
profile areas
The number of measured profile areas and the mean values of the samples are demonstrated in table 2. As can be seen the statistical tests did not reveal differences between swimming blastula or gastrula and prism stage. However, a statistically significant difference resulted when these data were compared with the profile areas of the mesenchyme blastula stage. Its enlarged values are also obvious if cumulative frequency distributions of profile areas are
Table 2. Mitochondrial profile areas in four different stages of S. granularis embryos In addition the ratios of cytoplasmic and mitochondrial
Stage Swimming blastula Mesenchyme blastula Gastrula Prism stage
areas are indicated. For further explanation see text
No. of mit. measured
Area of mit. mean &m”)
Significance
390
0.210+0.004
PQO.2
P90.2
14.0
E 401
0.229f0.004 0.203f0.004 0.214+0.004
P 0.05
P co.01 P >0.2
14.1 13.4 11.7
area of cytoplasm (table 1) no. of mit. (table 1) X area of mit. mean
(1 t-test.
b Kolmogorov-Smimov-test 13t-771807
for homogenicity. Exp
Cd
Rr.5 I I I (1978)
208
H. Bresch
I
I
?
50 OOSL
I
,
100 0128
Fig. 1. Abscissa:
200 0255
area; ordinate:
300 038L
560 Gnm4 06LO
[Id]
cumulative frequency
(%I. Cumulative frequency distributions of mitochondrial profile areas of four developmental stages of S. granufaris embryos. The almost straight lines indicate that raw-data have to be logarithmically transformed before entering the r-test. In addition the F-test allowed the evaluation of data by a t-test. Median value 0, +, A, 0,
Blastula Mes. blastula Gastnda Prism stage
(mm?
(rm”)
156 172 150 158
0.199 0.219 0.191 0.201
gests that the second hypothesis is correct. This is further supported by a study of Mtiller et al. [l l] according to which the S. granularis embryo demonstrates its largest volume in the mesenchyme blastula stage during development from fertilization to prism stage. The increased volume could be due to cytoplasmic expansion. DISCUSSION According to the presented results, no increase in mitochondrial number was registered in the S. granularis embryo from the swimming blastula up to the prism stage. Exp
Cell
Res
111 (1978)
These results do not support the pronounced increase in mitochondria observed in other species during these stages [3, 41. However, the results presented here do not necessarily contradict the earlier studies as different species might react differently. The data presented here nevertheless indicate that earlier studies should be repeated in the light of new technical methodology. Since mitochondrial activity is dependent not only on the number of the particles and their volumes, but also on other factors including the surface area of the inner membrane, tie attempted to count the number of cristae from the micrographs. The results consist of preliminary data, since for a precise counting a higher magnification is needed. The average number of cristae was found not to vary in the investigated stages. In conclusion, no significant mitochondrial changes were registered by morphological methods, with the exception of the increase in mitochondrial volume in the mesenthyme blastula stage. In these investigations the embryos of only one female were studied and it might be argued that in embryos of other females fluctuations in mitochondrial densities might occur. However, it this is the case such differences are probably not important for development at least up to the pluteus stage. The lack of mt (mitochondrial) DNA replication in S. granularis embryos [5] is in direct #accordance to the morphological data. As reported earlier, incorporation rates of glycerol into mtphosphatidylethanolamine and phosphatidylcholine were constant from blastula to prism stage, as was the incorporation rate of leucine into mitochondrial membranes [5]. These results are also in accordance with the interpretation of morphological data if it is assumed that the rates indicate a basic and constant turnover of the corresponding
Mitochondrial
profile
densities
macromolecules. Apart from this hypothesis, these biochemical data might also be related to intramitochondrial differentiation processes not manifested by the morphological measurements, The increased synthetic rates of cardiolipin and mtphosphatidylinositol measured in the mesenchyme blastula [5] is obviously not related to an increase in mitochondrial number. Therefore, the meaning of this requires further analysis in separate experiments. Results obtained from other sea urchin species demonstrated that synthetic rates of other mitochondrial components did not change in early development. Thus, as in S. granularis embryos, no mtDNA synthesis was detected in L. pictus and in Arbacia punctulata embryos [12, 131 and in S. purpuratus and L. pictus embryos polyA-containing mtRNA synthesis proceeded at constant rates [14]. In the latter study, measurement was started soon after fertilization, but unfortunately was only extended up to the 21 h embryo. Piko & Chase have investigated the morphology of mitochondria during the early development of the mouse embryo [15], another deuterostome, the egg of which also cleaves equally. In contrast to the sea urchin embryo, the mitochondrial volume increases continuously from the two cell stage and intramitochondrial differentiation has also been observed. It would be interesting to investigate the cause of this difference between the two species and to examine whether it might be linked to various metabolic activities in the early embryos. In order to complete such investigations, data regarding the matrix compounds would also be needed. Such in-
and areas in S. granularis
embryos
209
formation would also be necessary to find a correlation between mitochondrial activity and the S-shaped rates of oxygen uptake measured in embryos of different sea urchin species between fertilization and pluteus stage [ 161. I am greatly indebted to Professor R. Martin and his staff, the section of electronmicroscopy, University Ulm, for technical help, and to Drs W. Drommer and U. Veltmann, Tiermedizinische Hochschule, Hannover, who kindly allowed the use of the MOP-AM-01. I thank U. Arendt, K. Hoffmann and D. Kracke for skilful assistance, P. Schneider for advice in statistics, and Miss C. Murphy for correcting the translation of the manuscript. I am also indebted to Professor U. Mohr who sponsored this work. This research was carried out under contract 02174-1 ENVD of the European Communities Environmental Research Program.
REFERENCES 1. Borst, P, Ann rev biochem 41 (1972) 333 2. Bucher, T et al. (ed), Genetics and biogenesis of chloroplasts and mitochondria. Elsevier/NorthHolland Biomedical Press, Amsterdam (1976). 3. Gustafson, T & Lenicque, P, Exp cell res 8 (1955) 114. 4. Shaver. J R. EXD cell res 11 (1956) 548. 5. Bresch; H, Genetics and biogenesis of chloroplasts and mitochondria (ed T Bticher et al.) p. 83 1. Elsevier/North-Holland Biomedical Press,.Amsterdam (1976). 6. Massey jr, F J, J Am stat assoc 46 (1951) 68. 7. Berg, W E, Taylor, D A & Humphreys, W J, Dev bio14 (1%2) 165. 8. DeHoff, R T & Rhines, F N, Trans Am inst min metal1 pet eng 221 (l%l) 975. Bach, G, Z angew Math Phys 16 (1%5) 224. 1:: Loud, A V, J cell bio137 (1968) 27. 11. Miiller, W E G, Forster, W, Zahn, G & Zahn, R K, Wilhelm Roux arch Entwicklungsmech Org 167 (1971) 99. 12. Piko, L, Am zoo1 9 (1%9) 1118. 13. Matsumoto, L & Piko, L, Biol bull 141 (1971) 397. 14. Delvin, R, Dev biol50 (1976) 443. 15. Piko, L & Chase, D G, J cell biol58 (1973) 357. 16. Yanagisawa, T, The sea urchin embryo (ed G Czihak) p. 510. Springer Verlag Berlin, Heidelberg, New York (1975). Received June 14, 1977 Accepted July 13, 1977
Exp
Cd
Res
I I1 (1978)
