Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research91 (1974) 365-371
HETEROCHROMATIN
AND NUCLEOLAR
FIRST MEIOTIC A. STAHL, J. M. LUCIANI, Laboratoire
ORGANIZERS
PROPHASE IN QUAIL
M. DEVICTOR,
DURING
OOCYTES
A. M. CAPODANO
and M. HARTUNG
d’Histologie et Embryologic II, Faculte’ de Midecine, F-13385 Marseille Cedex 4, France
SUMMARY The study of chromosomes in oocytes of the quail shows, at the pachytene stage, that microchromosomes are made of a euchromatic segment and a heterochromatic juxtacentromeric region. The heterochromatic regions of the microchromosomes amalgamate between themselves so as to constitute bulky chromocentres from which radiate the euchromatic segments which remain free. At late pachytene, nucleoli appear at the contact of these chromocentres. When the oocytes reach the diplotene stage, the nucleoli become quite large. They are stuck against chromocentres and establish a very close relationship with the euchromatic segments of the microchromosomes which surround or penetrate them. These observations lead one to think that the euchromatic segments of microchromosomes could be bearing nucleolar organizers. The close relations that the nucleolar organizers develop with the bulk of the nucleolus could explain its Feulgen-positive character in the quail.
In quail somatic cell nuclei, the nucleolus presents the peculiarity of being Feulgenpositive, as shown by Le Douarin [l, 21. Ultrastructural cytochemistry of quail cell nucleoli reveals an intimate DNA-nucleolar RNA association, DNA either occupying a central position in the nucleolus, or associated with ribonucleoprotein structures [2, 31. Such an arrangement differs greatly from the majority of avian cell nuclei, and has not been established in mammal cells. It is known that in quail cell nuclei, as in those of chicken, the nucleolus organizers are carried on microchromosomes. These microchromosomes are constituted for the greater part by heterochromatin. The microchromosomal DNA is late replicating and contains 24-751806
a satellite rich in G-C bases [4]. Such DNA has been found to be repetitive and has been localized by in situ hybridization [5]. Studies of somatic cells in prophase reveal individualisation of microchromosomes in the form of multiple small heterochromatic masses at the nucleolar periphery [4]. In a preliminary study of meiotic prophase I our attention was drawn to a fusion process of microchromosomal heterochromatin concomitant with the role of the microchromosomes as nucleolus organizers. These observations have led us to wonder whether the above peculiarity could explain the original constitution of quail cell nucleoli. Consequently we have analysed the stages in prophase I with specific reference to the Exptl Cell Res 91 (1975)
366 Stahl et al. microchromosomes, the regions of constitutive heterochromatin in the macrochromosomes and their relationship with nucleolar synthesis. MATERIAL
AND
METHODS
The left gonad of quail embryos, from the 11th day of incubation until hatching, as well as the ovaries of 2-day-old quails were treated by the previously developed technique used for mammal ovaries [6]: one part of this sample was immersed in a 0.44% hypotonic KC1 solution, then in methanol-acetic acid (3 : 1); another part of the sample was placed directly in methanol-acetic acid. Dilaceration was carried out in this fixative. The fragments obtained were then placed into a 45 % acetic acid solution thus separating the cells from one another. The resulting suspension was spread on frozen slides which were then dried either by flame or by hot air. Most of the preparations were stained with a Giemsa solution (pH 6.7). Certain slides were treated by the Arrighi & Hsu technique [7] for constitutive heterochromatin. For fluorescence studv one groun of preparations were stained with acridine orange, another group was stained with Hoechst benzimidazol 33258 colorant. Observations and photomicrographs were performed on a Zeiss photomicroscope using an HBO 200 lamp, Zeiss excitation filter BG12 and Zeiss barrier filter 530.
OBSERVATIONS
Leptotene The chromosomes are individualized in the form of long, slender and entangled filaments. It is impossible to distinguish between microchromosomes and macrochromosomes. The presence of chromocentres is noticeable, varying from 4 to 18 according to the cell under examination. At this stage it is difficult to analyse the relationship of the chromocentres with the chromosomal filaments (fig. 1). Figs Id.
Zygotene The chromosomes present the general aspects characteristic of zygotene: contraction, polarisation and incipient pairing. Thus, the chromosomes constitute a very compact cluster at the periphery of which extend a few chromosomal loops (fig. 2). Distinction between euchromatin and heterochromatin is practically impossible during zygotene.
