Cytogenet. Cell Genet. 18: 309-319 (1977)
Preleptotene chromosome condensation in mouse oogenesis M. H artung and A. Stahl Laboratoire cl'Histologie et Embryologie II, Faculté de Médecine. Marseille
Abstract In the embryonic mouse ovary, studies of meiotic onset reveal the existence of a preleptotene stage of chromosome condensation. This stage begins with the appearance of very fine and irregular filaments, which gather around the chromo centers constituted by centromeric heterochromatin. At maximal condensation the nucleus contains 40 compact chromosomal masses. Decondensation engenders the reappearance of filaments more dense and regular than those seen at the onset of the condensation stage. The filaments elongate while progressively taking on the appearance of leptotene-stage chromosomes. The oocytes at different phases of condensation stage represent 41 “/ci of the germ cells in the 13-day-old mouse embryo. This proportion reaches 63 n/o at 14 days and then declines to 3 °/o at 17 days. Spectrophotometric studies of the condensation-stage nucleus, following staining by the Feulgen reaction, indicate that the nucleus contains 4C DNA. This observa tion, together with the morphologic characteristics of this stage, suggests that the oocyte is already engaged in prophase. Time studies indicate that the condensation stage occurs immediately prior to leptotene.
Request reprints from: Dr. A. Stahl, Laboratoire d'Histologie et Embryologie II. Faculté de Médecine, 27, boulevard Jean-Moulin. F-13385 Marseille, Cedex 4 (France).
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A preleptotene stage of chromosome contraction, or condensation, has been described in numerous plants (for review, see W alters , 1972. 1976). and a precise chronologic study has been performed in Lillium (W alters , 1970, 1972; B ennett and Stern , 1975). This stage has also been described in insects (for review, see W ilson , 1928. and W alters , 1976). It had either passed unobserved or been considered degenerative in mammals until its description in oogenesis of man (Stahl and L uciani, 1971), the rabbit (D evictor -V uillet et al., 1973), and sheep (M auleon et al., 1976).
310
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The cytologic study of early meiotic stages in the mouse oocyte demon strates the existence of a condensation stage whose morphologic charac teristics are particularly suitable to detailed analysis. Materials and methods The left gonad of female mouse embryos aged 13 through 20 days were removed and treated according to the technique of L uciani et al. (1974). Following fixation in mcthanol-acetic acid (3:1), the gonads were placed in 45 °/o acetic acid. Cellular dissociation was obtained by repeated pipetting. The cellular suspension was spread on precooled slides and stained with Giemsa. For spectrophotometric study, certain slides were stained by Feulgcn’s reaction. DNA content of the nuclei was measured using the densitometry module of a Quantimet 720 in monochromatic light at 570 nm (for details, see H artung et al., 1977). Preparations were also stained by freshly prepared 0.05 ug/ml 33258 Hoechst fluorochrome solution according to the H ilw ig and G ropp technique (1972). These slides were examined in a Zeiss fluorescence microscope with a 53 stop filter and BG 12 excitation filter. The right gonad from the same embryos was treated by double fixation. Fixa tion was carried out for 15 min at 4° C in 3 “/« glutaraldehyde in 0.1 m phosphate buffer (pH 7.2) containing 0.1 m saccharose. After washing in buffer, the gonads were fixed again in 2%> osmium tetroxide for 20 min in the same buffer. After cell dehydration in acetone-water solutions of increasing concentration, the ovaries were embedded in Epon. A diamond knife on a Reichert OMU 2 ultramicrotome produced ultrathin sections which were collected on copper grids and contrasted with uranyl acetate and lead citrate. The preparations were examined with a Siemens Elmiskop 101 electron microscope at 80 kV.
Cytologic examination shows many germinal cells at the preleptotene condensation stage in the mouse embryo at 13 days, with an increased number at 14 days. At 15 days, a high number of oocytes can be seen in different degrees of condensation, mixed with oocytes at the leptotene and zygotene stage. Numerous oocytes can still be seen in the condensation stage at 16 days, but zygotene and pachytene figures now predominate. The condensation stage almost disappears in the embryo at 17 days (table I). Sections of embryonic mouse ovaries at 15 days allow localization of the oocytes in condensation. They are grouped into clusters of synchronous cells, located a short distance from the surface of the ovary.
