基本情報

所属

• 理学研究院　生物科学部門　生殖発生生物学分野

職名

• 助教

学位

• 博士（農学）（名古屋大学）

ホームページURL

https://sites.google.com/site/kuroiwagroup/home

科研費研究者番号

• 20515382

J-Global ID

• 200901054278802966

プロフィール

• 鳥類や哺乳類で見られる特殊な現象に着目し、現在はウズラの多精受精・生殖腺分化、並びにY染色体を持たないトゲネズミ属の性分化機構に関する研究を行っています。

研究キーワード

• 細胞周期   性分化   ウズラ   トゲネズミ   多精受精   ゲノム編集   生殖細胞   

研究分野

• ライフサイエンス / 実験動物学
• ライフサイエンス / 動物生理化学、生理学、行動学

担当教育組織

•  学士課程　理学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2017年01月 - 現在 北海道大学 大学院理学研究院 生物科学部門 生殖発生生物学分野 助教
• 2017年01月 - 2019年03月 静岡大学大学院 農学研究科 客員准教授
• 2014年11月 - 2016年12月 富山大学 大学院理工学研究部（工学） 特命助教
• 2014年04月 - 2014年10月 静岡大学 大学院農学研究院 学術研究員
• 2011年04月 - 2014年03月 日本学術振興会 特別研究員・PD
• 2008年04月 - 2011年03月 早稲田大学 人間科学学術院 助手

所属学協会

• 日本動物学会   日本畜産学会   World Poultry Science Association   日本家禽学会   鳥類内分泌研究会   日本分子生物学会   

研究活動情報

論文

• FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species

  Mayuko Okamoto, Ryo Sasaki, Koki Ikeda, Kasumi Doi, Fumiya Tatsumi, Kenzi Oshima, Takaaki Kojima, Shusei Mizushima, Keisuke Ikegami, Takashi Yoshimura, Kyohei Furukawa, Misato Kobayashi, Fumihiko Horio, Atsushi Murai

  Frontiers in Immunology 15 2024年02月29日 [査読有り]
   

  Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal–newly-hatched IgY transfer are controlled by a single receptor.
• Identification of a New Enhancer That Promotes <i>Sox9</i> Expression by a Comparative Analysis of Mouse and <i>Sry</i>-Deficient Amami Spiny Rat

  Yurie Hirata, Shusei Mizushima, Shoichiro Mitsukawa, Masafumi Kon, Yoko Kuroki, Takamichi Jogahara, Nobuo Shinohara, Asato Kuroiwa

  Cytogenetic and Genome Research 1 - 10 2024年01月20日 [査読有り]
   

  <b><i>Introduction:</i></b> Testis differentiation is initiated by the <i>SRY</i> gene on the Y chromosome in mammalian species. However, the Amami spiny rat, <i>Tokudaia osimensis</i>, lacks both the Y chromosome and the <i>Sry</i> gene and acquired a unique <i>Sox9</i> regulatory mechanism via a male-specific duplication upstream of <i>Sox9</i>, without <i>Sry</i>. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote <i>SOX9</i> gene expression. Several enhancers located upstream of <i>Sox9</i>/<i>SOX9</i> have been reported in mice and humans. In particular, the binding of SRY to the highly conserved enhancer Enh13 is thought to be a common mechanism underlying testis differentiation and sex determination in mammals. <b><i>Methods:</i></b> Sequences of <i>T. osimensis</i> homologues of three <i>Sox9</i> enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in <i>Sox9</i> regulation in <i>T. osimensis</i>. <b><i>Results:</i></b> <i>T. osimensis</i> Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, <i>T. osimensis</i> Enh13 did not respond to SRY, even though the binding sites of SRY and NR5A1 were conserved. To identify the key sequence that is present in mouse but absent from <i>T. osimensis</i>, we performed reporter gene assays using vectors in which partial sequences of <i>T. osimensis</i> Enh13 were replaced with mouse sequences. For <i>T. osimensis</i> Enh13 in which the second half (approximately 430 bp) was replaced with the corresponding mouse sequence, activity in response to NR5A1 and SRY was recovered. Further, reporter assays revealed that multiple regions in the second half of the mouse Enh13 sequence are required for the response to NR5A1 and SRY. The latter 49 bp was particularly important and contained four binding sites for three transcription factors, POU2F1, HOXA3, and GATA1. <b><i>Conclusion:</i></b> We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of <i>Sry</i>-dependent and <i>Sry</i>-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination.
• Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens

  M. Matsuzaki, S. Mizushima, M. Tsudzuki, T. Maeda, T. Sasanami

  British Poultry Science 65 1 97 - 104 2024年01月02日 [査読有り]
  
• Research Note: Diethylstilbestrol reduces primordial germ cells in male Japanese quail

  Shusei Mizushima, Asato Kuroiwa

  Poultry Science 102 10 102910 - 102910 2023年10月
• The neo-X does not form a Barr body but shows a slightly condensed structure in the Okinawa spiny rat (Tokudaia muenninki).

  Ryoma Kudo, Ikuya Yoshida, Luisa Matiz Ceron, Shusei Mizushima, Yoko Kuroki, Takamichi Jogahara, Asato Kuroiwa

  Cytogenetic and genome research 2023年06月02日 [査読有り]
   

  X chromosome inactivation (XCI) is an essential mechanism for gene dosage compensation between male and female cells in mammals. The Okinawa spiny rat (Tokudaia muenninki) is a native rodent in Japan with XX/XY sex chromosomes, like most mammals; however, the X chromosome has acquired a neo-X region (Xp) by fusion with an autosome. We previously reported that dosage compensation has not yet evolved in the neo-X region; however, X-inactive-specific transcript (Xist) RNA (long non-coding RNA required for the initiation of XCI) is partially localized in the region. Here, we show that the neo-X region represents an early chromosomal state in the acquisition of XCI by analyses of heterochromatin and Barr body formation. We found no evidence for heterochromatin formation in the neo-X region by RBA (R-banding by acridine orange) assays and immunostaining of H3K27me3. Double-immunostaining of H3K27me3 and HP1, a component of the Barr body, revealed that the entire ancestral-X chromosome region (Xq) showed a bipartite folded structure. By contrast, HP1 was not localized to the neo-X region. However, BAC FISH revealed that the signals of genes on the neo-X region of the inactive X chromosome were concentrated in a narrow region. These findings indicated that although the neo-X region of the inactive X chromosome does not form a complete Barr body structure (e.g., it lacks HP1), it forms a slightly condensed structure. These findings combined with the previously reported partial binding of Xist RNA suggest that the neo-X region exhibits incomplete inactivation. This may represent an early chromosomal state in the acquisition of the XCI mechanism.
• Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing

  Shusei Mizushima, Tomohiro Sasanami, Tamao Ono, Asato Kuroiwa

  Genes 14 3 757 - 757 2023年03月20日 [査読有り]
   

  Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.
• Egg development after in vitro insemination in Japanese quail (Coturnix japonica)

  Yoshinobu Ichikawa, Shusei Mizushima, Noritaka Hirohashi, Tomohiro Sasanami

  The Journal of Poultry Science 60 1 n/a - n/a 2023年01月25日 [査読有り]
  
• Turnover of mammal sex chromosomes in the Sry -deficient Amami spiny rat is due to male-specific upregulation of Sox9

  Miho Terao, Yuya Ogawa, Shuji Takada, Rei Kajitani, Miki Okuno, Yuta Mochimaru, Kentaro Matsuoka, Takehiko Itoh, Atsushi Toyoda, Tomohiro Kono, Takamichi Jogahara, Shusei Mizushima, Asato Kuroiwa

  Proceedings of the National Academy of Sciences 119 49 2022年11月28日 [査読有り]
   

  Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry , leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis , in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9 . The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.
• Possible involvement of annexin A6 in preferential sperm penetration in the germinal disk region

  Yoshinobu Ichikawa, Mei Matsuzaki, Shusei Mizushima, Tomohiro Sasanami

  Reproduction and Fertility 3 3 152 - 161 2022年07月01日 [査読有り]
   

  Graphical abstract Abstract During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This phenomenon has been observed not only in domestic birds but also in wild birds; however, the mechanisms controlling sperm preference are still unclear. In this study, we investigated the possible involvement of annexin family protein in sperm–egg interaction in Japanese quail. Microscopic examination of fertilized eggs indicated that quail sperm penetration only occurred in the germinal disk region, and sperm localized outside the germinal disk were trapped in the perivitelline membrane. Western blot analysis and immunofluorescence microscopy revealed the presence of annexin A1 and A6 in the oocyte membrane, while annexin A6 localized in the perivitelline space of the germinal disk region. Further, our sperm binding assay using recombinant annexin A6 demonstrated that ejaculated sperm specifically bound to annexin A6 expressed in mammalian cell lines. These results suggest that annexin A6, which is expressed on the surface of oocytes, may function in sperm–egg interaction in the germinal disk region and that this binding may ensure sperm retention on the surface of the egg plasma membrane until fertilization takes place in Japanese quail. Lay summary In bird species, fertilization takes place immediately after ovulation of the egg. Sperm preferentially penetrate a specific area of the egg coating that covers the ‘germinal disk region’ – this area contains the cell that needs to be fertilized by a sperm. However, since the bird egg is extremely large in size and sperm must reach the ‘germinal disk region’ to achieve fertilization, it is unclear how this happens. Annexin proteins support fertilization in mammals, and we found that annexin A6 protein exhibits a unique localization in the germinal disk region in the eggs of Japanese quail. To test this interaction, we incubated quail sperm with cells that produced annexin A6 and found that ejaculated sperm bound to the cells. These results suggest that annexin A6 may have a role in the sperm–egg interaction in the germinal disk region in Japanese quail.
• Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca²⁺ During Quail Egg Activation

  Shusei Mizushima, Tomohiro Sasanami, Tamao Ono, Norio Kansaku, Asato Kuroiwa

  The Journal of Poultry Science 59 2 175 - 181 2022年04月 [査読有り]
  
