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A dormant nucleolus organizer in the guinea pig, Cavia cobaya
Experimenlul
498 A DORMANT
NUCLEOLUS
GUINEA S. OHNO, Department
PIG,
C. WEILER
of Experimental
Pathology,
Cdl Research 25, 49S.iO.3 (1961)
ORGANIZER
CAVIA
COBAYA’
and CHRISTINA City
of Hope
IN THE
Medical
STENIUS2 Center, Duarte, California,
U.S.A.
Received May 2, 1961
mammals, the first evidence that two homologous chromosomes within the same diploid nucleus did not function synchronously was obtained from regenerating liver cells of female rats [9]. One of the two X-chromosomes behaved as though made of euchromatin, remaining long and thread-like, while the other persistently demonstrated the precocious condensation inherent in all heterochromatic material, forming an interphase chromocenter known as the Barr sex chromatin body [a]. Significantly enough, the same behavior difference in the sex pair was also observed on female somatic cells are considered of highly inbred strains of mice, whose X-chromosomes essentially isogenic [S]. However, asynchronous behavior of two homologues within the same nucleus is not confined to the almost entirely heterochromatic X-chromosomes. Here we report a distinct difference in the nucleolus-organizing function of the largest pair of autosomes in the guinea pig, Cavia cobaya. IN
MATERIALS
AND
METHODS
Ten guinea pigs, which were not inbred, were acquired from two different sources. Mitotic figures of somatic cells were studied on adult bone marrow and fetal liver of both sexes, and ovarian follicles of adult females. Spermatogonial mitotic figures were obtained from testes of fetuses and newborns, and meiotic figures from adult testes. Preparations were made in accordance with our previously described Feulgen squash technique [lo]. Some preparations, however, after completion of hydrolysis with I N HCl, were stained with Giemsa solution instead of going through Schiff’s reagent. In addition to saving time, this procedure resulted in more intensely stained chromosomes. Most slides were counterstained with 1 per cent light green. 1 This work was supported in part by Grant C-5138 from the National Cancer Institute, Public Health Service. a The authors gratefully acknowledge the editorial assistance of Patricia A. Ray. Experimental
Cell Research 25
U.S.
Nucleolus-organizer
in guinea pig
499
OBSERVATIONS
Chromosome complement of Cavia cobaya The diploid chromosome number of this species has been determined as 64 [S, 71, and the morphological characteristics of individual chromosomes at somatic metaphase described in detail [l]. The X, which is submediocentric, corresponds in size to the second largest autosome, which has a subterminally-located kinetochore. Positive identilication of the Y at male somatic metaphase was difficult. Judging by its appearance at first and second meiotic metaphase, the Y is probably one of the very small acrocentric chromosomes.
Heteromorphic appearance of the largest pair at metaphase As shown in Figs. 1 and 2, the largest pair in this species consists of acrocentric autosomes. Invariably, they appeared heteromorphic, with one member of the pair demonstrating a distinct secondary constriction, or SAT-zone, at the middle of its second arm, while the second arm of the other member was in such an advanced state of condensation that the presence of the SAT-zone was completely concealed. As a consequence, the second arm of the former appeared much the longer of the two. Heteromorphism was clearly evident in all well-prepared metaphase figures, regardless of tissue origin, age, or sex. Furthermore, since all individuals examined were unrelated, the possibility of structural heterozygosity was ruled out. Obviously, this heteromorphism persists into meiosis. The substantial size
All photomicrographs shown in Figs. 1-5 were taken by Leitz Panphoto. Lenses used: 90 x 12. Fig. l.-Serial alignment of the 64 chromosomes of the guinea pig, obtained from an ovarian follicular cell of an adult female. The X-pair is underlined; an arrow indicates the satellited second arm of one member of the largest pair. Experimental
Cell Research 25
S. Ohno, C. Weiler and Christina Sfenius difference between the two second arms may be noted on the largest hiralent in a male diakinesis figure shown in Fig. 3. In squash preparations of adult testes, two second meiotic metaphase figures were often found in close proximity, indicating that they were daughter cells. Observations revealed no correlation between the segregation of the X from the Y, and the segregation of the largest autosome with a SAT-zone from that without a SAT-zone.