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Observations
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Age of the embryo (days) 13 14 15 16 17 18
Oogonia
(%) 59 16 11 2
Condensation stages (°/o) 41 63 55 41 3
(%)
I.eptotene
Zygotene (Vo)
Pachytene (Vo)
21 34 53 15 2
4 35 9
47 88
Nuclear processes characterizing the passage from oogonium to leptotene can be analyzed in detail only in spreads treated by cyto genetic techniques. Comparison with previously described figures in other mammals leads us to propose the following sequence: 1. The oogonium is characterized by its very large size. Chromatin is present as very fine filaments and numerous small chromocenters of unequal dimensions. One or two nucleoli can be observed (fig. 1). 2. The nuclear outline is distinguished by the increased density of the filaments, which become thicker, and by the increased size of the chromo centers, whose observable number approaches 40 (fig. 2). 3. In the following stage the filamentous structures gather around each chromocenter, thus constituting 39 entities of “hairy” appearance. These filaments are irregular in appearance, with blurred outlines. Furthermore, a structure outlined by its massively heteropycnotic aspect and its generally peripheral localization can be observed. The nucleoli are still present (fig. 3). This stage evolves by a progressive tightening of filaments around each chromocenter (fig. 4). 4. Condensation of the filaments increases, forming 39 compact entities, each corresponding to one chromosome. They are formed by a highly dense central mass, around which thin filaments radiate for a short distance. In addition, a very dense heteropycnotic chromosome, with smooth outlines, can be observed generally situated at the periphery. It probably corresponds to one of the two X chromosomes (fig. 5). 5. At maximum condensation, the nucleus contains 40 highly compact, angular-shaped masses, heteropycnotic in appearance. Occasionally, a few short filaments still project out from their edges (fig. 6).
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Table I. Percentage of condensation-stage oocytes in mouse embryos from day 13 to day 18.
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Fig. I. Oogonium nucleus with filamentous-appearing chromatin and numerous small chromocenters. Note presence of two nucleoli. Reproduced at 960X. Fig. 2. Oogonium nucleus at a later stage; the filaments are thicker and chromo centers slightly larger. Reproduced at 1280X.
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Fig. 3. Nucleus at onset of condensation stage; the filaments are gathered around each chromocenter. Note the presence of both a heteropycnotic chromosome peripherally situated (arrow) and a nucleolus (Nu). Reproduced at 1280X. Fig. 4. Slightly more advanced condensation stage demonstrating tightening of the filaments about each chromocenter. At the periphery note the presence of a hetero pycnotic chromosome (arrow). Reproduced at 1600X. Fig. 5. Progression of condensation leading to formation of 39 chromosomal entities, from which radiate fine filaments. A heteropycnotic chromosome is also visible at the periphery (arrow). Reproduced at 1600 X. Fig. 6. Maximal condensation. The chromosomes form 40 heterochromatic-appearing masses, from which emerge a few fine filaments. Reproduced at 1280X.
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6. The onset of decondensation is characterized by the reappearance and elongation of the filaments at the circumference of each heteropycnotic mass. These filaments are different in appearance from those that can be observed during condensation stages; they are thicker and show loops along their length (fig. 7). As decondensation gradually progresses, the volume of the heteropycnotic region decreases (fig. 8). At the end of this stage, only a heteropycnotic corpuscle remains, generally situated at the very end of a fila mentous cluster. The corpuscle position corresponds to that of the centro mere in mouse chromosomes. The filamentous cluster gradually loosens, so that the appearance of the filament approaches more and more that of a leptotene chromosome. Throughout this sequence, the entirely heteropycnotic chromosome situated at the nuclear periphery remains identifiable due to its allocyclic behavior (fig. 8). The nucleoli are still present. 7. Near the end of the decondensation stage, the nucleus contains chromosomes of filamentous appearance (fig. 9). The centromeric hetero chromatin is seen on one extremity of a large number of chromosomes. 8. The final decondensation phase ends at the onset of leptotene. The chromosomes become very long and are difficult to distinguish, as they are completely entangled. The centromeric heterochromatin is no longer observed (fig. 10). Fluorescence studies of 33258 Hoechst-stained preparations show that visible chromocenters in the oogonium nucleus correspond to centromeric heterochromatin. At maximum condensation, a bright fluorescent corpuscle can be seen, often situated on one edge of each chromosomal mass. When decondensation occurs, the centromeric heterochromatin is clearly situated at one end of the chromosomal filament, now undergoing individualization (fig. 11).