• Analysis of sex chromosome evolution in the clade Palaeognathae from phased genome assembly

  Miki Okuno, Shusei Mizushima, Asato Kuroiwa, Takehiko Itoh

  Genome Biology and Evolution 13 11 evab242  2021年11月 [査読有り]
  
• Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail

  Shusei Mizushima, Tomohiro Sasanami, Tamao Ono, Mei Matsuzaki, Norio Kansaku, Asato Kuroiwa

  Developmental Biology 476 249 - 258 2021年08月 [査読有り]
  
• Effect of sperm surface oligosaccharides in sperm passage into sperm storage tubules in Japanese quail (Coturnix japonica)

  Mei Matsuzaki, Noritaka Hirohashi, Shusei Mizushima, Tomohiro Sasanami

  Animal Reproduction Science 227 106731 - 106731 2021年04月 [査読有り]
   

  In birds, the ejaculated spermatozoa do not directly pass to the site of fertilization but rather are stored initially in specialized structures, referred to as sperm storage tubules (SSTs), located in the utero-vaginal junction (UVJ) of the oviduct. The fertilizing capacity of spermatozoa in the SSTs is maintained for an extended period (i.e., several days to months). Although many studies have been conducted to ascertain the mechanisms involved in sperm storage, the understanding of the phenomenon is limited. In this study, there was investigation of the effects of sperm surface oligosaccharides in sperm passage into SSTs in Japanese quail. Results from lectin staining of ejaculated spermatozoa indicated galactose/N-Acetylgalactosamine (Gal/GalNAc), N-Acetylglucosamine (GlcNAc) or mannose/glucose (Man/Glc) moieties were present on the sperm surface, indicating the presence of glycoproteins/glycolipids containing these oligosaccharides. When ejaculated spermatozoa were co-incubated with UVJ explants, the lectins derived from Agaricus bisporus and Canavalia ensiformis had marked inhibitory effects on sperm passage into SSTs. Preincubation of UVJ explants with these lectins, however, had no effect indicating there were no effects of UVJ oligosaccharides in this process. Furthermore, none of these lectin had effects on values of sperm motility variables. These results indicate that O-glycans with terminal β-Gal or GalNAc and N-glycans with terminal α-D-Man or α-D-Glc may have functions in the process of sperm passage into SSTs.
• Longer and faster sperm exhibit better fertilization success in Japanese quail

  Mei Matsuzaki, Noritaka Hirohashi, Masaoki Tsudzuki, Mohammad Ibrahim Haqani, Teruo Maeda, Shusei Mizushima, Tomohiro Sasanami

  Poultry Science 100 4 100980 - 100980 2021年04月 [査読有り]
   

  In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.
• ニホンウズラ(Coturnix japonica)を用いた発現様式に性的二型がみられる遺伝子の発現プロファイリング

  奥野 未来, 宮本 淳太郎, 伊藤 武彦, 関 真秀, 鈴木 穣, 水島 秀成, 黒岩 麻里

  比較内分泌学 47 174 1 - 4 日本比較内分泌学会 2021年
• Expression profiling of sexually dimorphic genes in the Japanese quail, Coturnix japonica

  Miki Okuno, Shuntaro Miyamoto, Takehiko Itoh, Masahide Seki, Yutaka Suzuki, Shusei Mizushima, Asato Kuroiwa

  Scientific Reports 10 1 20073 - 20073 2020年12月 [査読有り]
   

  Research on avian sex determination has focused on the chicken. In this study, we established the utility of another widely used animal model, the Japanese quail (Coturnix japonica), for clarifying the molecular mechanisms underlying gonadal sex differentiation. In particular, we performed comprehensive gene expression profiling of embryonic gonads at three stages (HH27, HH31 and HH38) by mRNA-seq. We classified the expression patterns of 4,815 genes into nine clusters according to the extent of change between stages. Cluster 2 (characterized by an initial increase and steady levels thereafter), including 495 and 310 genes expressed in males and females, respectively, contained five key genes involved in gonadal sex differentiation. A GO analysis showed that genes in this cluster are related to developmental processes including reproductive structure development and developmental processes involved in reproduction were significant, suggesting that expression profiling is an effective approach to identify novel candidate genes. Based on RNA-seq data and in situ hybridization, the expression patterns and localization of most key genes for gonadal sex differentiation corresponded well to those of the chicken. Our results support the effectiveness of the Japanese quail as a model for studies gonadal sex differentiation in birds.
• Expression of Transferrin and Albumin in the Sperm-Storage Tubules of Japanese Quail and their Possible Involvement in Long-Term Sperm Storage

  Mei Matsuzaki, Shusei Mizushima, Hideo Dohra, Tomohiro Sasanami

  The Journal of Poultry Science 57 1 88 - 96 2020年 [査読有り]
   

  Because of the presence of sperm storage tubules (SSTs) in the utero-vaginal junction (IJVJ) in the oviduct, once ejaculated sperm enter the female reproductive tract, they can survive for a prolonged period in domestic birds; however, the specific mechanisms involved in sperm maintenance within the SST remain to be elucidated. In this study, we showed that transferrin (TF) and albumin (ALB) are expressed in SSTs. When UVJ extracts were subjected to size-exclusion column chromatography, we obtained fractions that extend sperm longevity in vitro. LC-MS/MS analysis of the two major proteins in the fractions identified these proteins as TF and ALB. Immunohistochemical analysis using specific antisera against TF and ALB indicated that both proteins were localized not only in the SSTs, but also in the surface epithelium of the UVJ. When the ejaculated sperm were incubated with either purified TF or ALB, sperm viability increased after 24 h. These results indicated that oviductal TF and ALB are involved in the process of sperm storage in SSTs and may open a new approach for technological improvement to prolong sperm longevity in vitro.
• Spiny rat SRY lacks a long Q-rich domain and is not stable in transgenic mice.

  Ogata Y, Nishikata M, Kitada K, Mizushima S, Jogahara T, Kuroiwa A

  Developmental dynamics : an official publication of the American Association of Anatomists 2019年06月 [査読有り][通常論文]
  
• Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis.

  Kohei Washio, Shusei Mizushima, Takamichi Jogahara, Asato Kuroiwa

  Cytogenetic and genome research 159 3 143 - 150 2019年 [査読有り][通常論文]
   

  Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.
• Knockdown of DDX4 decreases the number of germ cells in male and female chicken embryonic gonads

  Aduma N, Izumi H, Mizushima S, Kuroiwa A

  Reproduction, Fertility and Development 2018年12月 [査読有り][通常論文]
  
• Female Japanese quail visually differentiate testosterone-dependent male attractiveness for mating preferences.

  Hiyama G, Mizushima S, Matsuzaki M, Tobari Y, Choi JH, Ono T, Tsudzuki M, Makino S, Tamiya G, Tsukahara N, Sugita S, Sasanami T

  Scientific reports 8 1 10012  2018年07月 [査読有り][通常論文]
   

  © 2018 The Author(s). Biased mating due to female preferences towards certain traits in males is a major mechanism driving sexual selection, and may constitute an important evolutionary force in organisms with sexual reproduction. In birds, although the role of male ornamentation, plumage coloration, genetic dissimilarity, and body size have on mate selection by females have been examined extensively, few studies have clarified exactly how these characteristics affect female mate preferences. Here, we show that testosterone (T)-dependent male attractiveness enhances female preference for males of a polygamous species, the Japanese quail. A significant positive correlation between female mating preference and circulating T in the male was observed. The cheek feathers of attractive males contained higher levels of melanin and were more brightly colored. The ability of females to distinguish attractive males from other males was negated when the light source was covered with a sharp cut filter (cutoff; < 640 nm). When females were maintained under short-day conditions, the expression of retinal red-sensitive opsin decreased dramatically and they became insensitive to male attractiveness. Our results showed that female preference in quail is strongly stimulated by male feather coloration in a T-dependent manner and that female birds develop a keen sense for this coloration due to upregulation of retinal red-sensitive opsin under breeding conditions.
• Unique XCI evolution in Tokudaia: initial XCI of the neo-X chromosome in Tokudaia muenninki and function loss of XIST in Tokudaia osimensis

  Hideki Zushi, Chie Murata, Shusei Mizushima, Chizuko Nishida, Asato Kuroiwa

  CHROMOSOMA 126 6 741 - 751 2017年12月 [査読有り][通常論文]
   

  X chromosome inactivation (XCI) is an essential mechanism to compensate gene dosage in mammals. Here, we show that XCI has evolved differently in two species of the genus Tokudaia. The Amami spiny rat, Tokudaia osimensis, has a single X chromosome in males and females (XO/XO). By contrast, the Okinawa spiny rat, Tokudaia muenninki, has XX/XY sex chromosomes like most mammals, although the X chromosome has acquired a neo-X region by fusion with an autosome. BAC clones containing the XIST gene, which produces the long non-coding RNA XIST required for XCI, were obtained by screening of T. osimensis and T. muenninki BAC libraries. Each clone was mapped to the homologous region of the X inactivation center in the X chromosome of the two species by BAC-FISH. XIST RNAs were expressed in T. muenninki females, whereas no expression was observed in T. osimensis. The sequence of the XIST RNA was compared with that of mouse, showing that the XIST gene is highly conserved in T. muenninki. XIST RNAs were localized to the ancestral X region (Xq), to the heterochromatic region (pericentromeric region), and partially to the neo-X region (Xp). The hybridization pattern correlated with LINE-1 accumulation in Xq but not in Xp. Dosage of genes located on the neo-X chromosome was not compensated, suggesting that the neo-X region is in an early state of XCI. By contrast, many mutations were observed in the XIST gene of T. osimensis, indicating its loss of function in the XO/XO species.
• Handling of gametes for in vitro insemination in birds

  Shusei Mizushima, Mei Matsuzaki, Tomohiro Sasanami

  Methods in Molecular Biology 1650 243 - 257 2017年 [査読有り][通常論文]
   

