102

Center for Experimental Medicine and Systems Biology

Laboratory of Reproductive Systems Biology
生殖システム研究分野

Project Professor
Masahito Ikawa, Ph.D.
Associate Professor Manabu Ozawa, Ph.D.

特任教授
准教授

博士（薬学）
博士（農学）

伊小川沢正人学

In the “post-genome project era,” genetically modified animals play a key role inbasic molecular biological investigations and act as models of human disease. Ourlaboratory studies the mechanisms underlying the mammalian reproductive system
in gene-manipulated mice. We are the first group in the world to generatetransgenic mice expressing GFP throughout the body (Green mice). We alsoestablished the ES cells that give green fluorescent spermatozoa to trace their
movement and acrosome reaction during fertilization. Another tool invented in ourlaboratory is the placenta-specific gene manipulation system using lentiviral (LV)
vectors. Using these techniques, we are trying to elucidate the mechanism
underlying gametogenesis, fertilization, implantation, and placentation. Our recent
interest is using the CRISPR/Cas9 system as a genome-editing tool. The
combination of GWAS studies with genome editing will pave the way to understandand control human fertility problems.

1.  Age-related decline in spermatogenic activity
accompanied with endothelial cell senescencein male mice.
Ozawa M, Mori H1, Endo T1, Ishikawa-Yamauchi Y,Motooka D¹, Emori C¹, Ikawa M1: ¹Research Institute for Microbial Diseases, Osaka University
Male fertility decreases with aging, with spermatogenic decline being one of its causes. Altered testis
environment is suggested as a cause of the phenotype; however, the associated mechanisms remain
unclear. Herein, we investigated the age-relatedchanges in testicular somatic cells on spermatogenic
activity. The number and proliferation of spermatogonia significantly reduced with aging in mice. Interestingly, senescence-associated β-galactosidase-positivecells appeared in testicular endothelial cell (EC) populations, but not in germ cell populations, with aging.
Transcriptome analysis of ECs indicated that senescence occurred in the ECs of aged mice. Furthermore,
the support capacity of ECs for spermatogonial pro-liferation significantly decreased with aging; however, the senolytic-induced removal of senescent cells
from aged ECs restored their supporting capacity to acomparable level as that of young ECs. Our results
suggest that the accumulation of senescent ECs in thetestis is a potential factor contributing to the age-related decline in spermatogenic activity.
2.  
Ptbp1 deletion does notcyte-to-neuron conversion.