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An estimation of DNA content in condensation-stage nuclei was per formed by comparing it with the DNA content of somatic nuclei present in the same preparations and stained by Feulgen’s reaction. Absorption of 40 germinal and 40 somatic cells was measured. The quantity of DNA in mouse somatic cells was measured at 4.98 pg by M c C arthy (1969). We found a mean integrated optical density of 4801 (arbitrary units) in the
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Fig. 7. Onset of decondensation. Reappearance and elongation of the filaments originating from each heteropycnotic mass can be seen. Reproduced at 960X. Loops can be seen in the inset (1920 X). Fig. 8. More advanced decondensation. The chromosomal entities form filaments that are both thicker than during previous stages and are terminated by a hetero pycnotic corpuscle. A heteropycnotic chromosome can still be seen at the periphery (arrow). Reproduced at 1760X. Fig. 9. Almost completed decondensation leading to individualization of the fil aments, whose appearance resembles that of leptotene chromosomes. Reproduced at 1760X. Fig. 10. Oocyte nucleus at leptotene. Reproduced at 1360X.
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Fig. 11. Oocyte nucleus at condensation stage, stained with 33258 Hoechst. A bright fluorescent corpuscle, often located on one edge of each chromosomal mass, can be seen, which corresponds to centromeric heterochromatin. Reproduced at 1440X.
somatic cells. At condensation stage the mean integrated optical density of the germinal cells was found to be 8980, which corresponds to an average DNA content of 9.32 pg. Theoretically, after the S-phase, the DNA content of germinal cells should be twice that of somatic cells, i.e., 4.98 X 2 = 9.96 pg. Taking into consideration the accidental loss of chromosomal material from a few cells resulting from the technique used, this finding reasonably indicates that the condensation stage oocytes contain 4C DNA. Studies of condensation-stage oocytes by electron microscopy demon strate the persistence of the nuclear membrane (fig. 12). The chromosomes are present as irregular masses formed by tangled fibrils. On the edges of the chromosomal masses, a few fibrils can be seen extending into the nucleoplasm, yet these fibrils can only be followed for a very short distance. The nucleolus is still present. The cytoplasm contains many ribosomes and mitochondria, a few of which are vacuolized. The Golgi complex is well developed. Neither centrioles nor micro tubules can be seen (fig. 13).
The onset of the condensation stage and its place in the evolutive chronology of the mammalian germ cell have been determined by Mauleon et al. (1976) in the sheep. Using tritiated thymidine, they showed that
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Discussion
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Prcleptotene chromosome condensation
Fig. 12. Electron micrograph of a mouse ovary at 14 days postfertilization, showing a cluster of oocytes at various stages of condensation. One of them (arrow) is probably close to maximum condensation. Reproduced at 4760X. Fig. 13. High magnification of the oocyte cytoplasm in fig. 12 (arrow). The Golgi complex is well developed. Neither centrioles nor microtubules are observed. Reproduced at 25,840 X.