  A characteristic biological property of avian gamete (e.g., extremely large egg and polyspermic fertilization) does not allow the direct observation of sperm-egg interactions in vitro, but recent research advances make it possible to manipulate the gamete in vitro. Here, we describe the techniques for the handling of gametes required for in vitro fertilization assay. In addition, we also introduce the procedures for sperm-perivitelline membrane assay, intracytoplasmic sperm injection, and ex ovo culture.
• Fertilization 2: Polyspermic fertilization

  Shusei Mizushima

  Advances in Experimental Medicine and Biology 1001 105 - 123 2017年 [査読有り][通常論文]
   

  During fertilization in animals, a haploid egg nucleus fuses with a haploid sperm nucleus to restore the diploid genome. In most animals including mammals, echinoderms, and teleostei, the penetration of only one sperm into an egg is ensured at fertilization because the entry of two or more sperm is prevented by polyspermy block systems in these eggs. On the other hand, several animals such as birds, reptiles, and most urodele amphibians exhibit physiological polyspermy, in which the entry of several sperm into one egg is permitted. However, in these polyspermic eggs, only one sperm nucleus is involved in zygotic formation with a female nucleus, thereby avoiding syngamy with multiple sperm nuclei. In the chicken, 20–60 sperm are generally found within the egg cytoplasm at fertilization and this number is markedly higher than that of other polyspermic species however, avian-specific events such as the degeneration and mitosis of supernumerary sperm nuclei during early embryo development allow a polyspermic egg to develop normally. This chapter describes current knowledge on polyspermy-related events in avian eggs during fertilization, and is characterized by a comparison to the fertilization modes of other vertebrates. The close relationship between sperm numbers and egg sizes, and the movement of supernumerary sperm nuclei towards the periphery of the egg cytoplasm and their degeneration are summarized. The molecular mechanisms by which polyspermy initiates egg activation to start embryo development are also discussed.
• Effects of a Protein Kinase Inhibitor on Sperm Motility in the Japanese Quail

  Mei Matsuzaki, Shusei Mizushima, Yoshinobu Ichikawa, Kogiku Shiba, Kazuo Inaba, Tomohiro Sasanami

  JOURNAL OF POULTRY SCIENCE 54 1 73 - 79 2017年01月 [査読有り][通常論文]
   

  Sperm motility is an essential trait for successful fertilization in animals. In birds, ejaculated sperm migrate into sperm storage tubules before fertilization and are stored in a quiescent state. We previously reported that this type of sperm's flagellar quiescence was induced by lactic acid through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). However, signal transduction in the sperm cells leading to motility inactivation is not well understood. The aim of the present study was to investigate the role of protein kinases in putative signal transduction in quail spermatozoa motility in vitro. Following incubation with bisindolylmaleimide II (BisII), a potent-competitive protein kinase C (PKC) inhibitor, sperm motility decreased in a dose related-manner. However, no such inhibitory effect was found in sperm exposed to bisindolylmaleimide V, H-89, or LY294002, a weak inhibitor of PKC, a potent inhibitor of protein kinase A (PKA) and a selective inhibitor of phosphatidylinositol 3-kinase, respectively. BisII-treated sperm exhibited no significant differences in pHi, mitochondrial activity, intracellular cAMP or ATP concentration, as well as dynein ATPase activity, compared to the control sperm. However, when the phosphorylated substrate proteins by PKC were detected by Western blot analysis, the intensity of the band in sperm incubated in the presence of BisII decreased. Moreover, immunoreactive PKCc and mu isoforms in the sperm lysates were also detected. These results indicated that the PKC signaling pathway may be involved in sperm motility regulation, and protein phosphorylation by PKC may be required to maintain flagellar movement in the Japanese quail.
• Egg Envelope Glycoproteins ZP1 and ZP3 Mediate Sperm-Egg Interaction in the Japanese Quail

  Yoshinobu Ichikawa, Mei Matsuzaki, Shusei Mizushima, Tomohiro Sasanami

  JOURNAL OF POULTRY SCIENCE 54 1 80 - 86 2017年01月 [査読有り][通常論文]
   

  Fertilization is indispensable for zygotic formation leading to the birth of animals and the species-specific sperm egg binding thought to be the initial step in this important process. In birds, the oocyte, which encounters the spermatozoa at the time of fertilization, is enclosed in a perivitelline membrane (pvm) constructed of several zona pellucida glycoproteins (ZP proteins: ZP1, ZP2, ZP3, ZP4 and ZPD). The aim of this study was to determine the ZP protein in the pvm responsible for sperm-pvm binding in Japanese quail. We tested the effects of anti-ZP protein antibodies on in vitro sperm perforation in the pvm. The results showed that the anti-ZP1 and ZP3 antibody significantly blocked hole formation by sperm, whereas anti-ZP2, ZP4 and ZPD as well as normal rabbit serum had no such effect. When the sperm acrosome reaction was inhibited in the presence of pertussis toxin, sperm-pvm binding was observed. This sperm-pvm binding was significantly prevented when the purified ZP1 or ZP3 was included in the reaction mixture. Moreover, both digoxigenin-labeled ZP1 and ZP3 were found to interact with the sperm head by immunocytochemical observation. Our results indicate that sperm binding to the pvm is, at least in part, mediated by the interaction of ZP1 and ZP3 with the sperm head during fertilization in Japanese quail.
• Attempt on the establishment of somatic cell nuclear transfer method in Japanese quail

  Mizushima S, Ono T, Sasanami T

  Proc of 17th AAAP Anim Sci Cong CM-ROM 2016年11月 [査読有り][通常論文]
  
• Sperm-Egg Interaction during Fertilization in Birds

  Yoshinobu Ichikawa, Mei Matsuzaki, Gen Hiyama, Shusei Mizushima, Tomohiro Sasanami

  JOURNAL OF POULTRY SCIENCE 53 3 173 - 180 2016年07月 [査読有り][通常論文]
   

  Fertilization in animals that employ sexual reproduction is an indispensable event for the production of the next generation. A significant advancement in our understanding of the molecular mechanisms of sperm-egg interaction in mammalian species was achieved in the last few decades. However, the same level of knowledge has not been accumulated for birds because of egg size and the difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. In this review, we summarize the current understanding of sperm-egg interaction mechanism during fertilization in birds, especially focusing on sperm-egg binding, sperm acrosome reaction and the authentic sperm protease required for the hole formation on the perivitelline membrane. We explain that the zona pellucida proteins (ZP1 and ZP3) in the perivitelline membrane play important roles in sperm-egg binding, induction of the acrosome reaction as well as sperm penetration by digestion of sperm protease. We anticipate that a deeper understanding of avian fertilization will open up new avenues to create powerful tools for a myriad of applications in the poultry industries including the production of transgenic and cloned birds.
• Expression of Prolactin Receptor on the Surface of Quail Spermatozoa

  Gen Hiyama, Shusei Mizushima, Mei Matsuzaki, Yoshinobu Ichikawa, Norio Kansaku, Tomohiro Sasanami

  JOURNAL OF POULTRY SCIENCE 53 2 157 - 164 2016年04月 [査読有り][通常論文]
   

  Prolactin receptor (PRLR) is expressed in a wide variety of tissues and mediates diverse biological actions of prolactin (PRL). In mammals, PRL signaling is thought to be involved not only in the process of spermatogenesis and steroidogenesis in the testis, but also in the survival of ejaculated sperm. In avian species, although the expression of PRLR with several variants in the testis was reported, the role of PRL in testicular function is still unclear. The aim of this study was to examine the expression of PRLR in the testis and mature sperm in quail. It is revealed that PRLR was mainly localized in the round- and elongated-spermatid by immunohistochemical analysis on the testis suggesting that PRL signaling may participate in the spermatogenesis. Western blot analysis confirmed the presence of PRLR in the plasma membrane of the ejaculated sperm (SPML), whereas the size of PRLR in the sperm was smaller than that in the hypothalamus. Moreover, PRLR was detected on the surface of the midpiece and flagellum of sperm by immunostaining. To evaluate the functionality of the sperm PRLR, the dot blot assay was performed to test the binding of pituitary PRL to PRLR in the SPML, and resulted in the detection of specific binding of PRL to the component of SPML, most likely to sperm PRLR. Furthermore, when the ejaculates were incubated with pituitary PRL to investigate the role of PRL on the sperm, the occurrence of spontaneous acrosome reaction was significantly decreased. In addition, the expression of PRL on the surface of utero-vaginal junction of oviduct was detected by immunohistochemistry. These results may suggest a novel system that the interaction between oviductal PRL and sperm PRLR is involved in the maintenance of the fertilizability of the spermatozoa through the prevention of the spontaneous acrosome reaction in Japanese quail.
• Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection

  Kyung Soo Kang, Tae Sub Park, Deivendran Rengaraj, Hyung Chul Lee, Hong Jo Lee, Hee Jung Choi, Shusei Mizushima, Tamao Ono, Jae Yong Han

  REPRODUCTION FERTILITY AND DEVELOPMENT 28 12 1974 - 1981 2016年 [査読有り][通常論文]
   

  Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70-120min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved-thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.
• Lactic acid is a sperm motility inactivation factor in the sperm storage tubules

  Mei Matsuzaki, Shusei Mizushima, Gen Hiyama, Noritaka Hirohashi, Kogiku Shiba, Kazuo Inaba, Tomohiro Suzuki, Hideo Dohra, Toshiyuki Ohnishi, Yoshikatsu Sato, Tetsuya Kohsaka, Yoshinobu Ichikawa, Yusuke Atsumi, Takashi Yoshimura, Tomohiro Sasanami

  SCIENTIFIC REPORTS 5 17643  2015年12月 [査読有り][通常論文]
   

  Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41 degrees C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). The long-term preservation of sperm morphology under hypoxic and high temperature conditions indicates that a combination of these factors enables sperm cells to survive during the ovulation cycles. Our findings suggested a novel physiological role for lactic acid in promoting sperm quiescence in SSTs and opened up a new opportunity for technological improvement in prolonging sperm longevity at ambient or body temperature.
• A unique mechanism of successful fertilization in a domestic bird

  Tomohiro Sasanami, Shunsuke Izumi, Naoki Sakurai, Toshifumi Hirata, Shusei Mizushima, Mei Matsuzaki, Gen Hiyama, Eriko Yorinaga, Takashi Yoshimura, Kazuyoshi Ukena, Kazuyoshi Tsutsui

  SCIENTIFIC REPORTS 5 7700  2015年01月 [査読有り][通常論文]
   

  Fertilization is an indispensable step for formation of a zygote in sexual reproduction, leading to species survival. When mating occurs, sperm is transported to the female reproductive tracts via the seminal plasma (SP). SP is derived from male accessory sex glands and it plays pivotal roles for fertilization in animals. However, molecular mechanisms of SP or a fluid derived from male accessory sex glands for successful fertilization remain unclear. Here, we report that in male quail the cloacal gland (CG) produces prostaglandin F-2 alpha (PGF(2 alpha)) that contributes to successful fertilization. PGF(2 alpha), as well as the secretion of CG (CGS), induced vaginal contractions and caused the opening of the entrance of the sperm storage tubules, the structures responsible for the long-term sperm storage and fertilization. The removal of CGS from the male before mating reduced the fertility, but the supplementation of CGS or PGF(2 alpha) rescued the subfertility. We further showed that male CG contains glucose that is utilized as energy source for the intrinsic sperm mobility after transportation to female vagina. This mechanism, in concert with the excitatory effects of PGF(2 alpha) enables successful fertilization in the domestic bird.
• The birth of quail chicks after intracytoplasmic sperm injection

  Shusei Mizushima, Gen Hiyama, Kogiku Shiba, Kazuo Inaba, Hideo Dohra, Tamao Ono, Kiyoshi Shimada, Tomohiro Sasanami

  DEVELOPMENT 141 19 3799 - 3806 2014年10月 [査読有り][通常論文]
   

  Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors - phospholipase C zeta (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) - all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca2+ rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca2+ oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.
• Simple Culture System for Bobwhite Quail and Japanese Quail Embryos from the Blastoderm Stage to Hatching using a Single Surrogate Eggshell

  Atsushi Kato, Shusei Mizushima, Kiyoshi Shimada, Hiroshi Kagami, Tamao Ono

  JOURNAL OF POULTRY SCIENCE 51 2 202 - 205 2014年04月 [査読有り][通常論文]
   

  The ex vivo culture of avian embryos is a technique for the long-term culturing of embryos outside of their own shell and shell membrane. It allows easy access to the developing embryos and embryo manipulation. The two-step system is widely applied when the culture is performed after oviposition. Japanese quail as well as bobwhite quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been more limited than those on the Japanese quail. We have developed a more simplified ex vivo culture protocol for the two species of quail embryos from the blastoderm stage through to hatching using a single surrogate eggshell. Hatchabilities of 31% and 27% were obtained in bobwhite quail and Japanese quail embryos, respectively. The simple system described in the present study is an easy and acceptable procedure.
• Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica)

  Gen Hiyama, Mei Matsuzaki, Shusei Mizushima, Hideo Dohra, Keisuke Ikegami, Takashi Yoshimura, Kogiku Shiba, Kazuo Inaba, Tomohiro Sasanami

  REPRODUCTION 147 2 167 - 178 2014年02月 [査読有り][通常論文]
   

  Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.
• Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds

  Kiyoshi Shimada, Tamao Ono, Shusei Mizushima

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 196 100 - 105 2014年01月 [査読有り][通常論文]
   

  Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24 h at 41 degrees C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12-18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37-50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37 degrees C after the 24 h incubation at 41 degrees C under 5% CO2 in air. It survived up to 2 days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24 h incubation at 41 degrees C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds. (C) 2013 Elsevier Inc. All rights reserved.
• New world quail and old world quail for germline transfer studies

  Ono T, Mizushima S, Tsuruta Y, Kato R, Kagami H

  Proceedings of the international Forum on Avian Germplasm 28 - 29 2013年10月 [査読有り][通常論文]
  
• Sperm Storage in the Female Reproductive Tract in Birds

  Tomohiro Sasanami, Mei Matsuzaki, Shusei Mizushima, Gen Hiyamm

  JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 4 334 - 338 2013年08月 [査読有り][通常論文]
   

  The ability to store sperm in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism of sperm storage in the female reproductive tract has been poorly understood until recently. In avian species, the specialized simple tubular invaginations referred to as sperm storage tubules (SSTs) are found in the oviduct as a sperm storage organ. In this review, we summarize the current understanding of the mechanism of sperm uptake into the SSTs, maintenance within it, and controlled release of the sperm from the SSTs. Since sperm storage in avian species occurs at high body temperatures (i.e., 41 C), elucidation of the mechanism for sperm storage may lead to the development of new strategies for sperm preservation at ambient temperatures, and these could be used in a myriad of applications in the field of reproduction.
• Culture System for Bobwhite Quail Embryos from the Blastoderm Stage to Hatching

  Atsushi Kato, Daichi Miyahara, Hiroshi Kagami, Yusuke Atsumi, Shusei Mizushima, Kiyoshi Shimada, Tamao Ono

  JOURNAL OF POULTRY SCIENCE 50 2 155 - 158 2013年04月 [査読有り][通常論文]
   

  Quail are divided phylogenetically into two groups, Old World quail and New World quail. Old World quail, such as the Japanese quail (Coturnix japonica), belong to the Phasianidae and distributed in the Palaearctic region (Europe, North Africa, and Asia), whereas New World quail, such as the bobwhite quail (Colinus virginianus), belong to the Odontophoridae and are restricted to North and South America. Both the bobwhite quail and the Japanese quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been limited compared with those on the Japanese quail. We have therefore now developed an ex vivo culture protocol for bobwhite quail embryos from the blastoderm stage through hatching. Of the various culture conditions examined in the present study, a good hatching rate (39%) was obtained when the embryos were cultured ex vivo in a two-step procedure. Unincubated embryos (with egg yolk) were first cultured inside the shell of a Japanese quail egg (11.5 to 13.0 g whole egg weight) together with chicken thin albumen for 63 to 65 h and were then transferred to the shell of a small-sized chicken egg (38 g whole egg weight) until hatching. This ex vivo culture system should provide to be widely applicable to the maintenance and generation of manipulated birds for basic and applied studies on the bobwhite quail.
• Establishment of Intracytoplasmic Sperm Injection Technique in Japanese Quail and its Possible Application for Poultry Resources and Transgenic Birds

  Shusei Mizushima

  JOURNAL OF POULTRY SCIENCE 49 4 225 - 230 2012年10月 [査読有り][通常論文]
   

  In mammals, a normal offspring can be obtained even from infertile male by intracytoplasmic sperm injection (ICSI). Although ICSI technique has permitted significant progress in clinical practice in humans and mouse to date, it has been established recently in birds. In addition, efficiency of fertility and developmental rates has been low and no chick has been produced by in vitro fertilization and culture. Furthermore, polyspermic fertilization and subsequent normal developmental processes remains unknown. The enhancement of fertility and developmental rates is the first step in the avian ICSI system to be applied for protection of endangered species and production of transgenic and clone birds. This review paper describes (1) the establishment of ICSI technique in Japanese quail, (2) molecular mechanisms whereby polyspermy activates development of quail oocyte, (3) improvement of ICSI efficiency by phospholipase C zeta cRNA. Also, possible application of ICSI for avian sex manipulation and transgenic birds was summarized.
• Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)

  Tomohiro Sasanami, Kenichi Sugiura, Toshinobu Tokumoto, Norio Yoshizaki, Hideo Dohra, Shunsuke Nishio, Shusei Mizushima, Gen Hiyama, Tsukasa Matsuda

  REPRODUCTION 144 4 423 - 431 2012年10月 [査読有り][通常論文]
   

  At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation. Reproduction (2012) 144 423-431
• Effects of utero-vaginal junction extracts on the in vitro sperm viability in Japanese quail (Coturnix japonica)

  Sasanami T, Hamano R, Matsuzaki M, Mizushima S

  Proceedings of 15th AAAP Animal Science Congress 3606 - 3708 2012年03月 [査読有り][通常論文]
  
• Effects of sperm pretreatment on efficiency of intracytoplasmic sperm injection (ICSI)-mediated gene transfer in quail

  Mizushima S, Sato A, Ono T, Shimada K, Sasanami T

  Proceedings of 15th AAAP Animal Science Congress 3003 - 3006 2012年03月 [査読有り][通常論文]
  
• Establishment of intracytoplasmic sperm injection (ICSI) method for green fluorescent protein (GFP) expression in quail blastoderms

  Shimada K, Mizushima S, Takagi S, Ono T, Atsumi Y, Tsukada A, Saito N, Sasanami T

  Proceedings of 9th Asia Pacific Poultry Conference CD 1 - 4 2011年03月 [査読有り][通常論文]
  
• Down-regulation of inositol 1,4,5-trisphosphate receptor type 1 (IP3R-1) for oocyte activation in quail

  Mizushima S, Kimura I, Kansaku N, Watanabe T, Ono T, Shimada K

  Proceedings of 9th Asia Pacific Poultry Conference CD 1 - 4 2011年03月 [査読有り][通常論文]
  
• 鳥類卵の受精の仕組み

  水島秀成, 島田清司

  比較内分泌学 37 140 14-20 (J-STAGE) - 20 Japan Society for Comparative Endocrinology 2011年02月28日 [査読無し][通常論文]
  
• Novel Method of Gene Transfer in Birds: Intracytoplasmic Sperm Injection for Green Fluorescent Protein Expression in Quail Blastoderms

