研究者詳細 - 木村　康二

論文 - 木村　康二

分割表示 124 件中 1 - 20 件目／全件表示 >>

Ovine conceptuses express phospholipase inhibitory genes on days 14-15 of pregnancy, interacting with IFNT pathways. 国際誌

Yuta Matsuno, Kazuya Kusama, Koji Kimura, Kazuhiko Imakawa

Reproduction (Cambridge, England) 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

In mammals, various molecules are involved in biochemical interaction between conceptus and endometrium for pregnancy recognition and establishment. In ruminants, interferon tau (IFNT) is the pregnancy recognition factor; however, IFNT alone does not explain corpus luteum maintenance. Although data on factors expressed during implantation has been accumulated, we hypothesized that the conceptus produces additional, uncharacterized molecules during the period of conceptus attachment. This study aimed to identify new conceptus secretory proteins involved in the biochemical interaction between conceptus and endometrium in sheep. We analyzed RNA-sequence data of ovine conceptuses from pregnant animals on days 12, 14, 15, 16, 17, 19, 20, and 21. To identify novel secretory proteins, we focused on highly expressed but uncharacterized genes and performed in silico protein function analysis, identifying genes encoding phospholipase inhibitory proteins expressed on days 14 and 15. Recombinant proteins from these genes were produced and the effects on cultured bovine endometrial epithelial cells (EECs) and stromal cells (STRs) were analyzed by RNA-sequence analysis. Differentially expressed gene (DEG) analysis demonstrated that the recombinant protein treatment upregulated 31 genes and downregulated 4 genes in EECs; is also upregulated 398 genes and downregulated 66 genes in STRs, including implantation-related genes such as ISG15, OAS1X, OAS1Y, PARP9, PARP14, MX1 and PTGS2. Gene set enrichment analysis revealed that DEGs were enriched in several implantation-related pathways, including ISG15 antivirus mechanisms. These results suggest that, in addition to numerous characterized molecules, phospholipase inhibitory protein is a new candidate molecule in enabling biochemical communication between the conceptus and endometrium.

DOI： 10.1530/REP-24-0286

Effects of intrauterine extracellular vesicle microRNAs on embryonic gene expression in low-fertility cows. 国際誌

Rei Ichikawa, Koji Kimura, Sho Nakamura, Satoshi Ohkura, Shuichi Matsuyama

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 38 ( 20 ) e70116 2024年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Embryo survival and pre-implantation development depend on uterine luminal fluid, which is believed to play a role in early embryonic death and infertility in cows. Extracellular vesicles (EVs) in the uterine luminal fluid contain microRNAs (miRNAs), crucial mediators of intercellular communication. miRNAs regulate conceptus-maternal interactions and participate in embryonic development by suppressing gene expression. Therefore, we hypothesized that miRNAs in the intrauterine EVs of low-fertility cows would hinder embryonic survival and development. EVs were collected from the bovine uterine luminal fluid of both normal- and low-fertility cows 7 days post-estrus. Small RNA-sequencing analysis of miRNAs isolated from these EVs identified eight miRNAs that were highly expressed in normal-fertility cows (normal-fertility miRNAs) and eight with elevated expression in low-fertility cows (low-fertility miRNAs). These two sets of miRNAs were transfected into hatched blastocysts via lipofection. RNA-seq following lipofection with low-fertility miRNAs identified 424 differentially expressed genes (DEGs) relative to the control; in contrast, following lipofection with normal-fertility miRNAs, seven DEGs were identified. Pathway analysis of the DEGs identified following lipofection with low-fertility miRNAs revealed substantial enrichment of mitogen-activated protein kinase (MAPK) signaling. Expression of activator protein 1 (AP1) and interferon-tau (IFNT) mRNA was significantly lower in the low-fertility miRNA transfection group than in the control. IFNT is essential for maternal pregnancy recognition. Therefore, miRNAs in intrauterine EVs from low-fertility cows at 7 days post-estrus may inhibit embryo development and suppress IFNT expression by altering MAPK signaling.

