Title ウシ発情周期中に発育する第１卵胞波主席卵胞の生理学的性質 ― 発育動態、機能および受胎性に及ぼす影響 ―( 本 文(Fulltext) ) Author(s) 三浦, 亮太朗 Report No.(Doctoral Degree) 博士(獣医学) 甲第440号 Issue Date 2015-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/51006 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
࢘ࢩⓎ࿘ᮇ୰Ⓨ⫱ࡍࡿ
➨
1 ༸⬊Ἴᖍ༸⬊ࡢ⏕⌮Ꮫⓗᛶ㉁
㸫Ⓨ⫱ືែࠊᶵ⬟࠾ࡼࡧཷ⫾ᛶཬࡰࡍᙳ㡪㸫
2014 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉
㸦ᖏᗈ␆⏘Ꮫ㸧
୕ᾆ ுኴᮁ
i ┠ḟ ➨㸯❶ ⥴ゝ 1 ➨㸰❶ ➨ 1 ༸⬊Ἴᖍ༸⬊㸦W1DF㸧ࡢⓎ⫱ືែ㸪༸๓ᚋࡢᶵ⬟࠾ࡼࡧ༸ᚋ ᙧᡂࡉࢀࡿ㯤యࡢᛶ㉁ 9 2㸫1. ᗎㄽ 10 2㸫2. ヨ㦂 1㸸༸⬊ἼⓎ⌧ࡽ 7 ᪥┠ࡲ࡛ࡢ W1DF W2DF ࡢⓎ⫱ືែ࠾ࡼࡧ ༸⬊ቨ⾑ὶࡢẚ㍑ 10 2㸫3. ヨ㦂 2㸸Ⓨ࠾ࡼࡧ༸ㄏ㉳࠾ࡅࡿ W1DF W2DF ࡢⓎ⫱ືែ㸪 ༸⬊ቨ⾑ὶ࠾ࡼࡧ⾑୰E2⃰ᗘࡢẚ㍑ 14 2㸫4. ヨ㦂 3㸸W1DF W2DF ࠾ࡅࡿ༸๓ࡢ༸⬊ෆࢫࢸࣟࢻ࣍ࣝࣔࣥ⃰ᗘ ࠾ࡼࡧ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿᅉᏊࡢẚ㍑ 16 2㸫5. ヨ㦂 4㸸W1DF W2DF ࡢ༸ᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែ㸪㯤య⾑ὶ ࠾ࡼࡧ⾑୰P4⃰ᗘ᥎⛣ࡢẚ㍑ 21 2㸫6. ⤖ᯝ 23 2㸫7. ⪃ᐹ 26 2㸫8. ᑠᣓ 32 ᅗ࣭⾲ 33
ii ➨㸱❶ ➨1 ༸⬊Ἴᖍ༸⬊㸦W1DF㸧ࡢⓎ⫱ࢆไᚚࡍࡿせᅉ 57 3㸫1. ᗎㄽ 58 3㸫2. ヨ㦂 1㸸༸⬊ἼⓎ⌧ࡽ 7 ᪥┠ࡢ W1DF ࡢ┤ᚄ⾑୰ P4⃰ᗘࡢ┦㛵㛵ಀ ࠾ࡼࡧW1DF 㯤యࡢඹᏑࡲࡓࡣ㠀ඹᏑ㛵ಀࡀ W1DF ࡢ┤ᚄཬࡰࡍ ᙳ㡪 58 3㸫3. ヨ㦂 2㸸W1DF 㯤యࡢඹᏑࡲࡓࡣ㠀ඹᏑ㛵ಀࡀ༸⬊ἼⓎ⌧᪥ࡽ 5 ᪥┠ ࡲ࡛ࡢW1DF 㯤యࡢⓎ⫱ືែ㸪⾑୰ P4⃰ᗘ㸪࠾ࡼࡧ༸⬊ᾮࢫࢸࣟࢻ࣍ ࣝࣔࣥ⃰ᗘཬࡰࡍᙳ㡪 60 3㸫4. ⤖ᯝ 63 3㸫5. ⪃ᐹ 64 3㸫6. ᑠᣓ 66 ᅗ࣭⾲ 68 ➨㸲❶ 㯤యྠ୍༸ᕢෆᏑᅾࡍࡿ➨1 ༸⬊Ἴᖍ༸⬊㸦W1DF㸧ࡢᛶ㉁࠾ࡼࡧ ཷ⫾ᛶཬࡰࡍᙳ㡪 77 4㸫1. ᗎㄽ 78 4㸫2. ヨ㦂 1㸸Ἢங∵࠾ࡅࡿ W1DF 㯤యࡢඹᏑ࠾ࡼࡧ㠀ඹᏑ㛵ಀࡀ W1DF 㯤యࡢⓎ⫱ືែ࠾ࡼࡧ⾑୰P4⃰ᗘ᥎⛣ཬࡰࡍᙳ㡪 78
iii 4㸫3. ヨ㦂 2㸸W1DF 㯤యࡢඹᏑ࠾ࡼࡧ㠀ඹᏑ㛵ಀࡀேᕤᤵ⢭ᚋࡢཷ⫾⋡ ཬࡰࡍᙳ㡪 80 4㸫4. ⤖ᯝ 82 4㸫5. ⪃ᐹ 84 4㸫6. ᑠᣓ 87 ᅗ࣭⾲ 88 ➨㸳❶ ⥲ྜ⪃ᐹ 92 ㅰ㎡ 100 ཧ⪃ᩥ⊩ 101
iv
␎ㄒ
P4㸸Progesterone㸦ࣉࣟࢪ࢙ࢫࢸࣟࣥ㸧
E2㸸Estradiol-17E㸦࢚ࢫࢺࣛࢪ࣮࢜ࣝ㸧
A4㸸Androstendion㸦ࣥࢻࣟࢫࢸࣥࢪ࢜ࣥ㸧
GnRH㸸Gonadotropin releasing hormone㸦ᛶ⭢่⃭࣍ࣝࣔࣥᨺฟ࣍ࣝࣔࣥ㸧 FSH㸸Follicle stimulating hormone㸦༸⬊่⃭࣍ࣝࣔࣥ㸧
LH㸸Luteinizing hormone㸦㯤యᙧᡂ࣍ࣝࣔࣥ㸧
W1DF㸸The first follicular wave dominant follicle㸦➨ 1 ༸⬊Ἴᖍ༸⬊㸧 W2DF㸸The second follicular wave dominant follicle㸦➨ 2 ༸⬊Ἴᖍ༸⬊㸧 PG㸸Prostaglandin㸦ࣉࣟࢫࢱࢢࣛࣥࢪࣥ㸧
BFA㸸Blood flow area㸦༸⬊ቨ⾑ὶ㠃✚㸧
BF%㸸The percentage of follicle circumference with blood flow signals㸦⾑ὶศᕸྜ㸧 EIA㸸Enzyme immuno assay㸦㓝⣲ච ᐃἲ㸧
BSA㸸Bovine serum albumin㸦∵⾑Ύࣝࣈ࣑ࣥ㸧 cDNA㸸complementary DNA㸦┦⿵ⓗ DNA㸧 PCR㸸Polymerase chain reaction
LHr㸸LH receptor㸦LH ཷᐜయ㸧
CYP19A1㸸Aromatase㸦࣐ࣟࢱ࣮ࢮ㸧 COX-2㸸Cyclooxygenase-2
NO㸸Nitric oxide㸦୍㓟❅⣲㸧
iNOS㸸Inducible nitric oxide synthase㸦ㄏᑟᆺ୍㓟❅⣲ྜᡂ㓝⣲㸧
eNOS㸸Endotherial nitric oxide synthase㸦⾑⟶ෆ⓶ᆺ୍㓟❅⣲ྜᡂ㓝⣲㸧 VEGF㸸Vascular endotherial growth factor㸦⾑⟶ෆ⓶ቑṪᅉᏊ㸧
v
StAR㸸Steroidogenic acute regulatory protein
P450-scc㸸Cytochrome P450 side chain cleavage enzyme㸦ࢳࢺࢡ࣮࣒ࣟ P450 ഃ㙐ษ᩿㓝⣲㸧 3E-HSD㸸3E-hydroxysteroid dehydrogenase㸦3E-Ỉ㓟ᇶ⬺Ỉ⣲㓝⣲㸧
GAPDH㸸Glyceraldehyde-3-phosphate dehydrogenase㸦ࢢࣜࢭࣝࣝࢹࣄࢻ 3 ࣜࣥ㓟⬺Ỉ⣲㓝⣲㸧 W1CL㸸Corpus luteum formed after ovulation of W1DF㸦W1DF ༸ᚋ㯤య㸧
W2CL㸸Corpus luteum formed after ovulation of W2DF㸦W2DF ༸ᚋ㯤య㸧 TMR㸸Total mixed ration
1
➨㸯❶
⥴ゝ
