@phdthesis{oai:obihiro.repo.nii.ac.jp:02000044,
 author = {MUKANGWA, Memory},
 month = {2023-10-17, 2023-10-17, 2023-10-17},
 note = {2023, application/pdf, Progesterone (P4) and cortisol production and expression of luteinizing markers: pentraxin 3 (PTX3), regulators of G-protein signaling 2 (RGS2), and vanin 2 (VNN2), increase in luteinized granulosa cells (LGCs), but their relationship is not well established. Therefore, this study investigated the effects of P4 and cortisol on each other’s production and their effects on the expression of the above genes in cultured bovine LGCs. In chapter 3, the functional and morphological changes associated with luteinization of granulosa cells (GCs) and the mechanism of cortisol production were investigated. GCs were collected from follicles of 2–5 mm diameter and cultured in DMEM/F-12 supplemented with 10% fetal calf serum (FCS) for up to 14 Days. P4 production and expression of steroidogenic acute regulatory protein (STAR), cholesterol side-chain cleavage enzyme (CYP11A1), and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1) rapidly increased until Day 10 and stayed high thereafter. The expression of 21-hydroxylase (CYP21A2), one of the two enzymes required in the conversion of P4 into cortisol, was low and decreased further after 4 Days, whereas the expression of 11β-hydroxylase (CYP11B1), which is responsible for the final stage of cortisol production, was not detectable. Moreover, cortisol production from P4 was not detected, although high P4 production was observed during the culture period. On the other hand, the expression of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts cortisone to cortisol, dramatically increased on Day 2, decreased until Day 8, and stayed relatively constant thereafter. Cortisol production was only observed when cortisone, a substrate for HSD11B1, was added to the culture medium. Because the LGCs expressed receptors for adrenocorticotrophin hormone (ACTH) and angiotensin II (Ang II), which are major factors that stimulate cortisol in the adrenal cortex, the effects of these factors on cortisol production by the LGCs were investigated on Day 6 of culture. A 24-h treatment with ACTH or Ang II downregulated P4 production by the LGCs but did not affect the expression of CYP21A2, CYP11B1,or HSD11B1, and cortisol production was undetected. In Chapter 4, the effects of P4 on cortisol production and the expression of factors related to luteinization were investigated. LGCs were treated with trilostane (a P4 synthesis inhibitor), nomegestrol acetate (NA, a synthetic progestogen), or P4 for 24 h on Days 6 and 12 of the culture. Trilostane suppressed P4 production and STAR expression while elevating HSD11B1 and HSD3B1, PTX3, RGS2, and VNN2 expression and cortisol production. Concomitant treatments with NA or P4 dosedependently decreased cortisol production, HSD11B1, HSD3B1, PTX3, RGS2, and VNN2 expression but xii elevated STAR and HSD11B2 expression in both Day 6 and Day 12 LGCs. In Chapter 