weheartspermatogenesis.png img_8831.jpgNext generation sequencer gene analysis... img_7564.jpgUltra-thin section (TEM sample) img_8834_20190703125010462.jpgNext generation sequencer gene analysis... img_8552.jpgMounting sample for SEM... img_4165_1024.png img_4170.pngNext generation sequencer gene analysis... cultures_20190703125011620.png img_8382.jpgSuper-resolution microscope observation... img_4155.pngUltra micotome... img_4283.png img_4157.png img_8681.jpg img_8263.jpgTransmission electron microscope observation... ngsanalitescreensnapz003.pngNext generation sequencer gene analysis... img_7349.jpgMaking sample for sorting... img_4161.png img_8832.jpg img_8804.jpgTime lapse observation (BZ-9000) img_5700.jpgSEM sample... img_5708.jpgScanning electron microscope observation... img_8837.jpgNext generation sequencer gene analysis...

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Mechanisms of male germline development from the stem cells towards the early embryonic level –understanding the part of the totipotent cycle.

Gametes, sperm and egg, are highly specialized and differentiated cells. However, through the unique event ‘fertilization’, they acquire new potency. A fertilized egg has totipotency, whereby individual cells transit to multipotent cells which, in the future, give rise to embryo proper and extraembryonic tissues. Germ cells, after being specified and become distinguishable from somatic cells, are accompanied by a number of epigenetic reprograming events towards the final gamete formation. Then, a sperm and an egg meet again to form a fertilized egg, a totipotent cell.  
Our team is studying the molecular mechanisms through which mammalian male germ cells differentiate to produce mature spermatozoa. Mouse male gametogenesis is supported by self-renewal and differentiation of spermatogonial stem cells (SSCs), which form syncytia connected by inter-cellular bridges due to incomplete division. Previous studies have identified many genes involved in SSC maintenance and differentiation, but the regulation of these genes is complex and much remains to be unanswered.
 
We have been particularly interested in how epigenetic machineries regulate SSC maintenance and differentiation. We have found the presence of an ‘epigenetic checkpoint’ during SSC differentiation: expression levels of representative epigenetic modifiers such as DNA methyltransferases and patterns of H3K9me2/3 change dynamically when spermatogonia start to express c-Kit, a differentiating spermatogonial marker. This finding led us to hypothesize that the c-Kit-negative spermatogonia may constitute a pool of stem cells, and this may be one of the key phenomena required for SSC differentiation (Development 2013 140(17):3565-76. doi: 10.1242/dev.094045).
 
Among a number of epigenetic modifiers, we found that Kmt2b, a histone H3K4 methyltransferase, is one of the enzymes required for SSCs to differentiate – Kmt2b knockout leads to a blockade of SSC differentiation towards the c-Kit-positive spermatogonia in mice. Furthermore, Kmt2b primes the SSC epigenome by monovalent and bivalent chromatin for spermiogenesis and embryonic development (Development 2018 145:dev169102. doi: 10.1242/dev.169102).

 
 

Proteinase inhibitor for a lung disease model

We recently identified a proteinase inhibitor which is expressed in undifferentiated cell populations of various adult tissues. We generated knock-out mice of this gene and found that the mice die due to respiratory distress with atelectasis. We are currently analyzing the pathogenesis of respiratory distress and observing other phenotypes in adult mice.

 
 

Actively using advanced technologies

We are actively using advanced technologies including super-resolution microscopy (STED), electron microscopy (TEM and SEM), next-generation sequencing (RNA-seq, ChIP-seq, BS-seq & single-cell multiomics) and bioinformatics to obtain high-resolution data to understand how different epigenetic and transcriptomic features give rise to germline or lung development.

 
 

Clinical Epigenetics 13, Article number: 132 (2021)

Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes.

Florence Naillat, Heba Saadeh, Joanna Nowacka-Woszuk, Lenka Gahurova, Fatima Santos, Shin-ichi Tomizawa & Gavin Kelsey.

Development (2021) 148, dev194605.

Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing.

Takada Y, Yaman-Deveci R, Shirakawa T, Shari J, Tomizawa S, Miura F, Ito T, Ono M, Nakajima K, Koseki Y, Shiotani F, Ishiguro K, Ohbo K, Koseki H.

Development (2021) 148 (8): dev196212.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

Kobayashi Y, Tomizawa S, Ono M, Kuroha K, Minamizawa K, Natsume K, Dizdarević S, Dočkal I, Tanaka H, Kawagoe T, Seki M, Suzuki Y, Ogonuki N, Inoue K, Matoba S, Anastassiadis K, Mizuki N, Ogura A, Ohbo K.

Dis Model Mech (2019) 12 (11): dmm040139.

Lack of whey acidic protein four disulphide core (WFDC) 2 protease inhibitor causes neonatal death from respiratory failure in mice.

Nakajima K, Ono M, Radović U, Dizdarević S, Tomizawa SI, Kuroha K, Nagamatsu G, Hoshi I, Matsunaga R, Shirakawa T, Kurosawa T, Miyazaki Y, Seki M, Suzuki Y, Koseki H, Nakamura M, Suda T, Ohbo K.

Development 145 (2018)

Kmt2b conveys monovalent and bivalent H3K4me3 in spermatogonial stem cells at germline and embryonic promoters.

Tomizawa S, Kobayashi Y, Shirakawa T, Watanabe K, Mizoguchi K, Hoshi I, Nakajima K, Nakabayashi J, Singh S, Dahl A, Alexopoulou D, Seki M, Suzuki Y, Royo H, Peters AHFM, Anastassiadis K, Stewart AF, Ohbo K

Mol Cell Biol. 37(19): e00082-17 (2017)

EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice.

