Makiko Tsutsumi

Age-Related Decrease of Meiotic Cohesins in Human Oocytes

Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.

Mutations of the SYCP3 Gene in Women with Recurrent Pregnancy Loss

Aneuploidy, a chromosomal numerical abnormality in the conceptus or fetus, occurs in at least 5% of all pregnancies and is the leading cause of early pregnancy loss in humans. Accumulating evidence now suggests that the correct segregation of chromosomes is affected by events occurring in prophase during meiosis I. These events include homologous chromosome pairing, sister-chromatid cohesion, and meiotic recombination. In our current study, we show that mutations in SYCP3, a gene encoding an essential component of the synaptonemal complex that is central to the interaction of homologous chromosomes, are associated with recurrent pregnancy loss. Two out of 26 women with recurrent pregnancy loss of unknown cause were found to carry independent heterozygous nucleotide alterations in this gene, neither of which was present among a group of 150 fertile women. Analysis of transcripts from minigenes harboring each of these two mutations revealed that both affected normal splicing, possibly resulting in the production of C-terminally mutated proteins. The mutant proteins were found to interact with their wild-type counterpart in vitro and inhibit the normal fiber formation of the SYCP3 protein when coexpressed in a heterologous system. These data suggest that these mutations are likely to generate an aberrant synaptonemal complex in a dominant-negative manner and contribute to abnormal chromosomal behavior that might lead to recurrent miscarriage. Combined with the fact that similar mutations have been previously identified in two males with azoospermia, our current data suggest that sexual dimorphism in response to meiotic disruption occurs even in humans.

The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements

Kurahashi H, Inagaki H, Ohye T, Kogo H, Tsutsumi M, Kato T, Tong M, Emanuel BS. The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements.

Recent advance in our understanding of the molecular nature of chromosomal abnormalities

The completion of the human genome project has enabled researchers to characterize the breakpoints for various chromosomal structural abnormalities including deletions, duplications or translocations. This in turn has shed new light on the molecular mechanisms underlying the onset of gross chromosomal rearrangements. On the other hand, advances in genetic manipulation technologies for various model organisms has increased our knowledge of meiotic chromosome segregation, errors which, contribute to chromosomal aneuploidy. This review focuses on the current understanding of germ line chromosomal abnormalities and provides an overview of the mechanisms involved. We refer to our own recent data and those of others to illustrate some of the new paradigms that have arisen in this field. We also discuss some perspectives on the sexual dimorphism of some of the pathways that leads to these chromosomal abnormalities.

Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2 deduced from mRNA nucleotide sequences.

Tol2 is a terminal‐inverted repeat transposable element of the medaka fish Oryzias latipes. It is one of a few elements of this class so far demonstrated to be active in vertebrates, thus providing a unique tool for establishing a gene tagging system. For the purpose of identifying its transposase, we analyzed the structures of mRNAs originating from the Tol2 element. The results indicated that transcription of Tol2 is initiated at several sites, the four open reading frames in Tol2 roughly corresponding to exons, and that two main forms of mRNAs, covering exons 1–4 and exons 2–4, are present in medaka fish cells. One or both of these mRNAs are likely to encode a transposase, the amino acid sequence of which was deduced.

HORMAD1‐dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes

Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1‐deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1‐deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11‐deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11‐deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11‐deficient testis and ovary, suggesting an involvement of novel DNA damage–independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1‐dependent checkpoint in response to asynapsis in mammalian meiosis.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab‐3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB‐3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior–dorsal telencephalon) is markedly reduced in this mutant at the 14‐somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes‐related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over‐expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior–dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior–dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior–dorsal telencephalic cell fate during somitogenesis.

Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations

Gross chromosomal rearrangements (GCRs), such as translocations, deletions or inversions, are often generated by illegitimate repair between two DNA breakages at regions with nucleotide sequences that might potentially adopt a non-B DNA conformation. We previously established a plasmid-based model system that recapitulates palindrome-mediated recurrent chromosomal translocations in humans, and demonstrated that cruciform DNA conformation is required for the translocation-like rearrangements. Here we show that two sequential reactions that cleave the cruciform structures give rise to the translocation: GEN1-mediated resolution that cleaves diagonally at the four-way junction of the cruciform and Artemis-mediated opening of the subsequently formed hairpin ends. Indeed, translocation products in human sperm reveal the remnants of this two-step mechanism. These two intrinsic pathways that normally fulfil vital functions independently, Holliday-junction resolution in homologous recombination and coding joint formation in rearrangement of antigen-receptor genes, act upon the unusual DNA conformation in concert and lead to a subset of recurrent GCRs in humans.

HORMAD2 is essential for synapsis surveillance during meiotic prophase via the recruitment of ATR activity

Meiotic chromosome segregation requires homologous pairing, synapsis and crossover recombination during meiotic prophase. The checkpoint kinase ATR has been proposed to be involved in the quality surveillance of these processes, although the underlying mechanisms remain largely unknown. In our present study, we generated mice lacking HORMAD2, a protein that localizes to unsynapsed meiotic chromosomes. We show that this Hormad2 deficiency hampers the proper recruitment of ATR activity to unsynapsed chromosomes. Male Hormad2‐deficient mice are infertile due to spermatocyte loss as a result of characteristic impairment of sex body formation; an ATR‐ and γH2AX‐enriched repressive chromatin domain is formed, but is partially dissociated from the elongated sex chromosome axes. In contrast to males, Hormad2‐deficient females are fertile. However, our analysis of Hormad2/Spo11 double‐mutant females shows that the oocyte number is negatively correlated with the frequency of pseudo–sex body formation in a Hormad2 gene dosage‐dependent manner. This result suggests that the elimination of Spo11‐deficient asynaptic oocytes is associated with the HORMAD2‐dependent pseudo–sex body formation that is likely initiated by local concentration of ATR activity in the absence of double‐strand breaks. Our results thus show a HORMAD2‐dependent quality control mechanism that recognizes unsynapsis and recruits ATR activity during mammalian meiosis.

Molecular basis of maternal age-related increase in oocyte aneuploidy.

Aneuploidy is one of the most common and serious pregnancy complications in humans. Most conceptuses with autosomal aneuploidy die in utero, resulting in early pregnancy loss. However, some fetuses with aneuploidy survive to term but suffer from disorders associated with congenital anomalies and mental retardation, such as Down syndrome with trisomy 21. Three general characteristics of this condition are well acknowledged: (i) in most cases the extra chromosome is of maternal origin; (ii) most cases are derived from a malsegregation event in meiosis I; and (iii) the frequency of these errors increases with maternal age. The basis for the age‐dependent increase in meiosis I errors has been a long‐standing enigma. Many investigators have addressed the nature of this biological phenomenon through genomic analyses of extra chromosome 21 using polymorphic markers to determine the frequency or location of crossovers that should ensure faithful chromosome segregation. Cytogenetic analyses of in vitro unfertilized oocytes have also been performed. However, no definitive conclusions regarding meiosis I errors have yet been reached from such studies. Recent findings in conditional knock‐out mice for meiosis‐specific cohesin have shed further light on this issue. The present review focuses on the current understanding of age‐related aneuploidy and provides an overview of the mechanisms involved. We refer to recent data to illustrate some of the new paradigms that have arisen in this field.