Heterochromatin (C-bands) in somatic and male germ cells in three species ofMicrotinae

The C-banding patterns in the chromosomes ofMicrotus oeconomus, M. arvalis andM. ochrogaster demonstrate differences in the amount and distribution of heterochromatin. Autosomal centromeric heterochromatin appears as conspicuous blocks or as small dots, and in several chromosomes no heterochromatin was detected; interstitial heterochromatin was observed in one autosome pair ofM. ochrogaster. The sex chromosomes also demonstrate differences in the C-banding pattern. InM. oeconomus, the X chromosome exhibits a block of centromeric heterochromatin which is larger than that of the autosomes; this characteristic helps to recognize the X chromosomes in the karyotype. InM. arvalis no heterochromatin was appreciated in the sex chromosomes. The Y chromosomes ofM. ochrogaster andM. oeconomus are entirely heterochromatic. During male meiosis heterochromatin shows condensation, association and chiasma prevention; the sex chromosomes pair end to end in the three species. At pairing, the Y chromosome ofM. arvalis is despiralized, but it appears condensed again shortly before separation of the bivalent.     

The mouse Y* chromosome involves a complex rearrangement, including interstitial positioning of the pseudoautosomal region.

Cytological analysis of the mouse Y* chromosome revealed a complex rearrangement involving acquisition of a functional centromere and centromeric heterochromatin and attachment of this chromosomal segment to the distal end of a normal Y* chromosome. This rearrangement positioned the Y* short-arm region at the distal end of the Y* chromosome and the pseudoautosomal region interstitially, just distal to the newly acquired centromere. In addition, the majority of the pseudoautosomal region was inverted. Recombination between the X and the Y* chromosomes generates two new sex chromosomes: (1) a large chromosome comprised of the X chromosome attached at its distal end to all of the Y* chromosome but missing the centromeric region (XY*) and (2) a small chromosome containing the centromeric portion of the Y* chromosome attached to G-band-negative material from the X chromosome (YX). Mice that inherit the XY* chromosome develop as sterile males, whereas mice that inherit the Y*X chromosome develop as fertile females. Recovery of equal numbers of recombinant and nonrecombinant offspring from XY* males supports the hypothesis that recombination between the mammalian X and Y chromosomes is necessary for primary spermatocytes to successfully complete spermatogenesis and form functional sperm.     

Localization of repetitive DNA in the chromosomes of Microtus agrestis by means of in situ hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.Supported in part by Research Grants DRG-1061 and 269 from the Damon Runyon Memorial Fund for Cancer Research, G-373 and G-267 from the Robert A. Welch Foundation.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

Sex chromosome-autosome translocations in the leaf-nosed bats, family Phyllostomidae. I. Mitotic analyses of the subfamilies Stenodermatinae and Phyllostominae

Mitotic analyses using RBA- and C-banding were performed on Stenodermatine bats with X-autosome (XY1Y2) and X- and Y- autosome (neo-XY) translocations. RBA-banded metaphases of females revealed differential replication of the inactive X chromosome. An early replicating band comprises the short arm of the X, and an intermediate replicating band is located interstitially on the long arm. The early replicating short arm has a homologous counterpart either in the form of a free autosome (the Y2) or as part of the Y. Both the "autosomal" short arm of the X and its homologue fused to the Y are C-band negative and behave autonomously from the remainder of the sex chromosomes. They are separated from X and Y chromatin by centromeric heterochromatin which presumably acts as a barrier. The intermediate replicating region of the long arm of the X is also present in the subfamily Phyllostominae. In both subfamilies this region lacks a homologous counterpart. However, it may also represent a translocated autosome which, unlike the short arm of the X, is not separated from the inactive X by centromeric heterochromatin. Its intermediate replication time may represent a retarded replication due to its juxtaposition to late replicating X chromatin. These data are discussed in light of the theory of the evolution of sex chromosome heteromorphism, specifically as it applies to mammals.     