Pachytene During early pachytene the bivalents are clearly visible as parallel ribbon-like structures and show distinguishable chromomeres. In early pachytene it is not uncommon to observe either dispersed microchromosomes (fig. 3) or small masses of microchromosomes retaining their individuality (fig. 4). One observes a heterochromatic region at one extremity of each microchromosome-probably corresponding to the centromeric zoneand a euchromatic region. The region of euchromatin is larger than the heterochromatic region and presents a chromomeric structure similar to that of macrochromosomes. Transition images indicate that the microchromosomes adhere to one another while still remaining individualized. Fluorescent preparations with either acridine orange or benzimidazol 33258 Hoechst colorant reveal the chromocentres in the process of constitution, their mass still appearing heterogeneous as the fusion process of the microchromosomes is not yet accomplished (fig. 7).
Different stages of the first meiotic prophase in quail oocyte showing morphological aspects of these stages and constitution of chromocentres. Fig. I, Leptotene stage, 1l-day-old embryo. x 3 000: fig. 2, zygolene stage, Il-day-old embryo. x 3 700; fig. 3, early pachytene stage. Microchromosomes are dispersed. Heterochromatic region is visible at one extremity (straight arrow); the remnant part is euchromatic (curved arrow). 13-day-old embryo. x 2 900; fig. 4, middle pachytene stage. Microchromosomes are joining together to constitute chromocentres. 13-day-old embryo. x 2 600; fig. 5, late pachytene stage. Chromocentres are formed by the fusion of the heterochromatic regions of the microchromosomes. Euchromatic regions radiate from chromocentres, 14-day-old embryo. x 2 000; fig. 6, diplotene stage. Note the large nucleolus associated with two chromocentres and surrounded by the euchromatic segments of microchromosomes. The euchromatic segments form chiasmata. Quail 2-day-old. x 1 300. Exptl Cell Res 91 (1975)
Heterochromatin and nucleolar organizers in quail oocytes
367
Exptl Cell Res 91 (1975)
368
Stahl et al.
Fig. 7. Constitution process of the chromocentres at
early pachytene stage. Chromocentres are formed by the union of the heterochromatic regions from 6 to 12 microchromosomes. Hoechst benzimidazol 33258 stain. 13-day-old embryo. x 2 500.
With the progression of pachytene, 4 to 6 large chromocentres are observed from which radiate short euchromatic segments of typical bivalent structure (fig. 5). These are euchromatic regions of microchromosomes whose heterochromatic portions have clearly fused to form compact chromocentres. One chromocentre is formed by the contribution of 6 to 12 microchromosomes. It is important to note that certain chromocentres are exclusively formed by fusion of heterochromatin of the microchromosomes while other chromocentres also contain macrochromosomal regions of heterochromatin (fig. 8). Certain macrochromosomes appear inserted by each one of their extremities into 2 distinct chromocentres; according to the chromosome examined either the two telomeres (chromosomes nos 1 and 2) or one telomere and the centromere (chromosomes nos 3, 4 and 5) participate. In the sex chromosomes W and Z, characteristic of avian females, chromosome W is formed of constitutive heterochromatin, while Exptl Cell Res 91 (1975)
chromosome Z is euchromatic [8,9]. Chromosome Z is frequently identifiable presenting a euchromatic univalent form which appears relatively despiralized compared to other macrochromosomes (fig. 8). Among certain cells we have observed the presence of a relatively short and deeply stained univalent, stretched between two chromocentres, which could correspond to chromosome W (fig. 9). During this stage it has never been possible simultaneously to identify chromosome W and Z in the same cell. Consequently we cannot speculate on the nature of any possible relationship between the two chromosomes. The nucleoli are extremely reduced during pachytene, most often in the form of one or two small spherules which are always associated with a chromocentre. Only after acridine orange staining are they clearly visible. Diplotene At this stage, homologous chromosomes begin to separate, yet remain united by chiasmata. The chromosomes elongate but the chromomeres remain clearly visible. The euchromatic portion of the microchromosomes always emerges from the periphery of the chromocentres with certain euchromatic portions manifesting characteristic chiasmata (figs 6, 10). Fairly often a heterochromatic chromosomal filament can be seen stretched between two chromocentres (fig. 10). Lacking chiasmata, this univalent structure could represent the W chromosome. Characteristic of diplotene as well is the occurrence of one or two voluminous nucleoli. As a rule, one or two chromocentres are associated with the nucleolus (figs 6, 10). The euchromatic part of the microchromosomes emerging from the chromocentre presents chiasmata the number of which is
Heterochromatin and nucleolar organizers in quail oocytes
369
Fig. 8. Pachytene stage. Arrow indicates a univalent corresponding to chromosome Z. Quail l-day-old. x 3 000; fig. 9, pachytene stage. Arrow indicates a heterochromatic univalent stretched between two chromocentres which could correspond to chromosome W. 13-day-old embryo. x 2 200; fig. IO, diplotene stage showing also the heterochromatic chromosome W (arrow) (n, nucleolus). 14-day-old embryo. x 2 600.
