研究者詳細 - 木村　康二

論文 - 木村　康二

分割表示 124 件中 41 - 60 件目／全件表示 >>

Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum 査読

Kei Horihata, Shin Yoshioka, Masahiro Sano, Yuki Yamamoto, Koji Kimura, Dariusz J. Skarzynski, Kiyoshi Okuda

REPRODUCTION FERTILITY AND DEVELOPMENT 29 ( 7 ) 1280 - 1286 2017年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

The corpus luteum (CL) synthesises and secretes progesterone (P4), which is essential for the establishment and maintenance of pregnancy in mammals. P4 is synthesised from cholesterol. Cholesterol is internalised by low-density lipoprotein receptor (LDLR) and/or scavenger receptor B1 (SR-BI), and is effluxed by ATP-binding cassette (ABC) transporter A1 (ABCA1) and G1 (ABCG1). To test the hypothesis that lipoprotein receptors and ABC transporters are involved in functional luteolysis, we examined the expression of LDLR, SR-BI, ABCA1 and ABCG1 in bovine CL during the luteal stages and after injection of prostaglandin (PG) F(2 alpha)on Day 10 after ovulation. Expression of LDLR and SR-BI mRNA and protein was lower in the regressed luteal than late luteal stage. Injection of cows with a PGF(2 alpha) did not affect LDLR mRNA and protein levels in the CL. Although expression of SR-BI mRNA did not change, SR-BI protein expression decreased 12 and 24 h after PGF(2 alpha) injection. The overall findings of the present study suggest that the decreased expression of SR-BI induced by PGF(2 alpha) is one of the factors responsible for the continuous decrease in P4 production during functional luteolysis.

DOI： 10.1071/RD15538

Local effect of lysophosphatidic acid on prostaglandin production in the bovine oviduct 査読

Yuka Yoshimoto, Yoshihiko Kobayashi, Izabela Woclawek-Potocka, Emilia Sinderewicz, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

REPRODUCTION FERTILITY AND DEVELOPMENT 29 ( 5 ) 1021 - 1028 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

The mammalian oviduct plays an important role in the fertilisation and transport of gametes and embryo. Prostaglandins (PGs) are local mediators of oviductal functions and are involved in fertilisation and the transport of gametes and embryo. Lysophosphatidic acid (LPA), a kind of phospholipid, is involved in various physiological actions. We hypothesised that LPA regulates PG production in the bovine oviduct. To test this hypothesis, we examined the mRNA expression of LPA receptors (LPAR1-6) and LPA-producing enzymes (ATX, PLA1, PLA1) in ampullary and isthmic tissues and in cultured epithelial and stromal cells isolated from the bovine oviduct. We also investigated the effects of LPA on PG synthase expression and PG production in cultured cells. The mRNA of LPAR1-4, 6, ATX and PLA1 were expressed in cultured epithelial and stromal cells. The expressions of LPAR1-3 were significantly lower and the expression of LPAR4 was significantly higher in the isthmic than in the ampullary tissues. Lysophosphatidic acid significantly stimulated PG production in the cultured isthmic stromal cells. The overall findings suggest that LPA stimulates PG production via LPAR4 in the bovine oviduct. Since PGs are important for fertilisation and the transport of gametes and embryo, these findings show that locally produced LPA regulates oviductal functions.

DOI： 10.1071/RD15409

ウシ初期および中期黄体細胞のプロジェステロン(P4)分泌に及ぼす低酸素環境の影響

長谷川啓喜, 西村亮, 菱沼貢, 山下真路, 伊藤典彦, 岡本芳晴, 窪友瑛, 伊賀浩輔, 木村康二, 奥田潔

日本畜産学会大会講演要旨集 122回 160 - 160 2017年3月

Hypoxia-inducible factor 1 mediates hypoxia-enhanced synthesis of progesterone during luteinization of granulosa cells 査読

Fadhillah, Shin Yoshioka, Ryo Nishimura, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

JOURNAL OF REPRODUCTION AND DEVELOPMENT 63 ( 1 ) 75 - 85 2017年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