DOI： 10.1096/fj.202401728R

IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes. 国際誌

Shabur Abdus Talukder, Ahmed Z Balboula, Hanako Bai, Manabu Kawahara, Koji Kimura, Masashi Takahashi

Reproduction (Cambridge, England) 166 ( 6 ) 411 - 421 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Interferon tau (IFNT) is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post-artificial insemination (AI), after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in non-pregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.

DOI： 10.1530/REP-23-0098

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows. 国際誌

Shuichi Matsuyama, Sho Nakamura, Shiori Minabe, Satoshi Ohkura, Koji Kimura

Biology of reproduction 108 ( 6 ) 936 - 944 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B, and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. Since neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

DOI： 10.1093/biolre/ioad039

Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows. 国際誌

Shuichi Matsuyama, Sho Nakamura, Shiori Minabe, Miki Sakatani, Naoki Takenouchi, Takuya Sasaki, Yuki Inoue, Hisataka Iwata, Koji Kimura

Molecular reproduction and development 90 ( 3 ) 141 - 152 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.

DOI： 10.1002/mrd.23670

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro 国際誌

Yuki Yamamoto, Maho Kurokawa, Taiji Ogawa, Sayaka Kubota, Koji Kimura

Reproduction 165 ( 3 ) 301 - 312 2023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：Bioscientifica

Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0–4 days after ovulation), at the mid-late luteal stage (10–17 days), and at the follicular stage (1–3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher at the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

DOI： 10.1530/rep-22-0174

その他リンク： https://rep.bioscientifica.com/downloadpdf/journals/rep/aop/rep-22-0174/rep-22-0174.xml

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles

Ahmad Farid RAWAN, Hikmatullah LANGAR, Maho MUNETOMO, Yuki YAMAMOTO, Kohei KAWANO, Koji KIMURA

Journal of Reproduction and Development 69 ( 6 ) 337 - 346 2023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：Japanese Society of Animal Reproduction

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17β (E2) assumes a central role in follicular development and selection by activating estrogen receptors β (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

DOI： 10.1262/jrd.2023-047

Expression of NFIL3 and CEBPA regulated by IFNT induced-PGE2 in bovine endometrial stromal cells during the pre-implantation period. 国際誌

Rulan Bai, Kazuya Kusama, Yuta Matsuno, Hanako Bai, Toshihiro Sakurai, Koji Kimura, Kazuhiko Imakawa

Frontiers in endocrinology 14 1075030 - 1075030 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

Prostaglandin E2 (PGE2) is considered as a luteoprotective factor, influencing the corpus luteum during the early pregnant period in the bovine species. Cyclic AMP (cAMP) is activated in response to PGE2 and plays a role in many physiological processes. The maternal recognition signal, interferon τ (IFNT), induces PGE2 secretion from the endometrial epithelial cells, the function of which in stroma cells has not been completely understood. In this study, PGE2 was found to activate cAMP in the bovine endometrial stromal cells (STRs). STRs were then treated with forskolin to activate the cAMP signaling, from which RNA extracted was subjected to global expression analysis. Transcripts related to transcription regulatory region nucleic acid binding of molecular function, nucleus of cellular component, and mitotic spindle organization of biological processes were up-regulated in cAMP-activated bovine STRs. An increase in the transcription factors, NFIL3, CEBPA, and HIF1A via the cAMP/PKA/CREB signaling pathway in the bovine STRs was also found by qPCR. Knockdown of NFIL3, CEBPA, or HIF1A blocked forskolin-induced PTGS1/2 and IGFBP1/3 expression. Moreover, NFIL3 and CEBPA were localized in endometrial stroma on pregnant day 17 (day 0 = estrous cycle), but not on cyclic day 17. These observations indicated that uterine PGE2 induced by conceptus IFNT is involved in the early pregnancy-related gene expression in endometrial stromal cells, which could facilitate pregnancy establishment in the bovine.