2 ࢘ࢩࡢᐙ␆ࡣ⣙ 9,000 ᖺ๓ࡽ㛤ጞࡋࡓ⪃࠼ࡽࢀ࡚࠾ࡾ㸪ᆅ⌫ୖࡢ࠸ࡓࡿࡇࢁ ⏕ᜥࡋ࡚࠸ࡓཎ∵㸦࣮࢜ࣟࢵࢡࢫ㸪Bos primigenius㸧ࢆᇶࡋ࡚㸪すࢪᆅᇦࢆ୰ᚰ ᐙ␆ࡀ㐍⾜ࡋࡓࡉࢀࡿࠋึᮇ࠾࠸࡚㸪࢘ࢩࡣ⫗ࡢ⏝ࡸ᐀ᩍⓗᘧ⏝࠸ࡿࡇ ࢆ┠ⓗᐙ␆ࡉࢀ㸪ࡑࡢᚋࡣ㎰⪔࠾࠸࡚ࡶᚲせྍḞ࡞ᐙ␆࡞ࡗ࡚࠸ࡗࡓࠋࡋ ࡋ࡞ࡀࡽ㸪ྂ௦㑇㊧ࡽⓎぢࡉࢀࡿ㔝⏕ື≀ࡢ㦵ࡀ࢘ࢩࡢᐙ␆ࢆቃࡋ࡚ῶᑡࡍࡿ ࡇࡽ㸪ᐙ␆࢘ࢩࡢせ࡞┠ⓗࡀ㣗ᩱࢆ☜ಖࡍࡿࡓࡵ࡛࠶ࡗࡓ⪃࠼ࡽࢀ࡚࠸ࡿࠋFAO ࡢ2007 ᖺࡢ⤫ィࡼࢀࡤ㸪ୡ⏺࡛㣫⫱ࡉࢀ࡚࠸ࡿ࢘ࢩࡢ㢌ᩘࡣ 13 ൨ 6,000 㢌࡛࠶ࡾ㸪 ࡑࡢศᕸࡣࢪᆅᇦ31㸣㸪୰ኸ࠾ࡼࡧ༡࣓ࣜ࢝ᆅᇦ 29㸣㸪ࣇࣜ࢝ 19㸣㸪 ࣮ࣚࣟࢵࣃ10㸣㸪࣓ࣜ࢝ 8㸣㸪࢜ࢭࢽ 3㸣࡞ࡗ࡚࠸ࡿࠋᅜ༢࡛ࡣࣈ ࣛࢪࣝ2 ൨㢌㸪ࣥࢻ 1 ൨ 8,000 㢌㸪࣓ࣜ࢝ྜ⾗ᅜ 9,700 㢌㸪୰ᅜ 8,200 㢌㸪ࣝࢮࣥࢳࣥࡀ5,100 㢌⥆ࡁ㸪᪥ᮏࡣ 440 㢌ࡀᏑᅾࡍࡿࠋࡇࡢࡼ࠺ୡ ⏺୰࡛ከࡃ㣫㣴ࡉࢀ࡚࠸ࡿ࢘ࢩࡽ㸪⫗㸪⏕ஙࡉࡽຍᕤங〇ရ࡞ࡀ⏕⏘ࡉࢀ㸪࢘ࢩ ࡣ⌧௦࠾࠸࡚᭱ࡶ㔜せ࡞ᐙ␆ࡢ୍ࡘ࡛࠶ࡿࡉࢀ࡚࠸ࡿࠋ ᪥ᮏ࠾ࡅࡿᐙ␆ࡋ࡚ࡢ࢘ࢩࡢከࡃࡣ㸪⫗⏕⏘ࢆ⾜࠺⫗⏝∵㸪࠶ࡿ࠸ࡣ⏕ஙࡸங〇 ရࢆ〇㐀ࡍࡿࡓࡵࡢஙࢆ⏕⏘ࡍࡿங⏝∵ࡋ࡚㣫㣴ࡉࢀ࡚࠸ࡿࠋ⫗⏝∵ࡢ⏕⏘ࡢࡓࡵ㸪 ࡲࡓங⏝∵ࡀἪஙࡋங⏕⏘ࢆ࠾ࡇ࡞࠺ࡓࡵࡣ㸪㞤∵ࡀཷ⫾ࡋศፔࡍࡿᚲせࡀ࠶ࡾ㸪␆ ⏘ᴗࡀ⤒῭ⓗᡂ❧ࡍࡿࡓࡵࡣ㸪㞤∵ࡀຠ⋡ⓗ㡰ㄪ࡞ࢧࢡ࡛ࣝཷ⫾ࡋศፔࡋ࡚࠸ ࡃᚲせࡀ࠶ࡿࠋ ㏆ᖺ㸪᪥ᮏࢆྵࡴඛ㐍ㅖᅜ࡛ࡣ␆⏘㎰ᐙ㸪≉㓗㎰ᐙ1 ᡞᙜࡓࡾࡢ㣫㣴㢌ᩘࡀቑຍࡋ ࡚࠸ࡿࠋ᪥ᮏࢆ࠶ࡆࡿ㸪1 ᡞᙜࡓࡾࡢங∵㣫㣴㢌ᩘࡣ 1960 ᖺࡣ 2.0 㢌࡛࠶ࡗࡓ ࡢᑐࡋ࡚㸪1980 ᖺࡣ 18.1 㢌㸪1990 ᖺࡣ 32.5 㢌㸪2000 ᖺࡣ 52.5 㢌ࡑࡋ࡚ 2012 ᖺࡣ72.1 㢌࡞ࡾ㸪ࡇࡢ 50 ᖺ࡛ᛴ⃭࡞㣫㣴つᶍᣑࡀࡳࡽࢀࡿ㸦␆⏘⤫ィㄪᰝ㸪㎰ ᯘỈ⏘┬㸧ࠋࡲࡓ㸪࢘ࢩࡢ㑇ఏᨵⰋࡸ㣫㣴⟶⌮ᢏ⾡ࡢྥୖࡼࡾ㸪ங⏝∵ࡢಶయங㔞ࡣ ୡ⏺ⓗቑຍഴྥ࠶ࡿࠋ࣓ࣜ࢝ྜ⾗ᅜ࡛ࡣ1951 ᖺࡽ 1996 ᖺࡅ࡚ 5,000 kg
3 ࡽ9,000 kg ௨ୖᖺ㛫ᖹᆒಶయங㔞ࡀቑຍࡋ࡚࠸ࡿ㸦18㸧ࠋ᪥ᮏ࠾ࡅࡿங⏝∵ࡢᖺ㛫 ᖹᆒಶయங㔞ࡣ㸪1975 ᖺࡣ 5,826 kg ࡛࠶ࡗࡓࡢᑐࡋ࡚㸪1980 ᖺࡣ 6,339 kg㸪1990 ᖺࡣ7,798 kg㸪2000 ᖺࡣ 8,794 kg ࡑࡋ࡚ 2010 ᖺࡣ 9,286 kg ࡞ࡾⴭࡋࡃቑຍࡋ࡚ ࠸ࡿ㸦ங⏝∵⩌⬟ຊ᳨ᐃᡂ⦼ࡢࡲࡵ㸫ᖹᡂ24 ᖺᗘ㸫㸪ᐙ␆ᨵⰋᴗᅋ㸧ࠋࡲࡓ㸪⫗⏝ ∵࠾࠸࡚ࡶ1 ᡞᙜࡓࡾࡢ㣫㣴㢌ᩘࡣ 1960 ᖺ࡛ࡣ 1.2 㢌࡛࠶ࡗࡓࡢᑐࡋ࡚㸪1980 ᖺ ࡣ5.9 㢌㸪1990 ᖺࡣ 11.6 㢌㸪2000 ᖺࡣ 24.2 㢌ࡑࡋ࡚ 2012 ᖺࡣ 41.8 㢌࡞ࡾ㸪ங ⏝∵ྠᵝ㣫㣴つᶍᣑࡀぢཷࡅࡽࢀࡿ㸦␆⏘⤫ィㄪᰝ㸪㎰ᯘỈ⏘┬㸧ࠋ ⌧௦࠾࠸࡚㸪ከࡃࡢ㞤∵ࡣேᕤᤵ⢭ࡼࡾཷ⫾㸪ศፔࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸪 ேᕤᤵ⢭ࡢᬑཬ⋡㛵ࡋ࡚ࡣ㸪࣓ࣜ࢝ྜ⾗ᅜ࡛ࡣங⏝∵ࡢ 72㸣㸦92㸧㸪᪥ᮏ࡛ࡣங⏝ ∵㸪⫗⏝∵ࡶ95㸣௨ୖ㸦44㸧࡞ࡗ࡚࠸ࡿࠋࡇࡢࡓࡵ㸪ຠ⋡ⓗࡘ㐺ษ࡞㞤∵ࡢⓎ Ⓨぢࡀ⦾Ṫᡂ⦼ྥୖࡢ㔜せ࡞せᅉ࡞ࡗ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽ㸪ᛴ⃭࡞㣫㣴つᶍࡢᣑ క࠺㸪ⓎⓎぢ⋡ࡢపୗࡀ✵⫾ᮇ㛫ࡢᘏ㛗ࡘ࡞ࡀࡾ㸪⦾Ṫᡂ⦼ࡢపୗࢆࡶࡓࡽࡋ ࡚࠸ࡿࠋࡲࡓ㸪ங⏝∵㛵ࡋ࡚ࡣ㧗Ἢஙక࠸㸪⫢⮚࡛ࡢࢫࢸࣟࢻ௦ㅰஹ㐍㸦100㸧 ࡀᣦࡉࢀ࡚࠾ࡾ㸪ࡑࢀక࠺Ⓨᚤᙅ࠾ࡼࡧⓎⓎ⌧㛫ࡢ▷⦰㸦55㸧㉳ᅉࡍࡿ ⓎⓎぢ⋡࡞ࡢపୗࡸ㸪㐺ᮇᤵ⢭ࡀᐇ࡛ࡁ࡞࠸ࡇࡼࡿཷ⫾ᛶࡢపୗࡀࡁ࡞ၥ 㢟࡞ࡗ࡚࠸ࡿࠋ ࡑࡢࡓࡵ㸪࣓ࣜ࢝ྜ⾗ᅜࢆ୰ᚰࡋࡓඛ㐍ᅜ࡛ࡣ㸪⦾Ṫᡂ⦼ྥୖࢆ┠ⓗྛ✀࣍ࣝ ࣔࣥ〇ࢆ⏝࠸࡚㸪࢜ࣈࢩࣥࢡἲ㸦72㸧࡞ⓎⓎぢࢆᚲせࡋ࡞࠸ᐃேᕤᤵ⢭ࣉࣟ ࢢ࣒ࣛࡢ㛤ⓎᨵⰋࢆ⾜ࡗ࡚ࡁࡓ㸦32㸪50㸪60㸪77㸪90㸧ࠋࡋࡋ࡞ࡀࡽ㸪ࡇࢀࡽࡢࣉࣟ ࢢ࣒ࣛࢆ⏝࠸ࡿࡇ࡛㸪ᤵ⢭⋡ࢆྥୖࡉࡏࡿࡇࡣᡂຌࡋࡓࡀ㸪ཷ⫾⋡ࡢྥୖ㛵ࡋ ࡚ࡣ⌧ᅾ⮳ࡿࡲ࡛༑ศ㐩ᡂ࡛ࡁ࡚࠸࡞࠸ࠋࡇࡢࡇࡣ㸪࢘ࢩࡢ⦾Ṫ⏕⌮ᑐࡍࡿ ⌮ゎࡸ㸪ዷፎᡂ❧㛵ࢃࡿせᅉࡢゎ᫂ࡉࡽࡣཷ⫾⋡ࡢྥୖࢆࡶࡓࡽࡍෆศἪ≧ែࢆస ฟࡍࡿᤵ⢭ࣉࣟࢢ࣒ࣛࡢ㛤Ⓨࡀ☜❧ࡋ࡚࠸࡞࠸ࡇࢆ♧၀ࡋ࡚࠸ࡿࠋࡑࡢࡓࡵ㸪࢘ࢩࡢ ⦾Ṫ⏕⌮ᑐࡋ࡚ࡲ࡛ὀ┠ࡉࢀ࡚࠸࡞ࡗࡓⅬࡘ࠸࡚ࡢᇶ♏ⓗ࡞ㄪᰝࡸ◊✲ࢆ⾜ࡗ
4 ࡚࠸ࡃᚲせࡀ࠶ࡿࠋ 㞤ࡀ㓄ࡢࡓࡵ㞝ࢆチᐜࡍࡿ≧ែࡀⓎᐃ⩏ࡉࢀ㸪ࡇࡢⓎࡣ࿘ᮇⓗ⧞ࡾ㏉ࡉ ࢀ࡚࠾ࡾ㸪ࡇࡢ࿘ᮇࢆⓎ࿘ᮇ࠸࠺ࠋ࢘ࢩࡣ࿘ᖺ⦾ṪࡢከⓎື≀࡛࠶ࡾ㸪6㹼12 ࣧ ᭶㱋࡛ᛶᡂ⇍㐩ࡋࡓᚋ㸦30㸧㸪ዷፎࡋ࡞࠸㝈ࡾ 1 ᖺࢆ㏻ࡋ࡚࿘ᮇⓗⓎࢆ⧞ࡾ㏉ࡍࡼ ࠺࡞ࡿࠋⓎ࿘ᮇ୰ࡣ༸ᕢෆᵓ㐀≀࡛࠶ࡿ㯤య༸⬊ࡢⓎ⫱㛢㙐࣭㏥⾜ࡢ࿘ᮇⓗ ኚࡀࡳࡽࢀࡿࠋ࢘ࢩࡢⓎ࿘ᮇࡢᖹᆒ᪥ᩘࡣ21 ᪥࡛࠶ࡿ㸦30㸧ࡀ㸪Ⓨ࿘ᮇࡢ᪥ᩘ ࡣ18㹼24 ᪥ᖜࡢ࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦30㸧ࠋⓎ࿘ᮇࡣࡁࡃ㯤యᮇ㸦14㹼18 ᪥ 㛫㸧༸⬊ᮇ㸦4㹼6 ᪥㛫㸧ࡢ 2 ࡘศ㢮ࡉࢀࡿ㸦30㸧ࠋ㯤యᮇࡣⓎᚋ༸ᕢෆ࡛ᡂ⇍ ༸⬊ࡀ༸ࡋࡓ┤ᚋࡽጞࡲࡾ㸪㯤యࡀᡂ⇍ࡋࡓᚋ㸪㯤య㏥⾜ࡀ㛤ጞࡍࡿࡲ࡛ࡢᮇ㛫࡛ ࠶ࡾ㸪㯤య㏥⾜ࡀ㛤ጞࡋ࡚ࡽ༸⮳ࡿࡲ࡛ࡢᮇ㛫ࢆ༸⬊ᮇ࠸࠺㸦30㸧ࠋ༸⬊ᮇࡢᡂ ⇍༸⬊ࡽ࢚ࢫࢺࣛࢪ࣮࢜ࣝ㸦E2㸧ࡀ⏘⏕࠾ࡼࡧศἪࡉࢀࡿ㸦30㸧ࡇ࡛⾑୰ E2⃰ᗘࡀ ୖ᪼ࡋ㸪ṇࡢࣇ࣮ࢻࣂࢵࢡస⏝ࡼࡾどᗋୗ㒊࡛ᛶ⭢่⃭࣍ࣝࣔࣥᨺฟ࣍ࣝࣔࣥ 㸦GnRH㸧ࡢࢧ࣮ࢪ≧ศἪࡀㄏ㉳ࡉࢀ㸪ࡑࢀࡀ⬻ୗᆶయ๓ⴥస⏝ࡍࡿࡇ࡛㸪㯤యᙧ ᡂ࣍ࣝࣔࣥ㸦LH㸧ࡢࢧ࣮ࢪ≧ࡢᨺฟࡀ㉳ࡇࡾ㸦80㸧㸪ᡂ⇍༸⬊ࡢ༸ࢆㄏᑟࡍࡿ㸦94㸧ࠋ ᡂ⇍༸⬊ࢆᵓᡂࡋ࡚࠸ࡿ㢛⢏ᒙ⣽⬊ෆ༸⬊⭷⣽⬊ࡀ LH ࢧ࣮ࢪࡼࡾ่⃭ࡉࢀ㸪ࡑࢀ ࡒࢀᆺ㯤య⣽⬊ᑠᆺ㯤య⣽⬊ኚࡋ㸪ࡑࢀࡽࡀቑṪࡍࡿࡇ࡛ᛴ㏿㯤యࡀᙧᡂ ࡉࢀ࡚࠸ࡃ㸦8㸧ࠋ㯤యࡣ㸪Ⓨ࿘ᮇࡢ 7 ᪥┠ࡈࢁࡲ࡛Ⓨ⫱㸪ᡂ㛗ࡋ࡚࠸ࡁ㸪ࡑࡢᚋࡣ㯤 య㏥⾜㛤ጞࡲ࡛ᶵ⬟ⓗ㯤యࡋ࡚Ꮡᅾࡍࡿࠋዷፎࡀᡂ❧ࡋ࡞࠸ሙྜ㸪Ꮚᐑࡽࣉࣟࢫࢱ ࢢࣛࣥࢪࣥ㸦PG㸧F2Dࡀᨺฟࡉࢀࡿ㸦54㸧ࡇ࡛㯤య㏥⾜ࡀ㛤ጞࡉࢀ㸪㯤యࡣᛴ⃭⦰ ᑠࡍࡿ㸦11㸪51㸪67㸧ࠋ㯤యࡽࡣ㸪ࣉࣟࢪ࢙ࢫࢸࣟࣥ㸦P4㸧ࡀ⏘⏕㸪ศἪࡉࢀ࡚࠾ࡾ㸦68㸧㸪 㯤యࡢⓎ⫱క࠸⾑୰P4⃰ᗘࡣୖ᪼ࡋ࡚࠸ࡁ㸪㯤యᙧᡂࡀࡍࡿ㯤య㏥⾜㛤ጞࡲ࡛ ⾑୰P4⃰ᗘࡣ㧗ࡃ⥔ᣢࡉࢀ㸪㯤య㏥⾜㛤ጞక࠸ᛴ⃭⾑୰P4⃰ᗘࡣపୗࡋ࡚࠸ࡃ㸦57㸧ࠋ ࡇࡢࡼ࠺࡞㯤యࡢⓎ⫱㸪⥔ᣢ࠾ࡼࡧ㏥⾜㸪༸⬊ࡢᡂ⇍༸ࡀ⧞ࡾ㏉ࡉࢀ㸪Ⓨ࿘ᮇ ࡀႠࡲࢀࡿࠋࡇࡢⓎ࿘ᮇᮇ㛫୰ࡣ༸ᕢෆ࡛ࡣ2㹼3 ᅇࡢ༸⬊⩌ࡢⓎ⫱ࡀほᐹࡉࢀ㸪ࡑ