Evidence for a highly differentiated sex chromosome heteromorphism in the salamander Necturus maculosus (Rafinesque)

The mitotic chromosomes of the neotenic (sensu Gould, 1977, and Alberch et al., 1979) salamander Necturus maculosus (Rafinesque) have been examined using a C-band technique to demonstrate the distribution of heterochromatin. The C-banded mitotic chromosomes provide evidence of a highly differentiated XY male/XX female sex chromosome heteromorphism, in which the X and Y chromosomes differ greatly in size and morphology, and in the amount and distribution of C-band heterochromatin. The X chromosome represents one of the largest biarmed chromosomes in the karyotype and is indistinguishable from similar sized autosomes on the basis of C-band heterochromatin. The Y chromosome, on the other hand, is diminutive, morphologically distinct from all other chromosomes of the karyotype, and is composed almost entirely of C-band heterochromatin. The discovery of an X/Y chromosome heteromorphism in this species is consistent with the observation by King (1912) of a heteromorphic spermatogenic bivalent. Karyological and phylogenetic implications are discussed.     

Sex chromosomes in the spiny eel (Mastacembelus aculeatus) revealed by mitotic and meiotic analysis

Lower vertebrates like fish exhibit tremendous diversity in sex determination. There are wide interplays between environment-dependent sex differentiation ranging from natural hermaphroditism to sex reversal and genetic sex determination. Diverse systems of male and female heterogamety coexist in fish and sex chromosomes are rarely distinguishable in morphology. Here we show that the spiny eel ((Mastacembelus aculeatus) of the Perciformes, has evolved highly heteromorphic X and Y chromosomes. The metacentric X and Y chromosomes are the largest among 24 homologous pairs, differ from each other in size and morphology, and become distinct after C-banding because of conspicuous heterochromatin blocks which exhibit alternate distribution around the centromeric region. Chromosome painting using probes from the microdissected X chromosome revealed sequence homology between X and Y. During the pachytene stage of meiosis the X and Y form a bivalent. However, their synapsis is delayed which is particularly evident in one terminus. Therefore, the X and Y have resulted from a pericentric inversion in the Y. We conclude that M. aculeatus represents an example of a highly advanced stage of sex chromosome evolution in fish.     

Cytomolecular analysis of the male sex chromosome of Tropidacris cristata grandis (Thunberg, 1824) (Orthoptera: Romaleidae)

Many species of grasshopper have an XX/XO sex chromosome system, including Tropidacris cristata grandis (23, XX/XO). The X chromosome behaves differently from the autosomes, but little is known about its origin and molecular composition. To better understand the genomic composition and evolutionary processes involved in the origin of the sex chromosomes, we undertook an analysis of its meiotic behavior, heterochromatin distribution and microdissection in T. c. grandis. Analysis of meiotic cells revealed a difference in the behavior of the X chromosome compared to the autosomes, with different patterns of condensation and cellular arrangement. Heterochromatic terminal blocks were predominant. The chromosome painting revealed a bright block in the centromeric/pericentromeric region of the X chromosome and slight markings in the other regions. In the autosomes, the X chromosome probe hybridized in the centromeric/pericentromeric region, and hybridization signals on terminal regions corresponding to the heterochromatic regions were also observed. The results showed that the X chromosome contains a significant amount of repetitive DNA. Based on the hybridization pattern, it is possible that the autosomes and sex chromosomes of T. c. grandis have a similar composition of repetitive DNAs, which could mean that the X chromosome has an autosomal origin.     

Variation of constitutive heterochromatin in the sex chromosomes of the rodent Bandicota bengalensis bengalensis (Gray)

Bandicota bengalensis bengalensis (Gray) trapped from different localities of India and Nepal exhibited a marked variation in the size and morphology of sex chromosomes. Three types of X's were found; A) simple acrocentric, B) composite subtelocentric and C) composite submetacentric X with their relative sizes 5.9%, 7.5% and 9.6% of the genome respectively. The autosomes remained unaltered. It was shown that this variation in the size of sex chromosomes was caused by deletion of constitutive heterochromatin. The Y chromosome was also found to be variable. Usually a large X was combined with a large Y. The preponderance of homozygotes for each type of X chromosome in populations, suggested the probable role of sex chromosomes heterochromatin in speciation.     