Hypoxia has been suggested to enhance progesterone (P4) synthesis in luteinizing granulosa cells (GCs), but the mechanism is unclear. The present study was designed to test the hypothesis that the hypoxia-induced increase in P4 synthesis during luteinization in bovine GCs is mediated by hypoxia-inducible factor 1 (HIF-1). GCs obtained from small antral follicles were cultured with 2 mu g/ml insulin in combination with 10 mu M forskolin for 24 h as a model of luteinizing GCs. To examine the influence of HIF-1 on P4 synthesis, we determined the effect of changes in protein expression of the alpha-subunit of HIF-1 (HIF1A) on P4 production and on the expression levels of StAR, P450scc, and 3 beta-HSD. CoCl2 (100 mu M), a hypoxia-mimicking chemical, increased HIF-l alpha protein expression in luteinizing GCs. After the upregulation of HIF-1 alpha, we observed an increase in P4 production and in the gene and protein expression levels of StAR in CoCl2-treated luteinizing GCs. In contrast, CoCl2 did not affect the expression of either P450scc or 3 beta-HSD. Echinomycin, a small-molecule inhibitor of HIF-1's DNA-binding activity, attenuated the effects of CoCl2 and of low oxygen tension (10% O-2) on P4 production and StAR. expression in luteinizing GCs. Overall, these findings suggest that HIF-1 is one of the factors that upregulate P4 in GCs during luteinization.

DOI： 10.1262/jrd.2016-068

ウシ過剰排卵処置時におけるニューロキニンB受容体作動薬の静脈内投与が回収胚の発育ステージおよび遺伝子発現に及ぼす影響

松山秀一, 中村翔, 美辺詩織, 大石真也, 大蔵聡, 木村康二

日本胚移植学雑誌 39 ( 1 ) 61 - 61 2017年1月

Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality 査読

Shun Takeo, Koji Kimura, Koumei Shirasuna, Takehito Kuwayama, Hisataka Iwata

REPRODUCTION FERTILITY AND DEVELOPMENT 29 ( 4 ) 759 - 767 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CSIRO PUBLISHING

Maternal age affects the quality of oocytes. The present study examined whether follicular fluid (FF) is a casual factor for age-associated decline in oocyte quality. First, we measured the concentration of advanced glycation end-products (AGE) in FF derived from young (21-45 months; Young-FF) and aged (120 months; Aged-FF) cows and found significantly higher concentrations of AGE in Aged-FF than Young-FF. Second, oocytes were collected from ovaries of young or aged cows and cultured in maturation medium containing 10% FF derived from young or aged cows. Regardless of oocyte origin, Aged-FF accelerated nuclear maturation progression and gap junction closure between oocytes and cumulus cells, increased reactive oxygen species (ROS) content and the rate of abnormal fertilisation of oocytes and decreased blastulation rate compared with Young-FF. Furthermore, supplementation of maturation medium with AGE induced similar age-associated events in oocytes derived from young cows, in that AGE accelerated the progression of nuclear maturation, increased ROS content in oocytes, increased the rate of abnormal fertilisation and decreased blastulation rate. In conclusion, maternal aging increased the concentration of AGE in FF, and both AGE and Aged-FF accelerated nuclear maturation and reduced the developmental competence of oocytes.

DOI： 10.1071/RD15228

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors 査読

Takamasa Kawaguchi, Dooseon Cho, Masafumi Hayashi, Tomoyuki Tsukiyama, Koji Kimura, Shuichi Matsuyama, Naojiro Minami, Masayasu Yamada, Hiroshi Imai

PLOS ONE 11 ( 12 ) e0167550 2016年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERR/3, and /IFN-tau) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy -related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

DOI： 10.1371/journal.pone.0167550

Remodeling of bovine oviductal epithelium by mitosis of secretory cells 査読

Sayaka Ito, Yoshihiko Kobayashi, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

CELL AND TISSUE RESEARCH 366 ( 2 ) 403 - 410 2016年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

DOI： 10.1007/s00441-016-2432-8

Superovulation with a single administration of FSH in aluminum hydroxide gel: a novel superovulation method for cattle 査読

Koji Kimura

JOURNAL OF REPRODUCTION AND DEVELOPMENT 62 ( 5 ) 423 - 429 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

