{"created":"2023-10-17T04:55:03.094530+00:00","id":2000046,"links":{},"metadata":{"_buckets":{"deposit":"300a8a1f-9992-45f5-b3bc-ffce520f8215"},"_deposit":{"created_by":12,"id":"2000046","owner":"12","owners":[12],"pid":{"revision_id":0,"type":"depid","value":"2000046"},"status":"published"},"_oai":{"id":"oai:obihiro.repo.nii.ac.jp:02000046","sets":["6:8"]},"author_link":[],"control_number":"2000046","item_3_alternative_title_1":{"attribute_name":"その他（別言語等）のタイトル","attribute_value_mlt":[{"subitem_alternative_title":"ウシの精子-子宮免疫クロストークを調節するTLR2の分子動力学のコンピューターモデリング","subitem_alternative_title_language":"ja"}]},"item_3_date_granted_12":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2023-09-29"}]},"item_3_degree_grantor_10":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_language":"ja","subitem_degreegrantor_name":"帯広畜産大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"10105","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_3_degree_name_9":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"Doctor of Agriculture","subitem_degreename_language":"en"},{"subitem_degreename":"博士（農学）","subitem_degreename_language":"ja"}]},"item_3_description_11":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_description":"2023","subitem_description_language":"ja","subitem_description_type":"Other"}]},"item_3_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_language":"en","subitem_description_type":"Other"}]},"item_3_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"In cattle, after artificial insemination (AI) or natural mating, a large number of sperm swim up into the female reproductive tract (FRT) toward the site of fertilization. During this journey, sperm interact with different compartments of the immune system of FRT. Bovine uterus has a well-regulated immune response to remove bacterial contamination after parturition, and it tolerates the allogenic sperm and accepts semi-allogenic embryos. Sperm generate transient proinflammatory response in the uterus which is required for the removal of dead/excess sperm with associated contaminants. TLR2 plays a central role in sperm-induced inflammation in the bovine uterus. In general, in immune cells, a dimerization of TLR2 with either TLR1 or TLR6 is required to activate intracellular signaling pathways, thereby inducing the innate immune response, but nothing is known about TLR2 dimerization in the bovine endometrial epithelium in response to sperm attachment. On the other hand, CD44 is a major cell surface receptor for hyaluronan (HA) involved in sperm attachment to the endometrial epithelium. In this study, using multiple approaches based on the computational modelling methods together with the in vitro experimental models, I was able to identify the major part of the molecular mechanism of sperm interaction with the bovine uterine immune system in cattle.\nIn chapter I, in order to test different TLR2 dimerization pathways in endometrium in an in-vitro model, 100 ng/mL TLR2 agonists (PAM3 as the TLR2/1 agonist, and PAM2 as the TLR2/6 agonist) were used to stimulate bovine endometrial epithelial cells (BEECs). Simultaneously, the expression of TLR1, 2 and 6 protein and gene in BEECs were investigated after exposure to sperm (5 million/mL). Further, sperm induced-inflammation was compared to PAM3 and PAM2 using the uterine explant ex-vivo model. The obtained data indicated that an activation of TLR2/1 signaling pathway in BEECs is involved in a weaker inflammation compared to TLR2/6. Moreover, similar to PAM3, sperm was able to induce TLR2 expression alongside with TLR1 in the uterus (gene and protein), particularly in uterine glands, but not TLR6. In the same way, PAM3 and sperm could induce similar and low gene expression of pro-inflammatory cytokines (TNFA, IL1B and IL8) and TNFA protein to a lesser extent than PAM2 in the bovine endometrium. Thus, it is highly possible that sperm trigger endometrial epithelia to induce a weak inflammatory response through activating TLR2/TLR1 signaling cascade, which is needed to prepare an ideal environment for embryo reception. Afterwards, in-silico approaches were employed to investigate and confirm TLR2 dimerization in bovine species (TLR2/1 or TLR2/6). Homology modeling methods were used to determine the 3D protein structure of bovine TLRs. The in-silico findings suggested that the stability of TLR2 dimerization is heavily depending on the presence of the bridging agonist in bovine, which is similar to human and mouse species.