Carmelit Richler

Meiotic association between the XY chromosomes and unpaired autosomal elements as a cause of human male sterility

Intimate association between autosomal translocation trivalents and XY bivalents at pachytene was observed in a majority of cells of two men ascertained through primary sterility and found to be heterozygous for a 14;21 Robertsonian translocation. The association, studied by light and electron microscopy of spread first spermatocytes, was between the unpaired short arms of the normal chromosomes of the translocation trivalent and the differential axes of the XY chromosomes. In a minority of cells, this contact was not established, or not maintained, as alternative combinations between the elements available for non-homologous pairing were realized. Following a suggestion of Lifschytz and Lindsley (1972), sterility in these patients was attributed to spermatogenic arrest caused by physical contact of sex chromosomes with autosomal material and consequent interference with the normal metabolism of the sex chromosomes. Autosomal aberrations and polymorphisms, which lead to the presence of unpaired segments at meiosis, may thus play a critical role in a general mechanism of chromosomally-derived male sterility. It is proposed that such a mechanism may also be instrumental in the initiation of reproductive barriers in nature.

Oncofetal H19 RNA promotes tumor metastasis.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

In situ nick-translation distinguishes between active and inactive X chromosomes

Template-active regions of chromatin are structurally distinct from nontranscribing segments of the genome. Recently, it was suggested that the conformation of active genes which renders them sensitive to DNase I may be maintained even in fixed mitotic chromosomes. We have developed a technique of mitotic cell fixation and DNase I-directed nick-translation which distinguishes between active and inactive X chromosomes. We report here that Gerbillus gerbillus (rodent) female cells contain easily identified composite X chromosomes each of which includes the original X chromosome flanked by two characteristic autosomal segments. After nick-translation the active X chromosome in each cell is labelled specifically in both the autosomal and X-chromosomal regions. The inactive X chromosome is labelled only in the autosomal regions and in a small early replicating band within the late replicating ‘original X’ chromosome. Our technique opens the possibility of following the kinetics of X-chromosome inactivation and reactivation during embryogenesis, studying active genes in the inactive X chromosome and mapping tissue-specific gene clusters.

Xist RNA is associated with the transcriptionally inactive XY body in mammalian male meiosis

Abstract.In eutherian mammals, X inactive-specific transcripts (Xist) are expressed in somatic cells possessing more than one X chromosome, and in germline cells of males, in which the single X chromosome is transcriptionally inactive. In early meiosis of males the sex chromosomes form an inactive XY nuclear compartment (XY body). We show by in situ reverse-transcribed polymerase chain reaction that Xist RNA is concentrated in the XY body. This fine localization suggests that Xist RNA is involved in inactivation of the male X chromosome, and that it has spreading capability, not only in cis but also in a quasi-cis mode, to juxtaposed non-X chromosomes. A hypothetical scheme links the evolution of heteromorphic sex chromosomes to the development of X condensation/inactivation in the male. The mechanism of X inactivation in somatic cells of mammalian females, resulting in male/female dosage compensation, has been recruited from the Xist-activated chromosome condensation machinery that developed in male meiosis earlier in evolution.

Histone macroH2A1.2 is concentrated in the XY compartment of mammalian male meiotic nuclei

We show here that histone macroH2A1.2 concentrates at the transcriptionally silent XY body, normally being formed during male meiosis in the mouse. A similar accumulation has earlier been observed on the inactive X chromosomes of somatic adult female mammalian cells by Costanzi and Pehrson (1998). This correspondence in the nature of heterochromatinization of the X chromosomes in males and females adds another property of X chromosome inactivation that is shared by males and females at different phases of their life cycle.

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress‐related infertility are not yet known. Overexpression of the “readthrough” variant of acetylcholinesterase (AChE‐R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE‐R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age‐matched control mice. Transgenic mice overexpressing AChE‐R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age‐matched nontransgenic controls. AChE‐R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE‐R transgenic mice. Head‐localized AChE‐R was characteristic of human sperm from fertile donors. In contrast, sperm head AChER staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE‐R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress‐related infertility.

REVISITING SPALAX: MITOTIC AND MEIOTIC CHROMOSOME VARIABILITY

ABSTRACT Further cytological studies on Israeli mole rats (Spalax) were motivated by their postulated active speciation. Four major chromosomal forms were characterized by Wahrman and collaborators in 1969. The differences between their chromosome numbers, 2n = 52, 54, 58 and 60, were then attributed to 1–4 Robertsonian changes, and the differences in the number of chromosome arms were assumed to be due to pericentric inversions. The new results obtained by differential staining techniques agree with the earlier interpretations. C-banding has shown that some of the evolutionary changes were accompanied by changes in the quantity and distribution of constitutive heterochromatin. All chromosomal forms also possess a considerable amount of chromosome micro-changes, including variation in the length of a C-negative, heterochromatic modification, at the base of the long arm of Chromosome 1. Four chromosomes may carry nucleolus-organizing regions (NORs), one of them in a distal position. Each of five individual...

Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

Background Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. Methodology/Principal Findings We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3′ overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. Conclusions/Significance Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease.

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11−/− males, which lack meiotic DSBs. In Atm−/− mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.

The origin of multiple sex chromosomes in the gerbil Gerbillus gerbillus (Rodentia: Gerbillinae)

The sex chromosomes of the partly sympatric species of gerbils Gerbillus pyramidum and G. gerbillus (Mammalia: Gerbillinae) were investigated by a variety of light- and electron-microscope methods, including DNA replication banding and synaptonemal complex (SC) techniques. The sex-chromosome mechanism of G. pyramidum is of the maleXY:femaleXX type, whereas that of G. gerbillus is of the less common maleXY1Y2:femaleXX system. The results include the demonstration that the X chromosomes of both species are compound. One segment is added to the X chromosome of G. pyramidum, leading to an increase in length from the standard 5% to approximately 7.3%, whereas two different extra segments increase the length of the X chromosome of G. gerbillus to approximately 11% of the length of the haploid genome. In both cases the extra material is autosomal and is also represented in the respective Y chromosomes. Classifying heterochromatin by the variation in staining quality was helpful in elucidating the possible origin of the different chromosome segments, including the pericentromeric regions. Observations on meiotic chromosome pairing and chiasma formation have confirmed the homologies established by band comparisons. The occurrence of chiasmata between the sex chromosomes supports the autosomal origin of the pairing segments. These and other findings have been interpreted in the framework of a multistep evolutionary model. This sequence starts from a hypothetical pair of sex chromosomes, the X element of which amounts to 5% of the haploid genome, and leads through three translocations involving two pairs of autosomes and one pericentric inversion to the most complex situation of this series, manifested in G. gerbillus. The adaptive value, if any, of autosome incorporation into the sex chromosomes repeatedly occurring here is unknown. It is, however, a remarkable fact that in one species, G. gerbillus, the complex sex-chromosome constitution is conserved over vast geographic distances, and in the other, G. pyramidum, the compound X and Y chromosomes withstand change in the face of extreme autosome restructuring.