5 ࢀࡒࢀࡢ༸⬊⩌ࡽ༸⮳ࡽ࡞࠸༸⬊ࡀ1㹼2 ࡘⓎ⫱ࡍࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸪ࡇࡢ༸ ⬊Ⓨ⫱ࡢ୍⩌ࢆ༸⬊Ἴ࠸࠺㸦41㸧ࠋ ࢘ࢩ࠾࠸࡚Ⓨ࿘ᮇ୰Ⓨ⌧ࡍࡿ᭱ึࡢ༸⬊Ἴࡣ㸪Ⓨ⤊ᚋࡢ༸ᕢෆ࡛ 3㹼4mm ⛬ࡢᑠ༸⬊ࡢ୍⩌㸦8㹼41 ಶ㸧ࡢⓎ⫱ࡀ㛤ጞࡉࢀ㸪ࡇࢀࢆ➨ 1 ༸⬊Ἴ࠸࠺㸦41㸧ࠋࡑࡢ ୍⩌ࡢ༸⬊ࡢ୰࡛᭱ࡢ༸⬊ࡀ1 ࡘࡔࡅ㑅ᢤࡉࢀ㸪ࡑࢀ௨እࡢᑠ༸⬊ࡣ㛢㙐ࡍࡿࠋࡇࡢ 㑅ᢤࡉࢀࡓ༸⬊ࢆᖍ༸⬊࠸࠸㸪➨1 ༸⬊Ἴࡢᖍ༸⬊ࢆ➨ 1 ༸⬊Ἴᖍ༸⬊㸦W1DF㸧 ࠸࠺ࠋW1DF ࡣⓎ࿘ᮇࡢ 8㹼11 ᪥㡭ࡲ࡛Ⓨ⫱ࡍࡿࡀ㸪㯤యࡽ P4ࡀ⏘⏕ศἪࡉࢀ࡚ ࠸ࡿᮇᙜࡓࡿࡓࡵ㸪ᡂ⇍ࡍࡿࡇࡀ࡛ࡁࡎ㸪ࡇࡢW1DF ࡣ༸⮳ࡽࡎ㛢㙐ࡋ࡚ࡋ ࡲ࠺㸦41㸧ࠋࡑࢀక࠸᪂ࡓ࡞༸⬊ἼࡢⓎ⌧ࡀ㛤ጞࡉࢀ㸪ࡇࢀࢆ➨ 2 ༸⬊Ἴ࠸࠺ࠋ࢘ࢩ ࡣ༸⬊Ἴࢆ2 ࡘᣢࡘࡶࡢ㸪3 ࡘᣢࡘࡶࡢࡀᏑᅾࡋ㸪ࡑࢀࡒࢀࡢⓎ⏕⋡ࡣヨ㦂⏝࠸ ࡿ࢘ࢩࡢရ✀࠾ࡼࡧᖺ㱋ࡼࡾ␗࡞ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦7㸪16㸪17㸪19㸪28㸪39㸪 69㸪74㸪88㸪108㸧ࠋ༸⬊Ἴࢆ 3 ࡘᣢࡘ࢘ࢩࡢẚ⋡ࡣపᰤ㣴≧ែ㸦66㸪76㸧ࡸࣄ࣮ࢺࢫࢺ ࣞࢫ᮲௳ୗ㸦10㸧࡛ቑຍࡍࡿ࠸࠺ሗ࿌ࡀ࠶ࡿࠋW1DF ࡢ⏕Ꮡᮇ㛫ࡀ༸⬊Ἴࡢᩘᙳ㡪 ࢆ࠼ࡿྍ⬟ᛶࢆ᥎ ࡋ࡚࠸ࡿሗ࿌㸦48㸧ࡶ࠶ࡿࡀ㸪ヲ⣽ࡣ᫂ࡽࡉࢀ࡚࠸࡞࠸ࠋ༸ ⬊Ἴࢆ2 ࡘᣢࡘ࢘ࢩ࡛ࡣ㸪➨ 2 ༸⬊ἼࡢⓎ⌧᪥ࡣⓎ࿘ᮇࡢ 9㹼11 ᪥㡭࡛࠶ࡾ㸪༸⬊Ἴ ࢆ3 ࡘᣢࡘ࢘ࢩ࡛ࡣ 8㹼9 ᪥㡭࡛࠶ࡿࡉࢀ࡚࠸ࡿ㸦4㸧ࠋ༸⬊Ἴࢆ 3 ࡘᣢࡘ࢘ࢩ࡛ࡣ㸪➨ 3 ༸⬊ἼࡢⓎ⌧᪥ࡀ 15㹼16 ᪥㡭࡛࠶ࡿ㸦4㸧ࠋࡲࡓ㸪༸⬊Ἴࢆ 2 ࡘᣢࡘ࢘ࢩ 3 ࡘᣢࡘ࢘ ࢩ࡛㯤య㏥⾜㛤ጞᮇⓎ࿘ᮇ᪥ᩘࢆẚ㍑ࡋࡓࡇࢁ㸪㯤య㏥⾜㛤ጞᮇࡣ༸⬊Ἴࢆ 2 ࡘᣢࡘ࢘ࢩ࡛᪩ᮇ㛤ጞࡉࢀ㸦2 ༸⬊Ἴ vs. 3 ༸⬊Ἴ㸻16 ᪥┠ vs. 19 ᪥┠㸧㸪ࡑࡢ⤖ᯝ㸪 Ⓨ࿘ᮇ᪥ᩘࡀ▷⦰ࡍࡿ㸦2 ༸⬊Ἴ vs. 3 ༸⬊Ἴ㸻19㹼20 ᪥┠ vs. 22㹼23 ᪥┠㸧㸦4㸧ࠋ༸ ⬊Ἴࢆ2 ࡘ࠶ࡿ࠸ࡣ 3 ࡘᣢࡘ࢘ࢩࡢࡕࡽࡢሙྜ࠾࠸࡚ࡶ㸪➨ 1 ༸⬊ἼࡢⓎ⌧ᮇࡣ ༸┤ᚋ࡛࠶ࡾ㸪㑅ᢤࡉࢀࡓᖍ༸⬊ࡣ༸ࡏࡎ㸪㛢㙐ࡍࡿ࠸࠺Ⅼ࡛ඹ㏻ࡋ࡚࠸ࡿࡓ ࡵ㸪➨1 ༸⬊Ἴࢆ⏝࠸ࡓ༸⬊Ⓨ⫱㛵ࡍࡿ◊✲ࡣከ࠸ࠋࡋࡋ㸪ࡇࡢ➨ 1 ༸⬊Ἴࡀ࢘ࢩ ࡢ⦾Ṫ⏕⌮࠾࠸࡚ᯝࡓࡍᙺࡸព⩏㛵ࡍࡿ◊✲ࡣᑡ࡞࠸ࠋ
6 㞤ࡢ⏕ṪෆศἪ⣔ࡣどᗋୗ㒊㸫ୗᆶయ㸫༸ᕢ㍈ࢆ୰ᚰᵓᡂࡉࢀ࡚࠾ࡾ㸪どᗋୗ㒊࡛ ࡣGnRH ࡢศἪࡀ⾜ࢃࢀ㸪ୗᆶయ๓ⴥ࡛ࡣ༸⬊่⃭࣍ࣝࣔࣥ㸦FSH㸧 LH ࡀศἪࡉࢀ ࡿ㸦80㸧ࠋどᗋୗ㒊ࡢṇ୰㝯㉳㒊ࡽᨺฟࡉࢀࡓ GnRH ࡣୗᆶయ㛛⬦ධࡾ㸦65㸧㸪ୗᆶ య๓ⴥస⏝ࡋ㸪FSH ࠾ࡼࡧ LH ࡢྜᡂศἪࡀಁ㐍ࡉࢀࡿࠋFSH ࡣ༸⬊ࡢⓎ⫱ࡸ༸⬊ ᩘࡢㄪ⠇㛵ࡍࡿ࡛࣍ࣝࣔࣥ࠶ࡾ㸪༸⬊ἼⓎ⌧㛵ࢃࡿ㸦6㸧ࠋLH ࡢࣃࣝࢫ≧ศἪࡣ GnRH ࡢᢿືᛶศἪ㐃ືࡋ࡚࠾ࡾ㸦107㸧㸪LH ࡣᖍ༸⬊ࡢ㑅ᢤᖍ༸⬊ࡢⓎ⫱㸪ࡉ ࡽᡂ⇍㛵ࢃࡿ࡛࣍ࣝࣔࣥ࠶ࡿ㸦36㸧ࠋどᗋୗ㒊ࡢάືࡣ㸪༸ᕢࡽศἪࡉࢀࡿᛶࢫࢸ ࣟࢻ࣍ࣝࣔࣥ㸦E2࠾ࡼࡧP4㸧ࡼࡾࣇ࣮ࢻࣂࢵࢡㄪ⠇ࡉࢀ࡚࠸ࡿࠋ㐣ཤࡢሗ࿌࠾ ࠸࡚㸪⾑୰P4⃰ᗘࡀప࠸LH ࡢࣃࣝࢫ㢖ᗘࡀቑຍࡍࡿࡇ㸪୍᪉࡛⾑୰ P4⃰ᗘࡀ㧗 ࠸LH ࡢࣃࣝࢫ㢖ᗘࡀῶᑡࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦15㸪52㸧ࠋLH ࡢࣃࣝࢫ≧ศἪ ࡣᖍ༸⬊ࡢⓎ⫱ᚲせ࡛࠶ࡿࡓࡵ㸪⾑୰P4⃰ᗘࡀ㧗⃰ᗘ࡞ࡿ㯤యᮇ࡛ࡣ㸪ᖍ༸⬊ ࡢⓎ⫱ࡣᢚไࡉࢀ༸⮳ࡿࡇࡣ࡞ࡃ㛢㙐ࡋ࡚࠸ࡃࠋࡋࡋ࡞ࡀࡽ㸪㯤యࡀ㏥⾜ࡋ⾑ ୰P4⃰ᗘࡀప࠸≧ἣ࡛ࡣ㸪LH ࣃࣝࢫ㢖ᗘࡀୖ᪼ࡍࡿࡇ࡛㸪༸⬊ࡢⓎ⫱ࡀಁࡉࢀ㸪ᡂ ⇍༸⬊࡛⏘⏕ࡉࢀࡓE2ࡀศἪࡉࢀ㸪⾑୰E2⃰ᗘࡀୖ᪼ࡍࡿࠋ⾑୰E2⃰ᗘࡢୖ᪼どᗋ ୗ㒊ࡀᛂࡋGnRH ࢧ࣮ࢪࡀᘬࡁ㉳ࡇࡉࢀ㸪ࡑࢀᛂࡋ࡚ୗᆶయ๓ⴥ࡛ࡣ LH ࢧ࣮ࢪ ࡀㄏ㉳ࡉࢀ㸪ᡂ⇍༸⬊ࡣ༸ࡍࡿ㸦81㸧ࠋ ⫾⏕ᮇ࠾ࡼࡧᶵⓎື๓ࡢ࢘ࢩࡢ༸ᕢ࠾࠸୍࡚ḟ༸ẕ⣽⬊ࡢ࿘ᅖ༸⬊ୖ⓶⣽⬊࡛ ⿕ࢃࢀࡓཎጞ༸⬊ࡀᙧᡂࡉࢀࡿࠋࡑࡢᚋ㸪ᶵⓎືక࠸㸪ཎጞ༸⬊ࡣⓎ⫱ࢆ㛤ጞࡋ㸪 ࡇࡢⓎ⫱ࢆ㛤ጞࡋࡓ༸⬊ࡣ୍ḟ༸⬊ࡼࡤࢀࡿࠋ୍ḟ༸ẕ⣽⬊ࢆྲྀࡾᅖࢇ࡛࠸ࡿ༸⬊ୖ ⓶⣽⬊ࡣయ✚ࡀቑࡋ❧᪉≧࡞ࡿ㸦63㸧ࠋࡉࡽ❧᪉ࡋࡓୖ⓶⣽⬊ࡀቑṪ㸪㔜ᒙࡍࡿ ࡇ࡛㢛⢏ᒙ⣽⬊࡞ࡾ㸪ࡉࡽࡑࡢእഃࢆෆ༸⬊⭷⣽⬊ࡀྲྀࡾᅖࢇࡔ≧ែ࡞ࡾḟ ༸⬊࡞ࡿ㸦49㸧ࠋࡇࡢẁ㝵ࡲ࡛ࡣ㸪༸⬊ࡣᛶ⭢่⃭࣍ࣝࣔࣥ㸦FSH ࠾ࡼࡧ LH㸧౫Ꮡ ࡍࡿࡇ࡞ࡃⓎ⫱ࡀ㐍⾜ࡍࡿࠋᆺ༸⬊ࡢᶵ⬟ࡢᾘኻ㸪ࡘࡲࡾⓎᚋࡢ༸ࡸ➨1 ࠶ࡿ ࠸ࡣ➨ 2 ༸⬊Ἴᖍ༸⬊㸦W2DF㸧ࡢ㛢㙐ࡀ㉳ࡇࡿ㸪ᖍ༸⬊ࡢ㢛⢏ᒙ⣽⬊࡛⏘⏕ࡉ
7 ࢀࡿࣥࣄࣅࣥࡢ⾑୰⃰ᗘࡀపୗࡍࡿࠋࣥࣄࣅࣥࡣୗᆶయ๓ⴥస⏝ࡋFSH ࡢᨺฟࢆ ᢚไࡍࡿࡓࡵ㸪༸ᚋࡸᖍ༸⬊㛢㙐㛤ጞࡶFSH ࡢࢧ࣮ࢪ≧ศἪࡀ㉳ࡇࡿ㸦62㸧ࠋ ࡑࢀࢆཷࡅ࡚ḟ༸⬊ࡢ୰ࡽᩘࡋ࡚8㹼41 ಶ㸪┤ᚄ 3㹼4mm ⛬ࡢ༸⬊ࡢⓎ⫱ࡀ㛤ጞ ࡉࢀ㸪༸⬊ἼࡢⓎ⌧࡞ࡿ㸦17㸪34㸧ࠋ༸⬊Ἴࡋ࡚ືဨࡉࢀࡓ༸⬊ࡣᛶ⭢่⃭࣍ࣝࣔࣥ ౫ᏑⓗⓎ⫱ࢆ⥆ࡅࡿࠋࡇࡢ୍⩌ࡢ༸⬊ࡣ1 ᪥ᙜࡓࡾ 1.5㹼2.0 mm ࡢ㏿ᗘ࡛Ⓨ⫱ࡋ࡚࠸ ࡃ㸦37㸧ࠋ༸⬊ἼࡢⓎ⌧ࡽ 2㹼3 ᪥⤒㐣ࡍࡿ㸪୍⩌ࡢ୰࡛᭱༸⬊ࡢ㢛⢏ᒙ⣽⬊࡚ LH ཷᐜయࡀⓎ⌧ࡋ㸪E2⏘⏕ࡀ⾜ࢃࢀࡿࡼ࠺࡞ࡿ㸦13㸧ࠋE2ࡣୗᆶయ๓ⴥస⏝ࡋFSH ࡢᨺฟࢆᢚไࡍࡿ㸦38㸧㸪ࡑࡢ⤖ᯝ㸪༸⬊Ἴࡢ༸⬊⩌ࡢ୰࡛ᖍ༸⬊ࡋ࡚㑅ᢤࡉࢀ࡞ ࡗࡓḟᖍ௨ୗࡢFSH ౫Ꮡᛶࡢ༸⬊ࡣⓎ⫱ࡀ⥔ᣢ࡛ࡁࡎ㛢㙐ࡋ࡚࠸ࡃࠋࡋࡋ࡞ࡀࡽ㸪᭱ ༸⬊ࡣ㢛⢏ᒙ⣽⬊LH ཷᐜయࢆⓎ⌧ࡍࡿࡇ࡛㸪ࡑࡢⓎ⫱ࢆ FSH ౫Ꮡᛶࡽ LH ౫ Ꮡᛶࢩࣇࢺࡍࡿࡓࡵ㸪LH ࣃࣝࢫࡢ่⃭ࢆཷࡅ࡚㸪㛢㙐ࡍࡿࡇ࡞ࡃⓎ⫱ࡀ⥔ᣢࡉࢀ ࡿࠋࡇࡢ㐣⛬ࢆ㑅ᢤ࠸࠸㸪㑅ᢤࡉࢀࡓ᭱༸⬊ࢆᖍ༸⬊࠸࠺㸦43㸧ࠋࡇࡢ㑅ᢤ ࠾ࡅࡿ᭱༸⬊ࡢᖹᆒ┤ᚄࡣ 8.5mm ࡛࠶ࡾ㸪ḟᖍ༸⬊ࡢᖹᆒ┤ᚄࡣ 7.7mm ࡉࢀ࡚࠸ ࡿ㸦35㸧ࠋW1DF ࡣ㸪㯤యࡀᙧᡂࡉࢀ㸪⾑୰ P4⃰ᗘࡀୖ᪼ࡍࡿᮇⓎ⫱ࡍࡿࡓࡵ㸪 ࢇࡀ༸⮳ࡿࡇ࡞ࡃ㛢㙐ࡋ࡚ࡋࡲ࠺ࠋ୍᪉࡛㸪W2DF ࡛ࡣ㸪Ⓨ⫱ࡋ࡚࠸ࡿᮇ㛫 㯤యࡢ㏥⾜ࡀ㉳ࡇࡾ⾑୰P4⃰ᗘࡀపୗࡋ㸪LH ࣃࣝࢫ㢖ᗘࡀୖ᪼ࡍࡿࡇ࡛ᖍ༸⬊ ࡢⓎ⫱ࡀ⥔ᣢࡉࢀ㸪༸⬊ࡽከ㔞ࡢE2ࡀ⏘⏕࠾ࡼࡧศἪࡉࢀࡿࠋࡑࡢ⤖ᯝ㸪LH ࢧ࣮ࢪ ࡀᘬࡁ㉳ࡇࡉࢀ㸪W2DF ࡣᡂ⇍ࡋ༸⮳ࡿࠋ ࡇࡢ➨1 ༸⬊Ἴ࠾࠸࡚㸪ࡑࡢⓎ⫱ືែࡸ࣍ࣝࣔࣥືែ㛵ࡋ࡚ヲ⣽ㄪᰝࡉࢀ࡚ࡣ ࠸ࡿࡀ㸪W1DF ⮬㌟ࡢᛶ㉁ࡸ㸪ࡑࢀࡀ⦾Ṫ⏕⌮ࡸཷ⫾ᛶཬࡰࡍᙳ㡪㛵ࡋ࡚ࡣ༑ศ ᳨ウࡉࢀ࡚ࡣ࠸࡞࠸ࠋ᪤㏙ࡓࡼ࠺㸪➨1 ༸⬊Ἴࡣ㯤యᙧᡂඹⓎ⫱ࡍࡿࡓࡵ㸪 W1DF ࡣ⾑୰ P4⃰ᗘࡀప್ࡽᚎࠎୖ᪼ࡍࡿᮇᡂ㛗ࡍࡿࠋ୍᪉㸪➨2 ༸⬊Ἴࡣ㯤 యࡀ༑ศᡂ⇍ࡋ࡚ࡽⓎ⫱ࢆ㛤ጞࡍࡿࡓࡵ㸪W2DF ࡣ⾑୰ P4⃰ᗘࡀ㧗್࡞ᮇᡂ㛗 ࡍࡿࠋᖍ༸⬊ࡢⓎ⫱ࡣ⬻ୗᆶయ๓ⴥࡽศἪࡉࢀࡿ LH ࡢࣃࣝࢫ≧ࡢศἪࡀᚲ㡲࡛