Superovulation (SOV) is a necessary technique to produce large numbers of embryos for embryo transfer. In the conventional methods, follicular stimulating hormone (FSH) is administered to donor cattle twice daily for 3 to 4 days. As this method is labor intensive and stresses cattle, improving this method has been desired. We previously developed a novel and simple SOV method, in which the intramuscular injection of a single dose of FSH in aluminum hydroxide gel (AH-gel) induced the growth of multiple follicles, ovulation and the production of multiple embryos. Here we show that AH-gel can efficiently adsorb FSH and release it effectively in the presence of BSA, a major interstitial protein. When a single intramuscular administration of the FSH and AH-gel mixture was performed to cattle, multiple follicular growth, ovulation and embryo production were induced. However, the treatments caused indurations at the administration sites in the muscle. To reduce the muscle damage, we investigated alternative administration routes and different amounts of aluminum in the gel. By administering the FSH in AH-gel subcutaneously rather than intramuscularly, the amount of aluminum in the gel could be reduced, thus reducing the size of the induration. Moreover, repeated administrations of FSH with AH-gel did not affect the superovulatory response. These results indicate that a single administration of FSH with AH-gel is an effective, novel and practical method for SOV treatment.

DOI： 10.1262/jrd.2016-066

Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers 査読

Ahmed Saad Ahmed Hassaneen, Yousuke Naniwa, Yuta Suetomi, Shuichi Matsuyama, Koji Kimura, Nahoko Ieda, Naoko Inoue, Yoshihisa Uenoyama, Hiroko Tsukamura, Kei-ichiro Maeda, Fuko Matsuda, Satoshi Ohkura

JOURNAL OF REPRODUCTION AND DEVELOPMENT 62 ( 5 ) 471 - 477 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOCIETY REPRODUCTION & DEVELOPMENT-SRD

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black x Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

DOI： 10.1262/jrd.2016-075

分娩後のウシ子宮内膜におけるSide population細胞の発現挙動

松山秀一, 美辺詩織, 中村翔, 古澤軌, 池田光美, 木村康二

The Journal of Reproduction and Development 62 ( Suppl. ) j143 - j143 2016年9月

記述言語：日本語出版者・発行元：(公社)日本繁殖生物学会

【目的】経産牛における空胎期間延長の一因として子宮回復不全が挙げられる。子宮は分娩時に胎盤が剥離して大きく損傷を受ける一方，その後速やかに子宮内膜が再生することで修復される。したがって経産牛における子宮回復不全には，分娩後の子宮内膜再生の不具合が関与していると考えられるが，ウシの分娩後における子宮内膜再生メカニズムは未だ解明されていない。我々はこれまでに，ウシ子宮内膜においても幹細胞活性を有すると報告されているSide population（SP）細胞が存在することを明らかにしており，子宮内膜幹細胞が子宮内膜の再生に寄与する可能性を示唆している。そこで本研究では，分娩後におけるSP細胞の経日的な発現パターンについて明らかにすることを目的とした。また，これまでの研究で，ウシ子宮内膜上皮細胞を上皮系の幹細胞用無血清培養液で培養することにより，明らかに形態学的に異なる小型の子宮内膜上皮細胞が得られており，これらの小型子宮内膜上皮細胞が前駆細胞として増殖し，分化することで，ウシの子宮内膜再生に寄与する可能性も考えられる。そこで，ウシ小型子宮内膜上皮細胞の分化能についても検討を行った。【方法および結果】分娩後の子宮内膜におけるSP細胞の割合について，分娩後10日，30日，50日，100日で子宮内膜組織をバイオプシーによって採取し，採取した子宮内膜組織から酵素処理によって得られた細胞をフローサイトメトリー解析によって検討した結果，SP細胞はウシ子宮内膜間質に存在し，子宮内膜上皮には含まれないことが示された。さらに，分娩後10日目にはSP細胞の割合が低く，その後徐々に増加することが明らかとなった。また，ウシ小型子宮内膜上皮細胞は，骨，脂肪，軟骨等の中胚葉由来組織に分化しないことが明らかとなった。以上の結果から，分娩後の子宮内膜再生にはウシ子宮内膜間質に存在するSP細胞が関与する可能性が考えられた。

