- Email: [email protected]
Presence of a chromatid core in mitotic and meiotic chromosomes of grasshoppers
Cell Biolog y lnterna tional Reports, Vol. 6, No. 3, March 1982
PRESENCE MEIOTIC J.S.
OF A CHROMATID CHROMOSOMES
Rufas, lnstituto Velszquez,
CORE
IN MITOTIC
261
AND
OF GRASSHOPPERS
G.Gimenez-Martin and P.Esponda de Biologia Celular, CSIC 144. Madrid-6, Spain
ABSTRACT After using a silver staining procedure two deeply stained strucfirst and second meiotic metaphase tures appear in spermatogonial, chromosomes of grasshoppers. First of all, rounded structures of about 0.4-0.6 urn in diameter, which represent kinetochores. Moreover a longitudinal axes, which runs the whole length of each a core or scaffold-like chromat id and which seems to represent structures.This axis is always in close relation with kinetochores. in first meiotic metaphase are The homologous axes of chromosomes very close together at the point were chiasmata take place. INTRODUCTION Chromosome structures have been extensively analysed from different points of view and by using different methods (Bostock and Sumner, 1978). But despite all these studies many problems remain unsolved. With the use of new techniques, other structures which form part of the chromosome but whose chemical composition does not seem to be chromosomal have been observed recently, as scaffold structures in (Paul son and Laemml i , 1977) and synaptonemal mitotic chromosomes pachytene chromosomes ( Moses, complexes which appear related to 1969; Westergaard and von Wettstein, 1972). Silver staining procedures applied to the study of chromosomes have been extensively developed in recent years. These methods have been specially used in the staining of nucleolar organizer regions(Goodpasture and Bloom, 1975). In recent papers it has been shown that a structure which seems to represent the scaffold appears in mi totic chromosomes of mammal ian after silver staining (Howell and Hsu,1979; Satya-Prakash et al. ,1979) . Some authors have shown that synaptoneimpregnation ma1 complex elements are also stained after silver (Fletcher, 1979; Pathak and Hsu, 1979). In the present paper we report the observations of kinetochores and an axial element in mitotic and meiotic metaphase chromatids of four species of grasshoppers after using silver staining procedure.
0309-1651/82/030261-07/$02.00/O
@ 1982Academic
Press Inc. (London)
Ltd.
262 MATERIAL
CellBiologylnternational
Reports, Vol. 6, No. 3, March 1982
AND METHODS
Adult males of Chorthippus jucundus, Chorthippus brunneus, Arcyptera tornosi and Anacridium aegyptium (Orthoptera, Acridida’Gllected from natural populations were used for this study. Testes were fixed in a 3:l ethanol acetic acid mixture. A single seminiferous tubule was squashed in a drop of 50% acetic acid. After removing the converslide, slides were air dried.For silver staining, 1 g AgNO was dissolved in 1 ml of distilled water previously adjusted t a pH 3 with formic acid. A drop of this solution was then placed on the slide and heated in a stove (60°C) from 5-15minutes, and then rinsed with distiled water and air dried. Slides were mounted with Euparal .
RESULTS When first meiotic metaphases are observed after silver staining the chromatin is poorly contrasted, and has a pale yellow colour. Two different darkly stained structures are clearly seen in these chromosomes when observed under both bright field and phase contrast (or phase-interference) optics (Fig. 1). Rounded dark points of about 0.4-0.6 urn in diameter can be seen in the centromeric regions. These points seem to correspond with kinetochores because of their position in the chromosome and the polarization they show during anaphase (Fig. 2a). Moreover their size corresponds to that of kinetochores of grasshoppers previously studied under electron microscopy (Bostock and Summer, 1978). A dark stained axis also appear associated to kinetochores and runs along the entire length of the chromosome (Fig. 1, 2). This regions axis attains a maximum width of about 0.4 urn. Chiasmatic are clearly observed, since the stained axes are very close together in such regions. Two or three of these points can be observed in long chromosomes, and only one or two in medium size and short chromosomes (Fig. 1, 2a-h) . On occasions the double nature of the axis can be seen (Fig. 29, h). This phenomenon is clearly visible in the sex chromosomes where it is possible to see two associated to kinetochores axes, -one per chromatidwhich appear (Fig. 2f) as it occurs in chromosomes from second meiotic meta phase (Fig. 2i). Spermatogonial metaphases observed after silver staining show the same structures: kinetochores and an axis which occupies all the chromatid length (Fig. 2j).