8 ࠶ࡿࡓࡵ㸪W1DF W2DF ࡀⓎ⫱ࡍࡿᮇ㛫࡛ࡢ⾑୰ P4⃰ᗘ᥎⛣ࡀ␗࡞ࡿࡇࡼࡿ㸪 LH ࣃࣝࢫ㢖ᗘࡢ㐪࠸ࡀⓎ⫱ࡍࡿᖍ༸⬊ࡢᙧែࡸᶵ⬟ᕪ␗ࢆࡶࡓࡽࡍ⪃࠼ࡽࢀࡿࠋ 㐣ཤࡢ◊✲ࢆࡳ࡚ࡶ㸪W1DF W2DF ࡢᛶ㉁ࡸᶵ⬟ࡢ㐪࠸ࢆ༑ศẚ㍑ࡋࡓ◊✲ࡣ ࢇ⾜ࢃࢀ࡚ࡇ࡞ࡗࡓࠋࡑࡇ࡛㸪W1DF ࡢ≉᭷ࡢᛶ㉁ࡸᶵ⬟ࢆ᫂ࡽࡍࡿࡓࡵ㸪 W1DF W2DF ࡢᛶ㉁ࢆẚ㍑ࡋࡓࠋ ࡲࡓ㸪W1DF ࡢⓎ⫱ࢆไᚚࡍࡿせᅉࢆ᫂ࡽࡍࡿࡓࡵ㸪➨ 1 ༸⬊ἼⓎ⫱ᮇ㛫୰ ࠾ࡅࡿᛶࢫࢸࣟࢻ࣍ࣝࣔࣥືែࡸ㸪༸ᕢෆ࡛Ⓨ⫱ࡍࡿ㯤యࡢ⨨㛵ಀ࡞ࡢせᅉ W1DF ࡢᛶ㉁ࢆẚ㍑ࡍࡿࡇࡋࡓࠋࡉࡽ㸪㐣ཤࡢሗ࿌ࡽ㯤యࡀᏑᅾࡍࡿ༸ᕢྠ ഃࡢᏊᐑゅࡢෆ⭷୰ࡢ P4⃰ᗘࡀ㧗࠸ࡇ㸦20㸧㸪༸๓༸⬊ࡀᏑᅾࡍࡿ༸ᕢྠഃࡢ༸ ⟶୰ࡢE2⃰ᗘࡀ㧗࠸ࡇ㸦99㸧ࡀ▱ࡽࢀ࡚࠾ࡾ㸪ᒁᡤ࡛ࡢ༸ᕢᏊᐑゅࡢᐦ᥋࡞㛵㐃ᛶ ࡀᏑᅾࡍࡿࡇࡀ♧၀ࡉࢀࡿࠋࡋࡋ࡞ࡀࡽ㸪W1DF ࡀྠ୍༸ᕢෆࡢ㯤యࡸᏊᐑゅᑐ ࡋ࡚ᒁᡤⓗࡢࡼ࠺࡞ᙳ㡪ࢆ࠼ࡿࡢࢆㄪᰝࡋࡓ◊✲ࡣぢᙜࡓࡽ࡞࠸ࠋ ᮏ◊✲࠾࠸࡚㸪➨2 ❶࡛ࡣ㸪W1DF ࡢᛶ㉁࠾ࡼࡧᶵ⬟ࢆ᫂ࡽࡍࡿࡓࡵ㸪㠀Ἢங ∵ࢆ⏝࠸࡚㸪༸⬊ࡢⓎ⫱ືែ㯤య㏥⾜ㄏ㉳ᚋࡢ༸⬊ࡢᡂ⇍࠾ࡼࡧ༸⮳ࡿ㐣⛬ࡢ༸ ⬊ቨ⾑ὶ㸪ࢫࢸࣟࢻ⃰ᗘ࠾ࡼࡧ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿᅉᏊ㸪ࡉࡽࡣ༸ᚋᙧ ᡂࡉࢀࡿ㯤యⓎ⫱ࢆW2DF ẚ㍑ࡋࡓࠋ ➨3 ❶࡛ࡣ㸪W1DF ࡢⓎ⫱ࢆไᚚࡍࡿせᅉࢆ᫂ࡽࡍࡿࡓࡵ㸪W1DF ࡢⓎ⫱ࡀ㸪Ⓨ ⫱ᮇ㛫୰ࡢ⾑୰ P4 ⃰ᗘ࠾ࡼࡧྠ୍༸ᕢෆ࡛ࡢ㯤యࡢ᭷↓ࡼࡿ༸ᕢᒁᡤෆ࡛ࡢᙳ㡪ࢆ ཷࡅࡿࡢࢆㄪࡓࠋ ➨4 ❶࡛ࡣ㸪Ἢங∵ࢆ⏝࠸㸪W1DF ࡀ⦾Ṫ⏕⌮ࡢࡼ࠺࡞ᙳ㡪ࢆ࠼ࡿࢆ┠ⓗ㸪 ᖍ༸⬊ࡀ㯤యྠഃࡢ༸ᕢᏑᅾࡍࡿሙྜᑐഃᏑᅾࡍࡿሙྜࡢཷ⫾ᛶࢆẚ㍑ࡋ ࡓࠋ
9
➨㸰❶
➨
1 ༸⬊Ἴᖍ༸⬊㸦W1DF㸧ࡢⓎ⫱ືែ㸪༸๓ᚋࡢ
10 2㸫1㸬ᗎㄽ ࢘ࢩࡢ➨1 ༸⬊ἼࡣⓎ࣭༸⤊┤ᚋࡽ༸ᕢෆ࡛Ⓨ⌧ࡍࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸪 ㏻ᖖࡣ㸪㯤యࡀᏑᅾࡍࡿ᮲௳ୗ࡛Ⓨ⫱ࡍࡿࡓࡵ㸪W1DF ࡣⓎ࿘ᮇࡢ 8㹼10 ᪥ࡲ࡛㛢 㙐ࡋ㸪༸⮳ࡿࡇࡣ࡞࠸㸦41㸧ࠋ࢘ࢩࡢ༸⬊ࡢⓎ⫱ືែ㸪Ⓨ⫱࠾ࡼࡧ㑅ᢤ㛵ࢃࡿෆ ศἪ࣍ࣝࣔࣥືែࢆ᫂ࡽࡍࡿࡇࢆ┠ⓗ㸪ࡇࢀࡲ࡛W1DF ὀ┠ࡋ࡚ヲ⣽◊✲ ࡀ⾜ࢃࢀ࡚ࡁࡓ㸦9㸪53㸪93㸪103㸧ࡀ㸪W1DF ⮬㌟ࡢᛶ㉁㸪ᶵ⬟࠾ࡼࡧ༸ㄏ㉳ࡢ≉ ᚩࡘ࠸࡚ࡣ༑ศ᳨ウࡉࢀ࡚ࡇ࡞ࡗࡓࠋ➨2 ༸⬊ἼࡣⓎ࿘ᮇࡢ 9㹼10 ᪥Ⓨ⌧࣭ Ⓨ⫱ࡋ㸪㯤యࡢ㏥⾜క࠸ᖍ༸⬊ࡣᡂ⇍㸪Ⓨࢆㄏ㉳ࡋ༸⮳ࡿ㸦88㸧ࠋ➨ 1 ༸⬊Ἴ ࡣ㯤యᙧᡂඹⓎ⫱ࡍࡿࡓࡵ㸪W1DF ࡣ P4⃰ᗘࡀప್ࡽᚎࠎୖ᪼ࡍࡿᮇᡂ㛗 ࡍࡿࠋ୍᪉㸪➨2 ༸⬊Ἴࡣ㯤యࡀ༑ศᡂ⇍ࡋ࡚ࡽⓎ⫱ࢆ㛤ጞࡍࡿࡓࡵ㸪W2DF ࡣ⾑ ୰ P4⃰ᗘࡀ㧗್࡞ᮇᡂ㛗ࡍࡿࠋࡘࡲࡾ㸪୧⪅ࡣ⾑୰ P4⃰ᗘ᥎⛣ࡀ␗࡞ࡿ≧ἣୗ࡛ Ⓨ⫱ࡍࡿࡓࡵ㸪ࡑࢀࡒࢀࡢᖍ༸⬊ࡢⓎ⫱ືែ㸪ࢫࢸࣟࢻ⏘⏕⬟㸪༸క࠺ኚ࠾ ࡼࡧ༸ᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែࡸᶵ⬟ࡀ␗࡞ࡿྍ⬟ᛶࡀ࠶ࡿࠋ ࡑࡇ࡛㸪ᮏ❶࡛ࡣ㸪W1DF ࡢᛶ㉁࠾ࡼࡧᶵ⬟ࢆ᫂ࡽࡍࡿࡓࡵ㸪㠀Ἢங∵ࢆ⏝࠸࡚㸪 ༸⬊ࡢⓎ⫱ືែ㯤య㏥⾜ㄏ㉳ᚋࡢᡂ⇍㸪༸⮳ࡿ㐣⛬ࡢ༸⬊ቨ⾑ὶ㸪ࢫࢸࣟࢻ⃰ ᗘ࠾ࡼࡧ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿᅉᏊࡉࡽࡣ༸ᚋᙧᡂࡉࢀࡿ㯤యⓎ⫱ࢆW2DF ẚ㍑ࡋㄪࡓࠋ 2㸫2㸬ヨ㦂 1㸸༸⬊ἼⓎ⌧ࡽ 7 ᪥┠ࡲ࡛ࡢ W1DF W2DF ࡢⓎ⫱ືែ࠾ࡼࡧ ༸⬊ቨ⾑ὶࡢẚ㍑ 2㸫2㸫1㸬౪ヨື≀
11 ⮫ᗋⓗᗣ࡛㸪ṇᖖ࡞Ⓨ࿘ᮇࢆ᭷ࡍࡿ࣍ࣝࢫࢱࣥ✀㠀Ἢங∵8 㢌㸦ᖹᆒᖺ㱋㸸 5.0 r 1.8 ṓ㸪ᖹᆒsᶆ‽ㄗᕪ㸧ࢆ⏝࠸ࡓࠋ ᑐ㇟∵ࡣᖏᗈ␆⏘Ꮫ␆⏘ࣇ࣮ࣝࢻ⛉Ꮫࢭ ࣥࢱ࣮ෆࡢࣃࢻࢵࢡ࡚㸪ࢥ࣮ࣥࢧ࣮ࣞࢪ㸪ⲡ࠾ࡼࡧ⃰ཌ㣫ᩱࡀΰྜࡉࢀࡓ TMR ࡀ⤥ࡉࢀ㸪⮬⏤㣧Ỉ࡛ࡁࡿ≧ἣ࡛㣫㣴ࡉࢀ࡚࠸ࡓࠋᐇ㦂ෆᐜࡣᖏᗈ␆⏘Ꮫࡢᐇ㦂 ⌮ጤဨ࡚ᢎㄆࡉࢀ࡚࠸ࡿࠋ 2㸫2㸫2㸬ᐇ㦂᪉ἲ 8 㢌ࡢ࢘ࢩࡣ➨ 1 ༸⬊Ἴヨ㦂⩌㸦n㸻4㸧➨ 2 ༸⬊Ἴヨ㦂⩌㸦n㸻4㸧ศࡅࡓࠋࡑࢀ ࡒࢀࡢ⩌ࡣヨ㦂⤊2 ᭶ᚋ㸪ࣛࢸࣥ᱁Ꮚἲࢆ⏝࠸࡚ࢡࣟࢫ࣮࢜ࣂ࣮ヨ㦂ࢆ⾜ࡗࡓࠋ ࡑࡢࡓࡵ㸪ࡑࢀࡒࢀࡢ⩌ࡣ8 㢌ࡢ࢘ࢩࡀ࡚ྵࡲࢀ࡚࠸ࡿ㸦n㸻8㸧ࠋ ➨1 ༸⬊Ἴヨ㦂⩌࡛ࡣ㸪⮬↛Ⓨ㛤ጞᚋ 12 㛫ࡈ㉸㡢Ἴ⏬ീデ᩿⨨ࢆ⏝࠸࡚༸ ᕢࢆほᐹࡋࡓࠋ༸๓༸⬊ࡢᾘኻࢆ☜ㄆࡋࡓⅬࢆDay 0 ࡋࡓࠋ༸⬊㑅ᢤࡀ⤊ࡋࡓ ⪃࠼ࡽࢀࡿDay 3 ࡽ 5 ࡲ࡛ࡢᮇ㛫㸪㉸㡢Ἴ⏬ീデ᩿⨨ࢆ⏝࠸࡚ᖍ༸⬊ࡢほᐹࢆ ⾜ࡗࡓࠋࡲࡓDay 6 ࡣ࣮࢝ࣛࢻࣉࣛ⏬ീデ᩿⨨ࢆ⏝࠸࡚ᖍ༸⬊ࡢᖹᆒ┤ᚄ࠾ࡼࡧ ༸⬊ቨ⾑ὶࢆ ᐃࡋࡓࠋࡲࡓDay 0㹼6 ࡢ㛫㸪1 ᪥ 1 ᅇṇ୰ᑿ㟼⬦ࡼࡾ᥇⾑ࢆ⾜ࡗࡓࠋ ➨2 ༸⬊Ἴ⩌࡛ࡣ㸪⮬↛Ⓨ⤊ᚋࡢ༸☜ㄆ᪥ࢆ Day 㸫7 ࡋࡓࠋ➨ 2 ༸⬊ἼⓎ⌧ ࢆㄏ㉳ࡍࡿࡓࡵ㸪Day 㸫2 W1DF ࡢ༸ㄏ㉳ࢆ┠ⓗ GnRH㸦100 μg 㓑㓟ࣇ࢙ࣝࢳ ࣞࣜࣥ㸪➽⫗ෆᢞ 㸹 ࢥࣥࢭ࣮ࣛࣝ㸹 ࣥࢱ࣮࣋ࢵࢺᰴᘧ♫㸪㜰㸪᪥ᮏ㸧ࢆᢞ ࡋࡓࠋGnRH ᢞ 36 㛫ᚋ㉸㡢Ἴ⏬ീデ᩿⨨ࢆ⏝࠸࡚ W1DF ࡀ༸ࡋ࡚࠸ࡿࡇ ࢆ☜ㄆࡋ㸪ࡑࡢⅬࢆDay 0 ࡋࡓࠋࡑࡢᚋ㸪➨ 1 ༸⬊Ἴ⩌ྠᵝ༸⬊㑅ᢤࡀ⤊ࡋ ࡓ⪃࠼ࡽࢀࡿDay 3 ࡽ 5 ࡲ࡛ࡢᮇ㛫㸪㉸㡢Ἴ⏬ീデ᩿⨨ࢆ⏝࠸࡚ᖍ༸⬊ࡢᖹᆒ ┤ᚄࢆ ᐃࡋࡓࠋࡲࡓDay 6 ࡣ࣮࢝ࣛࢻࣉࣛ⏬ീデ᩿⨨ࢆ⏝࠸࡚ᖍ༸⬊ࡢᖹᆒ┤