5, the effect of cortisol on P4 production and the expression of factors related to luteinization were examined. Cortisol increased HSD11B1, HSD3B1, PTX3, RGS2, and VNN2 expression while decreasing STAR and HSD11B2 without influencing P4 production. Altogether, these results indicate that: 1) bovine LGCs cannot produce cortisol using the locally produced P4 as a substrate but produce cortisol via the HSD11B1 pathway using the adrenal-sourced cortisone as a substrate; 2) progestogens suppress cortisol production by regulating HSD11B1 expression; and 3) progestogens and cortisol differentially regulate STAR, HSD3B1, HSD11B1, HSD11B2, PTX3, RGS2, and VNN2 expression in bovine LGCs. This suggests that both P4 and cortisol are produced by GCs during luteinization and that they regulate luteal function in an autocrine manner., ⻩体化顆粒層細胞（LGC）においてprogesterone（P4）とcortisol，および⻩体化の指標であるpentraxin 3（PTX3），regulator of G protein signaling 2（RGS2）とvanin 2（VNN2）の発現はともに上昇するが，それらの関係性についてはよく分かっていない。そこで本研究ではP4 とcortisolが互いの⽣産に及ぼす影響と，これらのステロイドが上記の遺伝⼦発現に与える影響をウシLGCの培養系を⽤いて調べた。Chapter 3 ではウシ顆粒層細胞の⻩体化に伴う機能的，形態的変化とcortisol⽣産機序について調べた。直径2-5 mmの卵胞から吸引採取した顆粒層細胞を10%ウシ胎児⾎清を含むDMEM/F-12培養液で14⽇まで培養した。P4⽣産とそれに関与するsteroidogenic acute regulatory protein （STAR），cholesterol side-chain cleavage enzyme （CYP11A1），および 3β-hydroxysteroid dehydrogenase type 1 （HSD3B1）の発現は培養開始後10⽇まで増加し，その後⾼いレベルで推移した。P4 からcortisol への変換を仲介する２つの酵素のうち21-hydroxylase（CYP21A2）の発現は低く，4 ⽇以降にさらに低下した。Cortisol ⽣産の最終段階を担う11β -hydroxylase（CYP11B1）の発現は認められなかった。また培養期間中に⾼いP4 の⽣産が認められたにも関わらず，P4 からのcortisol ⽣産は認められなかった。⼀⽅でcortisone からcortisol への変換を仲介する11β-hydroxysteroid dehydrogenase type 1 （HSD11B1）の発現は2⽇⽬に顕著に増加した後，8⽇⽬にかけて減少し，その後はほぼ⼀定に保たれた。Cortisol⽣産はHSD11B1の基質であるcortisoneを培地中に加えた時のみに認められた。LGCで副腎⽪質においてcortisol合成を刺激する主要な因⼦であるACTH とangiotensin II（Ang II）の受容体の発現が認められたことから，これらの因⼦がLGC のcortisol ⽣産に与える影響を6 ⽇⽬のLGC を⽤いて調べた。ACTH，もしくはAng IIによる24時間の処置はLGCのP4⽣産を抑制したが，cortisol⽣産に関与するCYP21A2，CYP11B1，HSD11B1 のいずれの発現にも影響を与えなかった。Chapter 4 ではP4 がcortisol の⽣産および⻩体化関連因⼦発現に与える影響を調べた。6 ⽇⽬，もしくは12 ⽇⽬のLGC をtrilostane（P4合成阻害剤），nomegestrol acetate （NA，合成プロジェスタージェン），もしくはP4の存在下で24 時間培養した。Trilostane によりP4 ⽣産 阻害された結果HSD11B1，HSD3B1，PTX3，RGS2，および VNN2 の発現とcortisol の⽣産の増加が認められた ⼀⽅，STAR 発現の低下が認められた。NA もしくはP4 の同時添加は容量依存的にtrilostane の効果を低減させ，HSD11B1，HSD3B1，PTX3，RGS2，および VNN2 の発現とcortisol の⽣産を低下させ，STAR とHSD11B2の発現を増加させた。Chapter 5 ではcortisol がP4 の⽣産および⻩体化関連因⼦発現に与える影響を調べた。cortisol は容量依存的にHSD11B1，HSD3B1，PTX3， RGS2，および VNN2 の発現を増加させ，STAR とHSD11B2 の発現を低下させたが，P4 合成には影響を与えなかった。以上のことから，１）ウシLGC は局所で⽣産されるP4 を基質としたcortisol ⽣産は⾏えないが，副腎⽪質由来のcortisone を基質としたHSD11B1 によるcortisol ⽣産を⾏っていること，２）プロジェスタージェンはHSD11B1 の発現を抑えることで局所的なcortisolの⽣産を抑制すること，３）プロジェスタージェンとcortisolはSTAR，HSD3B1，HSD11B1，HSD11B2, PTX3，RGS2，および VNN2の発現を異なる⽅向に調節することが明らかになった。このことは⻩体化の過程でP4 とcortisol が共に顆粒層細胞で⽣産され、⾃⼰分泌的に⻩体機能の調節を⾏っていることを⽰唆するものである。, 博士学位論文, 大学院畜産学研究科 畜産科学専攻, Doctoral Program of Animal Science and Agriculture},
 school = {帯広畜産大学},
 title = {Glucocorticoid production and action in bovine luteinized granulosa cells},
 year = {}
}