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317
the condensation stage precedes leptotene. The former follows both S and G2 phases, which mark the onset of meiosis. These conclusions are analogous to those provided by chronology studies in Lillium (W alters, 1970, 1972, 1976; B ennett and Stern , 1975). In the mouse, the condensation stage appears and develops from day 13 to day 15, prior to leptotene. In this species this stage is situated between premeiotic interphase and leptotene. The morphologic aspects of the condensation stage are analogous to those already described in the rat by H ilscher et al. (1974) as postmitotic stages. Nevertheless, the 4C DNA content of these nuclei in the mouse excludes their being either telophase or G1 nuclei. In the same way, if the condensation stage corresponded to the S phase, DNA content inter mediary between 2C and 4C would be found. Condensation-stage oocytes can then only be either in G2, which is highly unlikely based on their morphology, or engaged in prophase. The latter interpretation is in agreement with the observation of individualization of chromosome structures and their progressive condensation. The hypothesis that these aspects correspond to the prophase of the last oogonial mitotic division can be excluded. In this case, as pointed out by M auleon et al. (1976), groups of metaphase and anaphase cells should be found around the condensation-stage groups, yet such is not the case. The significance of the preleptotene condensation stage is unknown. As outlined by W alters (1972), this stage is absent in many species and may present great variability in others. It has been suggested that prelep totene chromosome condensation could represent a partial reversion to mitosis (W alters, 1970). According to B ennett and Stern (1975), preleptotene condensation and decondensation represent a true reversion to mitosis, but with suppression of metaphase and anaphase. Nevertheless, it must be pointed out that mitotic prophase or even prometaphase never attain the extreme degree of chromosome contraction observed in the mammalian preleptotene condensation stage. Moreover, ultrastructural study of this stage reveals the absence in the oocyte cyto plasm of centrioles and microtubules arranged in such a way as to form a spindle. This finding indicates that the oocyte at this stage is not prepared for a mitotic-type division. It is possible that the phases of condensation and decondensation correspond to a reorganization of the chromatin in preparation for lep totene. B ennett and Stern (1975) observed in Lillium that the condensa tion-stage chromosomes were formed by two chromatids. On the contrary,
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during decondensation the filaments are single, without visible daughter chromatids, as typically observed in leptotene. In the mouse, the degree of condensation is such that it is impossible to distinguish whether or not there exist two chromatids. On the other hand, it is evident that the appearance of the filaments is totally different during the condensation and decondensation phase. As condensation progresses, the filaments become relatively fine and irregular, with often poorly limited outlines, whereas by the onset of decondensation, they acquire the diameter, regularity of appearance, and sometimes even the chromomeric structure of leptotene filaments. This finding suggests that it is during the conden sation stage that an internal rearrangement of the chromatin fibres occurs in preparation for adaptation to the specific processes of meiosis. During the condensation stage an entirely heteropycnotic chromosome is visible, generally at the nuclear periphery. We believe that this corresponds to one of the two X chromosomes, based on the observations of O hno et al. (1959) and O hno and H auschka (I960). These authors observed that in the diploid somatic nuclei of the mouse and rat, one of the X chromosomes was allocyclic, and presented a positive heteropycnosis during prophase. The condensation stage corresponds to a prophase. During the following stages, the need for pairing of the two X chromosomes would require them to be at the same degree of despiralization. A cknowledgements We thank Dr. C. M irre for his assistance in electron microscopy data. We are also grateful to Dr. M. HuLTfeN for editorial assistance with the manuscript.
References
stage in Lilliuni hybrid c.v. Black Beauty. Proc. R. Soc. Lond. B 188: 477-493 (1975). D evictor -V uillet , M.: L uciani, J.M.. and Staiil , A.: Individualisation d'un stade préleptotène de condensation chromosomique dans l’ovocyte de la lapine. C.r. Acad. Sci., Paris 276: 2453-2456 (1973). H artung, M.; Souchier , C., and S tahl , A.: Teneur en ADN de l’ovocyte de souris au stade préleptotène de condensation chromosomique. Ann. Biol. anim. Biochim. Biophys. (1977, submitted for publication).