DOI： 10.3389/fendo.2023.1075030

A Chinese Medicine, Tokishakuyakusan, Increases Bovine Oviductal Tonus <i>via</i> G Protein-Coupled Estrogen Receptor 1

Sayaka Kubota, Yuki Yamamoto, Koji Kimura

Biological and Pharmaceutical Bulletin 45 ( 8 ) 1133 - 1141 2022年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pharmaceutical Society of Japan

Early embryo and sperm transport through the oviductal isthmus depends on the contraction and relaxation of the smooth muscle layers. Dysfunction of the oviduct transport is considered to be one of the causes of infertility. For human infertility, Chinese medicine is used in East Asia. Although there are many clinical reports regarding Tokishakuyakusan (TSS), there is little scientific evidence that it affects infertility. In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation via the G protein-coupled estrogen receptor 1 (GPER1). We collected bovine oviductal isthmic tissues at four stages of the estrous cycle, classified based on a macroscopic observation of the ovary. The Magnus method was used to monitor longitudinal contractility (frequency, contraction force, and tonus). The effects of TSS solution, GPER1 agonist (G-1), and antagonist (G-15) on oviductal contractility were examined. The protein expression level of GPER1 in the oviductal isthmic smooth muscle of each estrous stage was assessed by Western blotting. Although TSS did not affect frequency and contraction force, the tonus was significantly increased by TSS or G-1 at all stages (p < 0.05), and the effect was especially highest at days 1-4 after ovulation. The addition of G-15 significantly suppressed the TSS-induced increase of oviductal tonus at all stages (p < 0.05). There was no significant difference in GPER1 protein expression among the estrous stages. TSS affects oviductal contractility by increasing tonus via GPER1, and it may accelerate gamete and early embryo transport by contracting the oviducts longitudinally.

DOI： 10.1248/bpb.b22-00201

Evaluation of bovine uterine gland functions in 2D and 3D culture system

Yosuke SUGINO, Taiki SATO, Yuki YAMAMOTO, Koji KIMURA

Journal of Reproduction and Development 68 ( 4 ) 254 - 261 2022年

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Society of Animal Reproduction

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in isolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did not affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology between them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

DOI： 10.1262/jrd.2022-029

Immunohistochemical identification of epithelial cell types in the isthmus of bovine oviduct: comparison with the ampulla

Sayaka ITO, Yuna YAMAGUCHI, Sayaka KUBOTA, Yuki YAMAMOTO, Koji KIMURA

Journal of Reproduction and Development 69 ( 1 ) 18 - 24 2022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：Japanese Society of Animal Reproduction

The oviductal epithelium consists of ciliated and non-ciliated cells, and their numbers vary depending on the segment of the oviduct and stage of the estrous cycle. Compared with the ampulla, fewer cyclic changes in the number of the two types of cells occur in the isthmus. Recently, we have reported that the epithelium in the ampullary oviduct is composed of many types of cells during different translational/transcriptional states, and their numbers change during the estrous cycle. However, detailed information regarding the epithelial cell subtypes lining the isthmic oviductal epithelium has not yet been reported. In this study, we aimed to identify the epithelial subtypes in the isthmus of the oviduct using immunohistochemistry. Some similarities and differences were observed between the ampulla and isthmus. As observed in the ampulla, epithelial cells of the isthmus expressed either FOXJ1 (ciliogenesis marker) or PAX8 (non-ciliated cell marker). The estrous cycle affected the number of Ki67+ cells but not that of ciliated cells. A relatively high rate of Ki67+ cells (60%) was observed at 1-4 days after the ovulation. Interestingly, unlike the ampulla, Ki67+/FOXJ1+ cells (12.6 ± 1.1%) were discovered in the isthmus. Double staining for Ki67 with FOXJ1, PAX8, or Centrin-1 (a centriole marker) revealed that Centrin-1 was localized on the apical surface of some Ki67+/FOXJ1+ cells. In conclusion, some epithelial cell subtypes exist in the isthmus of the oviduct and isthmus-specific cell subtypes have been identified. These region-specific cells may provide functional and morphological differences between the ampulla and isthmus of the oviduct.