DOI： 10.14882/jrds.109.0_p-87

Possible Contribution of Alpha2,6-Sialylation to Luteolysis in Cows by Inhibiting the Luteotropic Effects of Galectin-1 査読

Kazuhisa Hashiba, Junko Nio-Kobayashi, Masahiro Sano, Megumi Maeda, Yoshinobu Kimura, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

BIOLOGY OF REPRODUCTION 95 ( 1 ) 17 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC STUDY REPRODUCTION

The corpus luteum (CL) is essential for establishing pregnancy. If pregnancy does not occur during the estrous cycle, luteolysis is induced by prostaglandin (PG) F2alpha secreted from the uterus. Galectin-1, a beta-galactose-binding protein, is expressed in the functional CL of cows and increases the viability of bovine luteal steroidogenic cells (LSCs) by modifying the functions of membrane glycoproteins. The binding of galectin-1 to glycoproteins is blocked by alpha2,6-sialylation of the terminal galactose residues of glycoconjugates, which is catalyzed by a sialyltransferase (ST6Gal-I). However, the physiological role of alpha2,6-sialic acid in bovine CL is unclear. The level of alpha2,6-sialylation of the bovine CL was higher during the regressed-luteal stage than in other luteal stages. Lectin histochemistry revealed that alpha2,6-sialylated glycoconjugates were localized to luteal endothelial cells throughout the estrous cycle. In addition, alpha2,6-sialylated glycoconjugates concentrated to the membrane of LSCs during the regressed-luteal stage. PGF2alpha treatment for 72 h enhanced the expression of ST6Gal-I mRNA and the level of alpha2,6-sialylated glycoproteins in mid-LSCs. The level of alpha2,6-sialylated glycoproteins of late-stage LSCs (Days 15-17 after ovulation) was higher than that of mid-stage LSCs (Days 8-12 after ovulation), and galectin-1 increased the viability of mid-LSCs but not that of late-stage LSCs. Furthermore, galectin-1 increased the viability of late-LSCs when alpha2,6-sialic acid residues were removed by neuraminidase. The overall findings suggest that alpha2,6-sialylation stimulated by PGF2alpha contributes to luteolysis by inhibiting the luteotropic effects of galectin-1 in bovine CL.

DOI： 10.1095/biolreprod.116.140194

Roles of EDNs in regulating oviductal NO synthesis and smooth muscle motility in cows 査読

Yoshihiko Kobayashi, Yuka Yoshimoto, Yuki Yamamoto, Koji Kimura, Kiyoshi Okuda

REPRODUCTION 151 ( 6 ) 615 - 622 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

Endothelins (EDNs) participate in various physiological events including smooth muscle contraction, nitric oxide (NO) synthesis, and embryonic development. In this study, we investigated the regional roles of EDNs produced by bovine oviductal epithelial cells in NO synthesis and smooth muscle motility. Quantification of mRNA expressions indicated that expression of EDN receptor B (EDNRB) in the ampullary region was higher after ovulation than before ovulation, whereas expression of FONRA in the isthmic region was higher after ovulation than before ovulation. Immunohistochemistry revealed that the EDN receptors (EDNRA and EDNRB) were expressed in the epithelium, whereas smooth muscle showed positive staining only for EDNRA. The expressions of inducible NO synthase (i NOS) protein and its mRNA (NOS2) in cultured epithelial cells isolated from the ampulla were stimulated by EDN1, but not by EDN2 or EDN3, after 1 h of incubation. In isthmic epithelial cells, none of the EDNs affected the expression of NOS2. Isometric contraction tests indicated that spontaneous waves were strong in the isthmic region but weak in the ampullary region. EDN1 modulated smooth muscle motility in both the regions. The overall findings suggest that EDN1 plays region-specific roles in smooth muscle motility and epithelial NO synthesis, providing an optimal oviductal microenvironment for transport of gametes, fertilization, and development/transport of early embryo.

