Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Background

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Results

In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.     

Absence of satellite DNA synthesis during meiotic prophase in mouse and human spermatocytes

Mouse spermatocytes were labelled in situ with ³H-thymidine at successive stages of meiosis. Isolated mouse as well as human spermatocytes were similarly labelled under in vitro conditions. DNA synthesis was followed either by tracking radioactivities in Cs₂SO₄ gradients or by measuring reassociation kinetics. Mouse satellite DNA and the 3 satellites of human DNA are labelled during S-phase but not during pachytene. In the mouse genome, there is a preferential labelling of regions containing foldbacks (human spermatocytes were not analyzed in this respect). The absence of detectable pachytene synthesis in satellite DNA is consistent with genetic evidence on the absence of crossing-over in constitutive heterochromatin.     

Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes

The meiotic prophase I to metaphase I transition (G2/MI) involves disassembly of synaptonemal complex (SC), chromatin condensation, and final compaction of morphologically distinct MI bivalent chromosomes. Control of these processes is poorly understood. The G2/MI transition was experimentally induced in mouse pachytene spermatocytes by okadaic acid (OA), and kinetic analysis revealed that disassembly of the central element of the SC occurred very rapidly after OA treatment, before histone H3 phosphorylation on Ser10. These events were followed by relocalization of SYCP3 and final condensation of bivalents. Enzymatic control of these G2/MI transition events was studied using small molecule inhibitors: butyrolactone I (BLI), an inhibitor of cyclin-dependent kinases (CDKs) and ZM447439 (ZM), an inhibitor of aurora kinases (AURKs). The formation of highly condensed MI bivalents and disassembly of the SC are regulated by both CDKs and AURKs. AURKs also mediate phosphorylation of histone H3 in meiosis. However, neither BLI nor ZM inhibited disassembly of the central element of the SC. Thus, despite evidence that the metaphase promoting factor is a universal regulator of the onset of cell division, desynapsis, the first and key step of the G2/MI transition, occurs independently of BLI-sensitive CDKs and ZM-sensitive AURKs.     

The chromosomes of Micromys minutus (Rodentia, Murinae). II. Pairing pattern of X and Y chromosomes in meiotic prophase

Both light and electron microscopy were used to study the pairing behavior of the sex chromosomes of the harvest mouse, Micromys minutus, in surface-spread pachytene spermatocytes. The XY pairing pattern is very exceptional in that the site of synaptic initiation is located interstitially in the short arms of the X and the Y, next to their centromeric regions. From this tiny euchromatic site, synapsis proceeds unidirectionally along the homologous heterochromatic short arms of the X and the Y toward the ends of the chromosomes. After pairing of the short arm is concluded, synapsis begins between the nonhomologous long arms of the X and the Y in the immediate vicinity of the centromeres and progresses unidirectionally toward the end of the long arm of the Y. A synaptic complex develops between the constitutive heterochromatin of the long arm of the Y and the euchromatin of the long arm of the X. Analysis of C-banded and distamycin A/DAPI-stained diakineses revealed a trefoil-like XY bivalent, which was interpreted to be the result of an interstitial chiasma occurring in the paired short arms of the X and the Y. A conspicuous, electron-dense body, about 1 micron in diameter, was found closely associated with the centromeres of the X and the Y in numerous pachytene spermatocytes. A review of the literature showed that comparable XY-associated bodies have been found in only eight other mammals to date.     

The behavior and morphology of the X and Y chromosomes during prophase I in the Sitka deer mouse (Peromyscus sitkensis)

Surface-spread, silver-stained primary spermatocytes from individuals of the Sitka deer mouse (Peromyscus sitkensis) were analyzed by electron microscopy. Pairing of the X and Y chromosomes is initiated at early pachynema and is complete by mid pachynema. The pattern of sex chromosome pairing is unique in that it is initiated at an interstitial position, with subsequent synapsis proceeding in a unidirectional fashion towards the telomeres of the homologous segments. One-third the length of the X and two-thirds the length of the Y are involved in the synaptonemal complex of the sex bivalent. Various morphological complexities develop in the heteropycnotic (unpaired) segments as pachynema progresses, but desynapsis is not initiated until diplonema. Analysis of C-banded diakinetic nuclei indicated that sex chromosome pairing involves the heterochromatic short arm of the X and the long arm of the heterochromatic Y. An interstitial chiasma between the X and Y was observed in the majority of the diakinetic nuclei. The observation of a substantial pairing region and chiasma formation between the sex chromosomes of these deer mice is interpreted as indicating homology between the short arm of the X and the long arm of the Y.     

Persistence of two Y chromosomes through meiotic prophase and metaphase I in an XYY man

Summary Studies of spermatogenesis in an XYY male, presenting at a subfertility clinic, confirm the tendency for the germ cells to lose the second Y chromosome but for some XYY cells to reach metaphase I (MI). Light microscope studies of MI revealed the presence of YY bivalents and EM studies of microspread, silver-stained pachytene stages showed 30% of the cells to have two Y chromosomes; 13 out of 16 of these showing a YY synaptonemal complex. Strikingly, the Y axes show only partial synapsis; in no case was synapsis of the long arm heterochromatic regions apparent.     

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

Background  

The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I.     