Localization of repetitive DNA in the chromosomes of <Emphasis Type="Italic">Microtus agrestis</Emphasis> by means of <Emphasis Type="Italic">in situ</Emphasis> hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.     

Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica

In the domestic pig (2n=38) two types of constitutive heterochromatin can be differentiated by fluorescence counterstaining techniques. All 24 biarmed autosomes and the X chromosome have chromomycin A₃-positive centromeric C-bands, whereas all 12 acrocentric chromosomes exhibit DA-DAPI-positive centromeric heterochromatin. Fluorescence analysis of male pachytene nuclei revealed that the DA-DAPI-positive C-bands form one or two large chromocentres per cell, while the chromomycin A₃-bright C-material is well scattered. Hence, the bivalents formed by the acrocentric chromosome pairs are centromerically associated, whilst the submetacentric bivalents are not. —Counce-Meyer spreading techniques were used to study the structure of synaptonemal complexes (SCs) both by light and electron microscopy. In general, the SCs of the domestic pig resemble those described for other mammals. The SC formed by the X and the Y may include up to 94.5% of the Y chromosome. In silver-stained microspreads each of the bivalents (nos. 8 and 10) bearing the nucleolus-organizer-regions (NORs) is connected to a pair of nucleoli, indicating that all four NORs are active during early meiotic stages. By contrast, in the majority of mitotic metaphases of phytohaemagglutinin-stimulated lymphocytes only one pair (no. 10) exhibited Ag-NOR staining. — The significance of the chromosome disposition in the pachytene nucleus is discussed with regard to heterochromatin composition and karyotype evolution.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

Human male meiotic sex chromosome inactivation

In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.     

白眉长臂猿(Hylobates hoolock leuconedys)的染色体研究

本文对两只雄性白眉长臂猿的染色体的C带、G带及Ag-NORs分布进行了较详细的分析,证实染色体数2n=38,并对该种的分类地位提出了一些新看法。     

Euchromatic inversions causing mosaic gene expression in Drosophila hydei: A study of forked and Zebra

In D. hydei two new mutants, In(1)f³ and IN(5)Z, show obvious mosaic gene expression. Their phenotypic expression is susceptible to the breeding temperature and to the addition of a supernumerary Y chromosome to the chromosome set. In this respect the mutants resemble standard cases of position-effect variegation based on the action of heterochromatin. However, since neither centromeric nor sex chromosomal heterochromatin apparently are involved, the mutations point to a new type of variegation provoked by euchromatic sections. The mosaic patterns of these mutants, in particular those of In(1)f³, will be described.     

Complex sex chromosome system in Eigenmannia sp. (Pisces,Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10.     

Chromosomes and DNA of Mus

Using C-banded preparations of Mus dunni it is possible to study the behavior of constitutive heterochromatin in early stages of meiotic prophase. The X and the Y chromosomes, both of which contain a large amount of heterochromatin, lie apart in leptotene but move toward each other during zygotene. They then form the sex vesicle at late zygotene. In autosomes zygotene pairing appears to start from the telomeric ends. The centromere of the Y chromosome associates end-to-end with the terminal end of the long arm of the X chromosome. The autosomal heterochromatic short arms show forked morphology in certain bivalents at pachytene, suggesting probable incomplete synapsis.     