\nIn chapter II, I hypothesized that HA may act as a bridging ligand between sperm and CD44/TLR2 of BEECs. To test the above hypothesis, I first determined the binding affinity of HA to CD44 and TLR2 molecules. my in-silico model revealed that low molecular weight HA molecules have a higher affinity to CD44- than TLR2 interaction. Next, HA existence in bovine endometrium was investigated via immunostaining using a biotinylated HA-binding protein. Notably, HA is localized in the luminal and glandular endometrial epithelia. Moreover, ELISA showed detectable levels of HA (16.05 ± 2.33 ng/ml) in BEECs-conditioned medium. As a result, BEECs were treated with different concentrations of low molecular weight HA (at 0, 0.1, 1, or 10 μg/mL) for 2 h prior to the co-culture with 106 sperm/mL for additional 3 h. Importantly, HA dose-dependently increased the number of sperm attached to BEECs. Besides, the quantitative real-time PCR data illustrated that supplementation of BEECs with HA (at 1 μg/mL) upregulate mRNA expressions of TLR2, pro-inflammatory- cytokines (TNFA and IL1B) and chemokines (IL8) as well as prostaglandins E synthesis (PGES) in BEECs in response to sperm. However, BEECs treatment with HA only (no sperm exposure) did not show any significant difference in transcriptional levels of the selected genes when compared to the non-treated BEECs. Collectively, the findings provide evidence that HA, primarily through CD44 interaction, has the capacity to facilitate sperm attachment to the endometrial epithelia with a subsequent TLR2-mediated immune response.\nOverall, my findings in chapter I revealed that sperm activate TLR2/1 heterodimerization, but not TLR2/6, to trigger a weak physiological inflammatory response in bovine endometrium. The data of chapter II suggested that sperm keep a higher affinity for attaching to the BEECs in presence of HA through interaction with CD44, consequently inducing proinflammatory response through TLR2 signaling pathway. Collectively, this weak inflammation triggered by sperm with the molecular network of TLR2/1, CD44 and hyaluronan must be the specific way to remove excess/dead sperm remaining in the bovine uterine lumen without tissue damage for providing the ideal environment for embryo implantation.","subitem_description_language":"en","subitem_description_type":"Abstract"},{"subitem_description":"ウシでは、人工授精（AI）または自然交配の後、多数の精子が雌の生殖器官（FRT）内を受精部位に向かって泳ぎ上がってゆく。この旅の間、精子は FRT の免疫系のさまざまな部分と相互作用する。ウシの子宮は、出産後に細菌汚染を除去するためによく調節された免疫応答を備えており、同種異系精子を一部は許容し、半同種異系胚を受け入れる。精子は、関連する汚染物質を伴う死んだ、あるいは過剰な精子の除去のために子宮内で一時的な炎症誘発反応を引き起こす。 TLR2 は、ウシ子宮における精子誘発性炎症において中心的な役割を果たす。一般に、免疫細胞では、細胞内シグナル伝達経路を活性化し、それによって自然免疫応答を誘導するには、 TLR1 または TLR6 による TLR2 の二量体化が必要であるが、精子の付着に応答したウシ子宮内膜上皮における TLR2 の二量体化については何もわかっていない。一方、 CD44 は、子宮内膜上皮への精子の付着に関与するヒアルロン酸 (HA) の主要な細胞表面受容体である。この研究では、コンピュータモデリング法と in vitro 実験モデルに基づく複数のアプローチを使用して、牛の精子とウシ子宮免疫系の相互作用の分子機構の主要部分を特定することができた。\n第 I 章では、 in vitro モデルで子宮内膜の様々な TLR2 二量体化経路をテストするために、 100 ng/mL の TLR2 アゴニスト （TLR2/1 アゴニストとしての PAM3 、および TLR2/6 アゴニストとしての PAM2) を使用してウシ子宮内膜上皮細胞 (BEEC）を刺激した。 同時に、精子 (500万/mL) への曝露後の BEEC における TLR1 、 2 、および 6 タンパク質および遺伝子の発現を調査した。さらに、子宮外植片の ex vivo モデル を使用して、精子による誘発性炎症を PAM3 および PAM によるものと比較した。得られたデータは、 BEEC における TLR2/1 シグナル伝達経路の活性化が、 TLR2/6 と比較して弱い炎症に関与していることを示した。さらに、 PAM3 と同様に、精子は子宮遺伝子およびタンパク質、特に子宮腺で TLR1 とともに TLR2 発現を誘導したが、 TLR6 は誘導しなかった。加えて、 PAM3 と精子はウシ子宮内膜において、炎症誘発性サイトカイン (TNFA 、IL1B 、および IL8) および TNFA タンパク質と遺伝子発現を、 PAM2 よりも低い程度で同様に誘導していた。したがって、精子が子宮内膜上皮を 刺激し、胚の受容に理想的な環境を準備するために必要な TLR2/TLR1 シグナル伝達カスケードの活性化を通じて弱い炎症反応を誘発する可能性が非常に高い。 これらの実験系に引き続き、 in silico アプローチを使用してウシにおける TLR2 二量体化 (TLR2/1 または TLR2/6) を調べた。相同性モデリング法を使用して、ウシ TLR の 3D タンパク質構造を決定した。 In silico 法での発見は、 TLR 二量体化の安定性が、ヒトやマウス種と同様にウシにおける架橋アゴニストの存在に大きく依存していることを示唆した。\n第 II 章では、 HA が精子と BEEC の CD44/TLR2 の間の架橋リガンドとして機能する可能性があるという仮説を立てた。