  Shusei Mizushima, Soichi Takagi, Tamao Ono, Yusuke Atsumi, Akira Tsukada, Noboru Saito, Tomohiro Sasanami, Masaru Okabe, Kiyoshi Shimada

  BIOLOGY OF REPRODUCTION 83 6 965 - 969 2010年12月 [査読有り][通常論文]
   

  This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6[42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.
• Phospholipase C zeta mRNA Expression and its Potency During Spermatogenesis for Activation of Quail Oocyte as a Sperm Factor

  Shusei Mizushima, Soichi Takagi, Tamao Ono, Yusuke Atsumi, Akira Tsukada, Noboru Saito, Kiyoshi Shimada

  MOLECULAR REPRODUCTION AND DEVELOPMENT 76 12 1200 - 1207 2009年12月 [査読有り][通常論文]
   

  This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase C zeta (PLC zeta C) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLC zeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLC zeta cRNA at 60 mu g/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLC zeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLC zeta cRNA can induce development. In addition, RT-PCR revealed that PLC zeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLC zeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.
• Expression of P450arom, AMH and ERα mRNA in gonads of turkey, duck and goose within one week of age

  Koba N, Ohfuji T, Ha Y, Mizushima S, Tsukada A, Saito N, Shimada K

  The Journal of Poultry Science 45 3 220 - 226 2008年07月 [査読有り][通常論文]
   

  Although many studies have shown that P450aromatase (P450arom), anti-Mullerian hormone (AMH) and estrogen receptor alpha (ER alpha) play pivotal roles in sexual differentiation of the gonads during early embryonic development in chickens and quail, few studies have been reported for other domestic birds. Furthermore, little information is available in relation to the mRNA expression in gonads after sexual differentiation in posthatching birds. The present study was conducted to assay mRNA expression of P450arom, AMH and ERa in gonads at day of hatch in turkey and duck and 2 days after hatching in goose using the real-time PCR. The mRNA expression was also determined at one week of age in gonads of these birds. At the time of collection of the left gonads for total RNA extraction, the external appearance of the left and right gonads of male and female was documented by digital camera. Clear asymmetry was observed in the female ovary in which the right ovary was regressed completely by 1-2 days in posthatching turkey, duck and goose. Although gonadal asymmetry was as remarkable as in females, the left testis was larger than the right one in males. Remarkable expression of P450arom mRNA was observed only in females in all the 3 species but substantially no expression was detected in males. Significantly higher expression of AMH mRNA was detected in males than females only in goose but there was no sex difference in turkey and duck at 1-2 days posthatching and one week of age. Weak ERa mRNA expression without a sex difference was detected in the 3 birds. These results suggest that estrogen plays a key role for ovarian development via P450arom mRNA expression after hatching, whereas absence of its expression in males leads to testis development in turkey, duck and goose.
• Developmental enhancement of intracytoplasmic sperm injection (ICSI)-generated quail embryos by phospholipase Cζ cRNA

  Mizushima S, Takagi S, Ono T, Atsumi Y, Tsukada A, Saito N, Shimada K

  The Journal of Poultry Science 45 2 152 - 158 2008年04月 [査読有り][通常論文]
   

  The present study was conducted to improve the rate and stage of blastoderm and embryo development of quail oocytes by intracytoplasmic sperm injection (ICSI) with quail phospholipase C zeta (PLC zeta) cRNA. Blastoderm development of quail oocytes cultured for 24 or 72 h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLC( cDNA clone was isolated from sperm by RT-PCR. The 1978 bp nucleotide sequence contained an ORF encoding 637 amino acids of protein. The deduced quail PLC zeta protein consists of EF-hand domain, X and Y catalytic domain and C2 domain, but lacked a plextrin-homology (PH) domain at the N-terminus. RT-PCR analysis revealed that PLC zeta mRNA is expressed only in the testis. ICSI into a quail oocyte without PLC zeta cRNA induced blastodermal development in 16% (4/25) of the oocytes which reached stages III-VI 24 h after culture. In contrast, ICSI together with PLC zeta cRNA (in 3 nl at 60 mu g/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.06: 7/19) with embryos reaching stages VI-VII Furthermore, 72 h after culture ICSI into a quail oocyte without PLC zeta cRNA induced embryo development in 15.4% (2/13) of the oocytes and reached stages V-VII. In contrast, ICSI together with PLCC cRNA (in 3 nl at 60 mu g/ml) into a quail oocyte induced 3 fold more embryo development (50516, 9/18) with embryos between stages VI and over X. The microinjection of PLC zeta cRNA significantly improved the conventional ICSI method by enhancing the proportion and the stages of embryo development. Accordingly, the present PLC zeta cRNA microinjection method in birds may contribute to assist in the production of transgenic birds and protection of endangered species of birds.
• Profiles of mRNA expression of FOXL2, P450arom, DMRT1, AMH, P450c17, SF1, ERα and AR, in relation to gonadal sex differentiation in duck embryo

  Koba N, Ohfuji T, Ha Y, Mizushima S, Tsukada A, Saito N, Shimada K

  The Journal of Poultry Science 45 2 132 - 138 2008年04月 [査読有り][通常論文]
   

  The present study was conducted to show a profile of mRNA expression of sex-differentiation related genes in the duck gonad. Fertile duck eggs (Cherry-valley) were incubated (hatching at day 28 of incubation) and the gonads were collected at days 7, 85 10, 12 and 15 of incubation after taking photos of the gonads. Left gonad was used for identification of genetic sex and for total RNA extraction. Reverse transcription-polymerase chain reaction (PCR) was performed using appropriate forward and reverse primers for each of FOXL2, P450arom, DMRT1, AMH, P450(c17), SF1, ER alpha and AR genes of the chicken and the corresponding cDNA fragments were sequenced for duck. Subsequently, real-time PCR was performed to detect mRNA expression of those genes in the duck embryo. Male bilateral gonads assumed symmetry in appearance throughout the examined period, whereas the female gonads were noticeably asymmetric at 10 days of incubation with a smaller size of the right. Female asymmetry was distinct by day 15 of incubation. In females, FOXL2 and P450arom mRNA expression began at day 8 of incubation and remained high thereafter, but in males only negligible expression was detected. In males. mRNA expression of DMRT1 and AMH was detected at day 7 of incubation and tended to increase gradually thereafter, whereas in females the expression remained low throughout the examined period. Though the slight expression of P450(c17) mRNA started at day 7 only in males, the expression was low throughout the examined period, whereas in female the expression was distinct at day 8 of incubation and remained high thereafter. Expressions of the other genes were variable with no differences in each expression between the sexes. The results indicate that FOXL2 and P450arom play an important role for the ovarian formation.
• Effects of aromatase inhibitor (Fadrozole)-induced sex-reversal on gonadal differentiation and mRNA expression of P450arom, AMH and ERα in embryos and growth in posthatching quail

  Koba N, Mori M, Ha Y, Mizushima S, Tsukada A, Saito N, Ono T, Shimada K

  The Journal of Poultry Science 45 2 116 - 124 2008年04月 [査読有り][通常論文]
   

  The present study was conducted to investigate (1) the effective day of treatment with Fadrozole, nonsteroidal aromatase inhibitor (AI), during incubation for sex reversal, (2) the mRNA expression of aromatase (P450arom), anti-Mullerian hormone (AMH) and estrogen receptor a (ER alpha) in quail gonad and gonadal structures after the AI treatment before hatching and (3) the effects of AI on gonad growth, left-right asymmetry of the gonad and body weight gain between I to 7 weeks posthatch. (1) Females AI-treated at 0, 2 and 4 days of incubation had symmetrical gonads at hatch similar to normal males, whereas those treated at 6 and 8 days of incubation had asymmetrical gonads. (2) In females AI-treated at day 0 of incubation, the left gonad became an ovotestis at day 15 of incubation, displaying seminiferous tubules with testicular-like cords and ovarian follicle with ovum. In the control group, P450arom mRNA expression was significantly higher in females than males. However,AI-treated females showed 2 response-patterns of either higher or lower expression. The latter was similar to that of control males. No significant differences in levels of AMH and ERa mRNA between sexes either in control or Al-treated groups was observed. (3) A gradual increase in body weight of the control males and females without sex difference was observed up to 5 weeks of age. However, at 6-7 weeks the average weight of females was heavier than males each in control and treated group. These results indicate that shortly before day 6 of incubation P450arom mRNA expression, at least in a part, is important for asymmetrical formation of gonads in female quail. In AI-treated females, the high responder show similar level of P450arom mRNA in control males and the low responder do not respond to the AI. Body weight is controlled non-gonadally in quail.
• Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica)

  Shusei Mizushima, Soichi Takagi, Tamao Ono, Yusuke Atsumi, Akira Tsukada, Noboru Saito, Kiyoshi Shimada

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 307A 11 647 - 653 2007年11月 [査読有り][通常論文]
   

  Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.
• Fertilization and blastoderm development of quail oocytes after intracytoplasmic injection of chicken sperm bearing the W chromosome

  S. Takagi, T. Ono, A. Tsukada, Y. Atsumi, S. Mizushima, N. Saito, K. Shimada

  POULTRY SCIENCE 86 5 937 - 943 2007年05月 [査読有り][通常論文]
   

  Our previous study demonstrated that elongated spermatids and sperm carrying the female-specific W-chromosome of the sex-reversed domestic fowl can activate the mouse oocyte, but whether they can fertilize the avian oocyte and lead to a developing zygote remains undetermined. A single sperm isolated from the semen and testis of normal rooster and from a testis of sex-reversed hen was microinjected into a quail oocyte and cultured for 20 to 24 h. Blastoderms were fixed, cleaved, nuclei stained by 4',6'-diamidino-2-phenylindole, and developmental stages were assessed. In the normal rooster group, ejaculated and testicular sperm induced blastodermal development in 22.6 and 20%, of the quail oocytes, respectively. The developmental stages ranged from IV to VII. In the sex-reversal group, 20% of injected testicular sperm induced blastodermal development. The blastodermal stages varied from stage III to VI. Blastoderms after 4',6'-diamidino-2-phenylindole staining were assayed by PCR to identify the W chromosome of either chicken sperm or quail oocyte. The PCR assay results showed that 2 out of 9 developed blastoderms microinjected with sperm of sex-reversed hen were identified containing the female-specific W chromosome derived from sex-reversed hen. From these results, it is concluded that chicken sperm bearing the W chromosome possess fertilizing ability and can function to stimulate blastoderm development similar to that of normal chicken sperm carrying the Z chromosome.
• Profiles of mRNA expression of sex-differentiation-related genes in relation to gonadal sexual differentiation in duck

