- Email: [email protected]
The attachment of pachytene chromosomes to the nuclear membrane in mammalian spermatocytes
Experimental
657
Cell Research 42, 657-661 (1966)
THE ATTACHMENT OF PACHYTENE CHROMOSOMES TO THE NUCLEAR MEMBRANE MAMMALIAN SPERMATOCYTES D. H.
M. WOOLLAM, Department
IN
E. H. R. FORD and J. W. MILLEN Cambridge University, England
of Anatomy,
Received December 24, 1965
SPERMATOCYTESat pachynema show on electron microscopic examination complex” [a], which unites the chromothe presence of a “synaptinemal somes of maternal and paternal origin. This complex appears therefore as a core forming the matrix of the paired bivalents. Since it is, at the present time, the only chromosomal element readily identifiable on electron microscopic examination, its presence provides an obvious method for the identification of the loci of the chromosomes in relation both to other nuclear elements and to each other. The object of the present investigation was to study the points of attachment of the complexes to the nuclear membranes of spermatocytes of three animals, the mouse, the field vole and the golden hamster, and to investigate whether statistical analysis of the results supported any general hypothesis concerning the attachment of pachytene chromosomes to the nuclear membrane. MATERIALS
Preparations
AND
METHODS
were made from fresh testis of mouse (Mus
musculus), field vole and golden hamster (Cricetus auratus); (a) Light microscopic preparations were made by a variation on the standard techniques for chromosome analysis which has previously been described in detail [4]; (b) Small pieces of testis were fixed in Zetterqvist’s buffered isotonic osmium tetroxide for 45-60 min, then dehydrated over a period of 30 min in an ice-cold ethanol series and embedded in araldite. Both thin and thick sections were cut with a Huxley ultramicrotome and mounted on bare grids. Sections cut at a thickness of 190-240 rnp and stained in lead hydroxide were used for counting the number of points of attachment of the synaptinemal complex to the nuclear membrane in individual cells. The criterion for including a cell in the series was the presence within the nucleus of a portion of synaptinemal complex. After a preliminary trial in which 200 cells were examined in both mouse and field vole, it was realized that it would be necessary for purposes of statistical analysis to know the diameter of each nuclear section examined for points of attachment. The diameters of 100 sections of spermatocytes at pachynema from each of the three mammals were then measured and, at the same time the presence of synaptinemal complex attachments to the nuclear membrane in each cell section was recorded. The data thus obtained was analysed at the Statistical Laboratory, Cambridge. (Microtus
agrestis)
Experimental
Cell Research 42
658
Il. H. M. Woollam, E. H. R. Ford and J. IV. Millen
RESULTS
Statistical
analysis
Statistical analysis of the data which took into account the variation in thickness of the sections as being from 190-240 m,u revealed that 95 per cent confidence intervals for the number of points of attachment of the pachytene chromosomes to the nuclear membrane were as follows: mouse, 36-52; field vole, 41-57; golden hamster 35-50. Cytological
studies
In rare sections, as in that from the field vole testis shown in Fig. 1 a, the entire length of synaptinemal complex could be studied from one point of attachment to the other. As the metaphase plate from vole testis (Fig. 1 b) indicates, the autosomes of the vole are all either clearly telocentric or possess only minute wisps of chromatin material which may possibly represent short arms. Considerations of the length of the synaptinemal complex shown in the electron micrograph (Fig. 1 a) suggest that it is probably from one of the smaller bivalents (arrowed in the pachytene bouquet, Fig. 1 c) and that the complex and thus the bivalent is attached at either end. Since these autosomes are telocentric, one end corresponds to the centromere and the other to the distal end of the autosome. In the mouse the autosomes are also telocentric and have heterochromatic regions adjacent to the kinetochore loci [3]. The presence of these heterochromatic regions made it possible to identify the position of the centromere in electron micrographs of the mouse spermatocyte but no similar structures could generally be found in the electron micrographs of the field vole and golden hamster. In Fig. 1 a from the field vole it is impossible therefore to say which end of the synaptinemal complex is which. After the hypothesis was set up that pachytene chromosomes are attached at either end to the nuclear membrane and at no other point along their length, a careful search was made in material from the three mammals for appearances which would contradict this hypothesis. Where long sections of synaptinemal complex were encountered they invariably seemed to be directed at right angles to the nuclear membrane. Only on rare occasions was Fig. l.-(a) Electron micrograph of primary spermatocyte of field vole at pachynema. x 21,000. The synaptinemal complex of a small bivalent is shown throughout its entire length. (b) Metaphase chromosomes from field vole testis. x 2000. (c) Pachytene chromosomes from field vole testis. x 2800. The small bivalent arrowed is of a size consonant with its corresponding to the chromosome area in (a). Experimental
Cell Research 42
Attachment
of pachytene
chromosomes to nuclear membrane
Experimental
659
Cell Research 42
660
D. H. M. Woollam, E. H. R. Ford and J. U’. Millen
a section of synaptinemal complex seen to run either closely parallel or tangential to the membrane and then only for short distances. No evidence was seen to suggest that the complex gained attachment to the membrane for a short distance and then immediately moved away from it. A finding of this kind might be expected under two circumstances, firstly, if the synaptinemal complex of a bivalent were to be attached to the nuclear membrane other than at the ends, secondly, and this applies in the present study only to the hamster material, if a meta- or acrocentric chromosome were to be attached to the membrane at the centromere. Examination of the situation and numbers of mitochondria encountered suggested that, in the sectioned material, these bore no specific relationship to the points of attachment of pachytene chromosomes. The conclusions drawn from the study of the sections under the electron microscope therefore were that the pachytene bivalents in the mammals studied are attached to the nuclear membrane at either end but at no point along the length of the chromosome.