DOI： 10.1262/jrd.2022-104

Hyperthermia alters interleukin-6 production in response to lipopolysaccharide via endoplasmic reticulum stress in bovine endometrial cells. 査読国際誌

Shunsuke Sakai, Yuki Inoue, Keisuke Tanaka, Yuki Yamamoto, Hisataka Iwata, Koji Kimura

Journal of cellular physiology 237 ( 1 ) 1087 - 1099 2021年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

In the postpartum period, cows experience the uterine bacterial infection and develop the endometritis. To eliminate bacteria and recover from endometritis, endometrial epithelial and stromal cells secrete the cytokine and chemokine, such as interleukin 6 (IL-6), IL-8, and monocyte chemotactic protein 1 (MCP1), to recruit immune cells. Moreover, the symptom of endometritis is prolonged in summer and we have recently indicated that hyperthermia suppresses and enhances the IL-6 production in response to lipopolysaccharide (LPS) challenge in endometrial epithelial and stromal cells, respectively. However, the mechanisms for the opposite reaction of IL-6 secretion in response to LPS challenge in both types of endometrial cells under hyperthermia conditions were still unclear. To reveal these mechanisms, both types of endometrial cells were cultured with LPS under the control (38.5°C) or hyperthermia (40.5°C) conditions and comprehensively analyzed differential gene expressions of them by RNA-seq. In addition, based on these results, we examined the effect of endoplasmic reticulum (ER) stress on the IL-6 production in both types of endometrial cells cultured with LPS under hyperthermia conditions. In comprehensive analysis, hyperthermia induced the ER stress in the endometrial stromal cells but not in the endometrial epithelial cells. Actually, we confirmed that hyperthermia increased the gene expression of BiP, ATF4, and sXBP1 and protein expression of BiP and phosphorylated inositol requiring 1, ER stress marker, in the endometrial stromal cells but not in the endometrial epithelial cells. Moreover, in the endometrial stromal cells exposed to LPS, activation and inhibition of ER stress enhanced the IL-6 production under control conditions and suppressed it under hyperthermia conditions, respectively. In this study, we could uncover the one of causes for the disruption of IL-6 production in response to LPS challenge in the endometrial cells under hyperthermia conditions. This finding might be a clue for the improvement of the symptom of endometritis in cows during summer.

DOI： 10.1002/jcp.30604

Effects of cortisol on prostaglandin F2α secretion and expression of genes involved in the arachidonic acid metabolic pathway in equine endometrium - In vitro study. 査読国際誌

Anna Z Szóstek-Mioduchowska, Haruki Shiotani, Yuki Yamamoto, Agnieszka Sadowska, Anna Wójtowicz, Keisuke Kozai, Takuo Hojo, Koji Kimura, Dariusz J Skarzynski, Kiyoshi Okuda

Theriogenology 173 221 - 229 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF2α by affecting the AA cascade in the equine endometrium during the estrous cycle.

DOI： 10.1016/j.theriogenology.2021.08.009

Heat stress influences the attenuation of prostaglandin synthesis by interferon tau in bovine endometrial cells. 査読国際誌

Shunsuke Sakai, Mami Yagi, Nao Fujime, Mariko Kuse, Ryosuke Sakumoto, Yuki Yamamoto, Kiyoshi Okuda, Koji Kimura

Theriogenology 165 52 - 58 2021年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heat stress (HS) reduces reproductive performance of cattle, possibly by disrupting endocrine regulation such as prostaglandin (PG) production from uterus and estradiol 17β production from the dominant follicle. Prostaglandin F2α (PGF2α) secretion from endometrium surges during the luteal phase due to tumor necrosis factor (TNF) α stimulation and a positive-feedback loop with oxytocin (OT) from the corpus luteum, ultimately triggering luteolysis, while interferon τ (IFNT) inhibits upregulation of PGF2α production by TNFα and OT, thereby preventing luteolysis and triggering recognition of pregnancy. In the present study, we investigated the effect of OT, TNFα, and IFNT on PGF2α production in both types of endometrial cells under HS conditions. Stimulation of PGF2α production in endometrial epithelial cells by OT was unaffected by HS, while stimulation of PGF2α production in endometrial stromal cells by TNFα was enhanced by HS, and this increased PGF2α production was not significantly suppressed by IFNT. These results suggest that HS disrupted the regulation of PGF2α production by TNFα and IFNT in bovine endometrial stromal cells and it might be one of causes for low conception rate of cattle in summer.