Cell Biology international
Reports, Vol. 6, No. 3, March 1982
263
meiotic metaphase of Ch. jucundus. a, phase la and b : First Fig. contrzt microscopy; b, bright field microscopy. The axi? is ?bservable in all the bFvalents (arrow). In the sex chromosome (X) only one axis is observed. Kinetochores are strongly stained (arrow heads) ~ The marker : 10 urn.
264
CeilBiologylnternationalRepotis,
Vol. 6, No. 3, March 1982
Fig. 2.a : Early first meiotic anaphase of Ch. brunneus. The axis and the-kinetochores can be observed in all the bivalents. One of the chiasmata of one of the bivalents is resolved (arrow). The marker, 5 urn. b-f : Five bivalents and the sex chromosome (f) from a fi rst mieotic metaphase of A. tornosi observed by contrast interference microscopy. The axis and the kinetochores (arros) are observable. Two axes are clearly visible in the sex chromosome. bivalents from first meiotic metaphase of Ch. jucung-h : Selected dus. The double nature of the axis can be seen (arrow). In the site rthe chiasmata, the axes are close toqether. The marker : -2.5 urn. i : Chromosomes of a second meiotic metaphase of Ch inetochores (arrow heads) as we1 1 as an axis oer chromatid can be seen. The marker: 2.5 urn. j : Spermatogbnial metaphase of A: aegyptium. The axis and the kinetochores are visible. The marker 2.5 urn.
Cell Biolog y In ternational
Reports, Vol. 6, No. 3, March 1982
DISCUSSION The silver method used here shows that two types of structures are differentially stained in spermatogonial, first and second meiotic metaphase chromosomes of four species of grasshoppers. The rounded structures undoubtly represent kinetochores due to their size and position. In other cases, by using similar staining procedures kinetochores have also been differentially stained in mammalian mitotic chromosomes (Ved Brat et al., 1979). This differential staining seems to indicate that kinetochores have a chemical component which differs from the rest of the chromosome, an idea which has already been suggested by using cytochemical methods under the electron microscopy (Esponda, 1978; Rieder, 1979). The stained axis could represent a remnant of synaptonemal complex elements. When using silver staining method the synaptonemal complexes are deeply stained under both light ( Fletcher, 1979) and electron microscopy (Westergaard and von Wettstein, 1972). In this regard one would suspect that remnants of these structures would show a similar reaction to silver stain. Remnants of the synaptonexist in different emal camp 1ex have been shown to species of grasshoppers during late prophase and first meiotic metaphase, and these remnants later transform into structures known as polycomplexes (Esponda and Krimer, 1979; Moens and Church, 1979). Neverbe seen theless, these remanants of the synaptonemal complex can in first meiotic chromosomes occupying the region which is located between the chromatids. On the other hand, the axes described in this paper appear as a central element in each chromatid.Additionin observations of the sex chromosome ultrastructure some al IY, grasshoppers demonstrate that such chromosomes have only one single lateral element of the synaptonemal complex (Moens, 1969; Solari and Counce, 1977). It therefore seems difficult to believe that the two stained axis in the sex chromosome are remnants of the synaptonemal complex. Different authors have reported structures which have been denominated scaffold (Paulson and Laemmli, 1977). These structures have been described up to the moment in mitotic chromosomes, appearing as a core of acid proteins. some papers have discussed Recently, the possibility of scaffold structures being the result of certain technical procedures (Hadlaczky et al.,I98la). But the presence of certain types of structures which would have a function in maintaining the chromosomal form cannot be overlooked (Hadlaczky et al., 1981b). In previous reports it has been shown that a core, stained with silver, which seems to represent a scaffold, appear in mitotic metaphase chromosomes of mammals (Howe1 1 and Hsu, 1979). Our observations on the silver axis in spermatogonial mitotic chromosomes as we1 1 as in the second meiotic metaphases suggest that it corresponds to the same structure dealt in these papers.
