- Email: [email protected]
Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster
404
Preliminary
notes
may be examined by conventional TEM at 100, or even at 80, keV, and show sufficient detail to be potentially usable for some ARG applications.
through the intercellular bridges and finally lie adjacent to the oocyte nucleus [l]. It is after this stage that the nurse cells become References polyploid [2]. Whitten [3] had observed flaI. Cleaver, J E, Thymidine metabolism and cell kinegellar basal bodies, presumed to arise from tics, p. 96. North-Holland, Amsterdam (1967). 2. Hodges, G M & Muir, M M, Nature 247 (1974) centrioles, in cuticular diploid cells of Sar383. cophaga but polyploid cuticular cells lack 3. Vrensen, G F J M, J histochem cytochem I8 (1970) basal bodies. She proposed that highly 278. 4. Salpeter, M M, Budd, C C & Mattimoe, S, J histopolyploid insect cells which no longer dithem cytochem 22 (1974) 217. vide may lack centrioles. It appeared to us 5. Blackett, N M & Parry, D M, J histochem cytochem 25(1977)206. that the follicular epithelium of insect ovaries would be an excellent tissue to test the Received July 13, 1978 Revised version received October 2, 1978 relationship between polyploidization and Accepted October 6, 1978 presence of centrioles. The insect ovary in general is advantageous for the study of timed events because Printed in Sweden developmental stages are organized in a Copyright @I I979 by Academic Presr. Inc All rights of reproduction in any form reserved linear sequence within the ovarioles [4]. As nol4-48?7/79/0?404-0780?.00/0 the oocyte-nurse cell complex grown in the LOSSof centrioles and polyploidization ovariole, the original monolayer of 50 folin follicle cells of Drosophila licle cells increases in number by mitosis until there are approx. 1000 cells. Subsemelanogaster quently, the follicle cells undergo a comA. P. MAHOWALD,’ J. H. CAULTON,’ M. K. plex series of migrations [5] and then beEDWARDS’, * and A. D. FLOYD.2 ‘Denartment of Biology and =Medical Science Pro&m, Ik&a Unicome polyploid [6, 71. Thus it should be versity, Bloomington, IN 47401, USA possible to determine the relationship beSummary. Centrioles of the nurse cells of Drosophilu tween polyploidization and centrioles in have been shown to move into the oocyte prior to polyploidization of the nurse cells. In order to deter- this system. mine whether or not centriolar loss alwavs occurs in polyploid insect cells, the follicular epithklium of the Drosophila ovary was studied. The DNA content of the cells was determined by cytophotometry of Feulgen-stained squash preparations. The first two endomitotic replications occur at stage 7 and 8. Two additional replications occur prior to stage 11, but the DNA content appears to be under-replicated. Centrioles are found in follicle cells until stage IO at which time they are no longer present. At the inception of polyploidization the centrioles are no longer closely associated with each other or the nuclear envelope. Instead, they are located adjacent to the plasma membrane at the basal surface. These results closely parallel the previous results found for the nurse cells. Hence, it may be a general observation that centrioles are gradually lost in polyploid insect cells.
Centrioles are normally located in a juxtanuclear position. However, during oogenesis in Drosophila melanogaster, the centrioles from the 15 nurse cells migrate
Materials
and Methods
Cytophoromerry. Ovaries were removed from 4-7 day old Drosophila melanogcuter flies in Ephrussi-Beadle
Ringers solution, individual follicles were dissected free, measured, and staged according to King [S]. Each follicle was then transferred to a slide where the follicle was teased apart with tungsten needles and allowed to air-dry. The slides were subsequently fixed in acetic acid-ethanol (3 : I) for IO min and processed through a standardized Feulgen procedure. Teased specimens of testis were processed in an identical manner to serve as diploid and haploid Feulgen-DNA standards. Analysis of stained preparations utilized a highly-stabilized two-wavelength microphotometer constructed around the Leitz Orthoplan microscope and MPV I photometer.
* Current address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Preliminary notes
itag.
I
TO itog.,
,.*ti,
io
io
1. Abscissa: arbitrary units DNA; ordinate: % nuclei. Distribution of nuclear DNA content in squash preparations of Drosophila melanogaster follicle cells (solid squares) and testis controls (hatched squares).
Fig.
