Preliminary notes
459
edge would result such that various portions of the cell membrane might at any time become dominant. Random changes in direction would occur more frequently than in a fibroblast with abundant lamellar cytoplasm and more numerous adhesion sites, especially if migration rates were high. Thus, the more deficient lamellar cytoplasm of the neoplastic cell with possibly fewer attachment sites and the high migration rate may account for the loss of a persistent walk observed in the present study.
25. Vasiliev, J M, Gelfand, I M, Domnina, L V, Ivanova, 0 Y, Komm, S G & Olshevskaja, L V, J embryo1 exp morphol24 (1970) 625. 26. Vasiliev, G M & Gelfand, I M, Ciba foundation symposium I4 (1973) 3I 1. 27. Weiss, P. J exp zool 100 (1945) 353. 28. Wetzel, B, Sanford, K K, Fox, C H, Jones, G M, Westbrook, E W & Tarone, R E, Cancer res 37 (1977) 831.
References
Effect of Distamycin chromosomes
1. Abercrombie, M, Heaysman, J E M & Karthauser, H M, Exp cell res 13 (1957) 276. 2. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 3. Allison. A C. Locomotion of tissue cells. D. 109. Ciba Foundation Symposium I4 (1973). 4. Berman. L D. Int i cancer I.5 (1975) 973. 5. Carter, S B, Nat&e 208 (1965) 1183. 6. Dipasquale, A, Exp cell res 95 (1975) 425. 7. Domnina, L V, Ivanova, 0 Y, Margolis, L B, Olshevskaja, L V, Rovensky, Y A, Vasiliev, J M & Gelfand, I M, Proc natl acad sci US 69 (1972) 248. 8. Evans, V J, Bryant, J C, Kerr, H A & Schilling, E L, Exv cell res 36 (1964) 439. 9. Fox, C *H, Chaconas, P G & Sanford, K K, J microsc 107 (1976) 79. 10. Fox, C H, Dvorak, J A & Sanford, K K, Cancer res 36 (1976) 1556. Il. Fox, C H, Caspersson, T, Kudynowski, J, Sanford, K K & Tarone, R E. Cancer res 37 (1977) 892.
12. Gail, M H & Boone, C W, Biophys j 10 (1970) 980. 13. - Exp cell res 65 (1971) 221. 14. Greenwood, J A & Durand, D, Ann mathematical statistics, vol. 26, p. 233 (1955). 15. Harris, A K, Ciba foundation svmoosium I4 _ _ (1973) 3. 16. - Exp cell res 77 (1973) 285. 17. - Dev biol35 (1973) 97. 18. Martz, E, Phillips, H M & Steinberg, M S, J cell sci I6 (1974) 401. 19. Meltzer, M S, Tucker, R E, Sanford, K K & Leonard, E J, J natl cancer inst 54 (1975) 1177. 20. Revel, J P, Hoch, P & Ho, D, Exp cell res 84 (1974) 207. 21. Sanford, K K, Handleman, S L & Jones, Cl M, Cancer res 37 (1977) 821. 22. Shin, S, Freedman, V H, Risser, R & Pollack, R, Proc natl acad sci US 72 (1975)4435. 23. Steinberg, M S. Ciba foundation svmuosium 14 _ _ (1973) 35. 24. Tucker, R W, Sanford, K K, Handleman, S L & Jones, Cl M, Cancer res 37 (1977) 1571.
Received April 13, 1977 Revised version received June 28, 1977 Accepted July 8, 1977
A on Chinese hamster
G. PRANTERA, M. DI CASTRO, E. MARCHETTI, and A. ROCCHI, Istituto di Genetica e Centro di Genetica Evoluzionistica Roma, I-00185 Rome, Italy
de1 CNR
Universita
di
Distamycin A, an oligopeptide antibiotic, has the property of binding tightly to double helical AT-rich DNA regions, but poorly to GC-rich regions, single-strand DNA and RNA [ 1, 21. The binding takes place, without intercalation, through hydrogen bonding and hydrophobic interactions [3]. In a previous study the cells of the Chinese hamster strain C-125 were treated with H33258 [4], a benzimidazole derivative fluorochrome, which binds to double-strand AT-rich DNA [5] and, like Distamycin A, seems to bind to DNA not by intercalation but by probable interactions hydrophobic in nature [6]. It is, however, possible that the binding geometries of the two compounds are not identical [7]. This fluorochrome induced a precise pattern of interspersed nonspiralized areas on the Chinese hamster chromosomes. The treatment of the chromosomes in the various stages of the cell cycle with substances whose specificity for given bases or base sequences is known and a comparison of the results could be a promising approach Exp Call
Res
109 (1977)
460
Preliminary notes
Table 1. Effect of Distamycin A on Chinese hamster cells treated for different periods of time % of cells
Expt no.
