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Fibrous Structures within the Matrix of Developing Chick Embryo Mitochondria
Experimental Cell Research 255, 1–3 (2000) doi:10.1006/excr.2000.4768, available online at http://www.idealibrary.com on
PRELIMINARY NOTES Fibrous Structures within the Matrix of Developing Chick Embryo Mitochondria Margit M. K. Nass and S. Nass The Wenner-Gren Institute for Experimental Biology, University of Stockholm, Sweden
Some observations of the characteristics of the fibers after various fixation and staining methods may be important to note. The fibers and rods have been observed after fixation with 2 per cent osmium tetroxide buffered between pH 7.0 to 9.0 and at osmolar concentrations estimated to vary between 0.34 and 0.45 M [16] as well as, in some instances, after fixation with calcium permanganate [1]. Unstained sections of mitochondria show the usual electron-lucid areas, but only traces of the fibrous material. Staining with saturated uranyl acetate as well as with alkalinized lead acetate [14] increased the contrast of these fibers. (The embedding medium used was Epon 812 [8].) Fibrous material has been persistently found within the electron-lucid areas of mitochondria in the amoeba Pelomyxa [10]. The authors stated that the fibrous material within the mitochondria resembled the fibers found in the nuclear membrane. Further, mitochondria of sea urchin blastulae typically contain electron-lucid areas [15], and fibrous mitochondrial structures are apparent in some sections. Similar clear areas and fibers have been observed in mouse oocyte mitochondria [11]. Some suggestions about the biochemical nature of these fibers may be obtained from the observations of Steinert [13], who has shown by cytochemistry and electron microscopy that kinetoplasts of Trypanosoma contain both DNA and the conventional mitochondrial structure within the same body. His micrographs show fibers and rod-like structures within the DNA-containing region, and these fibers are located in a very electron-lucid area. These observations may be related to those of Kellenberger et al. [7], who have studied the nucleoplasm of Escherichia coli as well as the DNA pool within T2 phage infected bacteria; both were shown to be composed of fibrillar material within an electron-lucid area. The fibers appear much more distinct after lanthanum and uranyl acetate treatment. Our observations of a marked increase in contrast of the fibers after staining with uranyl acetate and lead acetate appear to relate to the findings just described.
During a study of the mechanisms involved in the uptake and utilization of yolk by the developing chick embryo and its relationship to the differentiation of cytoplasmic organelles, a consistently observed fibrous material has been found within the mitochondria from all regions and at all stages of development thus far studied (up to head-process, stage 5 [6]). A short description of the methods employed is given below. The figures depict the shape and location of the fibrous components of the mitochondria, which are not found outside of these structures. Very fine fibers or rods of variable thickness (15–100 Å in diameter) can be seen extending within the mitochondrial matrix (Fig. 1). They are always seen within electron-lucid regions which are generally lighter than the surrounding cytoplasm. In a few mitochondria the fine fibers appear to extend the length of the mitochondrion. An inclusion body similar to that described as a dead cell by Bellairs [2] is shown in Fig. 2. The densely-packed mitochondrial profiles here also show many typical rods within their electron lucid areas. In mitochondria from the unincubated blastoderm, the filamentous material is present too, but usually not organized into thicker rods (Fig. 3). The “extra-embryonic” white yolk quite unexpectedly also has many mitochondria. In the same region, one mitochondrion has also been observed by Bellairs [3], but our observations indicate that they are quite abundant. Many of these mitochondria, which are of a somewhat different type (denser matrix, surrounded by 150 Å particles, close association to large vesicles), also contain the fibers concerned. The regular occurrence of these characteristic inclusions in the mitochondria may often be used as a diagnostic feature of mitochondria in this material when no cristae are recognizable in the plane of the section. Abbreviations: M, mitochondrion. F, mitochondrial rods and fibers. V, large vesicular body containing some electron-dense material. Sections in all figures shown are stained with alkaline lead acetate [14]. 1
0014-4827/00 $35.00 Copyright © 1962 by Academic Press All rights of reproduction in any form reserved.