Exptl Cell Res 91 (1975)
370 Stahl et al.
Figs 11-12. Parts of diplotene nuclei showing certain euchromatic segments of microchromosomes seeming to penetrate the nucleolar mass. Quail 2-day-old. x 2 200 and 2 800; fig. 13, somatic cell from the gonad at
metaphase treated by the Arrighi & Hsu technique. Constitutive heterochromatin is located at one extremity of several microchromosomes. 13-day-old embryo. x 2 900.
dependent on the length of the segment involved. In certain instances the euchromatic segments which point towards the nucleoli partially encircle them and strictly follow the nucleolar contour (fig. 6). In other cases these segments appear superposed on the nucleolus (figs 11, 12). It is possible that such an aspect is the result of the microchromosomes’ adhering to the nucleolar surface, as seen head-on, since the technique employed spreads all structures in the same plane. It is however, equally possible that such an aspect represents a veritable penetration into the nucleolar mass. In either case, adherence or penetration, the nucleolarassociated microchromosomes form a very fine filament whose extremity exhibits one or two punctiform enlargements. DISCUSSION Our observations indicate that microchromosomes are not heterochromatic throughout their entire length, contrary to previous descriptions [4]. Such a structure is already Exptl CeN Res 9I (1975)
visible in metaphase somatic cells of gonads treated by the Arrighi & Hsu technique [7]. Indeed, the heterochromatic zone is often observed to be situated at one of the extremities of the microchromosomes and most probably corresponds to the centromeric region (fig. 13). During pachytene the microchromosomes are longer than those during somatic mitosis; it is clearly seen that the heterochromatic zone is limited to one of the extremities which thus reinforces the opinion of its being a centromeric region. The euchromatic zone presents exactly the same bivalent structure and chromomeric aspect as do the macrochromosomes. The only observed difference is a lesser-microchromosomal affinity for basic stains, already described in chicken spermatocytes [lo]. During diplotene the euchromatic region of many microchromosomes manifests chiasmata. It has been postulated that avian microchromosomes were not true chromosomes [ll] but several works [lo, 12, 131 on the domestic fowl have already established the inexactitude of such an opinon. Our observations on the quail confirm that microchromosomes do
Heterochromatin and nucleolar organizers in quail oocytes not differ essentially from macrochromosomes and in effect represent genetically active structures. During pachytene, the quail microchromosomes manifest a remarkable peculiarity: the heterochromatic regions fuse to constitute a large chromocentre. This process reminds us of that described in the Saccostomus campestris [14]. In quail, it is at the contact of chromocentres that we have seen one or several nucleoli building up at late pachytene. During diplotene, the voluminous nucleoli are in close contact with the euchromatic region of certain microchromosomes. From the chromocentres which are always adjacent emanate slender filaments, punctuated by enlargements, which adhere to the nucleolar surface or penetrate into the nucleolar mass. These are clearly euchromatic segments of microchromosomes carrying nucleolar organizers. The propensity of these microchromosomes to fuse by their heterochromatic regions could explain the appearance and properties of somatic cell nucleoli. The latter is indeed characterized by a tight DNA-RNA association [3]. The tendency to a fusion of the microchromosomes bearing nucleolar organizers, the association of nucleolar RNA with the euchromatic segments, can most probably be found in the greater part of somatic cell types as indicated by the observations of quail fibroblasts cultured in vitro
WI. The W chromosome is heterochromatic in quail somatic cells [9] as in other avian somatic cells [8]. During pachytene a fairly short and thick univalent is sometimes observed, more darkly stained than other chromosomes, which probably represents the W chromosome. An analogous heteropyc-
371
notic structure has been described in chicken oocytes and interpreted as a Z chromosome in a univalent state, since the W chromosome had not yet been discovered in this species [12]. During diplotene, an isolated, thick and deeply stained heteropycnotic filament lacking chiasmata can be frequently observed stretched between two chromocentres. Such an appearance strongly argues in favour of the W chromosome. The Z chromosome is simple to identify during pachytene in the form of a weakly chromophilic, slender, and relatively despiralized univalent. We have not been able to observe a relationship between Z and W chromosomes. Neither of these two chromosomes appears to present any particular relation with the nucleolus.