Participation
of the largest autosome in nucleolar organization
Regardless of sex, each diploid prophase figure contained one or two nucleoli. Several chromosomes appeared to participate in nucleolar organization but there was no difficulty in distinguishing the largest element from the other nucleolus-associated chromosomes, which were of small size. The spermatogonial nucleus shown in Fig. 4 represents the situation encountered at prophase. One large nucleolus at the right is associated with three small chromosomes and one of the largest elements in the nucleus. At a glance, the largest element appears to terminate in the nucleolus. Actually, however, from the attached end, a thin, Feulgen-positive thread of chromonemata of the SAT-zone traverses the nucleolus, ending in a small knob of chromosomal material representing a satellite of the second arm. The nucleolus at the left is associated with four small chromosomes. The other member of the largest pair is apparently not participating in nucleolar organization, as it lies midway between the two nucleoli, unattached to either. Positive heteropycnosis of its second arm may be noted. In all diploid prophase nuclei examined, one member of the largest pair was invariably associated with a nucleolus, while the other was never found to participate in nucleolus organization. Male pachytene figures from adult testes revealed the peculiar configuraFig. 2.-The heteromorphic appearance of the largest extremely stretched pairs from three ovarian follicular satellited second arms.
autosomal homologues is shown on three cells of an adult female. Arrows indicate
Fig. 3.-A diakinesis figure from an adult testis. Heteromorphism is obvious on the largest autosomal bivalent; an arrow indicates the satellited second arm. An X and Y in end-to-end association are seen at the upper left corner. Fig. One lies may
4.-A spermatogonial prophase nucleus from a fetal testis. Dotted lines indicate two nucleoli. member of the largest pair is associated with a large nucleolus at the right. The other member midway between two nucleoli. Positive heteropycnosis of its non-participating second arm be noted. At the lower left, the positively heteropycnotic Y-chromosome is seen.
Fig. 5.-A pachytene figure from an adult testis. A nucleolus associated with the largest autosomal bivalent is outlined. The positively heteropycnotic XY-bivalent embedded within the sex vesicle is seen at the upper middle. Experimental
Cell Research 25
Nucleolus-organizer
33 - 61137267
in guinea pig
Experimental
501
Cell Research 25
S. Ohno, C. Weiler and Christina
Stenius
tion of the largest autosomal bivalent. As sholvn in the bottom center of Fig. 5, the largest bivalent is split into two at one end. One half carries a nucleolus with a satellite on top, which is isopycnotic to other regions of the bivalent. There is no nucleolar material on the other half, which is intensely heteropycnotic.
Fig. 6.-Heteromorphism of the largest pair of autosomes at various stages of mitosis and meiosis is schematically illustrated. Isopycnotic areas are shown as dotted and positively heteropycnotic areas are shown as solid black. Nucleoli are indicated as circles. A, diploid metaphase; B, diploid prophase; C, pachytene; and D, diakinesis.
Fig. 6 is a semi-schematic illustration of the heteromorphic appearance of members of the largest autosomal pair at somatic metaphase and prophase, and the unique configuration of the largest bivalent at pachytene and diplotene. DISCUSSION
Classical studies on nucleolus-organizers of the salamander, Ambystoma tigrinum, by Dearing [3] and of the saffron, Crocus sativus, by Gates and Pathak [5] have already shown that diploid nuclei may contain odd numbers of nucleolus-organizing chromosomes, suggesting that one member of such pairs may remain dormant in nucleolus organization. Similarly, the heteromorphism of homologous pairs due to a conspicuous secondary constriction on only one member has been described on the second largest autosomes of Rattus noruegicus [l l] and the X-chromosome pair of Cricetus griseus [12 1. The comprehensive study here reported of the largest autosomal pair of Cavia cobaya has revealed that although both members have an inherent capacity for nucleolus-organizing, only one member of the pair in any given nucleus demonstrates a SAT-zone on its second arm and actually participates Experimenfai
Cell Research 25
Nucleolus-organizer
in guinea pig
503