2
NASS AND NASS
FIG. 1. Mitochondrial rods and fibers. Hensen’s node at early head process stage. ⫻76,000. FIG. 2. Mitochondrial rods. Large inclusion body in Hensen’s node at early head process stage. ⫻56,000. FIG. 3. Mitochondrial fibers. Area pellucida of unincubated blastoderm. ⫻104,000.
With regard to the physiological role of the fibrous inclusions, a suggestion may be based upon the observations by Che`vremont et al. [4], who have concluded, from cytochemical studies of chick fibroblasts, that mitochondria accumulate DNA which is synthesized in the cytoplasm and transported to the nucleus by the mitochondria.
The yolk has been reported to have a relatively high nucleic acid content (both DNA and RNA [5, 12]) and high nucleotide levels (especially within the white yolk region [5] where we have observed many mitochondria and presumably RNP granules). The intimate association between mitochondria, yolk globules and large vesicles (which according to our observations is quite
3
MITOCHONDRIAL INCLUSIONS
striking) suggests that the mitochondria may be either utilizing or transporting the products concerned at very early stages of development. The frequently observed contacts of mitochondria with the nuclear membrane [9] at these early stages may also be suggestive of Che`vremont’s interpretation of mitochondria acting as a DNA transport system. It is suggested that the occurrence of fibrous material within the clear areas of mitochondria may be of general significance and not artefactual because these structures have been observed in published micrographs from a variety of species and many different fixation and staining techniques have been used. They have been observed primarily in unicellular organisms, oocytes and embryonic cells. The apparent absence of these characteristic structures in most adult mitochondria may be due to masking of the fibers by the increased density of the mitochondrial matrix, to dispersion of the fibers or to other unknown causes. These speculations are in the process of being tested further by the use of additional electron “staining” techniques and correlated histochemical studies. We are indebted to Dr. B. Afzelius and Dr. T. Gustafson for their encouragement, critical discussions and guidance. It is a pleasure to acknowledge the hospitality of Prof. O. Lindberg who has put the facilities of the Wenner-Gren Institute at our disposal. This investiReceived December 22, 1961
gation was carried out during the tenure of a National Science Foundation Postdoctoral Fellowship (to M.N.) and a Postdoctoral Fellowship from the National Cancer Institute, United States Public Health Service (to S.N.).
REFERENCES 1.
Afzelius, B. Symposia Soc. Exptl. Biol. In press.
2.
Bellairs, R. (1961) J. Anat. 95, 54.
3.
Bellairs, R. (1961) J. Biophys. Biochem. Cytol. 11, 207.
4.
Che`vremont, M., Che`vremont-Comhaire, S., and Baeckland, E. (1959) Arch. Biol. (Lie`ge) 70, 811.
5.
Emanuelsson, H. (1961) Acta Physiol. Scand. 53, 46.
6.
Hamburger, V., and Hamilton, H. L. (1951) J. Morphol. 88, 49.
7.
Kellenberger, E., Ryter, A., and Se´chaud, J. (1958) J. Biophys. Biochem. Cytol. 4, 671.
8.
Luft, J. H. (1961) J. Biophys. Biochem. Cytol. 9, 409.
9.
Nass, S., and Nass, M. M. K. In preparation.
10.
Pappas, G. D., and Brandt, P. W. (1959) J. Biophys. Biochem. Cytol. 6, 85.
11.
Parsons, D. F. (1961) J. Biophys. Biochem. Cytol. 11, 492.
12.
Solomon, J. B. (1957) Biochim. et Biophys. Acta 24, 584.
13.
Steinert, M. (1960) J. Biophys. Biochem. Cytol. 8, 542.
14.
Watson, M. L. (1958) J. Biophys. Biochem. Cytol. 4, 727.
15.
Wolpert, L., and Mercer, E. H., personal communication, 1961.
16.