REFERENCES 1. Le Douarin, N M, Bull biol franc belg 103 (1969) 435. 2. Ann embryo1 morohoa 4 (1971) 125. 3. -Exptl cell res 77 (i973 459. ’ 4. Comings. D E & Mattoccia. E, Chromosoma 30 (1970) 262. 5. Brown, J E & Jones, K W, Chromosoma 38 (1972) 313. 6. Luciani, J M, Devictor-Vuillet, M. Gag&, R & Stahl, A, J reprod fert 36 (1974) 409. 7. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971) 8. i:;?fos, K & Arrighi, F E, Exptl cell res 68 (1971) 228. 9. Hartung, M & Stahl, A, Compt rend acad sci 278 (1974) 2157. 10. Ford, E H R & Woollam, D H M, Chromosoma 15 (1964) 568. 11. Newcomer, E H, J hered 48 (1957) 227. 12. Ohno, S, Chromosoma 11 (1961) 484. 13. Owen. J J T. Chromosoma 16 (1965) 601. 14. Ford,.C E & Hamerton, J L, Nature 177 (1956) 140. 15. Vagner-Capodano, A M & Stahl, A, Experientia 30 (1974) 277. Received July 17, 1974 Revised manuscript received October 7, 1974
Exptl Cell Res 91 (1975)
Experimental Cell Research91 (1974) 365-371
HETEROCHROMATIN
AND NUCLEOLAR
FIRST MEIOTIC A. STAHL, J. M. LUCIANI, Laboratoire
ORGANIZERS
PROPHASE IN QUAIL
M. DEVICTOR,
DURING
OOCYTES
A. M. CAPODANO
and M. HARTUNG
d’Histologie et Embryologic II, Faculte’ de Midecine, F-13385 Marseille Cedex 4, France
SUMMARY The study of chromosomes in oocytes of the quail shows, at the pachytene stage, that microchromosomes are made of a euchromatic segment and a heterochromatic juxtacentromeric region. The heterochromatic regions of the microchromosomes amalgamate between themselves so as to constitute bulky chromocentres from which radiate the euchromatic segments which remain free. At late pachytene, nucleoli appear at the contact of these chromocentres. When the oocytes reach the diplotene stage, the nucleoli become quite large. They are stuck against chromocentres and establish a very close relationship with the euchromatic segments of the microchromosomes which surround or penetrate them. These observations lead one to think that the euchromatic segments of microchromosomes could be bearing nucleolar organizers. The close relations that the nucleolar organizers develop with the bulk of the nucleolus could explain its Feulgen-positive character in the quail.