DISCUSSION
Table I shows the number of attachments of the bivalents to the nuclear membrane as predicted by certain hypotheses as to their mode of attachment. The findings of the investigation reported in Table I fit the hypothesis that the bivalents are each attached at both ends and further suggests that in the hamster (where the chromosomes are mainly acre- or metacentric) the attachment is still at both ends only, and not additionally at the centromere. In the mouse, as has been reported elsewhere [5], the centromeric point of attachment is identifiable from the distal point of attachment by virtue of the heterochromatic basal knob which surrounds the centromeric end of the telocentric chromosomes. This enables the two types of ending to be separately identified in relation to the nuclear membrane. It has been found that the centromeric endings are, in the mouse, clustered around the sex vesicle with the distal ends more widely dispersed over the surface of the membrane. Unfortunately it has not been possible in the mouse to distinguish individually more than four pairs of autosomes even at mitotic metaphase [l]. It does not appear therefore that the mouse is a particularly suitable animal, in which to pursue the question as to whether each bivalent has a determined pair of points of attachment to the nuclear membrane, which is constant from cell to cell. It is hoped, however, that work now in progress with other mammals will throw some light on this problem, a positive answer to which would lend considerable support to the theory that each pair of autosomes has a set Experimental
Cell Research 42
Attachment
of pachytene
chromosomes to nuclear membrane
661
position in the nucleus, and would suggest the advisability of giving further consideration to the possibility that the observed cytological phenomena of both meiosis and mitosis may be explained on the basis of three processes only (a) replication of DNA, (b) the degree of coiling and uncoiling of the chromosomal elements, (c) sol-gel relationships. TABLE
Animal
No. of attachments assuming bivalents are each attached at one end only
I.
No. of attachments assuming bivalents are attached at both ends
No. of attachments assuming bivalents are attached at each end and at centromeres
No. of attachments revealed by present study
Mouse
19
38
38
36-52
Field vole
24
48
48
41-57
Golden hamster
21
42
58
35-50
SUMMARY
1. Electron microscopic studies and statistical analyses have been made of the points of attachment of the synaptinemal complex to the nuclear membrane of primary spermatocytes in the mouse, field vole and golden hamster. 2. The findings of the investigation support the view that the synaptinemal complex, and hence the bivalent, are attached at both ends and not intermediately, even when the chromosome possesses a sub-terminal centromere. We are grateful to Mr Rollo Davidson of the Statistical Laboratory, Cambridge, for the statistical analysis. Our thanks are also due to Professor J. D. Boyd for his encouragement and to Messrs. K. W. Thurley, J. F. Crane and G. Oakes for the preparation of electron micrographs and photographs.
REFERENCES 1. 2. 3. 4. 5.
FORD, E. H. R. and WOOLLAM, D. H. M., Expfl Cell Res. 32,320 (1963). MOSES, M. M., J. Cell Sol. 2, 215 (1956). OHNO, S., KAPLAN, W. D. and KINOSITA, FL, Exptl Cell Res. 13, 358 (1957). WOOLLAM, D. H. M. and FORD, E. H. R., J. Anat. Land. 98,163 (1964). WOOLLAM, D. H. M., MILLEN, J. W. and FORD, E. H. FL, Nature Land. In press.