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B ennett , M.D. and Stern , H.: The time and duration of preleptotene condensation
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H ilscher , B.; H ilscher , W .; Bülthofe -O hnolz, B.; D ramer, U.; Birke , A.; P elzer , H., and G auss , G.: Kinetics of gamctogcncsis. I. Comparative histological
and autoradiographic studies of oocytes and transitional prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue Res. 154: 443-470 (1974). H ilw ig , 1. and G ropp , A.: Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. Expl Cell Res. 75: 122-126 (1972). L uciani, J.M.; D evictor -V uillet , M.; G agne, R.. and Stahl, A.: An air drying method for first meiotic prophase preparations from mammalian ovaries. J. Reprod. Fert. 36: 409-411 (1974). McC arthy, B.J.: The evolution of base sequences in nucleic acids. In P. L ima de F aria, ed.: Handbook of molecular cytology, pp. 3-20 (North Holland Publ. Co., Amsterdam/London 1969). M auleon, P.; D evictor-V uillet , M., and L uciani, J.M.: The preleptotene chro mosome condensation and decondensation in the ovary of the sheep embryo. Ann. Biol. anim. Biochim. Biophys. 16: 293-296 (1976). O mno, S.; K aplan, W.D., and K inosita , R.: Formation of the sex chromatin by a single X-chromosomc in liver cells of Rat tus norvégiens. Expl Cell Res. 18: 415-418 (1959). O hno, S. and H auschka, T.S.: Allocycly of the X chromosome in tumors and normal tissues. Cancer Res. 20: 541-545 (1960). Stahl, A. and L uciani, J.M.: Individualisation d’un stade preleptotene dc conden sation chromosomique au début de la méiose chez l’ovocyte foetal humain. C.r. Acad. Sci., Paris 272: 2041-2044 (1971). W alters , M.S.: Evidence on the time of chromosome pairing from the preleptotene spiral stage in Lillitim longiflorum “Croft”. Chromosoma 29: 375-418 (1970). W alters , M.S.: Preleptotene chromosome contraction in Lillium longiflorum “Croft”. Chromosoma 39: 311-332 (1972). W alters , M.S.: Variation in preleptotene chromosome contraction among three cultivais of Lillium longiflorum. Chromosoma 57: 51-80 (1976). W ilson , E.B.: The cell in development and heredity, 3rd Ed. (Macmillan, New York 1928).
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Manuscript received 27 December 1976; accepted for publication 22 March 1977.
Preleptotene chromosome condensation in mouse oogenesis M. H artung and A. Stahl Laboratoire cl'Histologie et Embryologie II, Faculté de Médecine. Marseille
Abstract In the embryonic mouse ovary, studies of meiotic onset reveal the existence of a preleptotene stage of chromosome condensation. This stage begins with the appearance of very fine and irregular filaments, which gather around the chromo centers constituted by centromeric heterochromatin. At maximal condensation the nucleus contains 40 compact chromosomal masses. Decondensation engenders the reappearance of filaments more dense and regular than those seen at the onset of the condensation stage. The filaments elongate while progressively taking on the appearance of leptotene-stage chromosomes. The oocytes at different phases of condensation stage represent 41 “/ci of the germ cells in the 13-day-old mouse embryo. This proportion reaches 63 n/o at 14 days and then declines to 3 °/o at 17 days. Spectrophotometric studies of the condensation-stage nucleus, following staining by the Feulgen reaction, indicate that the nucleus contains 4C DNA. This observa tion, together with the morphologic characteristics of this stage, suggests that the oocyte is already engaged in prophase. Time studies indicate that the condensation stage occurs immediately prior to leptotene.
Request reprints from: Dr. A. Stahl, Laboratoire d'Histologie et Embryologie II. Faculté de Médecine, 27, boulevard Jean-Moulin. F-13385 Marseille, Cedex 4 (France).
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A preleptotene stage of chromosome contraction, or condensation, has been described in numerous plants (for review, see W alters , 1972. 1976). and a precise chronologic study has been performed in Lillium (W alters , 1970, 1972; B ennett and Stern , 1975). This stage has also been described in insects (for review, see W ilson , 1928. and W alters , 1976). It had either passed unobserved or been considered degenerative in mammals until its description in oogenesis of man (Stahl and L uciani, 1971), the rabbit (D evictor -V uillet et al., 1973), and sheep (M auleon et al., 1976).