In quail somatic cell nuclei, the nucleolus presents the peculiarity of being Feulgenpositive, as shown by Le Douarin [l, 21. Ultrastructural cytochemistry of quail cell nucleoli reveals an intimate DNA-nucleolar RNA association, DNA either occupying a central position in the nucleolus, or associated with ribonucleoprotein structures [2, 31. Such an arrangement differs greatly from the majority of avian cell nuclei, and has not been established in mammal cells. It is known that in quail cell nuclei, as in those of chicken, the nucleolus organizers are carried on microchromosomes. These microchromosomes are constituted for the greater part by heterochromatin. The microchromosomal DNA is late replicating and contains 24-751806
a satellite rich in G-C bases [4]. Such DNA has been found to be repetitive and has been localized by in situ hybridization [5]. Studies of somatic cells in prophase reveal individualisation of microchromosomes in the form of multiple small heterochromatic masses at the nucleolar periphery [4]. In a preliminary study of meiotic prophase I our attention was drawn to a fusion process of microchromosomal heterochromatin concomitant with the role of the microchromosomes as nucleolus organizers. These observations have led us to wonder whether the above peculiarity could explain the original constitution of quail cell nucleoli. Consequently we have analysed the stages in prophase I with specific reference to the Exptl Cell Res 91 (1975)
366 Stahl et al. microchromosomes, the regions of constitutive heterochromatin in the macrochromosomes and their relationship with nucleolar synthesis. MATERIAL
AND
METHODS
The left gonad of quail embryos, from the 11th day of incubation until hatching, as well as the ovaries of 2-day-old quails were treated by the previously developed technique used for mammal ovaries [6]: one part of this sample was immersed in a 0.44% hypotonic KC1 solution, then in methanol-acetic acid (3 : 1); another part of the sample was placed directly in methanol-acetic acid. Dilaceration was carried out in this fixative. The fragments obtained were then placed into a 45 % acetic acid solution thus separating the cells from one another. The resulting suspension was spread on frozen slides which were then dried either by flame or by hot air. Most of the preparations were stained with a Giemsa solution (pH 6.7). Certain slides were treated by the Arrighi & Hsu technique [7] for constitutive heterochromatin. For fluorescence studv one groun of preparations were stained with acridine orange, another group was stained with Hoechst benzimidazol 33258 colorant. Observations and photomicrographs were performed on a Zeiss photomicroscope using an HBO 200 lamp, Zeiss excitation filter BG12 and Zeiss barrier filter 530.
OBSERVATIONS
Leptotene The chromosomes are individualized in the form of long, slender and entangled filaments. It is impossible to distinguish between microchromosomes and macrochromosomes. The presence of chromocentres is noticeable, varying from 4 to 18 according to the cell under examination. At this stage it is difficult to analyse the relationship of the chromocentres with the chromosomal filaments (fig. 1). Figs Id.
Zygotene The chromosomes present the general aspects characteristic of zygotene: contraction, polarisation and incipient pairing. Thus, the chromosomes constitute a very compact cluster at the periphery of which extend a few chromosomal loops (fig. 2). Distinction between euchromatin and heterochromatin is practically impossible during zygotene.
Pachytene During early pachytene the bivalents are clearly visible as parallel ribbon-like structures and show distinguishable chromomeres. In early pachytene it is not uncommon to observe either dispersed microchromosomes (fig. 3) or small masses of microchromosomes retaining their individuality (fig. 4). One observes a heterochromatic region at one extremity of each microchromosome-probably corresponding to the centromeric zoneand a euchromatic region. The region of euchromatin is larger than the heterochromatic region and presents a chromomeric structure similar to that of macrochromosomes. Transition images indicate that the microchromosomes adhere to one another while still remaining individualized. Fluorescent preparations with either acridine orange or benzimidazol 33258 Hoechst colorant reveal the chromocentres in the process of constitution, their mass still appearing heterogeneous as the fusion process of the microchromosomes is not yet accomplished (fig. 7).
Different stages of the first meiotic prophase in quail oocyte showing morphological aspects of these stages and constitution of chromocentres. Fig. I, Leptotene stage, 1l-day-old embryo. x 3 000: fig. 2, zygolene stage, Il-day-old embryo. x 3 700; fig. 3, early pachytene stage. Microchromosomes are dispersed. Heterochromatic region is visible at one extremity (straight arrow); the remnant part is euchromatic (curved arrow). 13-day-old embryo. x 2 900; fig. 4, middle pachytene stage. Microchromosomes are joining together to constitute chromocentres. 13-day-old embryo. x 2 600; fig. 5, late pachytene stage. Chromocentres are formed by the fusion of the heterochromatic regions of the microchromosomes. Euchromatic regions radiate from chromocentres, 14-day-old embryo. x 2 000; fig. 6, diplotene stage. Note the large nucleolus associated with two chromocentres and surrounded by the euchromatic segments of microchromosomes. The euchromatic segments form chiasmata. Quail 2-day-old. x 1 300. Exptl Cell Res 91 (1975)
Heterochromatin and nucleolar organizers in quail oocytes
367
Exptl Cell Res 91 (1975)
368
Stahl et al.