  Koba N, Mizushima S, Ha Y, Ohfuji T, Tsukada A, Saito N, Shimada K

  Proceedings of 8th Asia Pacific Poultry Conference 505 - 506 2007年03月05日 [査読無し][通常論文]
  
• Effect of strontium on ICSI-associated fertilization in quail

  Mizushima S, Takagi S, Ono T, Atsumi Y, Tsukada A, Saito N, Shimada K

  Proceedings of 8th Asia Pacific Poultry Conference 503 - 504 2007年03月05日 [査読無し][通常論文]
  
• Fertilizing ability of chicken sperm bearing W chromosome,

  Takagi, S, Ono, T, Tsukada, A, Atsumi, Y, Mizushima, S, Saito, N, Shimada, K

  Poultry Science 86 730 - 737 2007年 [査読有り][通常論文]
  
• Z-chromosome specific primers for chicken-quail hybrid blastoderm

  Soichi Takagi, Tamao Ono, Akira Tsukada, Yusuke Atsumi, Shusei Mizushima, Noboru Saito, Kiyoshi Shimada

  Journal of Poultry Science 44 2 209 - 212 2007年 [査読有り][通常論文]
   

  A polymerase chain reaction (PCR) primer set for a chicken pkci gene on the Z-chromosome, chPKCI, amplified a 329 bp fragment of genomic DNA in chickens but not quail. Likewise, a PCR primer set for the quail pkci gene on the Z-chromosome, quPKCI, amplified a 95 bp fragment of genomic DNA in quail but not chickens. These chicken and quail primers validly identified chicken-quail hybrids derived from Z-chromosome carrying sperm of chicken and the hybrid derived from Z-chromosome carrying oocyte of quail, respectively.
• Possible role of intracellular calcium release for fertilization in quail oocyte

  Mizushima S, Takagi S, Ono T, Atsumi Y, Tsukada A, Saito N, Shimada K

  Proceedings of XIIth AAAP Animal Science Congress CD 1 - 2 2006年09月18日 [査読有り][通常論文]
  
• cDNA cloning and mRNA expression of androgen receptor in male Japanese quail (Coturnix coturnix japonica)

  Mizushima S, Saito N, Shimada K

  The Journal of Poultry Science 43 2 173 - 179 日本家禽学会 2006年04月 [査読有り][通常論文]
   

  The biological activities of androgen are largely mediated via androgen receptor (AR). Although a partial sequence AR cDNA was revealed in canaries and zebra finch, AR cDNA has not been cloned in quail and chickens. To understand the physiological function of androgen and to apply it for other purposes, AR cDNA is necessary. Hence, this study was aimed to isolate the cDNA of AR and to reveal changes in mRNA expression using RT-PCR analysis in male reproductive organs of quail under different day-lengths and castration treatment. We found that a partial length of quail AR cDNA contains a 1385bp sequence encoding 343 amino acid residues and had high homology to other vertebrates. Quail were raised under continuous light regimen from 4 to 6 weeks old (6LL) and they were divided into 3 groups under the following conditions up to 9 weeks old : (1) continuous light regimen group (24h light ; 9LL), (2) short days regimen group (8h light and 16h darkness ; 9SD) and (3) castration group in which testis were surgically removed at 6 weeks old and raised under continuous light regimen (9CAS). Although weekly changes in the cloacal gland protrusion area showed significantly progressive increase at 7-9 weeks old in 9LL group and a progressive decrease in 9SD group, there were no changes in AR mRNA levels in the gland. In contrast, in 9CAS group, although the cloacal gland protrusion area decreased after castration treatment, AR mRNA levels increased in the gland. On the other hand, AR mRNA levels in epididymis and vas deferens increased in 9LL group, but there were no differences in 9SD and 9CAS groups from 6LL. The testicular AR mRNA levels did not change after long or short days treatment. These results indicate there is tissue-specific regulation in AR mRNA expression in quail.
• Prostaglandin levels in plasma and follicular and uterine tissues of the quail in relation to midsequence oviposition

  Saito, N, Shimada, K

  Japanese Poultry Science 25 261 - 267 1988年 [査読有り][通常論文]
  

書籍

• Specific Mechanism of Storage in Avian Oviducts

  Matsuzaki M, Hiyama G, Mizushima S, Shiba K, Inaba K, Sasanami T (担当:共著範囲:Sexual Reproduction in Animals and Plants (Sawada H, Inoue N and Iwano M Eds))
  Springer 2014年01月
• 胚盤葉細胞核を用いたクローンウズラ胚作出の試み

  松本 拓也, 笹浪 知宏, 島田 清司, 小野 珠乙, 水島 秀成 
  東海畜産学会報第23巻 2012年
• Developmental engineering for sex manipulation in domestic fowls

  Shimada K, Takagi S, Mizushima S, Saito N, Tsukada A, Ono T, Atsugi Y 
  Molecular and Physiological Aspects of Regulatory Processes of the Organism.pp.420-421.pp.420-421. 2007年

講演・口頭発表等

• 内分泌攪乱物質暴露下における鳥類生殖細胞への影響の解析

  小川湧也, 水島秀成, 黒岩麻里

  日本動物学会北海道支部第68回大会 2024年03月
• オスウズラの生殖細胞分化に与えるジエチルスチルベストロールの影響

  水島秀成, 小川湧也, 黒岩麻里

  鳥類内分泌研究会 2023年12月
• 鳥類配偶子の保存と体外受精技術の開発に向けて  [招待講演]

  水島秀成

  Cryopreservation conference 2023 2023年11月
• ニホンウズラの始原生殖細胞に及ぼす知恵チルスチベストロールの影響

  水島秀成, 小川湧也, 黒岩麻里

  日本家禽学会秋季大会 2023年09月
• Detrimental assessment of ethinylestradiol on the stem cell properties of quail primordial germ cells.  [通常講演]

  Mizushima S, Kuroiwa A

  57th Congress of the European Societies of Toxicology 2023年09月
• 内分泌撹乱物質の卵黄内投与が初期胚のウズラ始原生殖細胞に与える影響

  水島秀成, 黒岩麻里

  第46回鳥類内分泌研究会 2022年12月
• ニホンウズラの雌雄生殖腺分化におけるSOX9の役割

  水島秀成, 黒岩麻里

  日本家禽学会2022年度秋季大会 2022年09月
• Two isoforms of DDX4 and their differential expression during early embryo development of Japanese quail

  Shusei MIZUSHIMA, Tomohiro SASANAMI, Tamao ONO, Asato KUROIWA

  26th World Poultry Congress, E-poster online release 2021年09月
• ウズラDAZL（deleted in azoospermia-like）mRNAの3’-UTRに結合するタンパク質の解析  [通常講演]

  水島秀成, 塚田光, 笹浪知宏, 小野珠乙, 黒岩麻里

  第45回鳥類内分泌研究会 2021年12月
• ウズラ初期胚の⺟性-胚性遷移期におけるretinoblastoma分解シグナリング解析

  水島秀成、笹浪知宏、小野珠乙、松崎芽衣、神作宜男、黒岩麻里

  第92回日本動物学会米子大会 2021年09月 口頭発表（一般）
• ウズラ初期胚におけるDAZL (deleted in azoospermia-like) の発現解析

  水島秀成, 塚田光, 笹浪知宏, 小野珠乙, 黒岩麻里

  第44回鳥類内分泌研究会 2020年12月
• 鳥類多精受精における主精子選択と余剰精子排除メカニズムに関する研究  [招待講演]

  水島秀成

  第21回日本畜産学会若手企画シンポジウム 2020年11月
• ウズラ胚の母性-胚性遷移期におけるサイクリンDの役割  [招待講演]

  水島秀成, 黒岩麻里

  第6回北大・部局横断シンポジウム 2020年10月
• 雄ウズラの生殖腺におけるDAZLの発現解析

  水島秀成, 塚田光, 笹浪知宏, 小野珠乙, 黒岩麻里

  日本家禽学会春季大会, 2020年03月
• ウズラの生殖腺における生殖細胞特異的遺伝子の発現解析

  水島秀成, 佐藤望, 塚田光, 笹浪知宏, 小野珠乙, 黒岩麻里

  第42回日本分子生物学会年会 2019年12月
• The role of inositol-trisphosphate receptors during egg activation in Japanese quail.