in nucleolus formation during interphase. The second arm of the other member remains heavily condensed, and its nucleolus-organizer inert. Ferguson-Smith recently reported that, in man, all five pairs of acrocentric autosomes exhibit nucleolar satellites at their second arms at one time or another in tissue culture [4]. Yet our study on fetal liver cells of man in viva (to be reported elsewhere) has shown that the number of chromosomes in association with nucleoli at prophase is, at the most, six. It may well be that here, too, one member of certain nucleolus-organizing pairs may not participate in this function. Present findings on the largest autosomal pair of the guinea pig, and previous reports on the X-chromosome pair of many mammalian species, weaken the long-held belief that heteromorphism indicates an inherent structural difference between the homologues. Perhaps many homologous regions of chromosome pairs function differently from each other in living diploid mammalian nuclei. SUMMARY
1. The largest elements in the karyotype of the guinea pig, Cavia cobaya, are a pair of acrocentric autosomes which can be easily distinguished at any stage of mitosis or meiosis. 2. At diploid metaphase, only one member of this pair demonstrates a secondary constriction on its second arm. The second arm of the other member remains positively heteropycnotic. 3. At diploid prophase as well as meiotic prophase, only one member of the pair participates in the organization of the nucleolus. 4. This behavior difference of the largest pair was consistently observed on all somatic and germ cells of all individuals studied. 5. It is possible that in mammals, many homologous regions of chromosome pairs within the same diploid nucleus do not function synchronously. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
AWA, A.. SASAKI, M. and TAKAYAMA, S., Jap. J. 2001. 12, 257 (1959). BAR& M. L. and.BERTRAM, L. F., Nbfure 163, 676 (1949): DEARING, W. H., JR., J. Morphof. 56, 157 (1934). FERGUSON-SMITH, M. A. and HANDMAKER, S. D., Lancet 1, 638 (1961). GATES, R. R. and PATHAK, G. N., Nature 142, 156 (1938). Hsu, T. C. and POMERAT, C. M., J. Heredity 43, 167 (1953). MAKINO, S., J. Fat. Sei., Hokkaido Univ., Ser. VI, 2001. 9, 345 (1947). OHNO, S. and HAUSCHKA, T. S., Cancer Research 20, 541 (1960). OHNO, S., KAPLAN, W. D. and KINOSITA, R., Expfl. Cell Research 18, 415 (1959).
10. ~ ibid. 13, 358 (1957). 11. OHNO, S. and KINOSITA, R., ibid. 8, 558 (1955). 12.
YERGANIAN, Physiology
G., KATO, of Neoplasia,
R., LEONARD, M. J., GAGNON, H J. and GRODZINS, pp. 49-96. Univ. of Texas Press, Austin, 1960.
Experimental
L. A., in Cell Cell Research 25
498 A DORMANT
NUCLEOLUS
GUINEA S. OHNO, Department
PIG,
C. WEILER
of Experimental
Pathology,
Cdl Research 25, 49S.iO.3 (1961)
ORGANIZER
CAVIA
COBAYA’
and CHRISTINA City
of Hope
IN THE
Medical
STENIUS2 Center, Duarte, California,
U.S.A.
Received May 2, 1961
mammals, the first evidence that two homologous chromosomes within the same diploid nucleus did not function synchronously was obtained from regenerating liver cells of female rats [9]. One of the two X-chromosomes behaved as though made of euchromatin, remaining long and thread-like, while the other persistently demonstrated the precocious condensation inherent in all heterochromatic material, forming an interphase chromocenter known as the Barr sex chromatin body [a]. Significantly enough, the same behavior difference in the sex pair was also observed on female somatic cells are considered of highly inbred strains of mice, whose X-chromosomes essentially isogenic [S]. However, asynchronous behavior of two homologues within the same nucleus is not confined to the almost entirely heterochromatic X-chromosomes. Here we report a distinct difference in the nucleolus-organizing function of the largest pair of autosomes in the guinea pig, Cavia cobaya. IN
MATERIALS
AND
METHODS
Ten guinea pigs, which were not inbred, were acquired from two different sources. Mitotic figures of somatic cells were studied on adult bone marrow and fetal liver of both sexes, and ovarian follicles of adult females. Spermatogonial mitotic figures were obtained from testes of fetuses and newborns, and meiotic figures from adult testes. Preparations were made in accordance with our previously described Feulgen squash technique [lo]. Some preparations, however, after completion of hydrolysis with I N HCl, were stained with Giemsa solution instead of going through Schiff’s reagent. In addition to saving time, this procedure resulted in more intensely stained chromosomes. Most slides were counterstained with 1 per cent light green. 1 This work was supported in part by Grant C-5138 from the National Cancer Institute, Public Health Service. a The authors gratefully acknowledge the editorial assistance of Patricia A. Ray. Experimental
Cell Research 25
U.S.