Time of treatment (hours)
Unaffected
Slightly affected
Markedly affected
1 2 3 4
3.5 11 15 30
;: 28 8
10 39 56 72
0 3 16 20
to the problem of the chromosome molecular organization. In the present work the possible effect of Distamycin A on specific chromosome regions was investigated by supplying this compound to the cells of the same Chinese hamster strain.
strain lasts about 15 h: stage S takes about 9 h and stages G2+M 3.5 h [9]. Colchicine was added 2 h before fixing. The slides were stained with aceto-orcein. The preparations examined were obtained with cells treated with Distamycin A at concentration of 100 pg/ ml. A concentration of 200 pg/ml caused a strong decrease in the number of metaphases and no particular increase in effect.
Cells of C-125 Chinese hamster strain [8] grown on coverslips in Petri dishes were supplied with Distamycin A at a concentration of 100 and 200 pg/ml for 3.5, 11, 15 and 30 h. The mean cell cycle of the cells
Results and discussion Distamycin A causes the chromosomes to appear in metaphase in an elongated shape with brief interspersed areas lacking spiralization (fig. la). These areas are always the
Fig. I. Metaphase chromosomes of Chinese hamster cell strain C-125 treated with Distamycin A. A large
number of despiralized areas may be observed. Bar, 10 /.L.
Materials and Methods
EXPCell Res 109 (1977)
Preliminary
-3. Composite partial karyotypes from two cells of Chinese hamster cell strain C-125 treated with Distamycin A (D) and Hoechst 33258 (H). Note that chromosomes from different cells and different treatments show the same despiralized areas.
Fig.
notes
461
by the two compounds are in almost all metaphases practically the same (fig. 2), but that the effect of Distamycin A on these regions is usually less drastic than that of H33258. However, a specific characteristic of Distamycin A is that of inducing in some metaphases a rich sequence of regions spiralized both to greater and lesser extent. Therefore, even though despiralization of the two compounds often coincides, most likely since both bind preferentially to ATrich DNA, Distamycin A seems to have a less drastic but more widespread effect (fig. 1 b). The difference of the effect of the two compounds could be due to a difference in the type of bond and/or a different specificity for different sequences of AT-rich DNA. It seems now to be clear, however, that even if light satellite bands have never been separated from DNA of Chinese hamster in the CsCl density gradient [lo], they do exist, in this DNA, AT-rich sequences scattered along the chromosomes. This hypothesis is supported by the data of Evenson et al. [l l] who observed brief denatured sections at 0.4 k intervals in DNA of Chinese hamster under partial denaturation.