  Mizushima S, Sasanami T, Ono T, Kuroiwa A

  The 2nd International conference on Tropical Animal Science and Production 2019
• ウズラの性染色体遺伝子ノックアウト技術の開発

  水島秀成

  遺伝研研究会「有性生殖にかかわる染色体・クロマチン・核動態に関する研究会」 2019年06月
• ウズラの配偶子に発現するDNase Iの受精における機能

  水島秀成, 笹浪知宏, 小野珠乙, 黒岩麻里

  日本家禽学会春季大会 2019年03月
• 鳥類の受精成立に関する核分解酵素の役割

  水島秀成, 黒岩麻里

  日本動物学会北海道支部第63回大会 2019年03月
• ウズラ卵の受精成立に関する分子機構  [通常講演]

  水島 秀成

  日本畜産学会主催、旗影会協賛第3回若手企画サイエンスキャンプ-アニマルサイエンスのフロンティア- 2018年08月
• 受精システムを介したゲノム改変家禽の作出  [招待講演]

  水島 秀成

  広島大学日本鶏資源開発プロジェクト研究センター第21回JAB特別セミナー 2018年04月
• ウズラ初期胚におけるゲノム活性化のタイミングと細胞周期関連遺伝子の発現解析  [通常講演]

  水島秀成, 黒岩麻里, 須田千晶, 小野珠乙, 笹浪知宏

  2018年日本家禽学会春季大会 2018年03月
• 鳥類における遺伝子操作  [招待講演]

  水島 秀成

  日本農芸化学会2018年度大会 2018年03月
• Application of intracytoplasmic sperm injection technique for avian genome modification technologies  [招待講演]

  Shusei Mizushima

  International Forum on Avian Germplasm and Genome Editing 2017 2017年10月
• ウズラ体細胞核移植胚の作出効率に及ぼす卵紫外線照射の影響  [通常講演]

  水島秀成, 小野珠乙, 黒岩麻里, 笹浪知宏

  日本家禽学会秋季大会 2017年09月
• 今後の鳥類顕微授精法―産業への応用に向けて―  [招待講演]

  水島秀成

  日本家禽学会誌 2016年09月
• ウズラ体細胞核移植胚の発生能  [通常講演]

  水島秀成, 小野珠乙, 笹浪知宏, 笹浪知宏

  日本家禽学会誌 2016年09月
• Attempt on the establishment of somatic cell nuclear transfer method in Japanese quail

  Mizushima S, Ono T, Sasanami T

  17th AAAP Animal Science Congress 2016年08月
• Establishment of intracytoplasmic sperm injection (ICSI) technique in quail and its possible application for transgenic and cloned birds

  Shusei Mizushima

  The 2nd tenure track system international symposium 2015年03月
• Full-term development of quail egg by intacytoplasmic sperm injection (ICSI) and use of ICSI for avian transgenesis  [招待講演]

  Mizushima S, Hiyama G, Ono T, Shimada K, Sasanami T

  10 th Asia Pacific Poultry Conference 2014年10月 口頭発表（招待・特別）
• ウズラの受精時に卵を活性化する新規精子ファクターの探索  [通常講演]

  水島秀成, 檜山源, 柴小菊, 稲葉一男, 道羅英夫, 小野珠乙, 島田清司, 笹浪知宏

  日本家禽学会誌大会号 2014年09月
• ウズラの胚発生に必須な卵細胞質内Ca²⁺波の動態解析  [通常講演]

  水島秀成, 檜山源, 柴小菊, 稲葉一男, 小野珠乙, 島田清司, 笹浪知宏

  日本家禽学会誌大会号 2014年03月
• ウズラの受精における精子核の挙動とγ-チューブリンの動態

  水島秀成, 久枝雅広, 檜山源, 笹浪知宏

  第8回領域会議（アロ認証機構） 2014年
• リゾレシチンによる精子処理がGFP発現ウズラ胚の発生能に及ぼす影響  [通常講演]

  水島秀成, 仲西宏樹, 檜山源, 柴小菊, 稲葉一男, 小野珠乙, 笹浪知宏

  日本畜産学会大会講演要旨 2013年09月
• ウズラの新規卵子活性化因子を利用したGFP発現顕微授精胚の作出  [通常講演]

  水島秀成, 檜山源, 柴小菊, 稲葉一男, 小野珠乙, 笹浪知宏

  日本家禽学会誌大会号 2013年08月
• 体細胞核を移植したウズラ胚の発生  [通常講演]

  水島秀成, 松本拓也, 檜山源, 小野珠乙, 柴小菊, 稲葉一男, 笹浪知宏

  日本動物学会大会予稿集 2013年08月
• ウズラ精子由来卵子活性化因子の同定  [通常講演]

  水島秀成, 佐藤暁, 檜山源, 柴小菊, 稲葉一男, 道羅英夫, 小野珠乙, 島田清司, 笹浪知宏

  日本畜産学会大会講演要旨 2013年03月
• 顕微授精法により作出したGFP発現ウズラの発生率と遺伝子導入効率  [通常講演]

  水島秀成, 佐藤暁, 柴小菊, 稲葉一男, 檜山源, 小野珠乙, 島田清司, 笹浪知宏

  日本家禽学会誌大会号 2013年03月
• ウズラ精子由来卵子活性化因子の探索

  水島秀成, 檜山源, 柴小菊, 稲葉一男, 笹浪知宏

  第7回領域会議（アロ認証機構） 2013年
• Pattern of calcium oscillation during fertilization and egg development in quail  [招待講演]

  Mizushima S, Hiyama G, Shimada K, Ono T, Sasanami T

  7th International Chick Meeting 2012年11月 口頭発表（招待・特別）
• Effects of sperm pretreatment on efficiency of intracytoplasmic sperm injection (ICSI)-mediated gene transfer in quail

  Mizushima S, Sato A, Ono T, Shimada K, Sasanami T

  15th AAAP Animal Science Congress 2012年11月
• 顕微授精法によるGFP発現ウズラ胚作出の試み  [通常講演]

  水島秀成, 佐藤暁, 小野珠乙, 島田清司, 笹浪知宏

  日本動物学会大会予稿集 2012年08月
• ウズラ卵活性化に対するクエン酸シンターゼとATPクエン酸リアーゼ投与の効果  [通常講演]

  水島秀成, 門奈修平, 佐藤暁, 島田清司, 小野珠乙, 笹浪知宏

  日本家禽学会誌大会号 2012年08月
• 家禽の顕微授精法の改良と受精機構に関する研究  [招待講演]

  水島 秀成

  日本家禽学会2012年度春季大会 2012年03月 口頭発表（招待・特別）
• 顕微授精ウズラ胚の発生能に及ぼす精子抽出物とIP3の効果  [通常講演]

  水島秀成, 笹浪知宏, 佐藤暁, 小野珠乙, 島田清司

  日本畜産学会大会講演要旨 2012年03月
• 家禽の顕微授精法の改良と受精機構に関する研究  [通常講演]

  水島秀成

  日本家禽学会誌大会号 2012年03月
• Analysis of cell cycle regulation during quail embryo development

  Mizushima S, Sasanami T, Kansaku K, Ono T, Kiyoshi Shimada K

  10th International Symposium on Avian Endocrinology 2012年03月
• Developmental enhancement of intracytoplasmic sperm injection-generated quail embryo by inositol trisphosphate

  Mizushima S, Sasanami T, Sato A, Ono T, Shimada K

  7th Intercongress Symposium of the Asia and Oceania for Comparative Endocrinology 2012年03月
• ウズラ顕微授精後の卵細胞質内カルシウム濃度の変化と発生に及ぼす効果

  水島秀成, 佐藤暁, 柴小菊, 稲葉一男, 笹浪知宏

  第4回領域会議（アロ認証機構） 2012年
• 凍結・融解処理精子を用いたICSI-SMGTウズラ胚の作出

  水島秀成, 佐藤暁, 笹浪知宏

  第5回領域会議（アロ認証機構） 2012年
• 鳥類精子由来卵子活性化因子の探索

  水島秀成, 笹浪知宏

  生殖若手の会 2012年
• ウズラ初期胚におけるretinoblastoma遺伝子の発現解析  [通常講演]

  水島秀成, 笹浪知宏, 神作宜男, 木村一郎, 西嶋傑, 小野珠乙, 島田清司

  日本畜産学会大会講演要旨 2011年08月
• ウズラ卵子活性化における卵細胞質内Ca²⁺濃度の変化  [通常講演]

  水島秀成, 笹浪知宏, 神作宜男, 木村一郎, 小野珠乙, 島田清司

  日本動物学会大会予稿集 2011年08月
• Molecular mechanism on intracellular Ca2+ rise triggered by sperm-borne oocyte activating factor in quail  [招待講演]

  Mizushima S

  Korea-Japan Joint Symposium on Avian Biology 2011年06月 口頭発表（招待・特別）
• 鳥類卵子活性化に対する卵細胞質内Ca²⁺とIP₃受容体の役割  [通常講演]

  水島秀成, 木村一郎, 渡邊岳史, 刀川紗綾, 神作宜男, 笹浪知宏, 小野珠乙, 島田清司

  日本家禽学会誌大会号 2011年03月
• Down-regulation of inositol 1,4,5-trisphosphate receptor type 1 (IP3R-1) for oocyte activation in quail

  Mizushima S, Kimura I, Kansaku N, Watanabe T, Ono T, Shimada K

  9th Asia Pacific Poultry Conference 2011年03月
• 卵細胞質内精子注入法による外来遺伝子導入ウズラ胚作出の試み

  水島秀成, 笹浪知宏, 高木惣一, 小野珠乙, 渥美優介, 塚田光, 齋藤昇, 岡部勝, 島田清司

  第3回領域会議（アロ認証） 2011年
• ウズラ初期胚における細胞周期関連遺伝子の発現解析  [通常講演]

  水島秀成, 笹浪知宏, 神作宜男, 小野珠乙, 島田清司

  Annu Meet Jpn Avian Endocrinol 2011年
• 鳥類に特有な多精子受精機構の解明と顕微授精法の改良  [通常講演]

  水島秀成

  日本畜産学会大会講演要旨 2010年03月
• ウズラ卵子活性化におけるIP3注入の効果  [通常講演]

  水島秀成, 島田清司, 小野珠乙, 木村一郎

  日本畜産学会大会講演要旨 2009年09月
• ウズラ卵子DNase I活性に及ぼす精子数の効果  [通常講演]

  杉本宰甫, 水島秀成, 古寺敏子, 島田清司, 小野珠乙, 木村一郎

  日本家禽学会誌大会号 2009年03月
• 顕微授精ウズラ卵の発生能に及ぼすPhospholipase Czeta cRNA注入の効果  [通常講演]

  水島秀成, 高木惣一, 小野珠乙, 渥美優介, 塚田光, 齋藤昇, 島田清司

  日本家禽学会誌大会号 2008年03月
• 鳥類絶滅危惧種の救済に精子顕微注射・体外培養孵化を応用する  [通常講演]