Fig. 7. Constitution process of the chromocentres at
early pachytene stage. Chromocentres are formed by the union of the heterochromatic regions from 6 to 12 microchromosomes. Hoechst benzimidazol 33258 stain. 13-day-old embryo. x 2 500.
With the progression of pachytene, 4 to 6 large chromocentres are observed from which radiate short euchromatic segments of typical bivalent structure (fig. 5). These are euchromatic regions of microchromosomes whose heterochromatic portions have clearly fused to form compact chromocentres. One chromocentre is formed by the contribution of 6 to 12 microchromosomes. It is important to note that certain chromocentres are exclusively formed by fusion of heterochromatin of the microchromosomes while other chromocentres also contain macrochromosomal regions of heterochromatin (fig. 8). Certain macrochromosomes appear inserted by each one of their extremities into 2 distinct chromocentres; according to the chromosome examined either the two telomeres (chromosomes nos 1 and 2) or one telomere and the centromere (chromosomes nos 3, 4 and 5) participate. In the sex chromosomes W and Z, characteristic of avian females, chromosome W is formed of constitutive heterochromatin, while Exptl Cell Res 91 (1975)
chromosome Z is euchromatic [8,9]. Chromosome Z is frequently identifiable presenting a euchromatic univalent form which appears relatively despiralized compared to other macrochromosomes (fig. 8). Among certain cells we have observed the presence of a relatively short and deeply stained univalent, stretched between two chromocentres, which could correspond to chromosome W (fig. 9). During this stage it has never been possible simultaneously to identify chromosome W and Z in the same cell. Consequently we cannot speculate on the nature of any possible relationship between the two chromosomes. The nucleoli are extremely reduced during pachytene, most often in the form of one or two small spherules which are always associated with a chromocentre. Only after acridine orange staining are they clearly visible. Diplotene At this stage, homologous chromosomes begin to separate, yet remain united by chiasmata. The chromosomes elongate but the chromomeres remain clearly visible. The euchromatic portion of the microchromosomes always emerges from the periphery of the chromocentres with certain euchromatic portions manifesting characteristic chiasmata (figs 6, 10). Fairly often a heterochromatic chromosomal filament can be seen stretched between two chromocentres (fig. 10). Lacking chiasmata, this univalent structure could represent the W chromosome. Characteristic of diplotene as well is the occurrence of one or two voluminous nucleoli. As a rule, one or two chromocentres are associated with the nucleolus (figs 6, 10). The euchromatic part of the microchromosomes emerging from the chromocentre presents chiasmata the number of which is
Heterochromatin and nucleolar organizers in quail oocytes
369
Fig. 8. Pachytene stage. Arrow indicates a univalent corresponding to chromosome Z. Quail l-day-old. x 3 000; fig. 9, pachytene stage. Arrow indicates a heterochromatic univalent stretched between two chromocentres which could correspond to chromosome W. 13-day-old embryo. x 2 200; fig. IO, diplotene stage showing also the heterochromatic chromosome W (arrow) (n, nucleolus). 14-day-old embryo. x 2 600.
Exptl Cell Res 91 (1975)
370 Stahl et al.
Figs 11-12. Parts of diplotene nuclei showing certain euchromatic segments of microchromosomes seeming to penetrate the nucleolar mass. Quail 2-day-old. x 2 200 and 2 800; fig. 13, somatic cell from the gonad at
metaphase treated by the Arrighi & Hsu technique. Constitutive heterochromatin is located at one extremity of several microchromosomes. 13-day-old embryo. x 2 900.