Ultrastructural studies. Ovaries were removed from 4-7 day old flies and either fixed in 3 % glutaraldehyde in 0. I M sodium cacodylate at pH 7.4 or with the trialdehyde fixative of Kalt & Tandler [lo] for 2 h. The ovaries were teased apart in the fixative and individual ovarioles or egg chambers were washed six times with 0.2 M sucrose in 0.1 M sodium cacodylate. Subsequently, the tissue was posttixed in I % 0~0, for 2 h, dehydrated and embedded in DER 732/332 plastic. Serial thin sections (90 mm) were picked up on formvar film and transferred to 0.2~ 1.O mm single hole grids. After staining the sections with uranyl acetate and lead citrate [I 11,the grids were carbon-coated and viewed in a Philips EM300 electron microscope.
Results Cytophotometry. The only previously published account of polyploidy in follicle cells of Drosophila is a brief mention by Schultz [6] that they attain a ploidy of 16~. Hence, a more detailed study was first undertaken in order to determine both the degree of polyploidy reached and the time of its in27 - 7x 1803
405
ception. Primary and secondary spermatoxytes provided both diploid and tetraploid modal values as standards in this assay (fig. 3). Attempts to measure spermatids to establish a haploid value were not successful due to the very low content of chromophore and the dispersed nature of the chromatin. Cytophotometry of Feulgenstained follicle cell nuclei produced a range of values for specific stages as shown in fig. 1. Prior to stage 7, we were unable to disperse follicle cells sufftciently to permit meaningful numbers of measurements. The few measurements which were made of the specimen shown in fig. 2 fell in the diploid range, as compared to the 2c and 4c values obtained for testicular germ cells. Stage 7 follicle cells appear to be clearly polyploid with a modal value of 8c. By stage 8 all follicle cells contained more than the 8c amount of DNA and the modal value was 16~. Although the incorporation of [3H]thymidine indicates that the endomitotic S phase for follicle cells is asynchronous [ 121, our data shows that considerable synchrony for each round of DNA replication must be present during these first endoreduplications. After stage 8, far greater asynchrony is evident. Stage 9 follicle cell nuclei spread over a range of 16~to more than 32c values. Stage 10 (limited numbers of measurements) and stage 11 nuclei exhibit modal values between 45 and 50 arbitrary units. This value represents an under-replication of the full 64c value (64 arbitrary units). Assuming full replication to the t6c level (stage 8) and under-replication for each succeeding round of DNA synthesis, these data suggest an approximate 22 % under-replication of DNA in stage 11 follicle cell nuclei. We were unable to demonstrate higher DNA ploidy levels in stage 12 and 14 nuclei \ (data not shown), suggesting that a DNA plateau is reached in stage 11 follicle cells.
406
Preliminary
notes
Fig. 2. Squash preparation of a Feulgen-stained germarium (G), and stages 2, 3 and 4 of an ovariole. x 300.
Fig. S. Squash preparation of testis used as controls for microphotometry. Secondary spermatocytes (40. Primary spermatocytes (2C). X450.
Ultrastructure of follicle cells. A number found between adjacent follicle cells (fig. 5). of follicles from stage 2-5 were analyzed These bridges were always located in the with the electron microscope and no major basal portion of the cells, often within 1 pm changes in centriole number or location of the edge of the cell. At stage 6, except for the fact that there were found during these stages. Follicle cells in mitosis were found at each stage. were no more dividing cells, the observaCentrioles were always found, either at the tions were the same as for earlier stages. poles of the mitotic spindle in which the plane of division was parallel to the surface of the nurse cell-oocyte, or between the fol- Fig. 4. Nurse cell (N)-follicle cell (FC) border showing proximity of centrioles (c) to the cell membranes licle cell nucleus and the nurse cell-oocyte the of a stage 4 follicle. Nucleus (n); procentriole is insurface. Although some centrioles were dicated by an arrow. x 23000. close to the nuclear envelope, many were Fig. 5. Intercellular bridges (IB) between adjacent follicle cells at stage 3. x25 000. found adjacent to the basal surface of the Fig. 6. Centrioles (c) located adjacent to the OOCYte cell (fig. 4). Many intercellular bridges were at stage 4. Lysosome (I). X22 000. E.\p
Cd
Rr.,
I IX f 1979)
408
Preliminary
notes
Fig. 7. Centriole (c) adjacent to intercellular bridge (IB) between adjacent follicle cells at stage 9. x54000.
