Kichiya Suzuki

Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.

Foxa1 and Foxa2 Interact with the Androgen Receptor to Regulate Prostate and Epididymal Genes Differentially

Abstract: Previous studies from our group have shown that Foxa1 is expressed in the prostate and interacts with the androgen receptor (AR) to regulate prostate‐specific genes such as prostate‐specific antigen (PSA) and probasin (PB). We report here that Foxa2 but not Foxa1 is expressed in the epididymis. Further, Foxa2 interacts with the AR to regulate the mouse epididymal retinoic acid binding protein (mE‐RABP) gene, an epididymis‐specific gene. Binding of Foxa2 to the mE‐RABP promoter was confirmed by gel‐shift and chromatin immunoprecipitation (ChIP) assays. Overexpression of Foxa2 suppresses androgen activation of the mE‐RABP promoter while overexpression of Foxa2 with prostate‐specific promoters activates gene expression in an androgen‐independent manner. GST pull‐down assays determined that both Foxa1 and Foxa2 physically interact with the DNA binding domain of the AR. The interaction between Foxa proteins and AR was further confirmed by gel‐shift assays where Foxa protein was recruited to AR binding oligomers even when Foxa binding sites were not present, and AR was recruited to Foxa binding oligomers even in the absence of an AR binding site. Given that Foxa1 and Foxa2 proteins are expressed differentially in the prostate and epididymis, these data suggest that the Foxa proteins have distinct effects on AR‐regulated genes in different male reproductive accessory organs.

The Tohoku Medical Megabank Project: Design and Mission

The Great East Japan Earthquake (GEJE) and resulting tsunami of March 11, 2011 gave rise to devastating damage on the Pacific coast of the Tohoku region. The Tohoku Medical Megabank Project (TMM), which is being conducted by Tohoku University Tohoku Medical Megabank Organization (ToMMo) and Iwate Medical University Iwate Tohoku Medical Megabank Organization (IMM), has been launched to realize creative reconstruction and to solve medical problems in the aftermath of this disaster. We started two prospective cohort studies in Miyagi and Iwate Prefectures: a population-based adult cohort study, the TMM Community-Based Cohort Study (TMM CommCohort Study), which will recruit 80 000 participants, and a birth and three-generation cohort study, the TMM Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study), which will recruit 70 000 participants, including fetuses and their parents, siblings, grandparents, and extended family members. The TMM CommCohort Study will recruit participants from 2013 to 2016 and follow them for at least 5 years. The TMM BirThree Cohort Study will recruit participants from 2013 to 2017 and follow them for at least 4 years. For children, the ToMMo Child Health Study, which adopted a cross-sectional design, was also started in November 2012 in Miyagi Prefecture. An integrated biobank will be constructed based on the two prospective cohort studies, and ToMMo and IMM will investigate the chronic medical impacts of the GEJE. The integrated biobank of TMM consists of health and clinical information, biospecimens, and genome and omics data. The biobank aims to establish a firm basis for personalized healthcare and medicine, mainly for diseases aggravated by the GEJE in the two prefectures. Biospecimens and related information in the biobank will be distributed to the research community. TMM itself will also undertake genomic and omics research. The aims of the genomic studies are: 1) to construct an integrated biobank; 2) to return genomic research results to the participants of the cohort studies, which will lead to the implementation of personalized healthcare and medicine in the affected areas in the near future; and 3) to contribute the development of personalized healthcare and medicine worldwide. Through the activities of TMM, we will clarify how to approach prolonged healthcare problems in areas damaged by large-scale disasters and how useful genomic information is for disease prevention.

Intramanchette transport during primate spermiogenesis : expression of dynein, myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein, and Rab27b in the manchette during human and monkey spermiogenesis

AIM To show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein (MyRIP), and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport (IMT). METHODS Spermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey (Macaca fascicularis). Immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the proteins were carried out. Samples were analyzed by light microscope. RESULTS Using RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis. CONCLUSION We speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport.

DNA Demethylation-Dependent AR Recruitment and GATA Factors Drive Rhox5 Homeobox Gene Transcription in the Epididymis

Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.

Immortalization by large T-antigen of the adult epididymal duct epithelium.

The SV40 large T-antigen has been widely used to convert various cell types to a transformed phenotype, and also to induce progressive tumours in transgenic animals. The objectives of this review are to compare and discuss three different approaches to generate epididymal epithelial cell lines using the large T-antigen. In the first approach, retroviral transfection of primary cultures was used to immortalize canine epididymal cells in vitro; the other two approaches used transgenic mice expressing the large T-antigen. In one of these in vivo approaches, a construct consisting of the coding sequence of a temperature sensitive (ts) SV40 large T-antigen was inserted in a mouse genome. When the cells are exposed to the permissive temperature of 33 degrees C, functional expression of the large T-antigen occurs and cells start to proliferate. In the second in vivo approach a tissue-specific promoter, the 5kb GPX5 promoter, was used to direct expression of the large T-antigen to the epididymal duct epithelium.