Dong Y, Isono KI, Ohbo K, Endo TA, Ohara O, Maekawa M, Toyama Y, Ito C, Toshimori K, Helin K, Ogonuki N, Inoue K, Ogura A, Yamagata K, Kitabayashi I, Koseki H.

Epigenetics & Chromatin 10: 25-44 (2017)

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes

Gahurova L*, Tomizawa S*, Smallwood SA, Stewart-Morgan KR, Saadeh H, Kim J, Andrews S, Chen T, Kelsey G *Contributed equally

GENES & DEVELOPMENT 29(23):2449-2462 (2015)

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes

Kathleen R. Stewart, Lenka Veselovska, Jeesun Kim, Jiahao Huang, Heba Saadeh, Shin-ichi Tomizawa, Sébastien A. Smallwood, Taiping Chen, and Gavin Kelsey

Genome Biology 16: 209-215 (2015)

Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape.

Lenka Veselovska, Sebastien A. Smallwood, ... Shin-ichi Tomizawa4, Simon Andrews and Gavin Kelsey*

BioMol Concepts 6(1): 1–9 2015

Epigenetic regulation in stem cell development, cell fate conversion, and reprogramming.

Kazuyuki Ohbo and Shin-ichi Tomizawa

BMC Genomics 16:624-40 2015

DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis.

Naoki Kubo, ……., Takayuki Shirakawa, Hidetoshi Sone, Yasuyuki Sato, Shin-ichi Tomizawa, Kazuyuki Ohbo

Biological Research. 48:48-62 2015

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells.

Taro Mikami, Keiichiro Yoshida, Hajime Sawada, Michiyo Esaki, Kazunori Yasumura and Michio Ono

Dev Cell., 34: 1-12 2015

The RNA binding protein Nanos2 organizes a post-transcriptional buffering system to retain primitive state of mouse spermatogonial stem cells.

Zhou, Z., Shirakawa, T., Ohbo, K., Sada, A., Wu, Q., Hasegawa, K., Saba, R. and Saga, Y.

Curr. Pathobiol. Rep., 2:1-9 2014

Stem Cell Epigenetics: Insights from Studies on Embryonic, Induced Pluripotent, and Germline Stem Cells.

Shin-ichi Tomizawa, Takayuki Shirakawa, Kazuyuki Ohbo

Development, 140:3565-3576. 2013

An epigenetic checkpoint controls the transition from a stem cell pool to a progenitor cell state in mouse male germ cells.

Shirakawa T., Yaman-Deveci R., Tomizawa S., Kamizato Y., Nakajima K., at.,al.

Cell Biochem. Func 30(1), 33-40 2012

Plasmodium induced by SU6656, an Src family kinase inhibitor, is accompanied by a contractile ring defect.

Yoshida K, Ono M, Bito H, Mikami T, Sawada H

The International journal of developmental biology 56 867-875 2012

DNA methylation establishment during oocyte growth: mechanisms and significance.

Tomizawa S, Nowacka-Woszuk J, Kelsey G

Nature genetics 43 811-814 2011

Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.

Smallwood SA, Tomizawa S, Krueger F, Ruf N, Carli N, Segonds-Pichon A, Sato S, Hata K, Andrews SR, Kelsey G

Development, 138:4207-4217. 2011

HP1γ links histone methylation marks to meiotic synapsis in mice.

Takada Y., Naruse C., Costa Y., Shirakawa S., Tachibana M., Sharif J., Kezuka-Shiotani F., Kakiuchi D., Masumoto H., Shinkai Y., Ohbo K., Peters A. H.F.M., Turner J. M.A., Asano M. and Koseki K.

Development 138 811-820 2011

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes.

Tomizawa S, Kobayashi H, Watanabe T, Andrews S, Hata K, Kelsey G, Sasaki H

Kazuyuki Ohbo

Professor

Shin-ichi Tomizawa

Assistant Professor

Michio Ono

Assistant Professor

Kazushige Kuroha

Assistant Professor

Kuniko Nakajima

Technician

Ikue Hoshi

Technician

Yuuki Kobayashi

Doctor's Programs (5th)

Keisuke Minamizawa

Doctor's Programs (3nd)

Aki Hayashi

Doctor's Programs (1nd)

Ivana Dockal

Master's Programs (2nd)

Monika Šafhauzer

Master's Programs (1st)

Kentaro Fujisawa

Undergraduate 4th
->

Koji Natsume

Master's Graduate (2020)
->

Keita Mizoguchi

Master's Graduate (2020)
->

Uros Radvic

Master's Graduate (2020)
->

Selma Dizdarevic

Master's Graduate (2021)
->

Yuuki Maekawa

Graduate (2020)

The testicular stem cells have an advantage in that they can be studied with a variety of approaches including a long-term ex-vivo culture, a transplantation assay for evaluation of stemness and histological experiments. We also use other methods such as electron microscopy and super-resolution microscopy to understand their maintenance and differentiation mechanisms.

Research interests
-Analysis of cell fate determination by epigenetic mechanisms
-Study of stem cell proliferation and niche formation
-Mechanistic study of lung diseases associated with alveolar epithelial disturbance

For more information about our graduate programs contact us by e-mail.
E-mail: soshiki@yokohama-cu.ac.jp  

Phone: +81-45-787-2567  Fax: +81-45-787-2568
URL: http://www-user.yokohama-cu.ac.jp/~finemorp

Outline

Department

Histology and Cell Biology, Yokohama City University School of Medicine
Address

3-9 Fukuura, Kanazawaku, Yokohama, Japan
Principal Investigator/Professor

Kazuyuki Ohbo, M.D., Ph.D.
Study areas

anatomy, histology, epigenetics, germ cells, cell biology
Technology

electron microscopy, super-resolution fluorescent microscopy, gene expression and epigenetic analysis (NGS)
Member

4 faculty instructors; 4 technical staff