Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus–oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18β-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

DOI： 10.1093/molehr/gaab038

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Other Link： http://academic.oup.com/molehr/article-pdf/27/7/gaab038/38860704/gaab038.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Animal Reproduction  

Iron is important for many cellular functions, including ATP synthesis and cell proliferation. Insufficient of iron in the diet causes iron deficiency anemia (IDA), which often occurs in people living in the world. Since 50% of women with IDA show amenorrhea, the relationship of between iron deficiency and reproductive function was assessed using mice fed a low Fe diet (LFD). The estrous cycle in the LFD mice was blocked at diestrus, which impair follicle development, and fertility. Further, even LFD mice were injected with exogenous pregnant mare serum gonadotropin (PMSG), follicular development was ceased at the secondary follicle stage, and preovulatory follicles were not observed. Amount of ATP decreased in the ovary of the LFD mice, and expression of follicle development markers (<i>Fshr</i>, <i>Cyp19a1</i>, <i>Ccnd2</i>) and estradiol-17β (E2) was low level compared to levels mice fed a normal diet. Feeding a normal diet with sufficient iron to the LFD mice for an additional 3 weeks completely reversed absence the effects of iron insufficient on the estrous cycle and infertility. Thus, iron restriction depresses ovary functions, especially follicular development from secondary follicle to antral follicles and infertility. These effects are fully reversible by supplementation of a normal diet containing iron.

DOI： 10.1262/jrd.2020-074

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

The in vitro maturation (IVM) technique is beneficial for producing animal offspring, but the blastocyst rate is low after IVM. In this technique, cumulus-oocyte complexes (COCs) are collected from medium-size follicles. The follicles are ultimately selected as large dominant follicles or atretic follicles; therefore it is possible that the COCs collected using IVM are contaminated by follicles that will develop into large follicles and induce atresia. In the dominant follicles, estradiol-17beta and progesterone induce the differentiation of follicular somatic cells that exhibit the ability to respond to ovulation during follicular development. Thus, we hypothesized that changes in the hormonal condition of healthy follicles are essential for oocyte maturation during IVM. In this study, we performed a comparative analysis of the steroid hormone concentrations in nonvascularized follicles (NVFs) and vascularized follicles (VFs). The estradiol-17β concentration increased in medium VFs, whereas the level was low in NVFs of the same size. The progesterone concentration increased with large follicular size in VFs, but the level remained low in follicles of any size among NVFs. To improve the oocyte quality derived from NVFs, NVF COCs were cultured with follicle-stimulating hormone (FSH) alone or FSH under the VF hormonal conditions. Cultivation under the VF hormonal conditions dramatically improved the proliferation and survival of cumulus cells, meiotic maturation of oocytes, cumulus expansion, and blastocyst rate following in vitro fertilization. Thus, the cultivation of NVF COCs under VF hormonal conditions improves the developmental potential to the blastocyst stage by NVF oocytes.

DOI： 10.1095/biolreprod.116.138982

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Endocrine Society  

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.

DOI： 10.1210/en.2013-1655

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_aw-1

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：日本畜産学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：日本IVF学会  

J-GLOBAL

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Language：Japanese   Publishing type：Research paper, summary (national, other academic conference)   Publisher：日本IVF学会  

J-GLOBAL

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Authorship：Lead author   Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.112.0_or2-28

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese   Publisher：(公社)日本畜産学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.109.0_p-11

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Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.108.0_or2-10