Cell Biolog y International
266
As the silver not represent gued that it
Reports, Vol. 6, No. 3, March 1982
axis in the first meiotic metaphase remnants of the synaptonemal complex, corresponds to a core or scaffold-like
chromosomes could it can be arstructure.
Despite all these posible explanations the axis described in this paper represents an interesting approach to some topics on cytogenetics, such as chiasma number, position and terminalization phenomena. Anyway more accurate studies of this structure are needed before arriving to any definite conclusions. ACKNOWLEDGMENTS We thank to Drs. J. Gosblvez, C. LGpez-Fernsndez and C. delaTorre for valuable cooperation. J.S.R. is a fellow from INAPE (Spain) and this work was partially done with a grant from the Comisi6n Asesora para la Investigaci6n Cientifica y TBcnica (Spain) and by theAcuerdo de Cooperaci&, CSIC-Universidad de Chile. REFERENCES Bostock, C.J. and Sumner, A.T. (1978) The eucaryotic chromosome. North Holland Publish, Co. Amsterdam. Esponda, P. (1978) Cytochemistry of kinetochores under electron microscopy. Experimental Cel I Research 114, 247-252. Esponda, P. and Krimer, D.B. (1979) Development of the synaptonemal complex and polycomplex formation in three species of grasshoppers. Chromosoma (Berl.) 73, 237-245. Fletcher,J.M. (1979) Light mGroscope analysis of meiotic prophase chromosomes bysilver staining. Chromosoma (Berl.) 72, 241-248. Goodpasture, C. and Bloom, S.E. (1975) Visual izationof nucleolar organizer regions in mammal ian chromosomes using silver staining. Chromosoma (Berl.) 53, 37-50. Hadlaczky, G., Sumner, A.T.and Ross, A. (1 j8Ia) Protein-depleted chromosomes . I. Structure of isolated protein-depleted chromosomes. Chromosoma (Berl .) 81, 537-555. Hadlaczky, G., Sumner, A.T. and Ross, A. (1981b) Protein-depleted chromosomes . I I. Experiments concerning the real i ty of chromosomes. Chromosoma (Berl.) 81, 557-561. Howe1 1 , W.M. and Hsu, T.C. (1979) Chromosome core structure revealed by silver staining. Chromosoma (Berl .) 73, 61-66. (1969) The fine structure of meioticchromosome polaMoens, P.B. rization and pairing in Locusta migratoria spermatocytes. Chromosoma (Berl) 28, l-25. Moens, P.B. and Church, K. 1979) The distribution of synaptonemal complex material in metaphase I bivalents of Locusta and Chloealtis (Orthoptera: Acrididae). Chromosoma (Bet-m 247-K Moses, M.J.D. (1969) Structure and function of the synaptonemal complex. Genetics (Suppl.) 61, 41-51. Pathak, S. and Hsu, T.C. (1 j7j)Silver stained structures in mammal ian meiotic prophase. Chromosoma (Bet-l.) 195-203. -’70
Cell Biolog y In ternational
Reports, Vol. 6, No. 3, March 7982
Paulson, J.R. and Laemmli, U.K. depleted metaphase chromosomes. C.L. (1979) Ribonucleoprotein Rieder, kinetochores in newt lung cell 80, l-9. Satya-Prakash, chromosome
K.L., core
in
Hsu, T.C. mitosis
267
(1977)
The structure of histone12, 817-828. staining of centrioles and spindles. Journal of Cell Biology Cell
and Pathak, and meiosis.
S. (1980) Chromosoma
Behaviour (Berl.)
1-8. Solari, A.J. and Counce, (1977) Synaptonemal complex S.J. typing in Melanoplus differencialis. J. Cell Sci. 26, Ved Brat, S., Verna, R.S. and Dosik, H. (1979) A simplified nique for simultaneous staining of nucleolar organizer and kinetochores. Stain Technology 52, 107-108. Westergaard, M. and Wettstein, D. von (1972) The synaptinemal complex. Ann. Rev. Genet. 6, 71-110.