Fig. 8. Normal cross-sectional morphology of a centriole (c) at stage 9. Oocyte border (0). x39000.
At stages 8-9, centrioles were found adjacent to the oocyte or nurse cell border (fig. 6) and frequently they were at the edge of the intercellular bridges (fig. 7). Although some centrioles were closely associated with these bridges, we did not find centrioles within the lumen of a bridge as was found for nurse cells in the germarium [l]. The two centrioles of a cell were not found together in the typical 90” orientation to each other. Instead they were 1-2 pm away without any special orientation. Mature centrioles found at stage 9 had a normal morphology (fig. 8), except that no procentrioles were found. At stage 10 no centrioles were found in spite of the fact that serial sections of the
basal region of over 100 follicle cells were searched. In addition, no examples of degenerating centrioles were found. Intercellular bridges between follicle cells are also totally absent at this stage.
h/l, (‘P// Re.> I I8 11979)
Discussion
Our results show that centrioles eventually disappear in polyploid follicle cells, but that they do not disappear prior to the inception of endomitosis. Thus it is clear that the developmental change of switching a mitotic cell to an endomitotic cell cycle is not effected by eliminating the centrioles. However, a number of changes relating to centrioles occur at the time of this switch. The centrioles become located near the plasma
Preliminary notes membrane of the cell rather than near the nucleus, No more procentrioles are found, suggesting that duplication of centrioles ceases. Finally, the 90” orientation of one centriole to the other within each follicle cell is lost and the centrioles become located 1-2 pm apart in a random pattern. These changes are similar to those which occur in presumptive nurse cells prior to the movement of the centrioles to the future oocyte and the inception of endomitosis [l]. It is impossible from these descriptive studies to determine any causal relationships among these observations, but the possibility exists that the dissociation of centrioles from the nucleus and to each other is related to the mechanism controlling the switch to the endomitotic cycle. The fate of these centrioles is unclear. We found no instances of degenerating centriales, but since the Drosophila centriole is very short, changes in its ultrastructure may quickly make it unrecognizable. In the case of the nurse cell centrioles which move into the oocyte, the centrioles aggregate and begin to fuse prior to their disappearance [l]. In this earlier study degenerating centrioles were found. The time of centriole disappearance from the oocyte as well as from the follicle cells occurs after the nuclei have completed a number of rounds of DNA replication. Cytophotometry suggests that until stage 8, follicle cell nuclei maintain a considerable degree of synchrony of DNA synthesis. Beyond stage 8, the spread of FeulgenDNA values is considerable, indicating an increasing degree of asynchrony. However, these later stages do display a continued shift toward higher DNA values. Beyond stage 8, modal values do not fit a standard polyploid series, and suggest that underreplication occurs. While our data clearly demonstrates this under-replication at stage
409
1I, it is not sufficiently precise to make conclusive statements about the percentage of replication during early stages. Because the ovariole is readily accessible and techniques are available for isolating mass quantities of specific oocyte stages [13], it may be possible to determine the mechanisms involved in the under-replication of DNA during endopolyploidy in Drosophila. Intercellular bridges have been previously described between follicle cells of a number of diptera [ 14, 1.51.Contrary to the report of Giorgi [ 151, we could not find intercellular bridges between adjacent follicle cells at stage 10 in spite of the extensive serial section analysis. If they are still present, they must be rare. The function of these bridges is not known. In Feulgen whole mounts occasionally 2-3 adjacent follicle cells are in mitosis simultaneously. This synchronization could be due to the cytoplasmic continuity through the bridge. The same data, however, suggests that these follicular cell interconnections are not extensive. Thus, it is doubtful that intercellular bridges are the source of integration of follicle function as suggested previously [15]. However, we do know that gap junctions are present between adjacent follicle cells and between follicle cells and the nurse cells [ 161so that these cells must have ionic continuity. Recent evidence [12] has shown that gap junctions are also present between follicle cells and the oocyte. Thus, the whole egg chamber appears to be in ionic continuity, which may be the integrating factor. These studies have been supported by grant number HD-7983 from the NIH.