Non-random distribution of the pericentromeric heterochromatin in meiotic prophase nuclei of mammalian spermatocytes

The central or peripheral distribution of condensed chromatin (CC) was studied in pachytene spermatocyte nuclei in Mus domesticus, 2n=40; Pudu puda, 2n=70; Ctenomys opimus, 2n=26 and Octodon degus, 2n=58. Species were chosen according to the morphological characteristics of their chromosomal complements and in particular, the terminal or medial chromosomal localisation of the pericentromeric constitutive heterochromatin. Counts were made by defining the areas corresponding to peripheral and central location in each nuclear section from a series. The null hypothesis (i.e. random distribution of CC) was rejected. In the nuclear sections of Mus domesticus and Pudu puda, 69% and 74% of CC, respectively, was found in the peripheral nuclear space, while in those of Octodon degus and Ctenomys opimus, 69% and 65% of CC, respectively, was found in the central nuclear space. We estimate that if the CC measured in spermatocyte nuclei corresponds mainly to pericentromeric constitutive heterochromatin, the distribution found is consistent with that expected in accordance with the nuclear architecture model for meiocytes (Fernández-Donoso, 1982; Fernández-Donoso & Berrios, 1985). This model proposes a peripheral nuclear localisation for pericentromeric heterochromatin of telocentric bivalents and a relatively central nuclear localisation for pericentromeric heterochromatin of metacentric bivalents. We also discuss some of the biological consequences that could arise from the conservation of such distributions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Ultrastructural studies of late meiotic prophase nuclei of spermatocytes in Ascaris suum

Post pachytene stages of meiotic prophase in males of Ascaris suum have been analyzed with the electron microscope. No synaptonemal-like polycomplexes (PCs) have been observed in the nucleoplasm or cytoplasm during the period from pachytene to diakinesis. From Serially sectioned diplotene nuclei it was found that the bivalents are located near the periphery of the nuclei, the central part of the nuclei being vacant. Each nucleus contains one nucleolus. Up to 1 m long stretches of unpaired lateral elements (LEs) are found in some of the diplotene bivalents. These LEs are morphologically similar to unpaired LEs in early zygotene nuclei. Partial 3-dimensional reconstruction of two nuclei shows that the bivalents contain some small stretches of synaptonemal complex (SC) up to 1.9 m long. Some bivalents at diakinesis show remnants of SCs. At this stage chromosomes are fibrous, condensed, attached to the nuclear envelope and mostly with a rounded profile in cross section. The synchronous development of the spermatocytes and small bivalents at diplotene in A. suum make this system a good object for the study of localization of SC remnants.     

Nucleoli and chromosomes: Their relationships during the meiotic prophase of the human fetal oocyte

Summary The number of nucleoli and the relations between them and chromosomes in the human fetal oöcyte have been investigated in this study. The differences existing between first oöcytes and spermatocytes have been emphasized. These differences concern essentially the number of nucleoli, the stage during which they appear and the quantity of heterochromatin associated with the nucleoli.The latter appear very early in the oöcyte: they are already present at the beginning of the preleptotene stage. This stage is characterized by the processes of spiralization and despiralization, heretofore not described.The first prophase is characterized by the presence of abundant nucleolar material, especially in the diplotene stage. This abundance is certainly in relation with the active protein synthesis which characterizes the growth of the oöcyte. As in the spermatocyte of the pachytene stage, the majority of nucleolar chromosomes, in the oöcyte, are acrocentric. But in the latter, the quantity of heterochromatin associated with the nucleolus greatly exceeds the quantity present in the nucleolar organizers of acrocentric chromosomes, particularly during the leptotene stage. This supports the opinion that there are multiple sites for the synthesis of the various nucleolar componnents. Also discussed are the roles of heterochromatin and nucleolar organizers in nucleogenesis.

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Zusammenfassung Die Zahl der Nucleolen sowie ihre Beziehungen zu den Chromosomen wurde an der menschlichen fetalen Oocyte untersucht. Dabei stellten sich deutliche Differenzen zu den Spermatocyten heraus. Diese Differenzen betreffen vor allem die Zahl der Nucleolen, das Stadium, in welchem sie erscheinen, und die Heterochromatinmenge, die mit den Nucleolen assoziiert ist.Diese erscheinen in der Oocyte sehr früh, schon zu Beginn des Präleptotäns. In diesem Stadium wurde erstmalig eine Spiralisation und Despiralisation beschrieben.Die erste Prophase ist durch die Anwesenheit von großen Mengen von Nucleolusmaterial charakterisiert, besonders im Diplotän. Dieser Befund steht sicher in Zusammenhang mit der aktiven Proteinsynthese während des Oocytenwachstums. Wie bei Pachytänspermatocyten sind auch in der Oocyte die meisten der mit dem Nucleolus verbundenen Chromosomen akrozentrisch. Doch übertrifft bei letzterer die Heterochromatinmenge beim Nucleolus die Menge der Nucleolusorganisatoren der akrozentrischen Chromosomen erheblich, besonders während des Leptotäns. Dadurch wird die Auffassung unterstützt, daß die Synthese verschiedener Nucleoluskomponenten an verschiedenen Stellen erfolgt. Die Bedeutung des Heterochromatins und der Nucleolusorganisatoren bei der Entstehung des Nucleolus wird diskutiert.