induceastro-Hoang T2, Kim DW2, Appel H2, Ozawa M, Zheng S3,Kim J4, Blackshaw S2: 2Solomon H. Snyder Department of Neuroscience, John Hopkins University
School of Medicine, Baltimore, MD, USA. 3Divisionof Biomedical Sciences, University of California,Riverside, Riverside, CA, USA. 4Department of Psychiatry and Behavioral Science, John Hopkins University School of Medicine, Baltimore, MD, USA.
Although in vivo reprogramming of astrocytes
into neurons holds promise for treating neurodegen-103
erative disorders, protocols to achieve it are complex
and inefficient. A recent study reported that knockdown of Ptbp1 in the cortex, striatum and substantia
nigra of the midbrain efficiently reprogrammed astrocytes into functional neurons that rescued motor defects in a mouse model of Parkinson’s disease1, but it
did not convincingly demonstrate that astrocyte-toneuron conversion had actually occurred. Using genetic disruption of Ptbp1 in combination with cell-lineage analysis, we observe no astrocyte-to-neuron conversion or substantial changes in gene expression in
Ptbp1-deficient astrocytes. We conclude that the results of astrocyte-to-neuron conversion in the previous study1 most likely reflect leaky neuronal expression of the Gfapcre mouse lines used to label astrocytes,
and more-rigorous experimental methods must beused to support their observations of glial reprogramming in vivo.
3.  The histone H3K36 demethylase Fbxl11 plays
pivotal roles in the development of retinal lateborn cell types.
Iwagawa T5, Fukushima M5, Takeuchi S6, Kawamura Y6, Aihara Y6, Ozawa M, Yakushiji-Kaminatsui
N7, Aihara M8, Koseki H9, Suzuki Y8, Watanabe S5:
5
Department of Retinal Biology and Pathology, University of Tokyo Hospital, University of Tokyo. 6Department of Molecular and Developmental Biology,
Institute of Medical Science, University of Tokyo.
7
Laboratory for Developmental Genetics, RIKEN
Center for Integrative Medical Sciences. 8Department of Ophthalmology, Graduate School of Medicine, University of Tokyo. 9Department of Computational Biology and Medical Sciences, Graduate
School of Frontier Sciences, University of Tokyo.
Histone methylation plays a vital role in retinaldevelopment. However, the role of histone H3K36
methylation in retinal development is not clear. We
examined the role of H3K36 methylation by loss-offunction analysis of H3K36me1/2 demethylases,Fbxl10, and Fbxl11. We analyzed the effect of knockout of these genes in the developing and mature retina on retinal development. Knockout of Fbxl10 specifically in the developing retina did not result ingross developmental abnormalities. Although adultrod photoreceptor-specific knockout of Fbxl11 in mature retinas did not result in morphological abnormalities, Fbxl11 knockout in developing retinas increased apoptosis, suppressed the proliferation ofretinal progenitor cells, and resulted in microphthalmia. Morphological analysis revealed perturbed differentiation of rod photoreceptor and bipolar cells.
RNA-seq of retinas at P7 showed markedly decreased
expression of genes characterizing rod photoreceptor
and bipolar cells in Fbxl11-knockout retinas. In addition, perturbation of alternative splicing increased intron retention in Fbxl11-knockout retinas. Ge-nome-wide evaluation of the H3K36 methylation
status revealed that Fbxl11 knockout altered the distribution of H3K36me2/3 in genes important for rod
photoreceptor development. Taken together, we showthat Fbxl11 plays pivotal roles in the development of
retinal late-born cell types and may contribute to tightcontrol of H3K36 methylation during retinal development.
4.  CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility.
Shimada K9, Ikawa M1: 9Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University.
Sperm flagellum plays a crucial role in male fertility. Here, we generated Ccdc183 knockout mice using
the CRISPR/Cas9 system to reveal the protein function of the testis-specific protein CCDC183 in spermiogenesis. We demonstrated that the absence ofCCDC183 causes male infertility with morphologicaland motility defects in spermatozoa. Owing to thelack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface
and nucleus during spermiogenesis, which causes
subsequent loss of cytoplasmic invagination aroundthe flagellum. As a result, the flagellar compartment
does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In
addition, ectopic localization of accessory structures,such as the fibrous sheath and outer dense fibers, andabnormal head shape as a result of abnormal sculpting by the manchette are observed in Ccdc183 knockout spermatids. Our results indicate that CCDC183
plays an essential role in cytoplasmic invagination
around the flagellum to form functional spermatozoa
during spermiogenesis.
5.  A small secreted protein NICOL regulates lumicrine-mediated sperm maturation and malefertility.
Kiyozumi D1, Shimada K1, Chalick M10, Emori C1,Kodani M1, Oura S1, Noda T1, Endo T1, Matzuk
MM11, Wreschner DH12, Ikawa M1: 10Shmunis
School for Biomedicine and Cancer Research, TelAviv University, 11Center for Drug Discovery andDepartment of Pathology & Immunology, Baylor
College of Medicine, Houston. 12Shmunis School for
Biomedicine and Cancer Research, Tel Aviv University.
The mammalian spermatozoa produced in the testis require functional maturation in the epididymis
for their full competence. Epididymal sperm maturation is regulated by lumicrine signalling pathways inwhich testis-derived secreted signals relocate to the104
epididymis lumen and promote functional differentiation. However, the detailed mechanisms of lumicrine regulation are unclear. Herein, we demonstratethat a small secreted protein, NELL2-interacting cofactor for lumicrine signalling (NICOL), plays a crucial role in lumicrine signalling in mice. NICOL is expressed in male reproductive organs, including thetestis, and forms a complex with the testis-secretedprotein NELL2, which is transported transluminallyfrom the testis to the epididymis. Males lacking Nicol
are sterile due to impaired NELL2-mediated lumicrine signalling, leading to defective epididymal differentiation and deficient sperm maturation but canbe restored by NICOL expression in testicular germcells. Our results demonstrate how lumicrine signalling regulates epididymal function for successful
sperm maturation and male fertility.