310
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The cytologic study of early meiotic stages in the mouse oocyte demon strates the existence of a condensation stage whose morphologic charac teristics are particularly suitable to detailed analysis. Materials and methods The left gonad of female mouse embryos aged 13 through 20 days were removed and treated according to the technique of L uciani et al. (1974). Following fixation in mcthanol-acetic acid (3:1), the gonads were placed in 45 °/o acetic acid. Cellular dissociation was obtained by repeated pipetting. The cellular suspension was spread on precooled slides and stained with Giemsa. For spectrophotometric study, certain slides were stained by Feulgcn’s reaction. DNA content of the nuclei was measured using the densitometry module of a Quantimet 720 in monochromatic light at 570 nm (for details, see H artung et al., 1977). Preparations were also stained by freshly prepared 0.05 ug/ml 33258 Hoechst fluorochrome solution according to the H ilw ig and G ropp technique (1972). These slides were examined in a Zeiss fluorescence microscope with a 53 stop filter and BG 12 excitation filter. The right gonad from the same embryos was treated by double fixation. Fixa tion was carried out for 15 min at 4° C in 3 “/« glutaraldehyde in 0.1 m phosphate buffer (pH 7.2) containing 0.1 m saccharose. After washing in buffer, the gonads were fixed again in 2%> osmium tetroxide for 20 min in the same buffer. After cell dehydration in acetone-water solutions of increasing concentration, the ovaries were embedded in Epon. A diamond knife on a Reichert OMU 2 ultramicrotome produced ultrathin sections which were collected on copper grids and contrasted with uranyl acetate and lead citrate. The preparations were examined with a Siemens Elmiskop 101 electron microscope at 80 kV.
Cytologic examination shows many germinal cells at the preleptotene condensation stage in the mouse embryo at 13 days, with an increased number at 14 days. At 15 days, a high number of oocytes can be seen in different degrees of condensation, mixed with oocytes at the leptotene and zygotene stage. Numerous oocytes can still be seen in the condensation stage at 16 days, but zygotene and pachytene figures now predominate. The condensation stage almost disappears in the embryo at 17 days (table I). Sections of embryonic mouse ovaries at 15 days allow localization of the oocytes in condensation. They are grouped into clusters of synchronous cells, located a short distance from the surface of the ovary.
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Age of the embryo (days) 13 14 15 16 17 18
Oogonia
(%) 59 16 11 2
Condensation stages (°/o) 41 63 55 41 3
(%)
I.eptotene
Zygotene (Vo)
Pachytene (Vo)
21 34 53 15 2
4 35 9
47 88
Nuclear processes characterizing the passage from oogonium to leptotene can be analyzed in detail only in spreads treated by cyto genetic techniques. Comparison with previously described figures in other mammals leads us to propose the following sequence: 1. The oogonium is characterized by its very large size. Chromatin is present as very fine filaments and numerous small chromocenters of unequal dimensions. One or two nucleoli can be observed (fig. 1). 2. The nuclear outline is distinguished by the increased density of the filaments, which become thicker, and by the increased size of the chromo centers, whose observable number approaches 40 (fig. 2). 3. In the following stage the filamentous structures gather around each chromocenter, thus constituting 39 entities of “hairy” appearance. These filaments are irregular in appearance, with blurred outlines. Furthermore, a structure outlined by its massively heteropycnotic aspect and its generally peripheral localization can be observed. The nucleoli are still present (fig. 3). This stage evolves by a progressive tightening of filaments around each chromocenter (fig. 4). 4. Condensation of the filaments increases, forming 39 compact entities, each corresponding to one chromosome. They are formed by a highly dense central mass, around which thin filaments radiate for a short distance. In addition, a very dense heteropycnotic chromosome, with smooth outlines, can be observed generally situated at the periphery. It probably corresponds to one of the two X chromosomes (fig. 5). 5. At maximum condensation, the nucleus contains 40 highly compact, angular-shaped masses, heteropycnotic in appearance. Occasionally, a few short filaments still project out from their edges (fig. 6).
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Table I. Percentage of condensation-stage oocytes in mouse embryos from day 13 to day 18.
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Fig. I. Oogonium nucleus with filamentous-appearing chromatin and numerous small chromocenters. Note presence of two nucleoli. Reproduced at 960X. Fig. 2. Oogonium nucleus at a later stage; the filaments are thicker and chromo centers slightly larger. Reproduced at 1280X.
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Fig. 3. Nucleus at onset of condensation stage; the filaments are gathered around each chromocenter. Note the presence of both a heteropycnotic chromosome peripherally situated (arrow) and a nucleolus (Nu). Reproduced at 1280X. Fig. 4. Slightly more advanced condensation stage demonstrating tightening of the filaments about each chromocenter. At the periphery note the presence of a hetero pycnotic chromosome (arrow). Reproduced at 1600X. Fig. 5. Progression of condensation leading to formation of 39 chromosomal entities, from which radiate fine filaments. A heteropycnotic chromosome is also visible at the periphery (arrow). Reproduced at 1600 X. Fig. 6. Maximal condensation. The chromosomes form 40 heterochromatic-appearing masses, from which emerge a few fine filaments. Reproduced at 1280X.