Nucleolus-organizer
in guinea pig
499
OBSERVATIONS
Chromosome complement of Cavia cobaya The diploid chromosome number of this species has been determined as 64 [S, 71, and the morphological characteristics of individual chromosomes at somatic metaphase described in detail [l]. The X, which is submediocentric, corresponds in size to the second largest autosome, which has a subterminally-located kinetochore. Positive identilication of the Y at male somatic metaphase was difficult. Judging by its appearance at first and second meiotic metaphase, the Y is probably one of the very small acrocentric chromosomes.
Heteromorphic appearance of the largest pair at metaphase As shown in Figs. 1 and 2, the largest pair in this species consists of acrocentric autosomes. Invariably, they appeared heteromorphic, with one member of the pair demonstrating a distinct secondary constriction, or SAT-zone, at the middle of its second arm, while the second arm of the other member was in such an advanced state of condensation that the presence of the SAT-zone was completely concealed. As a consequence, the second arm of the former appeared much the longer of the two. Heteromorphism was clearly evident in all well-prepared metaphase figures, regardless of tissue origin, age, or sex. Furthermore, since all individuals examined were unrelated, the possibility of structural heterozygosity was ruled out. Obviously, this heteromorphism persists into meiosis. The substantial size
All photomicrographs shown in Figs. 1-5 were taken by Leitz Panphoto. Lenses used: 90 x 12. Fig. l.-Serial alignment of the 64 chromosomes of the guinea pig, obtained from an ovarian follicular cell of an adult female. The X-pair is underlined; an arrow indicates the satellited second arm of one member of the largest pair. Experimental
Cell Research 25
S. Ohno, C. Weiler and Christina Sfenius difference between the two second arms may be noted on the largest hiralent in a male diakinesis figure shown in Fig. 3. In squash preparations of adult testes, two second meiotic metaphase figures were often found in close proximity, indicating that they were daughter cells. Observations revealed no correlation between the segregation of the X from the Y, and the segregation of the largest autosome with a SAT-zone from that without a SAT-zone.
Participation
of the largest autosome in nucleolar organization
Regardless of sex, each diploid prophase figure contained one or two nucleoli. Several chromosomes appeared to participate in nucleolar organization but there was no difficulty in distinguishing the largest element from the other nucleolus-associated chromosomes, which were of small size. The spermatogonial nucleus shown in Fig. 4 represents the situation encountered at prophase. One large nucleolus at the right is associated with three small chromosomes and one of the largest elements in the nucleus. At a glance, the largest element appears to terminate in the nucleolus. Actually, however, from the attached end, a thin, Feulgen-positive thread of chromonemata of the SAT-zone traverses the nucleolus, ending in a small knob of chromosomal material representing a satellite of the second arm. The nucleolus at the left is associated with four small chromosomes. The other member of the largest pair is apparently not participating in nucleolar organization, as it lies midway between the two nucleoli, unattached to either. Positive heteropycnosis of its second arm may be noted. In all diploid prophase nuclei examined, one member of the largest pair was invariably associated with a nucleolus, while the other was never found to participate in nucleolus organization. Male pachytene figures from adult testes revealed the peculiar configuraFig. 2.-The heteromorphic appearance of the largest extremely stretched pairs from three ovarian follicular satellited second arms.
autosomal homologues is shown on three cells of an adult female. Arrows indicate
Fig. 3.-A diakinesis figure from an adult testis. Heteromorphism is obvious on the largest autosomal bivalent; an arrow indicates the satellited second arm. An X and Y in end-to-end association are seen at the upper left corner. Fig. One lies may
4.-A spermatogonial prophase nucleus from a fetal testis. Dotted lines indicate two nucleoli. member of the largest pair is associated with a large nucleolus at the right. The other member midway between two nucleoli. Positive heteropycnosis of its non-participating second arm be noted. At the lower left, the positively heteropycnotic Y-chromosome is seen.
Fig. 5.-A pachytene figure from an adult testis. A nucleolus associated with the largest autosomal bivalent is outlined. The positively heteropycnotic XY-bivalent embedded within the sex vesicle is seen at the upper middle. Experimental
Cell Research 25
Nucleolus-organizer
33 - 61137267
in guinea pig
Experimental
501
Cell Research 25
S. Ohno, C. Weiler and Christina
Stenius
tion of the largest autosomal bivalent. As sholvn in the bottom center of Fig. 5, the largest bivalent is split into two at one end. One half carries a nucleolus with a satellite on top, which is isopycnotic to other regions of the bivalent. There is no nucleolar material on the other half, which is intensely heteropycnotic.