References 1. Mahowald, A P 8c Strassheim, J M, J cell biol 45 (1970) 306. 2. Chandley, A C, Exp cell res 44 (1966) 201. Exp Cd Rrs I18 f 1979)
4 10
Preliminary notes
3. Whitten, J M, Science 181(1973) 1066. 4. Kafatos, F C, Regier, J, Mazur, G, Nadel, M, Blau, H, Petri, W H, Wyman, A R. Gelinas, R P. Moore, M, Paul, M, Efstradiadis, A, Vournakis, J. Goldsmith, M, Hunsley, J, Baker, B & Nardi, J, Biochemical differentiation in insect glands (ed W Beerman) p. 45. Springer-Verlag. New York (1977). 5. King, R C & Vanoucek, E G, Growth 24 (1960) 333. 6. Schultz, J. Cold Spring Harb symp quant biol 21 (1956) 307. 7. Jacob, J & Sirlin, J L, Chromosoma (Berl.) 10 (1959) 210. 8. King, R C, Ovarian development in Drosophila melanogaster. Academic Press, New York (1970). 9. Leuchtenberger, C, General cytochemical methods (edJ F Danielli) p. 219. Academic Press, New York (1958). IO. Kalt, M R & Tandler, B, J ultrastruct res 36 (1971) 633. II. Frasca, J M &Parks, B R, J cell biol25 (1%5) 157. 12. Mahowald, A P. Unpublished data. 13. Jacobs-Lorena, M & Crippa, M, Devel biol 57 (1977) 385. 14. Meola, S M, Mollenhauer, H H & Thompson, J M, J morphol 153(1977) 81. IS. Giorgi, F, Cell tiss res 186(1978) 413. 16. Mahowald, A P, J morphol 137(1972) 29. Received July 13, 1978 Accepted October 18, 1978
Printed in Sweden Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved
0014-4827/79/0241005$02.00/0
The growth stimulation of SV3T3 cells by transferrin and its dependenceon biotin DELANO V. YOUNG, FRED W. COX 111, STEWART CHIPMAN and STANDISH C. HARTMAN, Department of Chemistry, MA 02215, USA
Boston University,
Boston,
A critical nutrient for the growth of SV3T3 cells is iron. Iron must be added in the ferrous form or, if in the ferric state, with a suitable complexing agent. Both transferrin and hemoglobin, as iron complexes, will stimulate cell growth in biotin supplemented medium either with low serum (0.15 % v/v) or serum-free. The growth stimulation by iron (free or in complexed form) is dependent on the presence of biotin in the medium. These results indicate the importance of transferrin as a serum growth factor.
Summary.
Although the serum requirement of animal cells in tissue culture is only poorly understood, it is clear that transformation of cells by chemical or viral means alters the serum requirement [ 11.Many transformed cells re-
quire much less serum for optimal growth than their untransformed counterparts and some appear to have dispensed completely with the need for an exogenous protein factor to provoke the onset of DNA synthesis. The mouse fibroblast cell line, 3T3, and its virally transformed derivative, SV3T3, are a typical illustration. Upon serum depletion, 3T3 cells arrest in GI and require Fibroblast Growth Factor (,FGF), insulin, dexamethasone, and a serum fraction for re-initiation of DNA synthesis [2]. On the other hand, if care is taken to remove completely all residual trypsin during routine transfers and if a nutritionally adequate medium is supplied, SV3T3 cells are capable not only of DNA initiation but also slow cellular proliferation in the apparent absence of serum [3]. Serum, however, still accelerates both the rate of DNA synthesis and the mitotic rate [3]. Among the recently discovered agents which promote the growth of SV3T3 cells in culture are biotin [4], cis-unsaturated fatty acids [4], and a low molecular weight, unidentified substance from serum (“Peak III” or “Serum Factor III”) which enhances the viability of these cells [5]. This present communication demonstrates that the iron-carrying serum protein, transferrin (or other appropriately solubilized forms of ionic iron), is essential for vigorous, sustained SV3T3 growth and that, furthermore, the expression of the iron effect is dependent upon the presence of biotin in the culture medium. Materials and Methods Cell culture. The Swiss SV3T3 cells (obtained from Dr Marguerite Vogt) were maintained in Dulbecco’s Modified Eagle Medium (DME, Gibco) containing 4500 mg glucose per liter and 10% calf serum in a 5% D. V. Y. and S. C. H. are members of the Cancer Research Center, Boston University School of Medicine. This paper was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy from Boston University, Boston, MA (F. W. C.).