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6. The onset of decondensation is characterized by the reappearance and elongation of the filaments at the circumference of each heteropycnotic mass. These filaments are different in appearance from those that can be observed during condensation stages; they are thicker and show loops along their length (fig. 7). As decondensation gradually progresses, the volume of the heteropycnotic region decreases (fig. 8). At the end of this stage, only a heteropycnotic corpuscle remains, generally situated at the very end of a fila mentous cluster. The corpuscle position corresponds to that of the centro mere in mouse chromosomes. The filamentous cluster gradually loosens, so that the appearance of the filament approaches more and more that of a leptotene chromosome. Throughout this sequence, the entirely heteropycnotic chromosome situated at the nuclear periphery remains identifiable due to its allocyclic behavior (fig. 8). The nucleoli are still present. 7. Near the end of the decondensation stage, the nucleus contains chromosomes of filamentous appearance (fig. 9). The centromeric hetero chromatin is seen on one extremity of a large number of chromosomes. 8. The final decondensation phase ends at the onset of leptotene. The chromosomes become very long and are difficult to distinguish, as they are completely entangled. The centromeric heterochromatin is no longer observed (fig. 10). Fluorescence studies of 33258 Hoechst-stained preparations show that visible chromocenters in the oogonium nucleus correspond to centromeric heterochromatin. At maximum condensation, a bright fluorescent corpuscle can be seen, often situated on one edge of each chromosomal mass. When decondensation occurs, the centromeric heterochromatin is clearly situated at one end of the chromosomal filament, now undergoing individualization (fig. 11).
314
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An estimation of DNA content in condensation-stage nuclei was per formed by comparing it with the DNA content of somatic nuclei present in the same preparations and stained by Feulgen’s reaction. Absorption of 40 germinal and 40 somatic cells was measured. The quantity of DNA in mouse somatic cells was measured at 4.98 pg by M c C arthy (1969). We found a mean integrated optical density of 4801 (arbitrary units) in the
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Fig. 7. Onset of decondensation. Reappearance and elongation of the filaments originating from each heteropycnotic mass can be seen. Reproduced at 960X. Loops can be seen in the inset (1920 X). Fig. 8. More advanced decondensation. The chromosomal entities form filaments that are both thicker than during previous stages and are terminated by a hetero pycnotic corpuscle. A heteropycnotic chromosome can still be seen at the periphery (arrow). Reproduced at 1760X. Fig. 9. Almost completed decondensation leading to individualization of the fil aments, whose appearance resembles that of leptotene chromosomes. Reproduced at 1760X. Fig. 10. Oocyte nucleus at leptotene. Reproduced at 1360X.
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Fig. 11. Oocyte nucleus at condensation stage, stained with 33258 Hoechst. A bright fluorescent corpuscle, often located on one edge of each chromosomal mass, can be seen, which corresponds to centromeric heterochromatin. Reproduced at 1440X.
somatic cells. At condensation stage the mean integrated optical density of the germinal cells was found to be 8980, which corresponds to an average DNA content of 9.32 pg. Theoretically, after the S-phase, the DNA content of germinal cells should be twice that of somatic cells, i.e., 4.98 X 2 = 9.96 pg. Taking into consideration the accidental loss of chromosomal material from a few cells resulting from the technique used, this finding reasonably indicates that the condensation stage oocytes contain 4C DNA. Studies of condensation-stage oocytes by electron microscopy demon strate the persistence of the nuclear membrane (fig. 12). The chromosomes are present as irregular masses formed by tangled fibrils. On the edges of the chromosomal masses, a few fibrils can be seen extending into the nucleoplasm, yet these fibrils can only be followed for a very short distance. The nucleolus is still present. The cytoplasm contains many ribosomes and mitochondria, a few of which are vacuolized. The Golgi complex is well developed. Neither centrioles nor micro tubules can be seen (fig. 13).