Zetterqvist, H., Thesis, from Karolinska Institutet, Department of Anatomy, Stockholm, 1956.
PRELIMINARY NOTES Fibrous Structures within the Matrix of Developing Chick Embryo Mitochondria Margit M. K. Nass and S. Nass The Wenner-Gren Institute for Experimental Biology, University of Stockholm, Sweden
Some observations of the characteristics of the fibers after various fixation and staining methods may be important to note. The fibers and rods have been observed after fixation with 2 per cent osmium tetroxide buffered between pH 7.0 to 9.0 and at osmolar concentrations estimated to vary between 0.34 and 0.45 M [16] as well as, in some instances, after fixation with calcium permanganate [1]. Unstained sections of mitochondria show the usual electron-lucid areas, but only traces of the fibrous material. Staining with saturated uranyl acetate as well as with alkalinized lead acetate [14] increased the contrast of these fibers. (The embedding medium used was Epon 812 [8].) Fibrous material has been persistently found within the electron-lucid areas of mitochondria in the amoeba Pelomyxa [10]. The authors stated that the fibrous material within the mitochondria resembled the fibers found in the nuclear membrane. Further, mitochondria of sea urchin blastulae typically contain electron-lucid areas [15], and fibrous mitochondrial structures are apparent in some sections. Similar clear areas and fibers have been observed in mouse oocyte mitochondria [11]. Some suggestions about the biochemical nature of these fibers may be obtained from the observations of Steinert [13], who has shown by cytochemistry and electron microscopy that kinetoplasts of Trypanosoma contain both DNA and the conventional mitochondrial structure within the same body. His micrographs show fibers and rod-like structures within the DNA-containing region, and these fibers are located in a very electron-lucid area. These observations may be related to those of Kellenberger et al. [7], who have studied the nucleoplasm of Escherichia coli as well as the DNA pool within T2 phage infected bacteria; both were shown to be composed of fibrillar material within an electron-lucid area. The fibers appear much more distinct after lanthanum and uranyl acetate treatment. Our observations of a marked increase in contrast of the fibers after staining with uranyl acetate and lead acetate appear to relate to the findings just described.
During a study of the mechanisms involved in the uptake and utilization of yolk by the developing chick embryo and its relationship to the differentiation of cytoplasmic organelles, a consistently observed fibrous material has been found within the mitochondria from all regions and at all stages of development thus far studied (up to head-process, stage 5 [6]). A short description of the methods employed is given below. The figures depict the shape and location of the fibrous components of the mitochondria, which are not found outside of these structures. Very fine fibers or rods of variable thickness (15–100 Å in diameter) can be seen extending within the mitochondrial matrix (Fig. 1). They are always seen within electron-lucid regions which are generally lighter than the surrounding cytoplasm. In a few mitochondria the fine fibers appear to extend the length of the mitochondrion. An inclusion body similar to that described as a dead cell by Bellairs [2] is shown in Fig. 2. The densely-packed mitochondrial profiles here also show many typical rods within their electron lucid areas. In mitochondria from the unincubated blastoderm, the filamentous material is present too, but usually not organized into thicker rods (Fig. 3). The “extra-embryonic” white yolk quite unexpectedly also has many mitochondria. In the same region, one mitochondrion has also been observed by Bellairs [3], but our observations indicate that they are quite abundant. Many of these mitochondria, which are of a somewhat different type (denser matrix, surrounded by 150 Å particles, close association to large vesicles), also contain the fibers concerned. The regular occurrence of these characteristic inclusions in the mitochondria may often be used as a diagnostic feature of mitochondria in this material when no cristae are recognizable in the plane of the section. Abbreviations: M, mitochondrion. F, mitochondrial rods and fibers. V, large vesicular body containing some electron-dense material. Sections in all figures shown are stained with alkaline lead acetate [14]. 1
0014-4827/00 $35.00 Copyright © 1962 by Academic Press All rights of reproduction in any form reserved.