12 ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶࡢ ᐃࢆ⾜ࡗࡓࠋࡲࡓDay 0㹼6 ࡢ㛫㸪1 ᪥ 1 ᅇṇ୰ᑿ㟼⬦ࡼࡾ᥇ ⾑ࢆ⾜ࡗࡓࠋヨ㦂1 ࡢࣉࣟࢺࢥ࣮ࣝࡢᶍᘧᅗࢆ Fig. 2.1.♧ࡋࡓࠋ 2㸫2㸫3㸬༸⬊ᙧែࡢほᐹ Day 3ࡽ5ࡲ࡛ࡢᖍ༸⬊ࡢᖹᆒ┤ᚄࡣ㸪5.0MHzࡢࣜࢽࣉ࣮ࣟࣈࡀᒓࡋࡓ⤒┤⭠㉸ 㡢Ἴ⏬ീデ᩿⨨㸦HS̺101V㸪ᮏከ㟁Ꮚ㸪ឡ▱㸪᪥ᮏ㸧ࢆ⏝࠸࡚ ᐃࡋࡓࠋࣉ࣮ࣟࣈࢆ ᡭᣦࡶ┤⭠ෆᤄධࡋ㸪ᕥྑ୧ഃ༸ᕢࡢ᩿ᒙീࢆほᐹࡋࡓࠋᖍ༸⬊ࡢ༸⬊᩿ᒙ⏬ ീࡀ᭱࡞ࡿࡼ࠺㟼Ṇ⏬ࢆグ㘓ࡋ㸪ࣃࢯࢥࣥୖ㟼Ṇ⏬ࢆ⛣ືࡉࡏ㸪ᖍ༸⬊ࡣ㛗ᚄ ▷ᚄࡢᖹᆒ್ࢆᖹᆒ┤ᚄࡋ㸪ࢹ࣮ࢱࡋ࡚⏝࠸ࡓࠋ Day 6࠾ࡅࡿᖍ༸⬊ࡢ┤ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶࡣ㸪8.0MHzࡢࣜࢽࣉ࣮ࣟࣈ㸦1739̺ RSࣉ࣮ࣟࣈ㸹GEᶓἙ࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ᥋⥆ࡋࡓ࣮࢝ࣛࢻࣉࣛ㉸㡢Ἴ ⏬ീデ᩿⨨㸦LOGIQ Book XP㸪GE࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝࠸࡚࣮࢝ࣛ ࣮ࣔࢻࡼࡾほᐹࢆ⾜ࡗࡓࠋ༸⬊ቨ⾑ὶࡢ᳨ᰝ᪉ἲࡣࣉ࣮ࣟࣈࢆᡭᣦࡶ┤⭠ෆᤄ ධࡋ㸪ᖍ༸⬊ࡢ᩿ᒙീࢆほᐹࡋࡓࠋ༸⬊┤ᚄࡀ࡛᭱ࡘ⾑ὶ㠃✚ࡀ᭱࡞ࡿࡼ࠺࡞ 㟼Ṇ⏬ࢆグ㘓ࡋ㸪ᖹᆒ┤ᚄ㸪༸⬊ቨ⾑ὶ㠃✚㸦BFA㸧࠾ࡼࡧ⾑ὶศᕸྜ㸦BF%㸧ࢆ⟬ฟ ࡋࡓࠋ
BFAࡢ⟬ฟࡣ㸪グ㘓ࡋࡓᙳ⏬ീࢆࣃࢯࢥࣥෆྲྀࡾ㎸ࡳ㸪Photoshop 5.5㸦Adobe Systems Inc.㸪San Jose㸪CA㸪USA㸧ࢆ⏝࠸࡚⾑ὶ㡿ᇦࢆⓑ㯮⏬ീኚࡋ㸪Pop Imaging㸦ࢹࢪࢱ ࣭ࣝࣅ࣮ࣥࢢ࣭࢟ࢵࢬ㸪⚄ዉᕝ㸪᪥ᮏ㸧ࢆ⏝࠸࡚⾑ὶ㠃✚ࢆ⟬ฟࡋ㸪BFAࡋ࡚ࢹ࣮ࢱ ࢆ⏝࠸ࡓ㸦Fig. 2.2.㸧ࠋ
BF㸣ࡣ㸪ᙳࡋࡓ⏬ീࢆ⏝࠸࡚㸪༸⬊࿘ᑐࡍࡿ⾑ὶศᕸࡢྜࢆホ౯ࡍࡿࡶࡢ࡛࠶ ࡾ㸪༸⬊ቨෆ࡛ࡢ⾑⟶ศᕸࡢ⛬ᗘࢆ♧ࡍᣦᶆࡋ࡚⏝࠸ࡓࠋPhotoshop 5.5ࢆ⏝࠸࡚༸⬊
13 ࿘⾑ὶศᕸࡀࡳࡽࢀࡿ༸⬊ቨࡢ㛗ࡉࢆ⟬ฟࡋ㸪⾑ὶศᕸࡀࡳࡽࢀࡿ༸⬊ቨࡢ㛗ࡉࢆ༸⬊ ࿘࡛㝖ࡋࡓࡶࡢࢆBF㸣ࡋ࡚ࢹ࣮ࢱࢆ⏝࠸ࡓࠋ 2㸫2㸫4㸬᥇⾑ ヨ㦂ᮇ㛫୰ࡢDay 0ࡽ621G᥇⾑㔪࣊ࣃࣜࣥຍ┿✵᥇⾑⟶㸦࣋ࣀࢪ࢙ࢡࢺϩ┿✵᥇ ⾑⟶㸪TERUMO㸪ᮾி㸧ࢆ⏝࠸࡚ᑿ㟼⬦ࡼࡾ10mlࡢ᥇⾑ࢆ⾜ࡗࡓࠋ᥇ྲྀࡋࡓ⾑ᾮࡣịỈ ୰࡚ᛴ෭ᚋ㸪4Υ࡛2,000 g㸪20ศ㛫㐲ᚰࡋ㸪⾑₢ศ㞳ᚋ㸪⾑୰P4⃰ᗘ ᐃࡲ࡛㸫30Υ ࡚⤖ಖᏑࡋࡓࠋ 2㸫2㸫5㸬⾑୰P4⃰ᗘࡢ ᐃ ⾑୰P4⃰ᗘࡢ ᐃࡣ2ᢠయἲࡼࡿ㓝⣲ච ᐃἲ㸦EIA㸧ࡼࡾ ᐃࡋࡓࠋEIAࡢᡭ㡰 ࡣ௨๓ࡢሗ࿌ᇶ࡙࠸࡚⾜ࡗࡓ㸦64㸧ࠋ⾑₢ࡼࡾP4ࢆᢳฟࡍࡿࡓࡵ㸪⾑₢200μlࢪ࢚ࢳ ࢚࣮ࣝࢸࣝ㸦㛵ᮾᏛ㸪ᮾி㸪᪥ᮏ㸧1.0mlࢆຍ࠼㸪ᨩᢾᶵ㸦Mix-EVR㸪ࢱࢸࢵࢡ㸪ᇸ ⋢㸪᪥ᮏ㸧ࢆ⏝࠸࡚30ศ㛫࣎ࣝࢸࢡࢫࡋ㸪ࡑࡢᚋᐊ ࡚40ศ㛫㟼⨨ࡋࡓࠋࡑࡢᚋ㸪㸫30Υ ࡢ෭ᗜ⛣ືࡋ㸪24㛫ࡅ࡚⤖ࡉࡏࡓࠋ⤖ᚋ㸪ᾮ┦㒊ศࢆ5mlヨ㦂⟶ࢹ ࢝ࣥࢺࡋ㸪ࢻࣛࣇࢺෆࡢ࢛࣮࢘ࢱ࣮࣭ࣂࢫᾐࡋ㸪40Υࡲ࡛ୖ᪼ࡉࡏ࡞ࡀࡽࢪ࢚ࢳ࢚࣮ࣝ ࢸࣝࢆⓎࡉࡏࡓᚋ㸪50Υࡲ࡛ୖ᪼ࡉࡏ↓⮯࡞ࡿࡲ࡛ⓎࡉࡏࡓࠋⓎᚋ㸪ヨ㦂 ⟶ࢵࢭ࣭ࣂࢵࣇ࣮㸦7.12g㸹NA2HPO4㸪8.5g㸹NaCl㸪1.0g㸹BSA㸪pH 7.2㸹1N-HCL
࡛ㄪᩚ㸧ࢆ200μlຍ࠼㸪20ศ㛫࣎ࣝࢸࢡࢫࡋࡓࠋࡇࡢ ᐃἲࡼࡿP4ࡢᢳฟຠ⋡84㸣࡛࠶ࡗ
ࡓࠋEIAࡣᢠ࢘ࢧࢠIgGᢠయ㸦12.5μg/ml㸹⏕Ꮫᕤᴗᰴᘧ♫㸪ᮾி㸪᪥ᮏ㸧࡛⿕ࢃࢀࡓ 96✰ELISAࣉ࣮ࣞࢺ㸦Corning Glass Works㸪Corning㸪New York㸪USA㸧 ࢆ⏝࠸࡚⾜ࡗࡓࠋ ⃰ᗘ ᐃࡣ㸪ࢫࢱࣥࢲ࣮ࢻ࠾ࡼࡧࢧࣥࣉࣝ15μl100μlࡢP4࣏ࣜࢡ࣮ࣟࢼࣝᢠయ⁐ᾮ㸦OK-1㸧
14 ୖ┞ࡉࡏࡓࠋP4ࡢᶆ‽᭤⥺ࡣ0.05㹼50ng/mlࡢ⠊ᅖ࡛㸪 ᐃෆኚື࠾ࡼࡧ ᐃ㛫ኚືࡣࡑ ࢀࡒࢀ5.2㸣࠾ࡼࡧ7.4%࡛࠶ࡗࡓࠋ 2㸫2㸫6㸬⤫ィฎ⌮ ⤖ᯝࡣࡍ࡚ᖹᆒ್sᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ⾑୰P4⃰ᗘ᥎⛣ࡢᕪࡢ᳨ᐃࢆ⾜࠺ᙜࡓࡗ ࡚㸪2 ඖ㓄⨨ศᩓศᯒࢆ⾜࠸㸪యࡢኚࡢ᭷ពᛶࢆㄪ㸪స⏝ࡀ᭷ព࡛࠶ࡗࡓሙ ྜ㸪Bartlet ᳨ᐃࢆ⾜࠸ศᩓࡢᆒ୍ᛶࢆ᳨ᐃࡋࡓᚋ㸪Post-hoc ᳨ᐃࡋ࡚ Schefe ࡢከ⩌ẚ ㍑᳨ᐃࢆ⏝࠸ࡓࠋDay 6 ࠾ࡅࡿ༸⬊ቨ⾑ὶࡢẚ㍑ࡣ㸪F ᳨ᐃࢆ⾜࠸ศᩓࡢᆒ୍ᛶࢆ☜ ㄆࡋࡓᚋ㸪Student ࡢ t ᳨ᐃࢆ⏝࠸ࡓࠋ༴㝤⋡ 10㸣ᮍ‶㸦P < 0.1㸧ࢆഴྥ࠶ࡾࡋࠊ༴㝤 ⋡5㸣ᮍ‶㸦P < 0.05㸧࡛᭷ពᕪ࠶ࡾࡋࡓࠋ⤫ィฎ⌮ࡣ⤫ィฎ⌮ࢯࣇࢺ࡛࠶ࡿ StatView 㸦Abacus Concepts㸪Inc.㸪Berkeley㸪CA㸪USA㸧ࢆ⏝࠸ࡓࠋ 2㸫3㸬ヨ㦂 2㸸Ⓨ࠾ࡼࡧ༸ㄏ㉳࠾ࡅࡿ W1DF W2DF ࡢⓎ⫱ືែ㸪༸⬊ቨ⾑ὶ ࠾ࡼࡧ⾑୰E2⃰ᗘࡢẚ㍑ 2㸫3㸫1㸬౪ヨື≀ 2㸫2㸬❶ྠᵝࡢື≀ࢆ౪ヨࡋࡓࠋ 2㸫3㸫2㸬ᐇ㦂᪉ἲ ヨ㦂1 ྠࡌࣉࣟࢺࢥ࣮࡛ࣝ➨ 1 ༸⬊Ἴヨ㦂⩌➨ 2 ༸⬊Ἴヨ㦂⩌ࢆ‽ഛࡋࡓࠋ ࡑࡢᚋ㸪Ⓨ࠾ࡼࡧ༸ㄏ㉳ࢆ⾜࠺ࡇࢆ┠ⓗ㸪Day 6 PGF2α 㸦25 mg ࢺ࣓ࣟࢱ ࣑ࣥࢪࣀࣉࣟࢫࢺ㸪➽⫗ෆᢞ㸪ࣉࣟࢼࣝࢦࣥ F㸪ࢰ࢚ࢸࢫ㸪ᮾி㸪᪥ᮏ㸧ࢆᢞࡋ 㯤య㏥⾜ࢆㄏ㉳ࡋ㸪48 㛫ᚋࡢ Day 8 GnRH ࢆᢞࡋ༸ㄏ㉳ࢆ⾜ࡗࡓࠋDay 6㸪8
15 ࠾ࡼࡧ9 ࣮࢝ࣛࢻࣉࣛ⏬ീデ᩿⨨ࢆ⏝࠸࡚ᖍ༸⬊ࡢᖹᆒ┤ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶࡢ ᐃࢆ⾜ࡗࡓࠋࡲࡓDay 6㹼9 ࡢ㛫㸪1 ᪥ 1 ᅇṇ୰ᑿ㟼⬦ࡼࡾ᥇⾑ࢆ⾜ࡗࡓࠋヨ㦂 2 ࡢ ࣉࣟࢺࢥ࣮ࣝࡢᶍᘧᅗࢆFig. 2.5.♧ࡋࡓࠋ 2㸫3㸫3㸬 ༸⬊ᙧែࡢほᐹ Day 6㸪8࠾ࡼࡧ9࠾ࡅࡿᖍ༸⬊ࡢ┤ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶࡣ㸪8.0MHzࡢࣜࢽࣉ࣮ࣟ ࣈ㸦1739̺RSࣉ࣮ࣟࣈ㸹GEᶓἙ࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ᥋⥆ࡋࡓ࣮࢝ࣛࢻ ࣉࣛ㉸㡢Ἴ⏬ീデ᩿⨨㸦LOGIQ Book XP㸪GE࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝ ࠸࡚࣮࣮࢝ࣛࣔࢻࡼࡾほᐹࢆ⾜ࡗࡓࠋ༸⬊ቨ⾑ὶࡢ᳨ᰝ᪉ἲࡣࣉ࣮ࣟࣈࢆᡭᣦࡶ ┤⭠ෆᤄධࡋ㸪ᖍ༸⬊ࡢ᩿ᒙീࢆほᐹࡋࡓࠋ༸⬊┤ᚄࡀ࡛᭱ࡘ⾑ὶ㠃✚ࡀ᭱ ࡞ࡿࡼ࠺࡞㟼Ṇ⏬ࢆグ㘓ࡋ㸪ヨ㦂1ྠᵝࡢ᪉ἲ࡛ᖹᆒ┤ᚄ㸪BFA࠾ࡼࡧBF%ࢆ⟬ฟࡋࡓࠋ 2㸫3㸫4㸬᥇⾑ ヨ㦂ᮇ㛫୰ࡢDay 6㹼921G᥇⾑㔪࣊ࣃࣜࣥຍ┿✵᥇⾑⟶㸦࣋ࣀࢪ࢙ࢡࢺϩ┿✵᥇⾑ ⟶㸪TERUMO㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝࠸࡚ᑿ㟼⬦ࡼࡾ10mlࡢ᥇⾑ࢆ⾜ࡗࡓࠋ᥇ྲྀࡋࡓ⾑ᾮࡣ ịỈ୰࡚ᛴ෭ᚋ㸪4Υ࡛2,000 g㸪20ศ㛫㐲ᚰࡋ㸪⾑₢ศ㞳ᚋ㸪⾑୰P4⃰ᗘ࠾ࡼࡧ⾑୰E2 ⃰ᗘ ᐃࡲ࡛㸫30Υ࡚⤖ಖᏑࡋࡓࠋ 2㸫3㸫5㸬⾑୰P4࠾ࡼࡧE2⃰ᗘࡢ ᐃ ⾑୰P4⃰ᗘࡣヨ㦂1ྠᵝࡢᡭἲ࡛EIAࡼࡾ ᐃࡋࡓࠋࡇࡢ ᐃἲࡼࡿP4ࡢᢳฟຠ⋡ ࡣ80㸣࡛࠶ࡗࡓࠋ ᐃෆኚື࠾ࡼࡧ ᐃ㛫ኚືࡣࡑࢀࡒࢀ4.7㸣࠾ࡼࡧ6.5㸣࡛࠶ࡗࡓࠋ⾑ ୰E2⃰ᗘࡣDay 8ࡢࡳ ᐃࢆ⾜ࡗࡓࠋ⾑₢ࡼࡾE2ࢆᢳฟࡍࡿࡓࡵ㸪⾑₢2.0mlࢪ࢚ࢳࣝ