same even if they almost never appear all at once in the same cell or with the same degree of despiralization in the different cells. The results obtained on supplying Distamycin A to cells in culture for a different number of hours are reported in table 1. Some 200 metaphases were examined in each experiment. It can be clearly seen that References during stage S the chromosomes are sensi1. Krey, A K, Allison, R G & Hahn, F E, FEBS lett 29 (1973) 58. tive to the effect of despiralization and that 2. Zimmer, C, Reinert, K E, Luck, G, Wahnert, U, the number of cells influenced increases Lober, G & Thrum, H J, J mol biol 58 (1971) 329. 3. Zimmer, C, Progress in nucleic acid research and with the increase of time of treatment. molecular biologv (ed W E Cohn) vol. 15. D. 285. Given the preferential binding of Dista. Academic Press,-New York (1975). 4. Rocchi, A, Prantera, G, Pimpinelli, S & Di Castro, mycin A for AT-rich regions, it is reasonM, Chromosoma 56 (1976) 41. able to assume that the chromosomal re5. Hilwig, J & Gropp, A, Exp cell res 81 (1973) 474. 6. Comings, D E , Chromosoma 52 (1975) 229. gions which are undercontracted in meta7. Latt, S A & Wohlleb, J C, Chromosoma 52 (1975) phase after treatment, contain AT-rich 291, 8. Pahtti, F, Rizzoni, M, Perticone, P, De Salvia, R DNA. & Olivieri, G, Caryologia 27 (1974) 85. The comparison between the effect in9. Palitti, F, Rocchi, A, Mercanti, A & Olivieri, G, 25 (1972) 365. duced by H33258 and that of Distamycin A 10. Caryologia Comings, D E & Mattoccia, E, Exp cell res 71 shows that chromosomal regions influenced 1972) 113. 30-771804
Exp Cell Res 109 (1977)
462
Preliminary notes
Il. Evenson, D P, Mego, W A & Taylor, J H, Chromosoma 39 (1972) 225. Received April 27, I977 Revised version received July 7, 1977 Accepted July 13, 1977
Microvilli
in sea urchin eggs
Differences in their formation and type EVELYN
SPIEGEL and MELVIN
SPIEGEL, De-
partment of Biological Sciences, Dartmouth Hanover, NH 03755, and Marine Biological tory, Woods Hole. MA 02543, USA
College, Labora-
The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types. Summary.
The surface of the sea urchin egg is covered with microvilli which are very small in the unfertilized egg and become elongated several-fold upon fertilization [14]. It has been suggested that the source of membrane used in the elongation of microvilli is derived from the membranes surrounding the cortical granules [2J]. These granules lie just below the egg plasma membrane and their contents are released upon fertilization by exocytosis [ 1, 5-71. Mazia and coworkers have shown that activation of the unfertilized egg by a variety of agents initiates the elongation of microvilli [8]. We have been able to elicit the same reaction with the enzyme, papain, in unfertilized eggs of Lytechinus pictus and Arbacia punctulata. In addition, using transmission electron microscope studies, these experiments demonstrate that the cortical granule membranes are not involved in microvillus formation in this case. E.rp Crll’Res
109 (1977)
Materials and Methods Gametes of Lytechinus pictus and Arbacia punctulata were used. Methods for handling the gametes and their subsequent preparation for scanning (SEM) and transmission (TEM) electron microscopy have been presented in detail in previous publications [9, IO]. Fertilization membranes were removed enzvmatitally by fertilizing eggs in a solution containing’0.2 % papain plus 0.2 % cysteine . HCI and returning them to normal-sea water after 8 min. The rate and extent of microvillus elongation were followed at I5 set intervals after fertilization in normal sea water controls and in papain-treated eggs. Specimens were examined with TEM and SEM. The possibility that papain might have an effect on the cell surface itself, in addition to the membrane surrounding it, was tested by treating unfertilized eggs with the papain-cysteine . HCI solution for 8 min, the same length of time as that used for removing the fertilization membranes. Thin sections were then observed with TEM.
Results Fig. 1 shows an unfertilized Lytechinus egg. The microvilli are very short and the cortical granules are intact. Figs 2 and 3 are of eggs at 45 and 90 set, respectively, after fertilization in normal sea water, i.e., without papain treatment. At 45 set after fertilization, the microvilli are partially elongated and the cortical granules have begun to exocytose. By 90 set after fertilization, there is approx. a 3- to 4-fold increase in the length of the microvilli as compared to the unfertilized egg. The microvilli vary from about 0.15-0.30 (urn in diameter and 0.3-0.6 pm in length before fertilization. After fertilization, the microvilli appear to be more uniform in appearance. They are usually
1. Lytechinus, unfertilized egg in normal sea water; microvilli are very short and cortical granules are intact. M, microvilli; CC, cortical granules.
Fig.
x7ooo. Fig. 2. Lytechinus
egg, 45 set after fertilization in normal sea water; microvilli are partially elongated and cortical granules have begun to exocytose. x7 000. Fig. 3. Lytechinus egg, 90 set after fertilization in normal sea water; microvilli are fully elongated and no intact cortical granules remain. X 7 000. Fig. 4. Unfertilized Lytechinus egg treated with papain for 8 min; microvilli are irregular in form and cortical granules are irregularly distributed. x7000.