Preliminary
notes
may be examined by conventional TEM at 100, or even at 80, keV, and show sufficient detail to be potentially usable for some ARG applications.
through the intercellular bridges and finally lie adjacent to the oocyte nucleus [l]. It is after this stage that the nurse cells become References polyploid [2]. Whitten [3] had observed flaI. Cleaver, J E, Thymidine metabolism and cell kinegellar basal bodies, presumed to arise from tics, p. 96. North-Holland, Amsterdam (1967). 2. Hodges, G M & Muir, M M, Nature 247 (1974) centrioles, in cuticular diploid cells of Sar383. cophaga but polyploid cuticular cells lack 3. Vrensen, G F J M, J histochem cytochem I8 (1970) basal bodies. She proposed that highly 278. 4. Salpeter, M M, Budd, C C & Mattimoe, S, J histopolyploid insect cells which no longer dithem cytochem 22 (1974) 217. vide may lack centrioles. It appeared to us 5. Blackett, N M & Parry, D M, J histochem cytochem 25(1977)206. that the follicular epithelium of insect ovaries would be an excellent tissue to test the Received July 13, 1978 Revised version received October 2, 1978 relationship between polyploidization and Accepted October 6, 1978 presence of centrioles. The insect ovary in general is advantageous for the study of timed events because Printed in Sweden developmental stages are organized in a Copyright @I I979 by Academic Presr. Inc All rights of reproduction in any form reserved linear sequence within the ovarioles [4]. As nol4-48?7/79/0?404-0780?.00/0 the oocyte-nurse cell complex grown in the LOSSof centrioles and polyploidization ovariole, the original monolayer of 50 folin follicle cells of Drosophila licle cells increases in number by mitosis until there are approx. 1000 cells. Subsemelanogaster quently, the follicle cells undergo a comA. P. MAHOWALD,’ J. H. CAULTON,’ M. K. plex series of migrations [5] and then beEDWARDS’, * and A. D. FLOYD.2 ‘Denartment of Biology and =Medical Science Pro&m, Ik&a Unicome polyploid [6, 71. Thus it should be versity, Bloomington, IN 47401, USA possible to determine the relationship beSummary. Centrioles of the nurse cells of Drosophilu tween polyploidization and centrioles in have been shown to move into the oocyte prior to polyploidization of the nurse cells. In order to deter- this system. mine whether or not centriolar loss alwavs occurs in polyploid insect cells, the follicular epithklium of the Drosophila ovary was studied. The DNA content of the cells was determined by cytophotometry of Feulgen-stained squash preparations. The first two endomitotic replications occur at stage 7 and 8. Two additional replications occur prior to stage 11, but the DNA content appears to be under-replicated. Centrioles are found in follicle cells until stage IO at which time they are no longer present. At the inception of polyploidization the centrioles are no longer closely associated with each other or the nuclear envelope. Instead, they are located adjacent to the plasma membrane at the basal surface. These results closely parallel the previous results found for the nurse cells. Hence, it may be a general observation that centrioles are gradually lost in polyploid insect cells.
Centrioles are normally located in a juxtanuclear position. However, during oogenesis in Drosophila melanogaster, the centrioles from the 15 nurse cells migrate
Materials
and Methods
Cytophoromerry. Ovaries were removed from 4-7 day old Drosophila melanogcuter flies in Ephrussi-Beadle
Ringers solution, individual follicles were dissected free, measured, and staged according to King [S]. Each follicle was then transferred to a slide where the follicle was teased apart with tungsten needles and allowed to air-dry. The slides were subsequently fixed in acetic acid-ethanol (3 : I) for IO min and processed through a standardized Feulgen procedure. Teased specimens of testis were processed in an identical manner to serve as diploid and haploid Feulgen-DNA standards. Analysis of stained preparations utilized a highly-stabilized two-wavelength microphotometer constructed around the Leitz Orthoplan microscope and MPV I photometer.
* Current address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Preliminary notes
itag.
I
TO itog.,
,.*ti,
io
io
1. Abscissa: arbitrary units DNA; ordinate: % nuclei. Distribution of nuclear DNA content in squash preparations of Drosophila melanogaster follicle cells (solid squares) and testis controls (hatched squares).
Fig.