16 ᮏ㸧ࢆ⏝࠸࡚60ศ㛫࣎ࣝࢸࢡࢫࡋ㸪ࡑࡢᚋᐊ ࡚60ศ㛫㟼⨨ࡋࡓࠋࡑࡢᚋ㸫30Υࡢ෭ ᗜ⛣ືࡋ㸪24㛫ࡅ࡚⤖ࡉࡏࡓࠋ⤖ᚋ㸪ᾮ┦㒊ศࢆ10mlヨ㦂⟶ࢹ࢝ࣥࢺ ࡋ㸪ࢻࣛࣇࢺෆࡢ࢛࣮࢘ࢱ࣮࣭ࣂࢫᾐࡋ㸪40Υࡲ࡛ୖ᪼ࡉࡏ࡞ࡀࡽࢪ࢚ࢳ࢚࣮ࣝࢸࣝࢆ Ⓨࡉࡏࡓᚋ㸪50Υࡲ࡛ୖ᪼ࡉࡏ↓⮯࡞ࡿࡲ࡛ⓎࡉࡏࡓࠋⓎᚋ㸪ヨ㦂⟶ ࢵࢭ࣭ࣂࢵࣇ࣮ࢆ200μlຍ࠼㸪20ศ㛫࣎ࣝࢸࢡࢫࡋࡓࠋࡇࡢᡭἲࡼࡾ⾑₢୰ࡢE2ࢆ10 ಸ⃰⦰ࡋࡓࠋࡇࡢ ᐃἲࡼࡿE2ࡢᢳฟຠ⋡ࡣ80㸣࡛࠶ࡗࡓࠋ⃰ᗘ ᐃࡣ㸪ࢫࢱࣥࢲ࣮ࢻ ࠾ࡼࡧࢧࣥࣉࣝ15μl100μlࡢE2࣏ࣜࢡ࣮ࣟࢼࣝᢠయ⁐ᾮ㸦AS-A㸧㸦1㸸20,000㸧ࢆΰࡋ4Υ ࡚24㛫┞ࡉࡏࡓࠋࡑࡢᚋ㸪100μlࡢhorseradish peroxidase㸦1㸸150,000㸧ࢆΰࡋ㸪4Υ ࡚2㛫┞ࡉࡏࡓࠋE2ࡢᶆ‽᭤⥺ࡣ2㹼2,000 pg/mlࡢ⠊ᅖ࡛㸪 ᐃෆኚື࠾ࡼࡧ ᐃ㛫 ኚືࡣࡑࢀࡒࢀ6.5㸣࠾ࡼࡧ7.6%࡛࠶ࡗࡓࠋ 2㸫3㸫6㸬⤫ィฎ⌮ ⤖ᯝࡣࡍ࡚ᖹᆒ್sᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ⾑୰P4⃰ᗘ㸪ᖍ༸⬊ࡢᖹᆒ┤ᚄ࠾ࡼࡧ༸ ⬊ቨ⾑ὶ᥎⛣ࡢẚ㍑ࡣ㸪2 ඖ㓄⨨ศᩓศᯒࢆ⾜࠸㸪యࡢኚࡢ᭷ពᛶࢆㄪ㸪 స⏝ࡀ᭷ព࡛࠶ࡗࡓࡶࡢᑐࡋ࡚㸪Bartlet ᳨ᐃࢆ⾜࠸ศᩓࡢᆒ୍ᛶࢆ᳨ᐃࡋࡓᚋ㸪 Post-hoc ᳨ᐃࡋ࡚ Schefe ࡢከ⩌ẚ㍑᳨ᐃࢆ⾜ࡗࡓࠋDay 8 ࠾ࡅࡿ⾑୰ E2⃰ᗘࡢ᳨ᐃ
ࡣF ᳨ᐃࡼࡾศᩓࡢᆒ୍ᛶࢆ᳨ᐃࡋ㸪Student ࡢ t ᳨ᐃࢆ⾜ࡗࡓࠋP < 0.1 ࢆഴྥ࠶ࡾ ࡋࠊP < 0.05 ࡛᭷ពᕪ࠶ࡾࡋࡓࠋ⤫ィฎ⌮ࡣ⤫ィฎ⌮ࢯࣇࢺ࡛࠶ࡿ StatView㸦Abacus
Concepts㸪Inc.㸪Berkeley㸪CA㸪USA㸧ࢆ⏝࠸ࡓࠋ
2㸫4㸬ヨ㦂 3㸸W1DF W2DF ࠾ࡅࡿ༸๓ࡢ༸⬊ෆࢫࢸࣟࢻ࣍ࣝࣔࣥ⃰ᗘ࠾ࡼࡧ 㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿᅉᏊࡢẚ㍑
17 2㸫4㸫1㸬౪ヨື≀ ⮫ᗋⓗᗣ࡛㸪ṇᖖ࡞Ⓨ࿘ᮇࢆ᭷ࡍࡿ࣍ࣝࢫࢱࣥ✀㠀Ἢங∵7 㢌㸦ᖹᆒᖺ㱋㸸 4.0 r 1.1 ṓ㸪ᖹᆒsᶆ‽ㄗᕪ) ࢆ⏝࠸ࡓࠋ ࢘ࢩࡣᖏᗈ␆⏘Ꮫ␆⏘ࣇ࣮ࣝࢻ⛉Ꮫࢭࣥ ࢱ࣮ෆࡢࣃࢻࢵࢡ࡚㸪ࢥ࣮ࣥࢧ࣮ࣞࢪ㸪ⲡ࠾ࡼࡧ⃰ཌ㣫ᩱࡀΰྜࡉࢀࡓ TMR ࡀ ⤥ࡉࢀ㸪⮬⏤㣧Ỉ࡛ࡁࡿ≧ἣ࡛㣫㣴ࡉࢀ࡚࠸ࡓࠋᐇ㦂ෆᐜࡣᖏᗈ␆⏘Ꮫࡢᐇ㦂⌮ ጤဨ࡚ᢎㄆࡉࢀ࡚࠸ࡿࠋ 2㸫4㸫2 㸬ᐇ㦂᪉ἲ ᐇ㦂ࡢࣉࣟࢺࢥ࣮ࣝࡣヨ㦂 2 ྠᵝ࡛࠶ࡿࠋᮏヨ㦂࡛ࡣ LH ࢧ࣮ࢪ┤๓㸦PGF2αᢞ 40 㛫ᚋ㸸Day 8㸧࠾ࡼࡧ LH ࢧ࣮ࢪᚋ㸦GnRH ᢞ 24 㛫ᚋ㸸Day 9㸧ࡢᖍ༸⬊ࡢᛶ ㉁ࢆヲ⣽ホ౯ࡍࡿࡓࡵ㸪༸⬊ᾮ࠾ࡼࡧ㢛⢏ᒙ⣽⬊ࢆ᥇ྲྀࡋホ౯ࡋࡓࠋᮏヨ㦂࡛ࡣ௨ୗ 4 ヨ㦂⩌ࢆタࡅࡓ㸪㸯㸧LH ࢧ࣮ࢪ᭚㟢๓➨ 1 ༸⬊Ἴ⩌㸦n㸻7㸧㸪㸰㸧LH ࢧ࣮ࢪ᭚㟢๓➨ 2 ༸⬊Ἴ⩌㸦n㸻7㸧㸪㸱㸧LH ࢧ࣮ࢪ᭚㟢ᚋ➨ 1 ༸⬊Ἴ⩌㸦n㸻6㸧㸪㸲㸧LH ࢧ࣮ࢪ᭚㟢ᚋ ➨2 ༸⬊Ἴ⩌㸦n㸻6㸧࡛࠶ࡿࠋ㸯㸧㸰㸧ࡢ⩌ࡣࢡࣟࢫ࣮࢜ࣂ࣮ヨ㦂ࡼࡾ 7 㢌ࡢ࢘ ࢩࡀ࡚ྵࡲࢀ࡚࠸ࡿࠋ㸱㸧㸲㸧ࡢ⩌㛵ࡋ࡚ࡣ1 㢌ࡢ࢘ࢩ࡛㸪㢛⢏ᒙ⣽⬊ࡢᅇ㔞 ࡀᑡ࡞ࡃࠊࡑࡢ࢘ࢩࢆヨ㦂ࡼࡾ㝖እࡋࡓࡓࡵࠊ6 㢌ࢆ⏝࠸ࡓࢡࣟࢫ࣮࢜ࣂ࣮ヨ㦂࡞ࡗ ࡚࠸ࡿࠋࡲࡓ㸪ࡑࢀࡒࢀࡢ⩌ࡢ࢘ࢩࡢᖍ༸⬊ࡣู࡚ࡢⓎ࿘ᮇ࡛᥇ᮦࢆ⾜࡞ࡗ࡚࠸ ࡿࠋヨ㦂3 ࡢࣉࣟࢺࢥ࣮ࣝࡢᶍᘧᅗࢆ Fig. 2.9.♧ࡋࡓࠋ 2㸫4㸫3㸬ᖍ༸⬊ࡢ྾ᘬ㢛⢏ᒙ⣽⬊ࡢᅇ ᖍ༸⬊ࡢ྾ᘬࡣ㸪㉸㡢Ἴ⏬ീデ᩿⨨㸦HS-1500㸪ᮏከ㟁Ꮚ㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝࠸࡚ ㉸㡢Ἴ⏬ീほᐹୗ࡛ࡢ⤒⭐༸⬊✸่ࡼࡾ⾜ࡗࡓࠋ㉸㡢Ἴ⏬ീほᐹࡣ㸪7.5MHzࡢ⭐⏝ࣉ ࣮ࣟࣈ㸦HCV-3710MV㸪ᮏከ㟁Ꮚ㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋࣉ࣮ࣟࣈࡢୖ㒊ࡣ㸪
18 ⣙40cmࡢ✸่⏝ࡢ㔪㸦ࢹࢫ࣏࣮ࢨࣈࣝ᥇༸㔪 A-ࢱࣉ㸪࣑ࢧ࣡་⛉ᕤᴗ㸪Ⲉᇛ㸪᪥ᮏ㸧 ࢆ╔࡛ࡁࡿࡼ࠺࡞ࡗ࡚࠸ࡿࠋ༸⬊྾ᘬࡢ๓‽ഛࡋ࡚㸪⭐ෆ࠾ࡼࡧእ㝜㒊࿘㎶ࢆࡼࡃ Ὑίࡋࡓࠋḟࣉ࣮ࣟࣈࢆ⭐ࡼࡾᤄධࡋ㸪༸ᕢࢆ┤⭠ࡼࡾᤄධࡋࡓᡭ࡛⭐ቨ㉺ࡋᢲࡋᙜ ࡚ࡓࠋ㉸㡢Ἴ⏬ീデ᩿⨨ࡢ⏬㠃ୖࡣ㸪᥇༸㔪ࡢ࢞ࢻࣛࣥࡀ♧ࡉࢀ࡚࠸ࡿࡢ࡛㸪ࡇ ࡢ࢞ࢻࣛࣥୖ྾ᘬࡍࡿ༸⬊ࡀᥥࡉࢀࡿࡼ࠺ࡋࡓࠋ⏬ീࢆほᐹࡋ࡞ࡀࡽ㸪18Gࡢ 㔪ࢆ⭐ቨࢆࡋ࡚༸⬊⭍่ධࡋ㸪༸⬊ᾮ࠾ࡼࡧ㢛⢏ᒙ⣽⬊ࢆ᥇༸㔪㐃⤖ࡉࡏࡓࢩࣜ ࣥࢪࡼࡾ྾ᘬࡋࡓࠋ ༸⬊ᾮࡣ5.0mlࢳ࣮ࣗࣈධࢀ㸪ịỈ୰࡚ᛴ෭ᚋ㸪ᐇ㦂ᐊෆࡢᐇ య㢧ᚤ㙾ୗ࡛㢛⢏ᒙ⣽⬊ࢆᅇࡋࡓࠋ㢛⢏ᒙ⣽⬊ࡣTriPure Isolation Reagent㸦Roche Diagnostics GmbH㸪Mannheim㸪Germany㸧1.0mlࢆຍ࠼࡚⁐ゎࡉࡏ㸪㸫80Υ࡚mRNA ᢳฟࡲ࡛ಖᏑࡋࡓࠋ㢛⢏ᒙ⣽⬊ᅇᚋ㸪༸⬊ᾮࡣ4Υ㸪500 g࡛10ศ㛫㐲ᚰࢆ⾜࠸ୖΎࢆศ 㞳ࡋࡓᚋ㸪EIAࡼࡿP4㸪E2࠾ࡼࡧࣥࢻࣟࢫࢸࣥࢪ࢜ࣥ㸦A4㸧⃰ᗘ ᐃࡲ࡛㸫30Υ࡚