459
edge would result such that various portions of the cell membrane might at any time become dominant. Random changes in direction would occur more frequently than in a fibroblast with abundant lamellar cytoplasm and more numerous adhesion sites, especially if migration rates were high. Thus, the more deficient lamellar cytoplasm of the neoplastic cell with possibly fewer attachment sites and the high migration rate may account for the loss of a persistent walk observed in the present study.
25. Vasiliev, J M, Gelfand, I M, Domnina, L V, Ivanova, 0 Y, Komm, S G & Olshevskaja, L V, J embryo1 exp morphol24 (1970) 625. 26. Vasiliev, G M & Gelfand, I M, Ciba foundation symposium I4 (1973) 3I 1. 27. Weiss, P. J exp zool 100 (1945) 353. 28. Wetzel, B, Sanford, K K, Fox, C H, Jones, G M, Westbrook, E W & Tarone, R E, Cancer res 37 (1977) 831.
References
Effect of Distamycin chromosomes
1. Abercrombie, M, Heaysman, J E M & Karthauser, H M, Exp cell res 13 (1957) 276. 2. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exp cell res 67 (1971) 359. 3. Allison. A C. Locomotion of tissue cells. D. 109. Ciba Foundation Symposium I4 (1973). 4. Berman. L D. Int i cancer I.5 (1975) 973. 5. Carter, S B, Nat&e 208 (1965) 1183. 6. Dipasquale, A, Exp cell res 95 (1975) 425. 7. Domnina, L V, Ivanova, 0 Y, Margolis, L B, Olshevskaja, L V, Rovensky, Y A, Vasiliev, J M & Gelfand, I M, Proc natl acad sci US 69 (1972) 248. 8. Evans, V J, Bryant, J C, Kerr, H A & Schilling, E L, Exv cell res 36 (1964) 439. 9. Fox, C *H, Chaconas, P G & Sanford, K K, J microsc 107 (1976) 79. 10. Fox, C H, Dvorak, J A & Sanford, K K, Cancer res 36 (1976) 1556. Il. Fox, C H, Caspersson, T, Kudynowski, J, Sanford, K K & Tarone, R E. Cancer res 37 (1977) 892.
12. Gail, M H & Boone, C W, Biophys j 10 (1970) 980. 13. - Exp cell res 65 (1971) 221. 14. Greenwood, J A & Durand, D, Ann mathematical statistics, vol. 26, p. 233 (1955). 15. Harris, A K, Ciba foundation svmoosium I4 _ _ (1973) 3. 16. - Exp cell res 77 (1973) 285. 17. - Dev biol35 (1973) 97. 18. Martz, E, Phillips, H M & Steinberg, M S, J cell sci I6 (1974) 401. 19. Meltzer, M S, Tucker, R E, Sanford, K K & Leonard, E J, J natl cancer inst 54 (1975) 1177. 20. Revel, J P, Hoch, P & Ho, D, Exp cell res 84 (1974) 207. 21. Sanford, K K, Handleman, S L & Jones, Cl M, Cancer res 37 (1977) 821. 22. Shin, S, Freedman, V H, Risser, R & Pollack, R, Proc natl acad sci US 72 (1975)4435. 23. Steinberg, M S. Ciba foundation svmuosium 14 _ _ (1973) 35. 24. Tucker, R W, Sanford, K K, Handleman, S L & Jones, Cl M, Cancer res 37 (1977) 1571.
Received April 13, 1977 Revised version received June 28, 1977 Accepted July 8, 1977
A on Chinese hamster
G. PRANTERA, M. DI CASTRO, E. MARCHETTI, and A. ROCCHI, Istituto di Genetica e Centro di Genetica Evoluzionistica Roma, I-00185 Rome, Italy
de1 CNR
Universita
di
Distamycin A, an oligopeptide antibiotic, has the property of binding tightly to double helical AT-rich DNA regions, but poorly to GC-rich regions, single-strand DNA and RNA [ 1, 21. The binding takes place, without intercalation, through hydrogen bonding and hydrophobic interactions [3]. In a previous study the cells of the Chinese hamster strain C-125 were treated with H33258 [4], a benzimidazole derivative fluorochrome, which binds to double-strand AT-rich DNA [5] and, like Distamycin A, seems to bind to DNA not by intercalation but by probable interactions hydrophobic in nature [6]. It is, however, possible that the binding geometries of the two compounds are not identical [7]. This fluorochrome induced a precise pattern of interspersed nonspiralized areas on the Chinese hamster chromosomes. The treatment of the chromosomes in the various stages of the cell cycle with substances whose specificity for given bases or base sequences is known and a comparison of the results could be a promising approach Exp Call
Res
109 (1977)
460
Preliminary notes
Table 1. Effect of Distamycin A on Chinese hamster cells treated for different periods of time % of cells
Expt no.