Ultrastructural studies. Ovaries were removed from 4-7 day old flies and either fixed in 3 % glutaraldehyde in 0. I M sodium cacodylate at pH 7.4 or with the trialdehyde fixative of Kalt & Tandler [lo] for 2 h. The ovaries were teased apart in the fixative and individual ovarioles or egg chambers were washed six times with 0.2 M sucrose in 0.1 M sodium cacodylate. Subsequently, the tissue was posttixed in I % 0~0, for 2 h, dehydrated and embedded in DER 732/332 plastic. Serial thin sections (90 mm) were picked up on formvar film and transferred to 0.2~ 1.O mm single hole grids. After staining the sections with uranyl acetate and lead citrate [I 11,the grids were carbon-coated and viewed in a Philips EM300 electron microscope.
Results Cytophotometry. The only previously published account of polyploidy in follicle cells of Drosophila is a brief mention by Schultz [6] that they attain a ploidy of 16~. Hence, a more detailed study was first undertaken in order to determine both the degree of polyploidy reached and the time of its in27 - 7x 1803
405
ception. Primary and secondary spermatoxytes provided both diploid and tetraploid modal values as standards in this assay (fig. 3). Attempts to measure spermatids to establish a haploid value were not successful due to the very low content of chromophore and the dispersed nature of the chromatin. Cytophotometry of Feulgenstained follicle cell nuclei produced a range of values for specific stages as shown in fig. 1. Prior to stage 7, we were unable to disperse follicle cells sufftciently to permit meaningful numbers of measurements. The few measurements which were made of the specimen shown in fig. 2 fell in the diploid range, as compared to the 2c and 4c values obtained for testicular germ cells. Stage 7 follicle cells appear to be clearly polyploid with a modal value of 8c. By stage 8 all follicle cells contained more than the 8c amount of DNA and the modal value was 16~. Although the incorporation of [3H]thymidine indicates that the endomitotic S phase for follicle cells is asynchronous [ 121, our data shows that considerable synchrony for each round of DNA replication must be present during these first endoreduplications. After stage 8, far greater asynchrony is evident. Stage 9 follicle cell nuclei spread over a range of 16~to more than 32c values. Stage 10 (limited numbers of measurements) and stage 11 nuclei exhibit modal values between 45 and 50 arbitrary units. This value represents an under-replication of the full 64c value (64 arbitrary units). Assuming full replication to the t6c level (stage 8) and under-replication for each succeeding round of DNA synthesis, these data suggest an approximate 22 % under-replication of DNA in stage 11 follicle cell nuclei. We were unable to demonstrate higher DNA ploidy levels in stage 12 and 14 nuclei \ (data not shown), suggesting that a DNA plateau is reached in stage 11 follicle cells.
406
Preliminary
notes
Fig. 2. Squash preparation of a Feulgen-stained germarium (G), and stages 2, 3 and 4 of an ovariole. x 300.
Fig. S. Squash preparation of testis used as controls for microphotometry. Secondary spermatocytes (40. Primary spermatocytes (2C). X450.
Ultrastructure of follicle cells. A number found between adjacent follicle cells (fig. 5). of follicles from stage 2-5 were analyzed These bridges were always located in the with the electron microscope and no major basal portion of the cells, often within 1 pm changes in centriole number or location of the edge of the cell. At stage 6, except for the fact that there were found during these stages. Follicle cells in mitosis were found at each stage. were no more dividing cells, the observaCentrioles were always found, either at the tions were the same as for earlier stages. poles of the mitotic spindle in which the plane of division was parallel to the surface of the nurse cell-oocyte, or between the fol- Fig. 4. Nurse cell (N)-follicle cell (FC) border showing proximity of centrioles (c) to the cell membranes licle cell nucleus and the nurse cell-oocyte the of a stage 4 follicle. Nucleus (n); procentriole is insurface. Although some centrioles were dicated by an arrow. x 23000. close to the nuclear envelope, many were Fig. 5. Intercellular bridges (IB) between adjacent follicle cells at stage 3. x25 000. found adjacent to the basal surface of the Fig. 6. Centrioles (c) located adjacent to the OOCYte cell (fig. 4). Many intercellular bridges were at stage 4. Lysosome (I). X22 000. E.\p
Cd
Rr.,
I IX f 1979)
408
Preliminary
notes
Fig. 7. Centriole (c) adjacent to intercellular bridge (IB) between adjacent follicle cells at stage 9. x54000.