The meiotic structure and behavior of the strongly heteromorphic X/Y sex chromosomes of neotropical plethodontid salamanders of the genus Oedipina

Plethodontid salamanders in the genus Oedipina are characterized by a strongly heteromorphic sex-determining pair of X/Y chromosomes. The telocentric X chromosome and the subtelocentric Y chromosome are clearly distinguished from the autosomes and their behavior during meiosis can be sequentially followed in squash preparations of spermatocytes. In Oedipina the sex chromosomes are not obscured by an opaque sex vesicle during early meiotic stages, making it possible to observe details of sex bivalent structure and behavior not directly visible in other vertebrate groups. The sex chromosomes can first be distinguished from autosomal bivalents at the conclusion of zygotene, with X and Y synapsed only along a short segment at their non-centromeric ends, forming a bivalent that contrasts sharply with the completely synapsed autosomes. During pachytene, the XY bivalent becomes progressively shortened and more compact, disappearing as a visible structure when pachytene progresses into the diffuse stage of male meiosis. Diplotene bivalents gradually emerge from the diffuse nuclei, presumably by the return of the loops of chromatin into their respective chromomeres. During early diplotene, the X/Y bivalent is clearly visible with a single chiasma within the synapsed segment. This chiasma is terminalized by first meiotic metaphase with the X and Y appearing either in end-to-end synaptic contact or as univalents separated at opposite poles relative to the equatorially distributed autosomal bivalents. In C-banded preparations, the Y is entirely heterochromatic while the X contains a large centromeric C-band and another block of heterochromatin located at the telomeric end, in the region of synapsis with the Y. We find no cytological evidence of dosage compensation, such as differential staining of the X chromosomes or Barr bodies, in mitotic or interphase cells from female animals.     

Heterogeneity of constitutive heterochromatin in somatic Syrian hamster chromosomes.

Syrian hamster constitutive heterochromatin was analyzed for C-band distribution and for BrU late-replication pattern. Characteristic for this species is relatively large amounts of sex-chromosome and autosomal heterochromatin. The distribution of constitutive heterochromatin was determined. The long term of the X chromosome, the whole Y, the short arms of 8 autosomal pairs, the long arm of the smallest metacentric pair, and the centromeric regions of 12 pairs stained intensely dark on C-band preparations. In contrast to the heterochromatin in the centromeric regions, the autosomal short-arm heterochromatin has an increased susceptibility to the denaturation process, as indicated by prolonged exposure to NaOH or Ba(OH)2. Such further exposure to denaturing agents results in an intense dark stain only on the sex-chromosome heterochromatin and centromeric regions of the autosomes. The BrdU late-replication pattern demonstrated that the late-replicating regions correspond to C-bands. Centromeric regions replicate late in the S phase; however, no centromeric region is among the latest replicating segments of the complement. Centromeric and noncentromeric heterochromatin are two distinct categories of constitutive heterochromatin.     

A Novel MS7 Repeat Specific for Heterochromatin of Sex Chromosomes in Voles of Genus Microtus, Group arvalis: Genomic Organization and Chromosomal Localization

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Cytogenetics of the South American Akodont rodents (Cricetidae) X. Akodon mollis: a species with XY females and B chromosomes

The morphology, G- and C-banding pattern of the Akodon mollis chromosome complement is analysed. Over a total of 14 males and 10 females studied, 8 males and 7 females had a modal chromosome number of 22, while 6 males and 3 females showed a modal number of 23 chromosomes. In the animals with 23 chromosomes the odd element was considered a B chromosome on the basis of: (a) its small size, (b) the lack of an homologous chromosome and the subsequent formation of univalents at diakinesis and metaphase I from testes, (c) the weak or null genetic action as evidenced by the lack of any obvious variation in the phenotype of carriers.Four females exhibited a sex-pair dimorphism indistinguishable from that observed in males. The G-banding analysis showed homology between the pattern found in the Y chromosome and that detected in the short arm of the X. The study of C-band distribution showed that several autosome pairs and the X chromosomes had small masses of centromeric heterochromatin. On the other hand, the Y and B chromosomes were C-band negative. The Y-like chromosome in females with dimorphism of the sex pair was also C-band negative. Accordingly these females were considered to be XY and not Xx (the x being an extensively deleted X chromosome).This work was supported by grants from UNESCO, OEA, CONICET and CIC. Requests for reprints should be addressed to N.O. Bianchi.