  水島 秀成

  日本家禽学会誌 2007年11月
• ウズラにおける精子数の卵子活性化に及ぼす影響  [通常講演]

  水島秀成, 高木惣一, 小野珠乙, 渥美優介, 塚田光, 齋藤昇, 島田清司

  日本畜産学会大会講演要旨 2007年03月
• Effect of strontium and calcium on ICSI-associated fertilization in quail

  Mizushima S, Takagi S, Ono, T, Atsumi, Y, Tsukada, A, Saito, N, Shimada, K

  8th Asian Pacific Poultry Conference 2007年03月
• 精子抽出物および精子特異的PLCzeta cRNAの顕微注に対するウズラ卵子の反応  [通常講演]

  水島秀成, 高木惣一, 小野珠乙, 渥美優介, 塚田光, 齋藤昇, 島田清司

  Annu Meet Jpn Avian Endocrinol 2007年
• ウズラ卵子の前核形成に及ぼすカルシウム顕微注入の効果

  水島秀成, 高木惣一, 小野珠乙, 渥美優介, 塚田光, 齋藤昇, 島田清司

  第31回鳥類内分泌研究会 2006年11月
• Possible role of intracellular calcium release for fertilization in quail oocyte

  Mizushima S, Takagi S, Ono T, Tsukada A, Saito N, Shimada K

  XIIth AAAP Animal Science Congress 2006年09月
• ウズラ卵子活性化におけるストロンチウムの効果

  水島秀成, 高木惣一, 小野珠乙, 塚田光, 齋藤昇, 島田清司

  第30回鳥類内分泌研究会 2005年
• ニワトリ精子-ウズラ卵子間の受精率に及ぼすICSI後の培養時間および配偶子の性差の影響

  水島秀成, 高木惣一, 小野珠乙, 塚田光, 齋藤昇, 島田清司

  第29回鳥類内分泌研究会 2004年
• ウズラのアンドロゲンレセプター(AR)cDNAのクローニングとARmRNA発現  [通常講演]

  水島秀成, 斎藤昇, 島田清司

  日本家禽学会誌大会号 2003年09月

その他活動・業績

• 今後の鳥類顕微授精法 : 産業への応用に向けて (秋季大会号)

  水島 秀成 日本家禽学会誌 53 3 -5 2016年09月
• Full-term development of quail chick by ICSI.

  Shusei Mizushima Development, The Node- Community Site for Developmental Biologists- 2014年10月
• トキの復活の可能性を秘めた鳥類卵の活用法

  水島 秀成 動植物アロ認証 News Letter 2014年
• 鳥類絶滅危惧種の救済に精子顕微注射・体外培養孵化を応用する

  水島 秀成 日本家禽学会誌 44 (4) J189 -J190 2007年11月25日
• mRNA EXPRESSION PATTERNS OF IGFBPS IN DEVELOPING LIMB

  YAMADA Kikumi, TAHARA Ken-ichi, YAMAMOTO Ichiro, TSUKADA Akira, TANAKA Minoru, MIZUSHIMA Shusei, ONO Tamao, SAITO Noboru, SHIMADA Kiyoshi Proceedings of the Japan Society for Comparative Endocrinology (22) 15 -15 2007年10月12日

特許

• 特開JPWO2016043185A:鳥類の体外受精方法及びクローン細胞又はクローン個体の作製方法、並びにキット、  2017年07月06日

  笹浪知宏, 水島秀成

受賞

• 2021年09月 The Journal of Poultry Science Outstanding Paper Award
   Expression of transferrin and albumin in the sperm-storage tubules of Japanese quail and their possible involvement in long-term sperm storage 

  受賞者: Matsuzaka M;Mizushima S;Dohra H;Sasanami T
• 2019年07月 Award for best poster presentation at 2nd TASP and ICONC
   Down-regulation of Inositol 1,4,5-trisphosphate receptor type 1 (IP3R-1) for oocyte activation in quail 

  受賞者: Mizushima S
• 2017年09月 The Journal of Poultry Science Outstanding Paper Award
   Expression of Prolactin Receptor on the Surface of quail spermatozoa 

  受賞者: Hiyama G;Mizushima S;Matsuzaki M;Ishikawa Y;Sasanami T
• 2014年10月 Asia and Pacific Poultry Association Award for best oral presentation at 10th Asia and Pacific Poultry Conference
   Full-term development of quail egg by intracytoplasmic sperm injection (ICSI) and use of ICSI for avian transgenesis 

  受賞者: Mizushima S
• 2012年03月 日本家禽学会 奨励賞第24号
   家禽の顕微授精法の改良と受精機構に関する研究 

  受賞者: 水島 秀成
• 2008年03月 日本家禽学会 優秀発表賞
   顕微授精ウズラ卵の発生能に及ぼすPhospholipase Czeta cRNA注入の効果 

  受賞者: 水島 秀成

共同研究・競争的資金等の研究課題

• ユニークな抗体受容体によるトリ血中IgYの延命機構の解明と免疫増強への応用

  日本学術振興会：科学研究費補助金 基盤研究（B）研究分担者

  研究期間 : 2023年04月 -2026年03月 

  代表者 : 村井 篤嗣
• ウズラ始原生殖細胞の初期性分化を制御する遺伝子ネットワークの解析

  日本学術振興会：科学研究費補助金 基盤研究（C）

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 水島 秀成
• ウズラ生殖腺の卵巣分化におけるW性染色体遺伝子の機能解析

  一般財団法人旗影会：

  研究期間 : 2022年04月 -2023年03月 

  代表者 : 水島秀成
• PLA2R受容体による鳥類卵黄への抗体輸送機構の解明と高IgY卵創出への応用

  日本学術振興会：科学研究費補助金 基盤研究（B）研究分担者

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 村井篤嗣
• 鳥類卵の受精における多精防止の分子機構の解明と鳥類卵の長期保存の開発

  日本学術振興会：科学研究費補助金基盤研究（C）

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 水島秀成
• 鳥類の性決定にはたらくnon-coding RNAの解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 2018年06月 -2021年03月 

  代表者 : 黒岩 麻里, 水島 秀成

   

  鳥類は古くからW染色体上に性決定因子があると考えられているが、未だ同定されていない。その理由として、W染色体上のnon-coding RNAが性決定に関与しているからではないかと着想した。本研究は鳥類の性決定メカニズムを明らかにするために、性決定に働くW染色体上のncRNAを同定し、ゲノム編集による機能解明を目指す。鳥類の性決定は、家禽産業に直結する重要な生命現象であり、その分子メカニズムを解明することは、当該分野の命題である。しかし、ニワトリを用いた実験は長い時間を要し、解析が極めて困難である。そこで、性成熟の早いニホンウズラを用いた研究を計画し、鳥類では困難とされている機能解析を実現する。
• 鶏卵アレルゲン除去卵の作出

  公益財団法人：ニッポンハム食の未来財団

  研究期間 : 2019年04月 -2020年03月 

  代表者 : 水島秀成
• 受精システムを介したゲノム編集家禽の作出

  日本学術振興会：科学研究費補助金基盤研究（C）

  研究期間 : 2016年04月 -2019年03月 

  代表者 : 水島 秀成
• 近縁種を介したキジ科鳥類の個体復元

  財団法人旗影会：研究助成

  研究期間 : 2015年04月 -2016年03月 

  代表者 : 水島 秀成
• 体細胞クローンウズラの胚発生に及ぼす核の初期化と卵子活性化のタイミング

  日本学術振興会：科学研究費補助金若手研究（B）

  研究期間 : 2014年04月 -2016年03月 

  代表者 : 水島 秀成
• 新旧両世界のウズラの遺伝資源の保存と活用

  日本学術振興会：科学研究費助成事業

  研究期間 : 2011年04月 -2015年03月 

  代表者 : 小野 珠乙, 鏡味 裕, 松田 洋一, 水島 秀成

   

  新世界ウズラと旧世界ウズラについて以下のことを明らかにした。① コリンウズラ（成体重約250ｇ）と肉用系ニホンウズラ（成体重約300ｇ）の繁殖用飼育ケージを新規開発した。② 代理卵殻を用いたコリンウズラ胚の培養法を樹立した。③ コリンウズラとニホンウズラの異科間キメラの作出をした。④ウロコウズラ(新世界ウズラ)とイワシャコ(旧世界ウズラ類縁)の動原体特異的反復配列を単離し，ニワトリ，ニホンウズラ，ヒメウズラ，コリンウズラおよびカンムリウズラと核型を比較した。⑤ 白色変異体コリンウズラの新規系統を造成した。⑥ 実験動物として扱いやすい肉用系のアルビノ系統と黒色羽装白色卵系統を造成した。
• 多精受精モデル、鳥類の新規卵子活性化機構の探索とクローン鳥類創出への挑戦

  科学研究費補助金：PD研究助成金

  研究期間 : 2011年04月 -2014年03月 

  代表者 : 水島 秀成
• 鳥類の多精受精の生理的意義の解明による顕微授精技術の改良

  公益信託乾太助記念動物科学：研究助成基金

  研究期間 : 2012年07月 -2013年06月 

  代表者 : 水島 秀成
• 鳥類の受精成立に必要な精子因子の網羅的解析

  財団法人住友財団：研究助成金

  研究期間 : 2011年10月 -2012年09月 

  代表者 : 水島 秀成
• 顕微授精法を介した外来遺伝子導入ウズラの発生に対する精子処理法の検討

  財団法人中部科学技術センター：研究奨励金

  研究期間 : 2011年07月 -2012年06月 

  代表者 : 水島 秀成
• 鳥類の受精成立に関わるカルシウムオシレーションパターンと過剰の雄性前核形成

  日本学術振興会：科学研究費補助金若手研究（B）

  研究期間 : 2010年04月 -2011年03月 

  代表者 : 水島 秀成

教育活動情報

主要な担当授業