Fig. 8. Normal cross-sectional morphology of a centriole (c) at stage 9. Oocyte border (0). x39000.
At stages 8-9, centrioles were found adjacent to the oocyte or nurse cell border (fig. 6) and frequently they were at the edge of the intercellular bridges (fig. 7). Although some centrioles were closely associated with these bridges, we did not find centrioles within the lumen of a bridge as was found for nurse cells in the germarium [l]. The two centrioles of a cell were not found together in the typical 90” orientation to each other. Instead they were 1-2 pm away without any special orientation. Mature centrioles found at stage 9 had a normal morphology (fig. 8), except that no procentrioles were found. At stage 10 no centrioles were found in spite of the fact that serial sections of the
basal region of over 100 follicle cells were searched. In addition, no examples of degenerating centrioles were found. Intercellular bridges between follicle cells are also totally absent at this stage.
h/l, (‘P// Re.> I I8 11979)
Discussion
Our results show that centrioles eventually disappear in polyploid follicle cells, but that they do not disappear prior to the inception of endomitosis. Thus it is clear that the developmental change of switching a mitotic cell to an endomitotic cell cycle is not effected by eliminating the centrioles. However, a number of changes relating to centrioles occur at the time of this switch. The centrioles become located near the plasma
Preliminary notes membrane of the cell rather than near the nucleus, No more procentrioles are found, suggesting that duplication of centrioles ceases. Finally, the 90” orientation of one centriole to the other within each follicle cell is lost and the centrioles become located 1-2 pm apart in a random pattern. These changes are similar to those which occur in presumptive nurse cells prior to the movement of the centrioles to the future oocyte and the inception of endomitosis [l]. It is impossible from these descriptive studies to determine any causal relationships among these observations, but the possibility exists that the dissociation of centrioles from the nucleus and to each other is related to the mechanism controlling the switch to the endomitotic cycle. The fate of these centrioles is unclear. We found no instances of degenerating centriales, but since the Drosophila centriole is very short, changes in its ultrastructure may quickly make it unrecognizable. In the case of the nurse cell centrioles which move into the oocyte, the centrioles aggregate and begin to fuse prior to their disappearance [l]. In this earlier study degenerating centrioles were found. The time of centriole disappearance from the oocyte as well as from the follicle cells occurs after the nuclei have completed a number of rounds of DNA replication. Cytophotometry suggests that until stage 8, follicle cell nuclei maintain a considerable degree of synchrony of DNA synthesis. Beyond stage 8, the spread of FeulgenDNA values is considerable, indicating an increasing degree of asynchrony. However, these later stages do display a continued shift toward higher DNA values. Beyond stage 8, modal values do not fit a standard polyploid series, and suggest that underreplication occurs. While our data clearly demonstrates this under-replication at stage
409
1I, it is not sufficiently precise to make conclusive statements about the percentage of replication during early stages. Because the ovariole is readily accessible and techniques are available for isolating mass quantities of specific oocyte stages [13], it may be possible to determine the mechanisms involved in the under-replication of DNA during endopolyploidy in Drosophila. Intercellular bridges have been previously described between follicle cells of a number of diptera [ 14, 1.51.Contrary to the report of Giorgi [ 151, we could not find intercellular bridges between adjacent follicle cells at stage 10 in spite of the extensive serial section analysis. If they are still present, they must be rare. The function of these bridges is not known. In Feulgen whole mounts occasionally 2-3 adjacent follicle cells are in mitosis simultaneously. This synchronization could be due to the cytoplasmic continuity through the bridge. The same data, however, suggests that these follicular cell interconnections are not extensive. Thus, it is doubtful that intercellular bridges are the source of integration of follicle function as suggested previously [15]. However, we do know that gap junctions are present between adjacent follicle cells and between follicle cells and the nurse cells [ 161so that these cells must have ionic continuity. Recent evidence [12] has shown that gap junctions are also present between follicle cells and the oocyte. Thus, the whole egg chamber appears to be in ionic continuity, which may be the integrating factor. These studies have been supported by grant number HD-7983 from the NIH.