DOI： 10.1530/REP-15-0586

Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins 査読

Yoshihiko Kobayashi, Yuki Yamamoto, Soichi Kageyama, Hiroki Hirayama, Koji Kimura, Kiyoshi Okuda

REPRODUCTION 151 ( 6 ) 577 - 587 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOSCIENTIFICA LTD

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2 alpha. increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradio1-17 beta decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptor a (ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

DOI： 10.1530/REP-15-0254

Evidence for a PGF2α auto-amplification system in the endometrium in mares. 査読国際誌

Kozai K, Tokuyama S, Szóstek AZ, Toishi Y, Tsunoda N, Taya K, Sakatani M, Takahashi M, Nambo Y, Skarzynski DJ, Yamamoto Y, Kimura K, Okuda K

Reproduction (Cambridge, England) 151 ( 5 ) 517 - 26 2016年5月

ウシ黄体におけるresistin発現制御

伊藤さやか, 小林芳彦, Rak Agnieszka, 山本ゆき, 美辺詩織, 松山秀一, 木村康二, Skarzynski Dariusz J., 奥田潔

日本畜産学会大会講演要旨集 121回 191 - 191 2016年3月

The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts 査読

Mark P. Green, Alexandra J. Harvey, Lee D. Spate, Koji Kimura, Jeremy G. Thompson, R. Michael Roberts

MOLECULAR REPRODUCTION AND DEVELOPMENT 83 ( 1 ) 50 - 60 2016年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

The preimplantation bovine embryo displays sexual dimorphism in glucose sensitivity and interferon-tau (IFNT) secretion that are negated by inhibition of the pentose phosphate pathway, suggesting that the association between glucose metabolism and IFNT likely underpins the selective loss of female embryos. The aim of this study was to determine if altered glucose metabolism, through glucose supplementation and/or uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP), affected embryo development. Bovine blastocyst development, sex, and IFNT production were examined in embryos cultured in the presence or absence of glucose (0, 1.5, 4mM) with or without exposure to DNP (0, 10, 100M) between Days 5 and 8 post-fertilization. The absence or presence of high (4mM) glucose reduced blastocyst development and favored the development of male embryos (P<0.001). DNP at 10M had no effect, whereas 100M had a negative impact on blastocyst development. Notably, in the presence or even absence of glucose, supplementation with 10M DNP further skewed the sex ratio toward males (P<0.05). Sexually dimorphic IFNT production was maintained in these conditions, although total production was reduced in the presence of high glucose and DNP, irrespective of embryo sex. These data suggest that the pentose phosphate pathway can modulate embryonic sex ratio and development. Therefore, bovine embryo culture should be undertaken in a low glucose (<2.5mM) medium to minimize potential embryonic stress, as higher concentrations have sexually dimorphic effects on development and an embryo's ability to signal to the maternal reproductive tract. Mol. Reprod. Dev. 83: 50-60, 2016. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/mrd.22590

肉用繁殖牛における分娩前の腟内温度および電気抵抗値の変動

松山秀一, 美辺詩織, 吉岡耕治, 木村康二

日本胚移植学雑誌 38 ( 1 ) 45 - 45 2016年1月

ウシ卵管峡部上皮細胞におけるアクチビンシグナルによるアポトーシス制御

山本ゆき, 伊藤さやか, 小林芳彦, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 109 ( 0 ) P - 65-P-65 2016年