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

【目的】我々はLH刺激を受けた顆粒層細胞(GC)がAmphiregulin(AREG)を分泌し，卵丘細胞(CC)のEGF受容体(EGFR)-ERK1/2系の活性化を介し，膨潤関連遺伝子を発現させ，卵丘膨化や卵成熟，排卵が生じることを報告した。しかし，AREG添加によるCCのERK1/2の活性化レベルは低く，卵の受精率及び胚盤胞形成率は体内のそれと比較して低い。このことからAREG以外の新規因子の存在が示唆された。本研究ではマウスのアレイデータベースからERK1/2を活性化する候補因子を探索し，発現・機能解析を行った。【方法】hCG投与したマウスで発現上昇した遺伝子から分泌因子を絞り込みNeurotensin(NTS)を候補化した。3週齢C57BL/6雌マウスにPMSG/hCGを投与後，卵巣，GC，CCを各時間に回収し<i>Nts</i>と<i>Nts</i>受容体(<i>Ntsr</i>)遺伝子発現，NTS量測定，NTSとNTSRの局在を調べた。次にPMSG投与48h後回収したCOCをNTS区，AREG区，AREG+NTS区，AREG+NTSR阻害剤（SR）区，AREG+ NTS+SR区で16hまで培養し，pERK1/2の検出，<i>Egfr</i>及び膨潤関連遺伝子(<i>Has2，Ptx3，Tnfaip6</i>)発現，ヒアルロン酸(HA)量測定，COCの直径測定，受精後の受精率と胚盤胞率を測定した。 【結果】排卵刺激後に発現上昇する遺伝子群から，<i>Nts</i>を同定した。<i>Nts</i>はGCとCCでPMSG投与後まで低値であったが，hCG投与4h後以降両細胞で有意に上昇した。NTS量も排卵刺激により増加した。一方<i>Ntsr</i>は両細胞で恒常的に発現し，NTSとNTSRも両細胞に局在していた。pEKR1/2はNTS区で不検出，AREG区では8hをピークに減少した一方，AREG+NTS区では16hまで強いシグナルが維持された。pERK1/2が脆弱したAREG区の12h後において，膨潤関連遺伝子の発現は低値であったが，AREG+NTS区では高発現を維持し，このときAREG+NTS区で<i>Egfr</i>発現はAREG区よりも高値であった。HA量，COC直径，受精率，胚盤胞率もAREG+NTS区はAREG区に比べ高値であった。

DOI： 10.14882/jrds.107.0_aw-2

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

【目的】家畜の未成熟卵から成熟卵を作製する体外成熟培養(IVM)法は，家畜の効率的増産，品種改良に応用可能であるが，IVMにより作出される成熟卵の発生率は，体内成熟に比べ低いことから，改良が必要である。当研究グループではマウスを用いてLH/hCG刺激後に顆粒膜細胞や卵丘細胞で発現するAmphiregulin(AREG)を介してNTSが発現し，卵丘細胞のNTS受容体(NTSR)を介して卵成熟を誘導することを見出した。本研究ではIVMの改善の基礎実験として，ブタCOCの体外培養時における，卵丘細胞のNTSの発現とその役割を検討した。【方法】食肉処理場由来のブタ卵巣からCOCを採取し，無添加（Free）区，FSH+AREG区でCOCを40 hまで培養し，<i>Nts</i>と<i>Ntsr1</i> mRNAの経時的発現変化を調べた。またCOCをFree区，FSH+AREG区，FSH+AREG+NTS区，FSH+AREG+NTS+SR区で30h及び48h培養し，卵丘膨化遺伝子群（<i>Has2，Tnfaip6，Ptx3</i>）のmRNA発現，COC直径，MII率を調べた。【結果】<i>Nts</i>および<i>Ntsr1</i>発現は，Free区では上昇しなかったが，FSH+AREG区ではそれぞれ30hおよび20hで発現上昇した。培養後30hにおける<i>Has2</i> mRNA発現は，Free区では低値を示したが，この低い値はFSH+AREG区で有意に上昇し，FSH+AREG+NTS区でさらに増加した。この値はSRの添加により有意に減少した。COC直径は，Free区では増加しなかったが，FSH+AREG区で増大し，FSH+AREG+NTS区でさらに増大した。この増加はSR添加で有意に抑制された。MII率についても同様の傾向を示した。以上の結果から，IVM時におけるNTSの培地への添加は，ブタ卵の成熟率向上に寄与すると考えられた。

DOI： 10.14882/jrds.107.0_p-8

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2018.7 - 2018.8

Language：English   Presentation type：Poster presentation  

Country：Italy  

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Event date： 2018.7

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Event date： 2018.3

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Event date： 2017.9

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Event date： 2016.9

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Event date： 2016.5

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Event date： 2016.3

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Event date： 2016.1

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Event date： 2015.9

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Event date： 2015.5

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Event date： 2015.3

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Event date： 2014.9

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Event date： 2014.8

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Event date： 2014.8

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Event date： 2014.5

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