Fig. 6.-Heteromorphism of the largest pair of autosomes at various stages of mitosis and meiosis is schematically illustrated. Isopycnotic areas are shown as dotted and positively heteropycnotic areas are shown as solid black. Nucleoli are indicated as circles. A, diploid metaphase; B, diploid prophase; C, pachytene; and D, diakinesis.
Fig. 6 is a semi-schematic illustration of the heteromorphic appearance of members of the largest autosomal pair at somatic metaphase and prophase, and the unique configuration of the largest bivalent at pachytene and diplotene. DISCUSSION
Classical studies on nucleolus-organizers of the salamander, Ambystoma tigrinum, by Dearing [3] and of the saffron, Crocus sativus, by Gates and Pathak [5] have already shown that diploid nuclei may contain odd numbers of nucleolus-organizing chromosomes, suggesting that one member of such pairs may remain dormant in nucleolus organization. Similarly, the heteromorphism of homologous pairs due to a conspicuous secondary constriction on only one member has been described on the second largest autosomes of Rattus noruegicus [l l] and the X-chromosome pair of Cricetus griseus [12 1. The comprehensive study here reported of the largest autosomal pair of Cavia cobaya has revealed that although both members have an inherent capacity for nucleolus-organizing, only one member of the pair in any given nucleus demonstrates a SAT-zone on its second arm and actually participates Experimenfai
Cell Research 25
Nucleolus-organizer
in guinea pig
503
in nucleolus formation during interphase. The second arm of the other member remains heavily condensed, and its nucleolus-organizer inert. Ferguson-Smith recently reported that, in man, all five pairs of acrocentric autosomes exhibit nucleolar satellites at their second arms at one time or another in tissue culture [4]. Yet our study on fetal liver cells of man in viva (to be reported elsewhere) has shown that the number of chromosomes in association with nucleoli at prophase is, at the most, six. It may well be that here, too, one member of certain nucleolus-organizing pairs may not participate in this function. Present findings on the largest autosomal pair of the guinea pig, and previous reports on the X-chromosome pair of many mammalian species, weaken the long-held belief that heteromorphism indicates an inherent structural difference between the homologues. Perhaps many homologous regions of chromosome pairs function differently from each other in living diploid mammalian nuclei. SUMMARY
1. The largest elements in the karyotype of the guinea pig, Cavia cobaya, are a pair of acrocentric autosomes which can be easily distinguished at any stage of mitosis or meiosis. 2. At diploid metaphase, only one member of this pair demonstrates a secondary constriction on its second arm. The second arm of the other member remains positively heteropycnotic. 3. At diploid prophase as well as meiotic prophase, only one member of the pair participates in the organization of the nucleolus. 4. This behavior difference of the largest pair was consistently observed on all somatic and germ cells of all individuals studied. 5. It is possible that in mammals, many homologous regions of chromosome pairs within the same diploid nucleus do not function synchronously. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
AWA, A.. SASAKI, M. and TAKAYAMA, S., Jap. J. 2001. 12, 257 (1959). BAR& M. L. and.BERTRAM, L. F., Nbfure 163, 676 (1949): DEARING, W. H., JR., J. Morphof. 56, 157 (1934). FERGUSON-SMITH, M. A. and HANDMAKER, S. D., Lancet 1, 638 (1961). GATES, R. R. and PATHAK, G. N., Nature 142, 156 (1938). Hsu, T. C. and POMERAT, C. M., J. Heredity 43, 167 (1953). MAKINO, S., J. Fat. Sei., Hokkaido Univ., Ser. VI, 2001. 9, 345 (1947). OHNO, S. and HAUSCHKA, T. S., Cancer Research 20, 541 (1960). OHNO, S., KAPLAN, W. D. and KINOSITA, R., Expfl. Cell Research 18, 415 (1959).
10. ~ ibid. 13, 358 (1957). 11. OHNO, S. and KINOSITA, R., ibid. 8, 558 (1955). 12.
YERGANIAN, Physiology
G., KATO, of Neoplasia,
R., LEONARD, M. J., GAGNON, H J. and GRODZINS, pp. 49-96. Univ. of Texas Press, Austin, 1960.
Experimental
L. A., in Cell Cell Research 25