Time of treatment (hours)
Unaffected
Slightly affected
Markedly affected
1 2 3 4
3.5 11 15 30
;: 28 8
10 39 56 72
0 3 16 20
to the problem of the chromosome molecular organization. In the present work the possible effect of Distamycin A on specific chromosome regions was investigated by supplying this compound to the cells of the same Chinese hamster strain.
strain lasts about 15 h: stage S takes about 9 h and stages G2+M 3.5 h [9]. Colchicine was added 2 h before fixing. The slides were stained with aceto-orcein. The preparations examined were obtained with cells treated with Distamycin A at concentration of 100 pg/ ml. A concentration of 200 pg/ml caused a strong decrease in the number of metaphases and no particular increase in effect.
Cells of C-125 Chinese hamster strain [8] grown on coverslips in Petri dishes were supplied with Distamycin A at a concentration of 100 and 200 pg/ml for 3.5, 11, 15 and 30 h. The mean cell cycle of the cells
Results and discussion Distamycin A causes the chromosomes to appear in metaphase in an elongated shape with brief interspersed areas lacking spiralization (fig. la). These areas are always the
Fig. I. Metaphase chromosomes of Chinese hamster cell strain C-125 treated with Distamycin A. A large
number of despiralized areas may be observed. Bar, 10 /.L.
Materials and Methods
EXPCell Res 109 (1977)
Preliminary
-3. Composite partial karyotypes from two cells of Chinese hamster cell strain C-125 treated with Distamycin A (D) and Hoechst 33258 (H). Note that chromosomes from different cells and different treatments show the same despiralized areas.
Fig.
notes
461
by the two compounds are in almost all metaphases practically the same (fig. 2), but that the effect of Distamycin A on these regions is usually less drastic than that of H33258. However, a specific characteristic of Distamycin A is that of inducing in some metaphases a rich sequence of regions spiralized both to greater and lesser extent. Therefore, even though despiralization of the two compounds often coincides, most likely since both bind preferentially to ATrich DNA, Distamycin A seems to have a less drastic but more widespread effect (fig. 1 b). The difference of the effect of the two compounds could be due to a difference in the type of bond and/or a different specificity for different sequences of AT-rich DNA. It seems now to be clear, however, that even if light satellite bands have never been separated from DNA of Chinese hamster in the CsCl density gradient [lo], they do exist, in this DNA, AT-rich sequences scattered along the chromosomes. This hypothesis is supported by the data of Evenson et al. [l l] who observed brief denatured sections at 0.4 k intervals in DNA of Chinese hamster under partial denaturation.