Received:
23rd
September
1981
of
81 -'
karyo-
229-250. techregions
Modified version accepted: 23rd October 1981
PRESENCE MEIOTIC J.S.
OF A CHROMATID CHROMOSOMES
Rufas, lnstituto Velszquez,
CORE
IN MITOTIC
261
AND
OF GRASSHOPPERS
G.Gimenez-Martin and P.Esponda de Biologia Celular, CSIC 144. Madrid-6, Spain
ABSTRACT After using a silver staining procedure two deeply stained strucfirst and second meiotic metaphase tures appear in spermatogonial, chromosomes of grasshoppers. First of all, rounded structures of about 0.4-0.6 urn in diameter, which represent kinetochores. Moreover a longitudinal axes, which runs the whole length of each a core or scaffold-like chromat id and which seems to represent structures.This axis is always in close relation with kinetochores. in first meiotic metaphase are The homologous axes of chromosomes very close together at the point were chiasmata take place. INTRODUCTION Chromosome structures have been extensively analysed from different points of view and by using different methods (Bostock and Sumner, 1978). But despite all these studies many problems remain unsolved. With the use of new techniques, other structures which form part of the chromosome but whose chemical composition does not seem to be chromosomal have been observed recently, as scaffold structures in (Paul son and Laemml i , 1977) and synaptonemal mitotic chromosomes pachytene chromosomes ( Moses, complexes which appear related to 1969; Westergaard and von Wettstein, 1972). Silver staining procedures applied to the study of chromosomes have been extensively developed in recent years. These methods have been specially used in the staining of nucleolar organizer regions(Goodpasture and Bloom, 1975). In recent papers it has been shown that a structure which seems to represent the scaffold appears in mi totic chromosomes of mammal ian after silver staining (Howell and Hsu,1979; Satya-Prakash et al. ,1979) . Some authors have shown that synaptoneimpregnation ma1 complex elements are also stained after silver (Fletcher, 1979; Pathak and Hsu, 1979). In the present paper we report the observations of kinetochores and an axial element in mitotic and meiotic metaphase chromatids of four species of grasshoppers after using silver staining procedure.
0309-1651/82/030261-07/$02.00/O
@ 1982Academic
Press Inc. (London)
Ltd.
262 MATERIAL
CellBiologylnternational
Reports, Vol. 6, No. 3, March 1982
AND METHODS
Adult males of Chorthippus jucundus, Chorthippus brunneus, Arcyptera tornosi and Anacridium aegyptium (Orthoptera, Acridida’Gllected from natural populations were used for this study. Testes were fixed in a 3:l ethanol acetic acid mixture. A single seminiferous tubule was squashed in a drop of 50% acetic acid. After removing the converslide, slides were air dried.For silver staining, 1 g AgNO was dissolved in 1 ml of distilled water previously adjusted t a pH 3 with formic acid. A drop of this solution was then placed on the slide and heated in a stove (60°C) from 5-15minutes, and then rinsed with distiled water and air dried. Slides were mounted with Euparal .
RESULTS When first meiotic metaphases are observed after silver staining the chromatin is poorly contrasted, and has a pale yellow colour. Two different darkly stained structures are clearly seen in these chromosomes when observed under both bright field and phase contrast (or phase-interference) optics (Fig. 1). Rounded dark points of about 0.4-0.6 urn in diameter can be seen in the centromeric regions. These points seem to correspond with kinetochores because of their position in the chromosome and the polarization they show during anaphase (Fig. 2a). Moreover their size corresponds to that of kinetochores of grasshoppers previously studied under electron microscopy (Bostock and Summer, 1978). A dark stained axis also appear associated to kinetochores and runs along the entire length of the chromosome (Fig. 1, 2). This regions axis attains a maximum width of about 0.4 urn. Chiasmatic are clearly observed, since the stained axes are very close together in such regions. Two or three of these points can be observed in long chromosomes, and only one or two in medium size and short chromosomes (Fig. 1, 2a-h) . On occasions the double nature of the axis can be seen (Fig. 29, h). This phenomenon is clearly visible in the sex chromosomes where it is possible to see two associated to kinetochores axes, -one per chromatidwhich appear (Fig. 2f) as it occurs in chromosomes from second meiotic meta phase (Fig. 2i). Spermatogonial metaphases observed after silver staining show the same structures: kinetochores and an axis which occupies all the chromatid length (Fig. 2j).