DOI： 10.1016/j.theriogenology.2021.02.005

Gene-expression profile and postpartum transition of bovine endometrial side population cells†. 査読国際誌

Ryoki Tatebayashi, Sho Nakamura, Shiori Minabe, Tadashi Furusawa, Ryoya Abe, Miki Kajisa, Yasuhiro Morita, Satoshi Ohkura, Koji Kimura, Shuichi Matsuyama

Biology of reproduction 104 ( 4 ) 850 - 860 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene Ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells.

DOI： 10.1093/biolre/ioab004

Alteration of chemokine production in bovine endometrial epithelial and stromal cells under heat stress conditions. 査読国際誌

Shunsuke Sakai, Toshimitsu Hatabu, Yuki Yamamoto, Koji Kimura

Physiological reports 8 ( 22 ) e14640 2020年11月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

After parturition, cows frequently develop uterine bacterial infections, resulting in the onset of endometritis. To eliminate the bacteria, bovine endometrial cells secrete chemokines, such as IL-6 and MCP1, which attract macrophages (MΦs) to the subepithelial stroma. These attracted MΦs are not only involved in bacterial elimination but also the orchestration of inflammation and tissue repair. These immune responses aid in the recovery from endometritis; however, the recovery from endometritis takes longer in summer than in any other season. Based on these findings, we hypothesized that heat stress (HS) affects the chemokine production in endometrial cells. To confirm this hypothesis, we compared IL-6 and MCP1 production induced by lipopolysaccharide (LPS) in bovine endometrial epithelial and stromal cells under normal (38.5°C) and HS conditions (40.5°C). In the endometrial epithelial cells, IL-6 production stimulated by LPS was significantly (p < .05) suppressed under HS conditions. MCP1 production in endometrial epithelial cells was not detected under both the control and HS conditions regardless of the presence of LPS. Moreover, LPS significantly (p < .05) stimulated IL-6 and MCP1 production in endometrial stromal cells. Moreover, HS significantly (p < .05) enhanced their production compared to that under the control conditions. In addition, HS did not affect the migration ability of MΦs; however, the supernatant of the endometrial stromal cells cultured under the HS condition significantly (p < .05) attracted the MΦs when compared to the control condition. These results suggest that HS disrupts chemokine production in two types of endometrial cells and alters the distribution of MΦs in the endometrium during the summer.