⤖ಖᏑࡋࡓࠋ 2㸫4㸫4㸬༸⬊ᾮP4㸪E2࠾ࡼࡧA4⃰ᗘࡢ ᐃ ༸⬊ᾮࡼࡾࢫࢸࣟࢻ࣍ࣝࣔࣥࢆᢳฟࡍࡿࡓࡵ㸪⾑₢40μlࢪ࢚ࢳ࢚࣮ࣝࢸࣝ㸦㛵ᮾ Ꮫ㸪ᮾி㸪᪥ᮏ㸧1.0mlࢆຍ࠼㸪ᨩᢾᶵ㸦Mix-EVR㸪ࢱࢸࢵࢡ㸪ᇸ⋢㸪᪥ᮏ㸧ࢆ⏝࠸ ࡚30ศ㛫࣎ࣝࢸࢡࢫࡋ㸪ࡑࡢᚋᐊ ࡚40ศ㛫㟼⨨ࡋࡓࠋࡑࡢᚋ㸫30Υࡢ෭ᗜ࡚⛣ື ࡋ24㛫ࡅ࡚⤖ࡉࡏࡓࠋ⤖ᚋ㸪ᾮ┦㒊ศࢆ5mlヨ㦂⟶ࢹ࢝ࣥࢺࡋ㸪ࢻࣛࣇ ࢺෆࡢ࢛࣮࢘ࢱ࣮࣭ࣂࢫᾐࡋ㸪40Υࡲ࡛ୖ᪼ࡉࡏ࡞ࡀࡽࢪ࢚ࢳ࢚࣮ࣝࢸࣝࢆⓎࡉࡏࡓ ᚋ㸪50Υࡲ࡛ୖ᪼ࡉࡏ↓⮯࡞ࡿࡲ࡛ⓎࡉࡏࡓࠋⓎᚋ㸪ヨ㦂⟶ࢵࢭ࣭ࣂ ࢵࣇ࣮ࢆ200μlຍ࠼㸪20ศ㛫࣎ࣝࢸࢡࢫࡋࡓࠋࡇࡢᡭἲࡼࡾ༸⬊ᾮ୰ࡢࢫࢸࣟࢻ࣍ࣝ ࣔࣥࢆ5ಸᕼ㔘ࡋࡓࠋࡇࡢ ᐃἲࡼࡿP4㸪E2࠾ࡼࡧA4ࡢᢳฟຠ⋡ࡣࡑࢀࡒࢀ82㸣㸪81㸣 ࠾ࡼࡧ85㸣࡛࠶ࡗࡓࠋP4࠾ࡼࡧE2ࡢ⃰ᗘ ᐃࡣ❶ࡢ2㸫2㸫5.࠾ࡼࡧ2㸫3㸫5.࡛グࡋࡓ࠾ࡾ
19 ࡛࠶ࡿࠋP4ࡢ ᐃෆኚື࠾ࡼࡧ ᐃ㛫ኚືࡣࡑࢀࡒࢀ5.5%࠾ࡼࡧ7.4%㸪E2ࡢ ᐃෆኚື࠾ ࡼࡧ ᐃ㛫ኚືࡣ6.2㸣࠾ࡼࡧ8.6%࡛࠶ࡗࡓࠋA4ࡢ⃰ᗘ ᐃࡣࢫࢱࣥࢲ࣮ࢻ࠾ࡼࡧࢧࣥࣉ ࣝ15μl100μlࡢA4࣏ࣜࢡ࣮ࣟࢼࣝᢠయ⁐ᾮ㸦A-3-CMO-BSA㸪ࢥࢫ࣭ࣔࣂ࢜㸪ᮾி㸪᪥ ᮏ㸧㸦1㸸750,000㸧100μlࡢA-3-CMO-horseradish peroxidase㸦ࢥࢫ࣭ࣔࣂ࢜㸪ᮾி㸪᪥ ᮏ㸧㸦1㸸500,000㸧ࢆΰࡋ4Υ࡚24㛫┞ࡉࡏࡓࠋA4ࡢᶆ‽᭤⥺ࡣ7.8㹼8,000 pg/ml ࡢ⠊ᅖ࡛㸪 ᐃෆኚື࠾ࡼࡧ ᐃ㛫ኚືࡣࡑࢀࡒࢀ4.3㸣࠾ࡼࡧ5.2%࡛࠶ࡗࡓࠋ 2㸫4㸫5㸬RNAᢳฟࣜࣝࢱ࣒PCR Total RNAࡢᢳฟࡣ௨ୗࡢᡭ㡰࡛⾜ࡗࡓࠋゎࡋࡓࢧࣥࣉࣝࢡ࣒ࣟࣟ࣍ࣝ⁐ᾮࢆ200μl ຍ࠼࡚㸪15⛊㛫࣎ࣝࢸࢡࢫࡋ3ศ㛫ᐊ ࡛㟼⨨ࡋࡓᚋ㸪4Υ㸪36,000 g࡚15ศ㛫㐲ᚰศ㞳 ࢆ⾜ࡗࡓࠋࢡ࣒ࣟࣟ࣍ࣝᾮ┦TriPure Isolation Reagent ⁐ᾮ┦ศ㞳ࡋ࡚࠸ࡿࡇࢆ☜ㄆ ࡋ㸪ࢡ࣒ࣟࣟ࣍ࣝᒙࢆูࡢ1.5mlࢳ࣮ࣗࣈ⛣ࡍࠋࡇࢀ500μlࡢࢯࣉࣟࣃࣀ࣮ࣝ⁐ᾮ 㸦3-Propanol㸧ࢆຍ࠼㸪15⛊㛫࣎ࣝࢸࢡࢫࡋ㸪10ศ㛫ᐊ ࡚㟼⨨ࡋࡓᚋ㸪4Υ㸪36,000 g ࡚15ศ㛫㐲ᚰศ㞳ࢆ⾜ࡗࡓࠋRNAࡢ࣌ࣞࢵࢺࡢᏑᅾࢆ☜ㄆࡋࡓᚋ㸪ࢯࣉࣟࣃࣀ࣮ࣝ⁐ ᾮࢆ㝖ཤࡋ㸪500μlࡢ80㸣࢚ࢱࣀ࣮ࣝࢆຍ࠼㸪Ὑίࡋ㸪4Υ㸪36,000 g࡚5ศ㛫㐲ᚰศ㞳ࢆ ⾜ࡗࡓࠋ࢚ࢱࣀ࣮ࣝࢆ㝖ཤᚋ㸪ᐊ ࡛5ศ㛫࣌ࣞࢵࢺࢆ⇱ࡉࡏ㸪6μlࡢࢪ࢚ࢳࣝࣆ࣮ࣟ࢝ ࣎ࢿ࣮ࢺ㸦DEPC㸧ฎ⌮Ỉࢆຍ࠼ࡓࠋ
㏫㌿ࡣSuperscript VIRO Reaction Mix 㸦Invitrogen.㸪Carlsbad㸪CA㸪USA㸧ࢆ⏝࠸ࡓࠋ ࡲࡎ㸪Nano-Drop 2000㸦Thermo scientific㸪Wilmington㸪DE㸪USA㸧ࢆ⏝࠸࡚RNA⃰ᗘࢆ⟬ ฟࡋࡓࠋḟ㸪RQ1 RNase-Free DNase (Promega㸪Madison㸪WI㸪USA) ࢆ⏝࠸࡚DNaseฎ ⌮ࢆ⾜ࡗࡓࠋRNAࡀ1μgྵࡲࢀࡿRNA⁐ᾮ㸪1μlࡢDNase 10×Reaction Buffer࠾ࡼࡧ2μlࡢ RNase-Free DNaseࢆΰࡋ㸪37Υ࡛30ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋ㸪1μlࡢDNase Stop Solutionࢆຍ࠼65Υ࡛10ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋDNaseฎ⌮ࡋࡓࢧࣥࣉࣝ5×VILO
20
Reaction Mixࢆ4μl㸪10×Super Script Enzymeࢆ2μlຍ࠼㸪⥲㔞ࡀ20μl࡞ࡿࡼ࠺DEPCฎ⌮ Ỉࢆຍ࠼ࡓᚋ㸪25Υ࡛10ศ㛫㸪42Υ࡛60ศ㛫࠾ࡼࡧ85Υ࡛5ศ㛫࣮ࣥ࢟ࣗ࣋ࢺࡋ㸪cDNA ࢆసᡂࡋࡓࠋ
2㸫4㸫6㸬ࣜࣝࢱ࣒PCRࡼࡿmRNAⓎ⌧㔞ࡢ ᐃ
ࣜࣝࢱ࣒PCRࡣTli RNaseH Plus㸦࢟ࢤࣥ㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝ࡋ㸪Roter-Gene Q 㸦࢟ ࢤࣥ㸪ᮾி㸪᪥ᮏ㸧 ࢆ⏝࠸࡚⾜ࡗࡓࠋ7.5μlࡢSYBR Green Master Mix㸪5.75μlࡢDEPCฎ ⌮Ỉ㸪ForwardReverseࡢࣉ࣐࣮ࣛࢆྛ0.375μlຍ࠼㸪᭱ᚋ1.0μlࡢࢧࣥࣉࣝࢆຍ࠼࡚ ᛂࡉࡏࡓࠋ ᐃࡍࡿmRNAࡣ㸪LHཷᐜయ㸦LHr㸧㸪࣐ࣟࢱ࣮ࢮ㸦CYP19A1㸧㸪ࢩࢡࣟ࢜ ࢟ࢩࢤࢼ࣮ࢮ̺2㸦COX-2㸧㸪PGE2ྜᡂ㓝⣲㸦PGES㸧㸪PGF2αྜᡂ㓝⣲㸦PGFS㸧㸪ㄏᑟᆺ
୍㓟❅⣲ྜᡂ㓝⣲㸦iNOS㸧㸪⾑⟶ෆ⓶ᆺ୍㓟❅⣲ྜᡂ㓝⣲㸦eNOS㸧㸪⾑⟶ෆ⓶ቑṪ ᅉᏊ㸦VEGF㸧120㸪VEGF164㸪⥺⥔ⱆ⣽⬊ቑṪᅉᏊ㸦FGF㸧-2㸪Steroidogenic acute regulatory protein㸦StAR㸧㸪ࢳࢺࢡ࣮࣒ࣟP450ഃ㙐ษ᩿㓝⣲㸦cytochrome P450 cholesterol side-chain cleavage enzymes㸹 P450-scc㸧㸪3β-Ỉ㓟ᇶ⬺Ỉ⣲㓝⣲㸦3β-hydroxy steroid dehydrogenase㸹 3β-HSD㸧࠾ࡼࡧࢢࣜࢭࣝࣝࢹࣄࢻ3ࣜࣥ㓟⬺Ỉ⣲㓝⣲㸦glyceraldehyde-3-phosphate dehydrogenase㸹GAPDH㸧࡛࠶ࡿࠋGAPDHࡣෆ㒊ᶆ‽㑇ఏᏊࡋ࡚⏝࠸㸪ࡑࡢⓎ⌧ࡢ ྜ࡛ồࡵࡓࠋࣉ࣐࣮ࣛࡣPrimer-3ࢆ⏝࠸࡚సᡂࡋࡓࠋࣜࣝࢱ࣒PCRࡢᛂ᮲௳ࡣ94Υ ࡛10ศ㛫ฎ⌮ࡋࡓᚋ㸪94Υ࡛30⛊㸪ྛࠎࡢࢽ࣮ࣜࣥࢢ ᗘ࡛30⛊࠾ࡼࡧ72Υ࡛30⛊ࡢࢧ ࢡࣝࢆ40ᅇ⾜ࡗࡓࠋࡑࢀࡒࢀࡢmRNAࡢࣉ࣐࣮ࣛࡢࢩ࣮ࢡ࢚ࣥࢫࢆ♧ࡋࡓ⾲ࢆTable 2.3.♧ࡋࡓࠋLHࢧ࣮ࢪ᭚㟢๓ࡢ㢛⢏ᒙ⣽⬊ᑐࡋ࡚ࡣLHr࠾ࡼࡧCYP19A1 mRNAⓎ⌧㔞 ࢆ ᐃࡋࡓࠋLHࢧ࣮ࢪ᭚㟢ᚋࡢ㢛⢏ᒙ⣽⬊ᑐࡋ࡚ࡣCOX-2㸪PGES㸪PGFS㸪iNOS㸪eNOS㸪 VEGF120㸪VEGF164㸪FGF-2㸪StAR㸪P450-scc࠾ࡼࡧ3β-HSD mRNAⓎ⌧㔞ࢆ ᐃࡋࡓࠋ