Epididymis-specific promoter-driven gene targeting: a transcription factor which regulates epididymis-specific gene expression.

Mammalian spermatozoa undergo several modification and finally acquire the ability to fertilize during epididymal transit. One of the distinct features of the epididymis is that it displays a highly regionalized pattern of gene expression. This tissue-, region-, and cell-specific pattern of gene expression is critical for the maintenance of a fully functional epididymis. One would hypothesize that disrupting this process provides an ideal approach to male contraception, since it would not interfere with testicular endocrine output or sperm production. To achieve this purpose, we studied a cluster of epididymis-specific lipocalin genes for understanding the specific mechanisms involved in the control of gene expression in the epididymis. We have identified six epididymis-specific lipocalin genes that are differently regulated and regionalized in the epididymis. Lipocalin 5 [Lcn5 or epididymal retinoic acid-binding protein (E-RABP)] is a member of this epididymis-specific lipocalin gene cluster, which binds hydrophobic molecules such as retinoic acid. We have previously shown that the 5kb promoter fragment of the Lcn5 gene confers both androgen-dependent regulation and epididymis-specific gene expression in transgenic mice whereas 0.6 kb promoter fragment does not. To further narrow down the important cis-regulatory elements that regulate gene expression in the epididymis, we studied the Lcn5 promoter in both transgenic mice and immortalized epididymal cells. We have found that 1.8kb promoter fragment of the Lcn5 gene was sufficient for tissue- and region-specific expression in transgenic mice, and that a transcription factor Forkhead box A2 (Foxa2) interacts with the androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 and 1.3 kb. Our finding provides a framework for further analysis of the epididymal lipocalin gene regulation and modulated control of epididymis-specific expression.

Retinoids and Epididymal Function

Retinoids are highly potent molecules with pleiotropic action in a wide range of biological processes including vision, reproduction, maintenance of epithelial tissues, bone development, and pattern formation during embryogenesis (for review, Sporn et al., 1994). Inappropriate temporal or spatial concentration of retinoic acid causes serious defects in the numerous organs that require retinoids (Maden, 2000). Vitamin A deficiency (VAD) in the adult is characterized by keratinizing and non-keratinizing squamous metaplasia of numerous epithelia (Wolback and Howe, 1925; Mason, 1939). Retinol administration prevents or reverses the effect of the VAD syndrome. Retinoic acid, in the absence of retinol, also prevents or reverses the VAD syndrome with the exception of the spermatogenic epithelium and retina (Thompson et al., 1964; Dowling and Wald, 1960). Pregnancies are also not carried to term if retinoic acid is the only retinoid provided (Howell etal., 1964).

Organic cation/carnitine transporter, OCTN2, transcriptional activity is regulated by osmotic stress in epididymal cells

Protection of cells from osmotic stress is crucial for their survival. Exposure to high osmolarity promotes rapid diffusion of water across cell membranes, dramatically increasing cellular ionic strength, leading to disruption of key proteins/DNA resulting in cell‐cycle arrest and apoptosis. The luminal microenvironment of the epididymis is hypertonic; therefore, epididymal cells adapt to the higher osmolarity by accumulating organic osmolytes, such as l‐carnitine. Osmolytes do not perturb cells when accumulated in high concentrations, nor do they affect key proteins or damage DNA. Therefore, osmolytes and their transporters are crucial for cell survival. Transporters that are responsible for the accumulation of organic osmolytes have been shown to be regulated at the transcriptional level by hypertonicity. The present study examines the gene expression of known osmoprotective/stress genes in epididymal cells exposed to changes in tonicity. We demonstrate that the osmoprotective/stress pathways present in other organs, such as the kidney, operate in the epididymis, potentially aiding in the protection of its luminal cells and spermatozoa. Further, it was also seen that OCTN2, a transporter that is thought to be responsible for the accumulation of l‐carnitine in the epididymal lumen, is regulated in response to changes in tonicity. Mol. Reprod. Dev. 77: 114–125, 2010.

The shape of the sperm midpiece in intracytoplasmic morphologically selected sperm injection relates sperm centrosomal function

PurposeTo evaluate whether the morphology of the sperm midpiece observed by high magnification microscopy relates to sperm centrosomal function.MethodsSperm selected by conventional microscopy were defined as controls. By high magnification microscopy, sperm with straight midpieces were defined as Group 1, while those with tapering midpieces were defined as Group 2. Heterologous ICSI of human sperm into bovine oocytes was used to assess human sperm centrosomal function and analysis of sperm aster formation.ResultsThe total rate of sperm aster formation was 80.5% in Group 1, which was significantly higher (P < 0.05) than the rate of 33.3% seen for Group 2. Furthermore, sperm aster formation rates tended to be higher for Group 1 than for the controls.ConclusionsThis study demonstrates improvement of sperm aster formation rates by selecting sperm on the basis of midpiece morphology. The injection of selected sperm bearing morphologically straight midpieces may contribute to improved expression of sperm centrosomal function, providing a positive effect on fertilization after ICSI.