References 1. Mahowald, A P 8c Strassheim, J M, J cell biol 45 (1970) 306. 2. Chandley, A C, Exp cell res 44 (1966) 201. Exp Cd Rrs I18 f 1979)
4 10
Preliminary notes
3. Whitten, J M, Science 181(1973) 1066. 4. Kafatos, F C, Regier, J, Mazur, G, Nadel, M, Blau, H, Petri, W H, Wyman, A R. Gelinas, R P. Moore, M, Paul, M, Efstradiadis, A, Vournakis, J. Goldsmith, M, Hunsley, J, Baker, B & Nardi, J, Biochemical differentiation in insect glands (ed W Beerman) p. 45. Springer-Verlag. New York (1977). 5. King, R C & Vanoucek, E G, Growth 24 (1960) 333. 6. Schultz, J. Cold Spring Harb symp quant biol 21 (1956) 307. 7. Jacob, J & Sirlin, J L, Chromosoma (Berl.) 10 (1959) 210. 8. King, R C, Ovarian development in Drosophila melanogaster. Academic Press, New York (1970). 9. Leuchtenberger, C, General cytochemical methods (edJ F Danielli) p. 219. Academic Press, New York (1958). IO. Kalt, M R & Tandler, B, J ultrastruct res 36 (1971) 633. II. Frasca, J M &Parks, B R, J cell biol25 (1%5) 157. 12. Mahowald, A P. Unpublished data. 13. Jacobs-Lorena, M & Crippa, M, Devel biol 57 (1977) 385. 14. Meola, S M, Mollenhauer, H H & Thompson, J M, J morphol 153(1977) 81. IS. Giorgi, F, Cell tiss res 186(1978) 413. 16. Mahowald, A P, J morphol 137(1972) 29. Received July 13, 1978 Accepted October 18, 1978
Printed in Sweden Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved
0014-4827/79/0241005$02.00/0
The growth stimulation of SV3T3 cells by transferrin and its dependenceon biotin DELANO V. YOUNG, FRED W. COX 111, STEWART CHIPMAN and STANDISH C. HARTMAN, Department of Chemistry, MA 02215, USA
Boston University,
Boston,
A critical nutrient for the growth of SV3T3 cells is iron. Iron must be added in the ferrous form or, if in the ferric state, with a suitable complexing agent. Both transferrin and hemoglobin, as iron complexes, will stimulate cell growth in biotin supplemented medium either with low serum (0.15 % v/v) or serum-free. The growth stimulation by iron (free or in complexed form) is dependent on the presence of biotin in the medium. These results indicate the importance of transferrin as a serum growth factor.
Summary.
Although the serum requirement of animal cells in tissue culture is only poorly understood, it is clear that transformation of cells by chemical or viral means alters the serum requirement [ 11.Many transformed cells re-
quire much less serum for optimal growth than their untransformed counterparts and some appear to have dispensed completely with the need for an exogenous protein factor to provoke the onset of DNA synthesis. The mouse fibroblast cell line, 3T3, and its virally transformed derivative, SV3T3, are a typical illustration. Upon serum depletion, 3T3 cells arrest in GI and require Fibroblast Growth Factor (,FGF), insulin, dexamethasone, and a serum fraction for re-initiation of DNA synthesis [2]. On the other hand, if care is taken to remove completely all residual trypsin during routine transfers and if a nutritionally adequate medium is supplied, SV3T3 cells are capable not only of DNA initiation but also slow cellular proliferation in the apparent absence of serum [3]. Serum, however, still accelerates both the rate of DNA synthesis and the mitotic rate [3]. Among the recently discovered agents which promote the growth of SV3T3 cells in culture are biotin [4], cis-unsaturated fatty acids [4], and a low molecular weight, unidentified substance from serum (“Peak III” or “Serum Factor III”) which enhances the viability of these cells [5]. This present communication demonstrates that the iron-carrying serum protein, transferrin (or other appropriately solubilized forms of ionic iron), is essential for vigorous, sustained SV3T3 growth and that, furthermore, the expression of the iron effect is dependent upon the presence of biotin in the culture medium. Materials and Methods Cell culture. The Swiss SV3T3 cells (obtained from Dr Marguerite Vogt) were maintained in Dulbecco’s Modified Eagle Medium (DME, Gibco) containing 4500 mg glucose per liter and 10% calf serum in a 5% D. V. Y. and S. C. H. are members of the Cancer Research Center, Boston University School of Medicine. This paper was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy from Boston University, Boston, MA (F. W. C.).