Cell Biology international
Reports, Vol. 6, No. 3, March 1982
263
meiotic metaphase of Ch. jucundus. a, phase la and b : First Fig. contrzt microscopy; b, bright field microscopy. The axi? is ?bservable in all the bFvalents (arrow). In the sex chromosome (X) only one axis is observed. Kinetochores are strongly stained (arrow heads) ~ The marker : 10 urn.
264
CeilBiologylnternationalRepotis,
Vol. 6, No. 3, March 1982
Fig. 2.a : Early first meiotic anaphase of Ch. brunneus. The axis and the-kinetochores can be observed in all the bivalents. One of the chiasmata of one of the bivalents is resolved (arrow). The marker, 5 urn. b-f : Five bivalents and the sex chromosome (f) from a fi rst mieotic metaphase of A. tornosi observed by contrast interference microscopy. The axis and the kinetochores (arros) are observable. Two axes are clearly visible in the sex chromosome. bivalents from first meiotic metaphase of Ch. jucung-h : Selected dus. The double nature of the axis can be seen (arrow). In the site rthe chiasmata, the axes are close toqether. The marker : -2.5 urn. i : Chromosomes of a second meiotic metaphase of Ch inetochores (arrow heads) as we1 1 as an axis oer chromatid can be seen. The marker: 2.5 urn. j : Spermatogbnial metaphase of A: aegyptium. The axis and the kinetochores are visible. The marker 2.5 urn.
Cell Biolog y In ternational
Reports, Vol. 6, No. 3, March 1982
DISCUSSION The silver method used here shows that two types of structures are differentially stained in spermatogonial, first and second meiotic metaphase chromosomes of four species of grasshoppers. The rounded structures undoubtly represent kinetochores due to their size and position. In other cases, by using similar staining procedures kinetochores have also been differentially stained in mammalian mitotic chromosomes (Ved Brat et al., 1979). This differential staining seems to indicate that kinetochores have a chemical component which differs from the rest of the chromosome, an idea which has already been suggested by using cytochemical methods under the electron microscopy (Esponda, 1978; Rieder, 1979). The stained axis could represent a remnant of synaptonemal complex elements. When using silver staining method the synaptonemal complexes are deeply stained under both light ( Fletcher, 1979) and electron microscopy (Westergaard and von Wettstein, 1972). In this regard one would suspect that remnants of these structures would show a similar reaction to silver stain. Remnants of the synaptonexist in different emal camp 1ex have been shown to species of grasshoppers during late prophase and first meiotic metaphase, and these remnants later transform into structures known as polycomplexes (Esponda and Krimer, 1979; Moens and Church, 1979). Neverbe seen theless, these remanants of the synaptonemal complex can in first meiotic chromosomes occupying the region which is located between the chromatids. On the other hand, the axes described in this paper appear as a central element in each chromatid.Additionin observations of the sex chromosome ultrastructure some al IY, grasshoppers demonstrate that such chromosomes have only one single lateral element of the synaptonemal complex (Moens, 1969; Solari and Counce, 1977). It therefore seems difficult to believe that the two stained axis in the sex chromosome are remnants of the synaptonemal complex. Different authors have reported structures which have been denominated scaffold (Paulson and Laemmli, 1977). These structures have been described up to the moment in mitotic chromosomes, appearing as a core of acid proteins. some papers have discussed Recently, the possibility of scaffold structures being the result of certain technical procedures (Hadlaczky et al.,I98la). But the presence of certain types of structures which would have a function in maintaining the chromosomal form cannot be overlooked (Hadlaczky et al., 1981b). In previous reports it has been shown that a core, stained with silver, which seems to represent a scaffold, appear in mitotic metaphase chromosomes of mammals (Howe1 1 and Hsu, 1979). Our observations on the silver axis in spermatogonial mitotic chromosomes as we1 1 as in the second meiotic metaphases suggest that it corresponds to the same structure dealt in these papers.