DOI： 10.14814/phy2.14640

Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows. 査読国際誌

Yuki Yamamoto, Sayaka Ito, Kiyoshi Okuda, Koji Kimura

Theriogenology 153 143 - 150 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Activin (ACV) A induces various cellular functions via activin receptor type 2 (ACVR2A/2B)-activin receptor-like kinase (ALK) 4 -Smad 2/3 pathway. Although the production of ACVA is indicated in bovine oviducts, its role on the oviduct is unclear. Oviductal isthmus needs to change its function rapidly at peri-fertilization, however, the mechanism is unknown. This study was aimed to clarify the role of ACVA in the morphological changes of oviductal isthmus in cows. First, mRNA expressions of INHBA (ACVA component) and its receptors (ALK4, ACVR2A and ACVR2B) in the isthmic tissues were examined throughout the estrous cycle. INHBA was the highest, however, ACVR2A was the lowest on the day of ovulation, suggesting reduced ACV signal transduction in the isthmus just after ovulation. Proteins of ACVRs and Smad2/3 were clearly detected in the cultured epithelial cells. It is known that ACVA regulates cellular apoptosis. Our data showed that the number of cleaved caspase-3-positive epithelial cells was largest at 2-3 days after ovulation in the isthmus. Interestingly, our study demonstrated that follistatin (ACV/TGFB/BMP inhibitor) significantly decreased the BCL2/BAX ratio in the cultured isthmic epithelial cells. To clarify which ALK pathway is involved in the regulation of BCL2/BAX ratio, the effects of SB431542 (ACV signaling (ALK4) and TGFB signaling (ALK5) inhibitor), SB525334 (ALK5 inhibitor) and LDN193189 (BMP signaling (ALK2/3) inhibitor) were investigated in the next study. The results showed that only SB431542 significantly decreased BCL2/BAX and the others had no effects. These results suggest that decreased ACVA-ACVR2A-ALK4 signal at the post-ovulation induces cyclic apoptosis of isthmic epithelial cells in bovine oviducts.

DOI： 10.1016/j.theriogenology.2020.05.009

ウシ子宮内膜side population(SP)細胞の遺伝子発現解析と分娩後におけるSP細胞割合の推移

舘林亮輝, 中村翔, 美辺詩織, 古澤軌, 阿部良哉, 加治佐実希, 森田康広, 大蔵聡, 木村康二, 松山秀一

The Journal of Reproduction and Development 66 ( Suppl. ) j41 - j41 2020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

経産牛の受胎性低下は，分娩後の子宮内膜再生時に生じる不具合が一因である可能性が考えられるが，その詳細なメカニズムは未だ明らかでない。ヒトにおいては，月経周期毎に起こる子宮内膜更新に子宮内膜の間葉系幹細胞が関与することが示唆されており，ウシの分娩後の子宮内膜修復においても子宮内膜幹細胞が関与する可能性が考えられる。そこで本研究では，組織幹細胞の集団として考えられているside population（SP）細胞がウシ子宮内膜にも存在することを示すとともに，ウシ子宮内膜SP細胞の特性および分娩後の動態を明らかにすることを目的とした。食肉センター由来のウシ子宮から単離した子宮内膜細胞をHoechst33342で染色し，フローサイトメトリー解析を行った結果，子宮内膜細胞にSP細胞が含まれることが明らかとなった。次に，ウシ子宮内膜SP細胞（n=3）とSP細胞以外の細胞（main population, MP細胞）（n=3）をセルソーターにより分取し，遺伝子発現をRNA-seqにより解析した。その結果，SP細胞とMP細胞では遺伝子発現パターンが異なることが示された。さらに，SP細胞とMP細胞間の発現変動遺伝子（FDR＜0.05）についてGOおよびKEGGパスウェイ解析（p＜0.05）を行ったところ，MP細胞と比較してSP細胞で高発現であった遺伝子には，核酸代謝，血管発達といった機能や幹細胞の自己複製や分化のシグナル経路に関与する遺伝子が多く含まれており，幹細胞マーカーの遺伝子も含まれていた。また，分娩後10，30，50，100日目にバイオプシーによってウシの子宮内膜組織を採取し（n=8），子宮内膜細胞に対するSP細胞の割合を算出した結果，子宮内膜細胞に対するSP細胞の割合は，分娩後10日目で最も低く，その後徐々に増加した。以上の結果より，ウシ子宮内膜に存在するSP細胞は幹細胞/前駆細胞様な遺伝子発現パターンを示し，分娩後に子宮内膜細胞に分化，増殖することで子宮修復に寄与する可能性が示された。

DOI： 10.14882/jrds.113.0_aw1-4

Analysis of ciliogenesis process in the bovine oviduct based on immunohistochemical classification. 査読国際誌