2
NASS AND NASS
FIG. 1. Mitochondrial rods and fibers. Hensen’s node at early head process stage. ⫻76,000. FIG. 2. Mitochondrial rods. Large inclusion body in Hensen’s node at early head process stage. ⫻56,000. FIG. 3. Mitochondrial fibers. Area pellucida of unincubated blastoderm. ⫻104,000.
With regard to the physiological role of the fibrous inclusions, a suggestion may be based upon the observations by Che`vremont et al. [4], who have concluded, from cytochemical studies of chick fibroblasts, that mitochondria accumulate DNA which is synthesized in the cytoplasm and transported to the nucleus by the mitochondria.
The yolk has been reported to have a relatively high nucleic acid content (both DNA and RNA [5, 12]) and high nucleotide levels (especially within the white yolk region [5] where we have observed many mitochondria and presumably RNP granules). The intimate association between mitochondria, yolk globules and large vesicles (which according to our observations is quite
3
MITOCHONDRIAL INCLUSIONS
striking) suggests that the mitochondria may be either utilizing or transporting the products concerned at very early stages of development. The frequently observed contacts of mitochondria with the nuclear membrane [9] at these early stages may also be suggestive of Che`vremont’s interpretation of mitochondria acting as a DNA transport system. It is suggested that the occurrence of fibrous material within the clear areas of mitochondria may be of general significance and not artefactual because these structures have been observed in published micrographs from a variety of species and many different fixation and staining techniques have been used. They have been observed primarily in unicellular organisms, oocytes and embryonic cells. The apparent absence of these characteristic structures in most adult mitochondria may be due to masking of the fibers by the increased density of the mitochondrial matrix, to dispersion of the fibers or to other unknown causes. These speculations are in the process of being tested further by the use of additional electron “staining” techniques and correlated histochemical studies. We are indebted to Dr. B. Afzelius and Dr. T. Gustafson for their encouragement, critical discussions and guidance. It is a pleasure to acknowledge the hospitality of Prof. O. Lindberg who has put the facilities of the Wenner-Gren Institute at our disposal. This investiReceived December 22, 1961
gation was carried out during the tenure of a National Science Foundation Postdoctoral Fellowship (to M.N.) and a Postdoctoral Fellowship from the National Cancer Institute, United States Public Health Service (to S.N.).
REFERENCES 1.
Afzelius, B. Symposia Soc. Exptl. Biol. In press.
2.
Bellairs, R. (1961) J. Anat. 95, 54.
3.
Bellairs, R. (1961) J. Biophys. Biochem. Cytol. 11, 207.
4.
Che`vremont, M., Che`vremont-Comhaire, S., and Baeckland, E. (1959) Arch. Biol. (Lie`ge) 70, 811.
5.
Emanuelsson, H. (1961) Acta Physiol. Scand. 53, 46.
6.
Hamburger, V., and Hamilton, H. L. (1951) J. Morphol. 88, 49.
7.
Kellenberger, E., Ryter, A., and Se´chaud, J. (1958) J. Biophys. Biochem. Cytol. 4, 671.
8.
Luft, J. H. (1961) J. Biophys. Biochem. Cytol. 9, 409.
9.
Nass, S., and Nass, M. M. K. In preparation.
10.
Pappas, G. D., and Brandt, P. W. (1959) J. Biophys. Biochem. Cytol. 6, 85.
11.
Parsons, D. F. (1961) J. Biophys. Biochem. Cytol. 11, 492.
12.
Solomon, J. B. (1957) Biochim. et Biophys. Acta 24, 584.
13.
Steinert, M. (1960) J. Biophys. Biochem. Cytol. 8, 542.
14.
Watson, M. L. (1958) J. Biophys. Biochem. Cytol. 4, 727.
15.
Wolpert, L., and Mercer, E. H., personal communication, 1961.
16.
Zetterqvist, H., Thesis, from Karolinska Institutet, Department of Anatomy, Stockholm, 1956.