Cell Biolog y International
266
As the silver not represent gued that it
Reports, Vol. 6, No. 3, March 1982
axis in the first meiotic metaphase remnants of the synaptonemal complex, corresponds to a core or scaffold-like
chromosomes could it can be arstructure.
Despite all these posible explanations the axis described in this paper represents an interesting approach to some topics on cytogenetics, such as chiasma number, position and terminalization phenomena. Anyway more accurate studies of this structure are needed before arriving to any definite conclusions. ACKNOWLEDGMENTS We thank to Drs. J. Gosblvez, C. LGpez-Fernsndez and C. delaTorre for valuable cooperation. J.S.R. is a fellow from INAPE (Spain) and this work was partially done with a grant from the Comisi6n Asesora para la Investigaci6n Cientifica y TBcnica (Spain) and by theAcuerdo de Cooperaci&, CSIC-Universidad de Chile. REFERENCES Bostock, C.J. and Sumner, A.T. (1978) The eucaryotic chromosome. North Holland Publish, Co. Amsterdam. Esponda, P. (1978) Cytochemistry of kinetochores under electron microscopy. Experimental Cel I Research 114, 247-252. Esponda, P. and Krimer, D.B. (1979) Development of the synaptonemal complex and polycomplex formation in three species of grasshoppers. Chromosoma (Berl.) 73, 237-245. Fletcher,J.M. (1979) Light mGroscope analysis of meiotic prophase chromosomes bysilver staining. Chromosoma (Berl.) 72, 241-248. Goodpasture, C. and Bloom, S.E. (1975) Visual izationof nucleolar organizer regions in mammal ian chromosomes using silver staining. Chromosoma (Berl.) 53, 37-50. Hadlaczky, G., Sumner, A.T.and Ross, A. (1 j8Ia) Protein-depleted chromosomes . I. Structure of isolated protein-depleted chromosomes. Chromosoma (Berl .) 81, 537-555. Hadlaczky, G., Sumner, A.T. and Ross, A. (1981b) Protein-depleted chromosomes . I I. Experiments concerning the real i ty of chromosomes. Chromosoma (Berl.) 81, 557-561. Howe1 1 , W.M. and Hsu, T.C. (1979) Chromosome core structure revealed by silver staining. Chromosoma (Berl .) 73, 61-66. (1969) The fine structure of meioticchromosome polaMoens, P.B. rization and pairing in Locusta migratoria spermatocytes. Chromosoma (Berl) 28, l-25. Moens, P.B. and Church, K. 1979) The distribution of synaptonemal complex material in metaphase I bivalents of Locusta and Chloealtis (Orthoptera: Acrididae). Chromosoma (Bet-m 247-K Moses, M.J.D. (1969) Structure and function of the synaptonemal complex. Genetics (Suppl.) 61, 41-51. Pathak, S. and Hsu, T.C. (1 j7j)Silver stained structures in mammal ian meiotic prophase. Chromosoma (Bet-l.) 195-203. -’70
Cell Biolog y In ternational
Reports, Vol. 6, No. 3, March 7982
Paulson, J.R. and Laemmli, U.K. depleted metaphase chromosomes. C.L. (1979) Ribonucleoprotein Rieder, kinetochores in newt lung cell 80, l-9. Satya-Prakash, chromosome
K.L., core
in
Hsu, T.C. mitosis
267
(1977)
The structure of histone12, 817-828. staining of centrioles and spindles. Journal of Cell Biology Cell
and Pathak, and meiosis.
S. (1980) Chromosoma
Behaviour (Berl.)
1-8. Solari, A.J. and Counce, (1977) Synaptonemal complex S.J. typing in Melanoplus differencialis. J. Cell Sci. 26, Ved Brat, S., Verna, R.S. and Dosik, H. (1979) A simplified nique for simultaneous staining of nucleolar organizer and kinetochores. Stain Technology 52, 107-108. Westergaard, M. and Wettstein, D. von (1972) The synaptinemal complex. Ann. Rev. Genet. 6, 71-110.
Received:
23rd
September
1981
of
81 -'
karyo-
229-250. techregions
Modified version accepted: 23rd October 1981