Sayaka Ito, Yuki Yamamoto, Koji Kimura

Molecular biology reports 47 ( 2 ) 1003 - 1012 2020年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The oviductal epithelium is composed of ciliated and non-ciliated cells. The proportions of these cells change during the estrous cycle. However, the mechanism underlying this cyclic change in the cell proportions remains unclear. Our previous study indicated that ciliated cells are derived from non-ciliated cells. Here, we aimed to investigate the mechanism regulating the changes in the populations of ciliated and non-ciliated cells during the estrous cycle. To this end, we examined the numbers of cells that were positive for acetylated-α-tubulin (cilia marker), Ki67 (proliferation marker), PAX8 (non-ciliated cell marker), and FOXJ1 and MYB (ciliogenesis markers) in the epithelial cells at four different estrous stages (Stage I: days 1-4 after ovulation, Stage II: days 5-10, Stage III: days 11-17, and Stage IV: days 18-20) by immunohistochemistry. The oviductal epithelial cells expressed either FOXJ1 or PAX8. All the acetylated-α-tubulin+ cells were positive for FOXJ1, although there were a few acetylated-α-tubulin-/FOXJ1+ cells. MYB was expressed in both the FOXJ1+ and PAX8+ cells, but it was not expressed in the Ki67+ cells. The numbers of Ki67+ and MYB+ cells were the highest in Stage IV, while the numbers of FOXJ1+ and acetylated-α-tubulin+ cells were the highest in the following Stage I, suggesting that ciliogenesis is associated with the estrous cycle. Thus, based on immunological classification, the oviductal epithelium contains at least seven types of cells at different translational/transcriptional states, and their number is regulated by the estrous cycle. This cyclic event might provide an optimal environment for gamete transport, fertilization, and embryonic development.

DOI： 10.1007/s11033-019-05192-w

妊娠ウシ子宮頸管におけるISGs発現誘導へのIFNTの直接関与

國井宏樹, 窪友瑛, 浅岡那月, 嶋崎知哉, 古山敬祐, 木村康二, 唄花子, 川原学, 高橋昌志

日本繁殖生物学会講演要旨集 113 ( 0 ) P - 65-P-65 2020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】ウシの着床過程では，授精後18日前後をピークとして，胚の栄養外胚葉からインターフェロン・タウ（IFNT）が分泌される。IFNTは母体の子宮内膜に作用し，JAK-STAT経路を介してIFN誘導性遺伝子（ISGs）の発現を誘導する。我々はこれまでに，子宮外組織である子宮頸部粘膜組織（CMM）においても妊娠特異的なISGsの高発現を見出した。しかし，IFNTの存在が直接的に示されているのは子宮内のみであり，IFNTがCMMにおけるISGs発現にどのように関与するのかは不明である。そこで本研究では，CMMへのIFNT移行の有無，ならびにCMMにおけるISGsの発現機序を明らかにすることを目的とした。【方法】非妊娠（np）および妊娠（p）ホルスタイン種搾乳ウシ由来CMMを，AI実施後14日目（d14），18日目（d18）および25日目（d25）において低侵襲的に採取後，RNAおよびタンパク質を抽出した。IFNTの検出は，抗IFNT抗体を用いウェスタンブロッティング（WB）にて行った。加えて，np-, p-CMMと共培養したnp-ウシ末梢血白血球（PBL）におけるISG15発現を解析し，p-CMMのI型IFN活性を評価した。次に，定量PCRによりJAK-STAT経路関連因子の継時的な遺伝子発現動態を，またWBにより着床前におけるSTAT1活性化状態を評価した。【結果】p-d18のCMM由来タンパク質成分より，IFNTのバンドが検出された。加えて，CMM-PBL共培養実験では，np-d18のCMMと比較して，p-d18のCMMと共培養したPBLにおけるISG15発現が顕著に増加した。さらに，p-d18のCMMでは，STAT1, STAT2, IRF9発現および，JAK-STAT経路の活性化を示すリン酸化STAT1の発現が顕著に増加した。以上の結果から，子宮頸部における着床前特異的ISGs発現は，胚由来因子IFNTが子宮外へ移行し，JAK-STAT経路を活性化することにより誘導されることが明らかとなった。</p>

DOI： 10.14882/jrds.113.0_P-65

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