Experimental
Cell Research 42
657
Cell Research 42, 657-661 (1966)
THE ATTACHMENT OF PACHYTENE CHROMOSOMES TO THE NUCLEAR MEMBRANE MAMMALIAN SPERMATOCYTES D. H.
M. WOOLLAM, Department
IN
E. H. R. FORD and J. W. MILLEN Cambridge University, England
of Anatomy,
Received December 24, 1965
SPERMATOCYTESat pachynema show on electron microscopic examination complex” [a], which unites the chromothe presence of a “synaptinemal somes of maternal and paternal origin. This complex appears therefore as a core forming the matrix of the paired bivalents. Since it is, at the present time, the only chromosomal element readily identifiable on electron microscopic examination, its presence provides an obvious method for the identification of the loci of the chromosomes in relation both to other nuclear elements and to each other. The object of the present investigation was to study the points of attachment of the complexes to the nuclear membranes of spermatocytes of three animals, the mouse, the field vole and the golden hamster, and to investigate whether statistical analysis of the results supported any general hypothesis concerning the attachment of pachytene chromosomes to the nuclear membrane. MATERIALS
Preparations
AND
METHODS
were made from fresh testis of mouse (Mus
musculus), field vole and golden hamster (Cricetus auratus); (a) Light microscopic preparations were made by a variation on the standard techniques for chromosome analysis which has previously been described in detail [4]; (b) Small pieces of testis were fixed in Zetterqvist’s buffered isotonic osmium tetroxide for 45-60 min, then dehydrated over a period of 30 min in an ice-cold ethanol series and embedded in araldite. Both thin and thick sections were cut with a Huxley ultramicrotome and mounted on bare grids. Sections cut at a thickness of 190-240 rnp and stained in lead hydroxide were used for counting the number of points of attachment of the synaptinemal complex to the nuclear membrane in individual cells. The criterion for including a cell in the series was the presence within the nucleus of a portion of synaptinemal complex. After a preliminary trial in which 200 cells were examined in both mouse and field vole, it was realized that it would be necessary for purposes of statistical analysis to know the diameter of each nuclear section examined for points of attachment. The diameters of 100 sections of spermatocytes at pachynema from each of the three mammals were then measured and, at the same time the presence of synaptinemal complex attachments to the nuclear membrane in each cell section was recorded. The data thus obtained was analysed at the Statistical Laboratory, Cambridge. (Microtus
agrestis)
Experimental
Cell Research 42
658
Il. H. M. Woollam, E. H. R. Ford and J. IV. Millen
RESULTS
Statistical
analysis
Statistical analysis of the data which took into account the variation in thickness of the sections as being from 190-240 m,u revealed that 95 per cent confidence intervals for the number of points of attachment of the pachytene chromosomes to the nuclear membrane were as follows: mouse, 36-52; field vole, 41-57; golden hamster 35-50. Cytological
studies
In rare sections, as in that from the field vole testis shown in Fig. 1 a, the entire length of synaptinemal complex could be studied from one point of attachment to the other. As the metaphase plate from vole testis (Fig. 1 b) indicates, the autosomes of the vole are all either clearly telocentric or possess only minute wisps of chromatin material which may possibly represent short arms. Considerations of the length of the synaptinemal complex shown in the electron micrograph (Fig. 1 a) suggest that it is probably from one of the smaller bivalents (arrowed in the pachytene bouquet, Fig. 1 c) and that the complex and thus the bivalent is attached at either end. Since these autosomes are telocentric, one end corresponds to the centromere and the other to the distal end of the autosome. In the mouse the autosomes are also telocentric and have heterochromatic regions adjacent to the kinetochore loci [3]. The presence of these heterochromatic regions made it possible to identify the position of the centromere in electron micrographs of the mouse spermatocyte but no similar structures could generally be found in the electron micrographs of the field vole and golden hamster. In Fig. 1 a from the field vole it is impossible therefore to say which end of the synaptinemal complex is which. After the hypothesis was set up that pachytene chromosomes are attached at either end to the nuclear membrane and at no other point along their length, a careful search was made in material from the three mammals for appearances which would contradict this hypothesis. Where long sections of synaptinemal complex were encountered they invariably seemed to be directed at right angles to the nuclear membrane. Only on rare occasions was Fig. l.-(a) Electron micrograph of primary spermatocyte of field vole at pachynema. x 21,000. The synaptinemal complex of a small bivalent is shown throughout its entire length. (b) Metaphase chromosomes from field vole testis. x 2000. (c) Pachytene chromosomes from field vole testis. x 2800. The small bivalent arrowed is of a size consonant with its corresponding to the chromosome area in (a). Experimental
Cell Research 42
Attachment
of pachytene
chromosomes to nuclear membrane
Experimental
659
Cell Research 42
660
D. H. M. Woollam, E. H. R. Ford and J. U’. Millen
a section of synaptinemal complex seen to run either closely parallel or tangential to the membrane and then only for short distances. No evidence was seen to suggest that the complex gained attachment to the membrane for a short distance and then immediately moved away from it. A finding of this kind might be expected under two circumstances, firstly, if the synaptinemal complex of a bivalent were to be attached to the nuclear membrane other than at the ends, secondly, and this applies in the present study only to the hamster material, if a meta- or acrocentric chromosome were to be attached to the membrane at the centromere. Examination of the situation and numbers of mitochondria encountered suggested that, in the sectioned material, these bore no specific relationship to the points of attachment of pachytene chromosomes. The conclusions drawn from the study of the sections under the electron microscope therefore were that the pachytene bivalents in the mammals studied are attached to the nuclear membrane at either end but at no point along the length of the chromosome.