Publication
1.		Ozawa M, Mori H, Endo T, Ishikawa-Yamauchi Y,Motooka D, Emori C, Ikawa M. Age-related decline in spermatogenic activity accompanied withendothelial cell senescence in male mice. iScience
2023 Nov 13;26:108456.
2.		Shibata T, Sato R, Taoka M, Saitoh SI, Komine M,
Yamaguchi K, Goyama S, Motoi Y, Kitaura J, Izawa K, Yamauchi Y, Tsukamoto Y, Ichinohe T, Fujita E, Hiranuma R, Fukui R, Furukawa Y, Kitamura
T, Takai T, Tojo A, Ohtsuki M, Ohto U, Shimizu T,Ozawa M, Yoshida N, Isobe T, Latz E, Mukai K,Taguchi T, Hemmi H, Akira S, Miyake K. TLR7/8
stress response drives histiocytosis in SLC29A3disorders. J Exp Med. 2023 Sep 4;220:e20230054.
3.		Li D, Johmura Y, Morimoto S, Doi M, Nakanishi K,Ozawa M, Tsunekawa Y, Inoue-Yamauchi A,Naruse H, Matsukawa T, Takeshita Y, Suzuki N,Aoki M, Nishiyama A, Zeng X, Konishi C, Suzuki
N, Nishiyama A, Harris AS, Morita M, Yamaguchi
K, Furukawa Y, Nakai K, Tsuji S, Yamazaki S,
Yamanashi Y, Shimada S, Okada T, Okano H, Toda
T, Nakanishi M. LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes
late-onset neurological deficits. Nat Aging. 2023
Aug;3:1001-1019.
4.		Hoang T, Kim DW, Appel H, Ozawa M, Zheng S,
Kim J, Blackshaw S. Ptbp1 deletion does not induce astrocyte-to-neuron conversion. Nature 2023
Jun;618:E1-E7.
5.		Iwagawa T, Fukushima M, Takeuchi S, Kawamura
Y, Aihara Y, Ozawa M, Yakushiji-Kaminatsui N,Aihara M, Koseki H, Suzuki Y, Watanabe S. Thehistone H3K36 demethylase Fbxl11 plays pivotal
roles in the development of retinal late-born celltypes. Genes Cells 2023 Jul;28:482-495.
6.		Emori C, Kodani M, Abbasi F, Mori M, Ikawa M.
PABPN1L is required for maternal mRNA degradation after meiosis resumption. J Reprod Dev.
2023 Dec 7. Online ahead of print.
7.		Ogawa Y, Lu Y, Kiyozumi D, Chang H, Ikawa M.
CRISPR/Cas9-mediated genome editing reveals
seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice. Andrology 2023 Dec 12. Online ahead of print.
8.		Shimada K, Ikawa M. CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility. Development 2023 Nov 1;150:dev201724.
9.		Kiyozumi D, Ikawa M. ADGRG2 is dispensable
for lumicrine signalling regulating epididymal initial segment differentiation and gene expression.
Biol Reprod. 2023 Oct 13;109:474-481.
10.	Shimada K, Lu Y, Ikawa M. Disruption of testis-enriched cytochrome c oxidase subunit
COX6B2 but not COX8C leads to subfertility. ExpAnim. 2023 Jul 10. Online ahead of print.
11.	Kiyozumi D, Shimada K, Chalick M, Emori C,
Kodani M, Oura S, Noda T, Endo T, Matzuk MM,Wreschner DH, Ikawa M. A small secreted protein
NICOL regulates lumicrine-mediated sperm maturation and male fertility. Nat Commun. 2023
Apr24;14:2354.