記述言語：日本語出版者・発行元：日本繁殖生物学会

<p>アクチビンA（ACVA）はアポトーシスや細胞分化など様々な生理現象に関与する局所因子で，ウシ卵管上皮でも遺伝子発現が確認されている。哺乳動物の卵管峡部は，受精前は精子の通路であるとともに貯留や受精能獲得の場であり，受精後は初期胚の発育と輸送の場となる。本研究では，卵管峡部におけるACVAの役割を明らかにする目的で以下について検討した。1）ウシ卵管液中ACVAタンパク質をwestern blotによって確認したところ，発情周期を通じて検出された。2） ACV受容体（ActR）1Bおよび2A，シグナル因子Smad2/3のタンパク質発現が培養上皮細胞で確認されたが，培養間質細胞ではActRsタンパク質は検出されなかった。また卵管峡部組織において，排卵日のActR2A遺伝子発現は排卵後8–12日と比較し有意に低かった。3）発情周期中の峡部上皮細胞におけるcleaved caspase-3（CCP3：アポトーシス誘導因子）局在を免疫組織化学で検討したところ，CCP3陽性細胞数は排卵後2–3日で排卵後5–6日に対し有意に多かった。4）アポトーシス関連因子の遺伝子発現に及ぼすACVAの影響を確認するため，培養峡部上皮細胞にACVAおよびフォリスタチン（FST: ACVA作用阻害因子）またはSB431542（SB:ACVシグナル伝達阻害剤）を単独又は組み合わせで添加し，4時間培養した。ACVA単独添加では影響が認められなかったが，FST単独およびACVAとの組み合わせ，ならびにSB単独添加において，Bcl2（抗アポトーシス因子）/Bax （アポトーシス促進因子）の遺伝子発現の比が有意に減少した。以上より，ウシ卵管峡部上皮ではACVA 作用の阻害によりBcl2/Bax比が減少すること，受精前にはActR2A発現の減少に伴いACVA作用が減弱することが示唆されたため，受精後における峡部上皮細胞のアポトーシス増加にACVAシグナルが関与している可能性が示された。ウシ卵管峡部は，受精前後の数日間で精子の輸送・貯留から初期胚輸送・発育のサポートへとその役割を変えることから，ACVAはアポトーシス制御因子として受精前後の峡部上皮機能調節に関与している可能性が考えられる。</p>

DOI： 10.14882/jrds.109.0_P-65

ウシ卵管分泌上皮細胞の増殖と分化による卵管機能の制御

伊藤さやか, 小林芳彦, 山本ゆき, 木村康二, 奥田潔

日本繁殖生物学会講演要旨集 109 ( 0 ) OR1 - 4-OR1-4 2016年

記述言語：日本語出版者・発行元：日本繁殖生物学会

<p>【目的】卵管は受精の場，配偶子および初期胚の輸送経路である。膨大部で受精した初期胚は，卵管上皮の繊毛運動により子宮へと輸送される。卵管上皮は卵母細胞および初期胚の輸送を促す繊毛細胞ならびに，初期胚発育に必要な物質を分泌する分泌細胞の主に2種類から構成される。繊毛細胞の数は排卵時に多い一方，分泌細胞の数は黄体の生育に応じて徐々に増加する。この繊毛および分泌細胞数の変化は卵管上皮の機能を制御していると考えられるが，両細胞比の変化が生じるメカニズムは不明である。本研究では卵管の機能制御機構を明らかにするため，排卵周期にともなう細胞分裂の増減とともに，繊毛および分泌細胞の比率を制御するメカニズムをウシをモデルに検討した。【方法】排卵周期を通じたウシ卵管膨大部および峡部組織における1） FOXJ1（繊毛細胞マーカー）および 2） Ki67（細胞分裂マーカー）陽性細胞の割合を検討した。3） Ki67とFOXJ1またはPAX8（分泌細胞マーカー）との共染色により繊毛ならびに分泌細胞における分裂能を評価した。4）分泌細胞における繊毛細胞への分化能を評価するために，分泌細胞の表面抗原であるSSEA1の抗体を結合させた磁気ビーズを用いて単離した細胞をAir-liquid interface（ALI）法で培養後，acetylated tubulin（繊毛マーカー）陽性細胞を調べた。【結果】1）膨大部においてFOXJ1陽性細胞は排卵前後に増加する一方，峡部では変化がなかった。 2） Ki67陽性細胞は膨大部では卵胞期および排卵日に，峡部では排卵前後に多かった。3）膨大部および峡部においてKi67 陽性細胞は全てFOXJ1陰性であった。4） SSEA1陽性細胞をALI法で培養するとacetylated tubulin陽性細胞へと分化した。以上より，排卵周期を通じた繊毛および分泌細胞の比は分泌細胞の増殖・分化によって制御されることが示唆された。また，排卵前には繊毛細胞が増加する一方，分泌細胞が排卵後に増加することで，卵母細胞の輸送，受精，初期胚の輸送および発育などの，卵管内で起こる現象に対応した適切な環境を提供する可能性が示された。</p>

DOI： 10.14882/jrds.109.0_OR1-4

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