DISCUSSION
Table I shows the number of attachments of the bivalents to the nuclear membrane as predicted by certain hypotheses as to their mode of attachment. The findings of the investigation reported in Table I fit the hypothesis that the bivalents are each attached at both ends and further suggests that in the hamster (where the chromosomes are mainly acre- or metacentric) the attachment is still at both ends only, and not additionally at the centromere. In the mouse, as has been reported elsewhere [5], the centromeric point of attachment is identifiable from the distal point of attachment by virtue of the heterochromatic basal knob which surrounds the centromeric end of the telocentric chromosomes. This enables the two types of ending to be separately identified in relation to the nuclear membrane. It has been found that the centromeric endings are, in the mouse, clustered around the sex vesicle with the distal ends more widely dispersed over the surface of the membrane. Unfortunately it has not been possible in the mouse to distinguish individually more than four pairs of autosomes even at mitotic metaphase [l]. It does not appear therefore that the mouse is a particularly suitable animal, in which to pursue the question as to whether each bivalent has a determined pair of points of attachment to the nuclear membrane, which is constant from cell to cell. It is hoped, however, that work now in progress with other mammals will throw some light on this problem, a positive answer to which would lend considerable support to the theory that each pair of autosomes has a set Experimental
Cell Research 42
Attachment
of pachytene
chromosomes to nuclear membrane
661
position in the nucleus, and would suggest the advisability of giving further consideration to the possibility that the observed cytological phenomena of both meiosis and mitosis may be explained on the basis of three processes only (a) replication of DNA, (b) the degree of coiling and uncoiling of the chromosomal elements, (c) sol-gel relationships. TABLE
Animal
No. of attachments assuming bivalents are each attached at one end only
I.
No. of attachments assuming bivalents are attached at both ends
No. of attachments assuming bivalents are attached at each end and at centromeres
No. of attachments revealed by present study
Mouse
19
38
38
36-52
Field vole
24
48
48
41-57
Golden hamster
21
42
58
35-50
SUMMARY
1. Electron microscopic studies and statistical analyses have been made of the points of attachment of the synaptinemal complex to the nuclear membrane of primary spermatocytes in the mouse, field vole and golden hamster. 2. The findings of the investigation support the view that the synaptinemal complex, and hence the bivalent, are attached at both ends and not intermediately, even when the chromosome possesses a sub-terminal centromere. We are grateful to Mr Rollo Davidson of the Statistical Laboratory, Cambridge, for the statistical analysis. Our thanks are also due to Professor J. D. Boyd for his encouragement and to Messrs. K. W. Thurley, J. F. Crane and G. Oakes for the preparation of electron micrographs and photographs.
REFERENCES 1. 2. 3. 4. 5.
FORD, E. H. R. and WOOLLAM, D. H. M., Expfl Cell Res. 32,320 (1963). MOSES, M. M., J. Cell Sol. 2, 215 (1956). OHNO, S., KAPLAN, W. D. and KINOSITA, FL, Exptl Cell Res. 13, 358 (1957). WOOLLAM, D. H. M. and FORD, E. H. R., J. Anat. Land. 98,163 (1964). WOOLLAM, D. H. M., MILLEN, J. W. and FORD, E. H. FL, Nature Land. In press.
Experimental
Cell Research 42