The onset of the condensation stage and its place in the evolutive chronology of the mammalian germ cell have been determined by Mauleon et al. (1976) in the sheep. Using tritiated thymidine, they showed that
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Discussion
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Fig. 12. Electron micrograph of a mouse ovary at 14 days postfertilization, showing a cluster of oocytes at various stages of condensation. One of them (arrow) is probably close to maximum condensation. Reproduced at 4760X. Fig. 13. High magnification of the oocyte cytoplasm in fig. 12 (arrow). The Golgi complex is well developed. Neither centrioles nor microtubules are observed. Reproduced at 25,840 X.
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the condensation stage precedes leptotene. The former follows both S and G2 phases, which mark the onset of meiosis. These conclusions are analogous to those provided by chronology studies in Lillium (W alters, 1970, 1972, 1976; B ennett and Stern , 1975). In the mouse, the condensation stage appears and develops from day 13 to day 15, prior to leptotene. In this species this stage is situated between premeiotic interphase and leptotene. The morphologic aspects of the condensation stage are analogous to those already described in the rat by H ilscher et al. (1974) as postmitotic stages. Nevertheless, the 4C DNA content of these nuclei in the mouse excludes their being either telophase or G1 nuclei. In the same way, if the condensation stage corresponded to the S phase, DNA content inter mediary between 2C and 4C would be found. Condensation-stage oocytes can then only be either in G2, which is highly unlikely based on their morphology, or engaged in prophase. The latter interpretation is in agreement with the observation of individualization of chromosome structures and their progressive condensation. The hypothesis that these aspects correspond to the prophase of the last oogonial mitotic division can be excluded. In this case, as pointed out by M auleon et al. (1976), groups of metaphase and anaphase cells should be found around the condensation-stage groups, yet such is not the case. The significance of the preleptotene condensation stage is unknown. As outlined by W alters (1972), this stage is absent in many species and may present great variability in others. It has been suggested that prelep totene chromosome condensation could represent a partial reversion to mitosis (W alters, 1970). According to B ennett and Stern (1975), preleptotene condensation and decondensation represent a true reversion to mitosis, but with suppression of metaphase and anaphase. Nevertheless, it must be pointed out that mitotic prophase or even prometaphase never attain the extreme degree of chromosome contraction observed in the mammalian preleptotene condensation stage. Moreover, ultrastructural study of this stage reveals the absence in the oocyte cyto plasm of centrioles and microtubules arranged in such a way as to form a spindle. This finding indicates that the oocyte at this stage is not prepared for a mitotic-type division. It is possible that the phases of condensation and decondensation correspond to a reorganization of the chromatin in preparation for lep totene. B ennett and Stern (1975) observed in Lillium that the condensa tion-stage chromosomes were formed by two chromatids. On the contrary,
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during decondensation the filaments are single, without visible daughter chromatids, as typically observed in leptotene. In the mouse, the degree of condensation is such that it is impossible to distinguish whether or not there exist two chromatids. On the other hand, it is evident that the appearance of the filaments is totally different during the condensation and decondensation phase. As condensation progresses, the filaments become relatively fine and irregular, with often poorly limited outlines, whereas by the onset of decondensation, they acquire the diameter, regularity of appearance, and sometimes even the chromomeric structure of leptotene filaments. This finding suggests that it is during the conden sation stage that an internal rearrangement of the chromatin fibres occurs in preparation for adaptation to the specific processes of meiosis. During the condensation stage an entirely heteropycnotic chromosome is visible, generally at the nuclear periphery. We believe that this corresponds to one of the two X chromosomes, based on the observations of O hno et al. (1959) and O hno and H auschka (I960). These authors observed that in the diploid somatic nuclei of the mouse and rat, one of the X chromosomes was allocyclic, and presented a positive heteropycnosis during prophase. The condensation stage corresponds to a prophase. During the following stages, the need for pairing of the two X chromosomes would require them to be at the same degree of despiralization. A cknowledgements We thank Dr. C. M irre for his assistance in electron microscopy data. We are also grateful to Dr. M. HuLTfeN for editorial assistance with the manuscript.
References
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H ilscher , B.; H ilscher , W .; Bülthofe -O hnolz, B.; D ramer, U.; Birke , A.; P elzer , H., and G auss , G.: Kinetics of gamctogcncsis. I. Comparative histological
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Manuscript received 27 December 1976; accepted for publication 22 March 1977.