dependent on the length of the segment involved. In certain instances the euchromatic segments which point towards the nucleoli partially encircle them and strictly follow the nucleolar contour (fig. 6). In other cases these segments appear superposed on the nucleolus (figs 11, 12). It is possible that such an aspect is the result of the microchromosomes’ adhering to the nucleolar surface, as seen head-on, since the technique employed spreads all structures in the same plane. It is however, equally possible that such an aspect represents a veritable penetration into the nucleolar mass. In either case, adherence or penetration, the nucleolarassociated microchromosomes form a very fine filament whose extremity exhibits one or two punctiform enlargements. DISCUSSION Our observations indicate that microchromosomes are not heterochromatic throughout their entire length, contrary to previous descriptions [4]. Such a structure is already Exptl CeN Res 9I (1975)
visible in metaphase somatic cells of gonads treated by the Arrighi & Hsu technique [7]. Indeed, the heterochromatic zone is often observed to be situated at one of the extremities of the microchromosomes and most probably corresponds to the centromeric region (fig. 13). During pachytene the microchromosomes are longer than those during somatic mitosis; it is clearly seen that the heterochromatic zone is limited to one of the extremities which thus reinforces the opinion of its being a centromeric region. The euchromatic zone presents exactly the same bivalent structure and chromomeric aspect as do the macrochromosomes. The only observed difference is a lesser-microchromosomal affinity for basic stains, already described in chicken spermatocytes [lo]. During diplotene the euchromatic region of many microchromosomes manifests chiasmata. It has been postulated that avian microchromosomes were not true chromosomes [ll] but several works [lo, 12, 131 on the domestic fowl have already established the inexactitude of such an opinon. Our observations on the quail confirm that microchromosomes do
Heterochromatin and nucleolar organizers in quail oocytes not differ essentially from macrochromosomes and in effect represent genetically active structures. During pachytene, the quail microchromosomes manifest a remarkable peculiarity: the heterochromatic regions fuse to constitute a large chromocentre. This process reminds us of that described in the Saccostomus campestris [14]. In quail, it is at the contact of chromocentres that we have seen one or several nucleoli building up at late pachytene. During diplotene, the voluminous nucleoli are in close contact with the euchromatic region of certain microchromosomes. From the chromocentres which are always adjacent emanate slender filaments, punctuated by enlargements, which adhere to the nucleolar surface or penetrate into the nucleolar mass. These are clearly euchromatic segments of microchromosomes carrying nucleolar organizers. The propensity of these microchromosomes to fuse by their heterochromatic regions could explain the appearance and properties of somatic cell nucleoli. The latter is indeed characterized by a tight DNA-RNA association [3]. The tendency to a fusion of the microchromosomes bearing nucleolar organizers, the association of nucleolar RNA with the euchromatic segments, can most probably be found in the greater part of somatic cell types as indicated by the observations of quail fibroblasts cultured in vitro
WI. The W chromosome is heterochromatic in quail somatic cells [9] as in other avian somatic cells [8]. During pachytene a fairly short and thick univalent is sometimes observed, more darkly stained than other chromosomes, which probably represents the W chromosome. An analogous heteropyc-
371
notic structure has been described in chicken oocytes and interpreted as a Z chromosome in a univalent state, since the W chromosome had not yet been discovered in this species [12]. During diplotene, an isolated, thick and deeply stained heteropycnotic filament lacking chiasmata can be frequently observed stretched between two chromocentres. Such an appearance strongly argues in favour of the W chromosome. The Z chromosome is simple to identify during pachytene in the form of a weakly chromophilic, slender, and relatively despiralized univalent. We have not been able to observe a relationship between Z and W chromosomes. Neither of these two chromosomes appears to present any particular relation with the nucleolus.
REFERENCES 1. Le Douarin, N M, Bull biol franc belg 103 (1969) 435. 2. Ann embryo1 morohoa 4 (1971) 125. 3. -Exptl cell res 77 (i973 459. ’ 4. Comings. D E & Mattoccia. E, Chromosoma 30 (1970) 262. 5. Brown, J E & Jones, K W, Chromosoma 38 (1972) 313. 6. Luciani, J M, Devictor-Vuillet, M. Gag&, R & Stahl, A, J reprod fert 36 (1974) 409. 7. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971) 8. i:;?fos, K & Arrighi, F E, Exptl cell res 68 (1971) 228. 9. Hartung, M & Stahl, A, Compt rend acad sci 278 (1974) 2157. 10. Ford, E H R & Woollam, D H M, Chromosoma 15 (1964) 568. 11. Newcomer, E H, J hered 48 (1957) 227. 12. Ohno, S, Chromosoma 11 (1961) 484. 13. Owen. J J T. Chromosoma 16 (1965) 601. 14. Ford,.C E & Hamerton, J L, Nature 177 (1956) 140. 15. Vagner-Capodano, A M & Stahl, A, Experientia 30 (1974) 277. Received July 17, 1974 Revised manuscript received October 7, 1974
Exptl Cell Res 91 (1975)