same even if they almost never appear all at once in the same cell or with the same degree of despiralization in the different cells. The results obtained on supplying Distamycin A to cells in culture for a different number of hours are reported in table 1. Some 200 metaphases were examined in each experiment. It can be clearly seen that References during stage S the chromosomes are sensi1. Krey, A K, Allison, R G & Hahn, F E, FEBS lett 29 (1973) 58. tive to the effect of despiralization and that 2. Zimmer, C, Reinert, K E, Luck, G, Wahnert, U, the number of cells influenced increases Lober, G & Thrum, H J, J mol biol 58 (1971) 329. 3. Zimmer, C, Progress in nucleic acid research and with the increase of time of treatment. molecular biologv (ed W E Cohn) vol. 15. D. 285. Given the preferential binding of Dista. Academic Press,-New York (1975). 4. Rocchi, A, Prantera, G, Pimpinelli, S & Di Castro, mycin A for AT-rich regions, it is reasonM, Chromosoma 56 (1976) 41. able to assume that the chromosomal re5. Hilwig, J & Gropp, A, Exp cell res 81 (1973) 474. 6. Comings, D E , Chromosoma 52 (1975) 229. gions which are undercontracted in meta7. Latt, S A & Wohlleb, J C, Chromosoma 52 (1975) phase after treatment, contain AT-rich 291, 8. Pahtti, F, Rizzoni, M, Perticone, P, De Salvia, R DNA. & Olivieri, G, Caryologia 27 (1974) 85. The comparison between the effect in9. Palitti, F, Rocchi, A, Mercanti, A & Olivieri, G, 25 (1972) 365. duced by H33258 and that of Distamycin A 10. Caryologia Comings, D E & Mattoccia, E, Exp cell res 71 shows that chromosomal regions influenced 1972) 113. 30-771804
Exp Cell Res 109 (1977)
462
Preliminary notes
Il. Evenson, D P, Mego, W A & Taylor, J H, Chromosoma 39 (1972) 225. Received April 27, I977 Revised version received July 7, 1977 Accepted July 13, 1977
Microvilli
in sea urchin eggs
Differences in their formation and type EVELYN
SPIEGEL and MELVIN
SPIEGEL, De-
partment of Biological Sciences, Dartmouth Hanover, NH 03755, and Marine Biological tory, Woods Hole. MA 02543, USA
College, Labora-
The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types. Summary.
The surface of the sea urchin egg is covered with microvilli which are very small in the unfertilized egg and become elongated several-fold upon fertilization [14]. It has been suggested that the source of membrane used in the elongation of microvilli is derived from the membranes surrounding the cortical granules [2J]. These granules lie just below the egg plasma membrane and their contents are released upon fertilization by exocytosis [ 1, 5-71. Mazia and coworkers have shown that activation of the unfertilized egg by a variety of agents initiates the elongation of microvilli [8]. We have been able to elicit the same reaction with the enzyme, papain, in unfertilized eggs of Lytechinus pictus and Arbacia punctulata. In addition, using transmission electron microscope studies, these experiments demonstrate that the cortical granule membranes are not involved in microvillus formation in this case. E.rp Crll’Res
109 (1977)
Materials and Methods Gametes of Lytechinus pictus and Arbacia punctulata were used. Methods for handling the gametes and their subsequent preparation for scanning (SEM) and transmission (TEM) electron microscopy have been presented in detail in previous publications [9, IO]. Fertilization membranes were removed enzvmatitally by fertilizing eggs in a solution containing’0.2 % papain plus 0.2 % cysteine . HCI and returning them to normal-sea water after 8 min. The rate and extent of microvillus elongation were followed at I5 set intervals after fertilization in normal sea water controls and in papain-treated eggs. Specimens were examined with TEM and SEM. The possibility that papain might have an effect on the cell surface itself, in addition to the membrane surrounding it, was tested by treating unfertilized eggs with the papain-cysteine . HCI solution for 8 min, the same length of time as that used for removing the fertilization membranes. Thin sections were then observed with TEM.
Results Fig. 1 shows an unfertilized Lytechinus egg. The microvilli are very short and the cortical granules are intact. Figs 2 and 3 are of eggs at 45 and 90 set, respectively, after fertilization in normal sea water, i.e., without papain treatment. At 45 set after fertilization, the microvilli are partially elongated and the cortical granules have begun to exocytose. By 90 set after fertilization, there is approx. a 3- to 4-fold increase in the length of the microvilli as compared to the unfertilized egg. The microvilli vary from about 0.15-0.30 (urn in diameter and 0.3-0.6 pm in length before fertilization. After fertilization, the microvilli appear to be more uniform in appearance. They are usually
1. Lytechinus, unfertilized egg in normal sea water; microvilli are very short and cortical granules are intact. M, microvilli; CC, cortical granules.
Fig.
x7ooo. Fig. 2. Lytechinus
egg, 45 set after fertilization in normal sea water; microvilli are partially elongated and cortical granules have begun to exocytose. x7 000. Fig. 3. Lytechinus egg, 90 set after fertilization in normal sea water; microvilli are fully elongated and no intact cortical granules remain. X 7 000. Fig. 4. Unfertilized Lytechinus egg treated with papain for 8 min; microvilli are irregular in form and cortical granules are irregularly distributed. x7000.