この仮説を検証するために、私はまず、 CD44 および TLR2 分子に対する HA の結合親和性を決定した。私の in silico モデルでは、低分子量 HA 分子が TLR2 との相互作用よりも 、むしろ CD44 との相互作用に対して高い親和性を持っていることが明らかになった。次に、ビオチン化 HA 結合タンパク質を用いた免疫染色により、ウシ子宮内膜における HA の存在を調べた。 注目すべきことに、HA は管腔および腺の子宮内膜上皮に局在していた 。さらに ELISA によって、 BEEC 培養上清中に検出可能なレベルの HA (16.05 ± 2.33 ng/ml) が検出できた。さらに、 BEEC を様々な濃度の低分子量 HA (0 、 0.1 、 1 、または 10 μg/mL) で 2 時間処理した後、 10 6 精子 /mL とさらに 3 時間共培養した。 重要なことに、 HA は BEEC に付着する精子の数を用量依存的に増加させた。さらに、定量的リアルタイム PCR データから、 BEEC に HA (1 µg/mL) を添加すると、 TLR2 、炎症誘発性サイトカイン (TNFA および IL1B) 、ケモカイン (IL8) の mRNA 発現、およびプロスタグランジン E 合成酵素が増加することがわかった。しかし、 HA 添加だけでは BEEC ( 精子曝露なし ) の焦点とした遺伝子の転写レベルに有意な差は示されなかった。まとめると、これらの発見は、 HA が主に CD44 との相互作用を通じて、その後の TLR2 媒介による免疫応答を伴う子宮内膜上皮への精子の付着を促進する能力があるという証拠を提供した。\n全体として、第 1 章での私の発見は、精子が TLR2/1 ヘテロ二量体化を活性化するが、 TLR2/6 は活性化せず、この経路がウシ子宮内膜における弱い生理的炎症反応を引き起こすことを明らかにした。 第 ２ 章のデータは、精子が HA の存在下で CD44 との相互作用を通じて BEEC への結合に対してより高い親和性を維持し、その結果、 TLR2 シグナル伝達経路を通じて炎症誘発性反応を誘導することを示唆した。以上をまとめると、 TLR2/1 、 CD44 、およびヒアルロン酸の分子ネットワークに支えられた精子によって引き起こされる弱い炎症は、胚の着床に理想的な環境を提供するために、ウシの子宮内腔に残っている過剰または死滅した精子を組織損傷なしに除去するための特別なシステムであると考えられた。","subitem_description_language":"ja","subitem_description_type":"Abstract"}]},"item_3_description_8":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"博士学位論文","subitem_description_language":"ja","subitem_description_type":"Other"},{"subitem_description":"大学院畜産学研究科 畜産科学専攻","subitem_description_language":"ja","subitem_description_type":"Other"},{"subitem_description":"Doctoral Program of Animal Science and Agriculture","subitem_description_language":"en","subitem_description_type":"Other"}]},"item_3_dissertation_number_13":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第135号"}]},"item_3_publisher_5":{"attribute_name":"公開者","attribute_value_mlt":[{"subitem_publisher":"帯広畜産大学","subitem_publisher_language":"ja"}]},"item_3_rights_14":{"attribute_name":"権利","attribute_value_mlt":[{"subitem_rights":"MANSOURI Alireza","subitem_rights_language":"en"}]},"item_3_subject_20":{"attribute_name":"日本十進分類法","attribute_value_mlt":[{"subitem_subject":"649","subitem_subject_language":"ja","subitem_subject_scheme":"NDC"}]},"item_3_version_type_19":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"MANSOURI, Alireza","creatorNameLang":"en"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-17"}],"filename":"MANSOURI.pdf","filesize":[{"value":"3 MB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"全文　Full text","objectType":"fulltext","url":"https://obihiro.repo.nii.ac.jp/record/2000046/files/MANSOURI.pdf"},"version_id":"335d4f7b-37c6-4ec0-bc86-b3274081d082"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-17"}],"filename":"Abstract(MANSOURI).pdf","filesize":[{"value":"144 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"全文の要旨　Abstract","objectType":"abstract","url":"https://obihiro.repo.nii.ac.jp/record/2000046/files/Abstract(MANSOURI).pdf"},"version_id":"fa47799a-0518-44aa-8f33-f65ecbd193c1"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-10-17"}],"filename":"Evaluation(MANSOURI).pdf","filesize":[{"value":"196 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"label":"審査の要旨　Evaluation","objectType":"other","url":"https://obihiro.repo.nii.ac.jp/record/2000046/files/Evaluation(MANSOURI).pdf"},"version_id":"146cae45-ac28-4847-9bc8-332d906b7dd8"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis","resourceuri":"http://purl.org/coar/resource_type/c_db06"}]},"item_title":"Computational modelling for molecular dynamics of TLR2 that regulates sperm-uterine immune crosstalk in cattle","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Computational modelling for molecular dynamics of TLR2 that regulates sperm-uterine immune crosstalk in cattle","subitem_title_language":"en"}]},"item_type_id":"3","owner":"12","path":["8"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2023-10-17"},"publish_date":"2023-10-17","publish_status":"0","recid":"2000046","relation_version_is_last":true,"title":["Computational modelling for molecular dynamics of TLR2 that regulates sperm-uterine immune crosstalk in cattle"],"weko_creator_id":"12","weko_shared_id":-1},"updated":"2023-10-30T00:11:47.957777+00:00"}