21 2㸫4㸫7㸬⤫ィฎ⌮ ⤖ᯝࡣࡍ࡚ᖹᆒ್sᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ༸⬊ᾮ୰ࡢᛶࢫࢸࣟࢻ࣍ࣝࣔࣥ⃰ᗘ࠾ࡼ ࡧ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡍࡿmRNA Ⓨ⌧㔞ࡢẚ㍑㛵ࡋ࡚ࡣ㸪ࡲࡎ F ᳨ᐃࡼࡾศᩓࡢᆒ୍ ᛶࢆ᳨ᐃࡋ㸪➼ศᩓࡀㄆࡵࡽࢀࡓሙྜ㸪Student ࡢ t ᳨ᐃࢆ⾜࠸㸪➼ศᩓࡀㄆࡵࡽࢀ࡞ ࡗࡓሙྜࡣMann–Whitney ࡢ U ᳨ᐃࢆ⾜ࡗࡓࠋP < 0.1 ࢆഴྥ࠶ࡾࡋࠊP < 0.05 ࡛᭷ព ᕪ࠶ࡾࡋࡓࠋ⤫ィฎ⌮ࡣ⤫ィฎ⌮ࢯࣇࢺ࡛࠶ࡿ StatView㸦Abacus Concepts㸪Inc.㸪 Berkeley㸪CA㸪USA㸧ࢆ⏝࠸ࡓࠋ 2㸫5㸬ヨ㦂 4㸸W1DF W2DF ࡢ༸ᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែ㸪㯤య⾑ὶ࠾ࡼࡧ⾑୰ P4⃰ᗘ᥎⛣ࡢẚ㍑ 2㸫5㸫1㸬౪ヨື≀ ⮫ᗋⓗᗣ࡛㸪ṇᖖ࡞Ⓨ࿘ᮇࢆ᭷ࡍࡿ࣍ࣝࢫࢱࣥ✀㠀Ἢங∵6 㢌㸦ᖹᆒᖺ㱋㸸 4.2 r 2.1 ṓ㸪ᖹᆒsᶆ‽ㄗᕪ) ࢆ⏝࠸ࡓࠋ ࢘ࢩࡣᖏᗈ␆⏘Ꮫ␆⏘ࣇ࣮ࣝࢻ⛉Ꮫࢭࣥ ࢱ࣮ෆࡢࣃࢻࢵࢡ࡚㸪ࢥ࣮ࣥࢧ࣮ࣞࢪ㸪ⲡ࠾ࡼࡧ⃰ཌ㣫ᩱࡀΰྜࡉࢀࡓ TMR ࡀ ⤥ࡉࢀ㸪⮬⏤㣧Ỉ࡛ࡁࡿ≧ἣ࡛㣫㣴ࡉࢀ࡚࠸ࡓࠋᐇ㦂ෆᐜࡣᖏᗈ␆⏘Ꮫࡢᐇ㦂⌮ ጤဨ࡚ᢎㄆࡉࢀ࡚࠸ࡿࠋ 2㸫5㸫2 㸬ᐇ㦂᪉ἲ 6 㢌ࡢ࢘ࢩࡣ W1DF ༸ᚋ㯤య㸦W1CL㸹n㸻3㸧 W2DF ༸ᚋ㯤య㸦W2CL㸹n㸻3㸧 ศࡅࡓࠋࡑࢀࡒࢀࡢ⩌ࡣヨ㦂⤊2 ᭶ᚋ㸪ࣛࢸࣥ᱁Ꮚἲࢆ⏝࠸࡚ࢡࣟࢫ࣮࢜ࣂ࣮ ヨ㦂ࢆ⾜ࡗࡓࠋࡑࡢࡓࡵ㸪ࡑࢀࡒࢀࡢ⩌ࡣ6 㢌ࡢ࢘ࢩࡀ࡚ྵࡲࢀ࡚࠸ࡿ㸦n㸻6㸧ࠋ
22
ヨ㦂ࡢࣉࣟࢺࢥ࣮ࣝࡣヨ㦂2 ྠᵝ࡛࠶ࡿࡀ㸪ᮏヨ㦂࡛ࡣᖍ༸⬊ࢆ྾ᘬࡏࡎ㸪༸ ࡉࡏ㸪ࡑࡢᚋᙧᡂࡉࢀࡿ㯤యࢆほᐹࡋࡓࠋ
W1DF W2DF ࡶ㸪Day 8 GnRH ࢆᢞࡋ㸪ࡑࡢ 24 㛫࠾ࡼࡧ 36 㛫ᚋ㉸ 㡢Ἴ⏬ീデ᩿⨨࡛༸☜ㄆࢆ⾜࠸㸪24 㛫ᚋ༸ࡏࡎ㸪36 㛫ᚋࡲ࡛༸ࡀ☜ㄆ ࡉࢀࡓⅬࢆDay 10 ࡋࡓࠋDay 9 ࠾ࡅࡿᖍ༸⬊ࡢᖹᆒ┤ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶ㸪Day 12㸪15㸪18 ࠾ࡼࡧ 21 ࠾ࡅࡿ㯤యࡢ㯤య᩿㠃✚࠾ࡼࡧ㯤య⾑ὶࢆ࣮࢝ࣛࢻࣉࣛ㉸㡢Ἴ ⏬ീデ᩿⨨ࢆ⏝࠸࡚ ᐃࡋࡓࠋ ࡲࡓDay 10㹼21 ࡢ㛫㸪1 ᪥ 1 ᅇṇ୰ᑿ㟼⬦ࡼࡾ᥇⾑ࢆ⾜ࡗࡓࠋヨ㦂 4 ࡢࣉࣟࢺࢥ࣮ ࣝࡢᶍᘧᅗࢆFig. 2.15.♧ࡋࡓࠋ 2㸫5㸫3㸬༸⬊࠾ࡼࡧ㯤యᙧែࡢほᐹ Day 9࠾ࡅࡿᖍ༸⬊ࡢᖹᆒ┤ᚄ࠾ࡼࡧ༸⬊ቨ⾑ὶ㸪Day 12㸪15㸪18࠾ࡼࡧ21࠾ࡅ ࡿ㯤యࡢ᩿㠃✚࠾ࡼࡧ㯤య⾑ὶࡣ㸪8.0MHzࡢࣜࢽࣉ࣮ࣟࣈ㸦1739̺RSࣉ࣮ࣟࣈ㸹GEᶓ Ἑ࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ᥋⥆ࡋࡓ࣮࢝ࣛࢻࣉࣛ㉸㡢Ἴ⏬ീデ᩿⨨㸦LOGIQ Book XP㸪GE࣓ࢹ࢝ࣝࢩࢫࢸ࣒㸪ᮾி㸪᪥ᮏ㸧ࢆ⏝࠸࡚࣮࣮࢝ࣛࣔࢻࡼࡾほᐹࢆ⾜ࡗ ࡓࠋ༸⬊ࡢホ౯ࡣヨ㦂1ྠᵝࡢ᪉ἲ࡛⾜࠸㸪༸⬊ቨ⾑ὶࡢホ౯ࡣBF㸣ࢆ⏝࠸ࡓࠋ㯤య ࡢ᩿㠃✚ࡣ㸪㯤యࡢ᩿ᒙ⏬ീࡀ᭱࡞ࡿࡼ࠺㟼Ṇ⏬ࢆグ㘓ࡋ㸪ࣃࢯࢥࣥୖ㟼Ṇ⏬ࢆ⛣ ືࡉࡏ㸪㯤యࡢ㛗ᚄ▷ᚄࡢᖹᆒ್ࡽᖹᆒ┤ᚄࢆ⟬ฟࡋ㸪㸦ᖹᆒ┤ᚄ㸭2㸧2×3.14ᙜ࡚ ࡣࡵ㯤య᩿㠃✚ࢆ⟬ฟࡋ㸪ࢹ࣮ࢱ⏝࠸ࡓࠋ 㯤య⾑ὶࡢ᳨ᰝ᪉ἲࡣ㸪㯤యࡢ᩿ᒙീࢆほᐹࡋ㸪㯤య᩿㠃✚ࡀ࡛᭱ࡘ⾑ὶ㠃✚ࡀ᭱ ࡞ࡿࡼ࠺࡞㟼Ṇ⏬ࢆグ㘓ࡋ㸪㯤య⾑ὶ㠃✚ࢆ⟬ฟࡋࡓࠋ 㯤య⾑ὶ㠃✚ࡢ⟬ฟࡣ㸪グ㘓ࡋࡓᙳ⏬ീࢆࣃࢯࢥࣥෆྲྀࡾ㎸ࡳ㸪Photoshop 5.5 㸦Adobe Systems Inc.㸪San Jose㸪CA㸪USA㸧ࢆ⏝࠸࡚⾑ὶ㡿ᇦࢆⓑ㯮⏬ീኚࡋ㸪Pop
23 Imaging㸦ࢹࢪࢱ࣭ࣝࣅ࣮ࣥࢢ࣭࢟ࢵࢬ㸪⚄ዉᕝ㸧ࢆ⏝࠸࡚⾑ὶ㠃✚ࢆ⟬ฟࡋ㸪㯤య⾑ ὶ㠃✚ࡋ࡚ࢹ࣮ࢱࢆ⏝࠸ࡓࠋ 2㸫5㸫4㸬᥇⾑ ヨ㦂ᮇ㛫୰ࡢDay 10㹼2121G᥇⾑㔪࣊ࣃࣜࣥຍ┿✵᥇⾑⟶㸦࣋ࣀࢪ࢙ࢡࢺϩ┿✵᥇ ⾑⟶㸪TERUMO㸪ᮾி㸧ࢆ⏝࠸࡚ᑿ㟼⬦ࡼࡾ10mlࡢ᥇⾑ࢆ⾜ࡗࡓࠋ᥇ྲྀࡋࡓ⾑ᾮࡣịỈ ୰࡚ᛴ෭ᚋ㸪4Υ࡛2,000 g㸪20ศ㛫㐲ᚰࡋ㸪⾑₢ศ㞳ᚋ㸪⾑୰P4⃰ᗘ ᐃࡲ࡛㸫30Υ ࡚⤖ಖᏑࡋࡓࠋ 2㸫5㸫5㸬⾑୰P4⃰ᗘࡢ ᐃ ⾑୰P4⃰ᗘࡢ ᐃࡣヨ㦂1ྠᵝࡢ᪉ἲ࡛⾜ࡗࡓࠋ ᐃෆኚື࠾ࡼࡧ ᐃ㛫ኚືࡣࡑࢀ ࡒࢀ6.4㸣࠾ࡼࡧ8.6%࡛࠶ࡗࡓࠋ 2㸫5㸫6㸬⤫ィฎ⌮ ⤖ᯝࡣࡍ࡚ᖹᆒ್sᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ㯤య᩿㠃✚㸪㯤య⾑ὶ㠃✚࠾ࡼࡧ⾑୰ P4 ⃰ᗘ᥎⛣ࢆẚ㍑ࡍࡿᙜࡓࡗ࡚ࡣ㸪2 ඖ㓄⨨ศᩓศᯒࢆ⾜࠸㸪యࡢኚࡢ᭷ពᛶࢆㄪ 㸪స⏝ࡀ᭷ព࡛࠶ࡗࡓሙྜ㸪Bartlet ᳨ᐃࢆ⾜࠸ศᩓࡢᆒ୍ᛶࢆ᳨ᐃࡋࡓᚋ㸪Post-hoc ᳨ᐃࡋ࡚Schefe ࡢከ⩌ẚ㍑᳨ᐃࢆ⏝࠸ࡓࠋ༸๓༸⬊ࡢᖹᆒ┤ᚄࢆẚ㍑ࡍࡿࡣ㸪ࡲ ࡎF ᳨ᐃࡼࡾศᩓࡢᆒ୍ᛶࢆ☜ㄆࡋࡓᚋ㸪Student ࡢ t ᳨ᐃࢆ⾜ࡗࡓࠋP < 0.1 ࢆഴྥ࠶ ࡾࡋࠊP < 0.05 ࡛᭷ពᕪ࠶ࡾࡋࡓࠋ⤫ィฎ⌮ࡣ⤫ィฎ⌮ࢯࣇࢺ࡛࠶ࡿ StatView 㸦Abacus Concepts㸪Inc.㸪Berkeley㸪CA㸪USA㸧ࢆ⏝࠸ࡓࠋ 2㸫6㸬⤖ᯝ
24 2㸫6㸫1㸬ヨ㦂1ࡢ⤖ᯝ ⾑୰P4⃰ᗘ᥎⛣ࢆFig. 2.3.♧ࡋࡓࠋヨ㦂ᮇ㛫୰࠾࠸࡚W2⩌࡛W1⩌ẚ㍑ࡋ࡚᭷ព 㧗ࡃ᥎⛣ࡋࡓࠋ ᖍ༸⬊ࡢᖹᆒ┤ᚄࡢ᥎⛣ࢆFig. 2.4.♧ࡋࡓࠋW1DFࡢᖹᆒ┤ᚄࡣヨ㦂ᮇ㛫୰㸪W2DF ẚ㍑ࡋ࡚ࡁࡃⓎ⫱ࡋ㸪≉Day 6᭷ពࡁࡗࡓࠋࡲࡓ㸪W1DF⩌ෆ࡛ࡣ㸪Day 3 ࠾ࡼࡧ4ࡽDay 6ࡅ࡚᭷ពࡁࡃⓎ⫱ࡋࡓࡀ㸪W2DF⩌ෆ࡛ࡣ㸪Day 3ࡽDay 6 ࡅ࡚ࡢࡳ᭷ពࡁࡃ࡞ࡗࡓࠋ
Day 6࠾ࡅࡿBFA࠾ࡼࡧBF㸣ࢆTable 2.1.♧ࡋࡓࠋBFA࠾ࡼࡧBF%ࡣW1DF࡛㧗࠸ഴ ྥࢆ♧ࡋࡓࠋ 2㸫6㸫2㸬ヨ㦂2ࡢ⤖ᯝ ᖍ༸⬊ࡢᖹᆒ┤ᚄࡢ᥎⛣ࢆFig. 2.6.♧ࡋࡓࠋᖹᆒ┤ᚄࡢ᥎⛣ࡣ㸪W1DFࡀW2DFẚ ㍑ࡋ࡚ヨ㦂ᮇ㛫୰ࢆ㏻ࡋ࡚᭷ព㧗ࡃ᥎⛣ࡋࡓࠋヨ㦂ᮇ㛫୰ࡢ⾑୰P4⃰ᗘ᥎⛣ࢆFig. 2.7. ♧ࡋࡓࠋDay 6࠾ࡅࡿ⾑୰P4⃰ᗘࡣW2DF࡛W1DFẚ㍑ࡋ࡚᭷ព㧗ࡗࡓࡀ㸪ࡑࡢ ᚋࡢ⾑୰P4⃰ᗘ᥎⛣୧⩌㛫࡛ᕪࡣ࡞ࡃ㸪PGF2Dᢞᚋᛴ⃭పୗࡋࡓࠋ ヨ㦂ᮇ㛫୰ࡢBFA࠾ࡼࡧBF%ࡢ᥎⛣ࢆFig. 2.8.♧ࡋࡓࠋBFAࡣ㸪W1DF࠾࠸࡚Day 9 ࡛W2DFẚ㍑ࡋ࡚᭷ព㧗ࡗࡓࡀ㸪Day 6࠾ࡼࡧ8࡛ࡣ୧⩌㛫࡛ᕪࡣ࡞ࡗࡓࠋࡲࡓ㸪 BFAࡢ᥎⛣ࢆ⩌ෆ࡛ẚ㍑ࡋࡓࡇࢁW1DF࠾ࡼࡧW2DFࡶDay 6ࡽ9ࡅ࡚᭷ព ቑຍࡋࡓࠋࡲࡓ㸪W1DF࠾࠸࡚ࡢࡳDay 8ࡽ9ࡅ࡚᭷ពቑຍࡋࡓࡀ㸪W2DF࡛ࡣ ᭷ព࡞ቑຍࡣࡳࡽࢀ࡞ࡗࡓࠋBF㸣ࡣ㸪W1DF࠾࠸࡚Day 9࡛W2DFẚ㍑ࡋ࡚᭷ព㧗 ࡗࡓࡀ㸪Day 6࠾ࡼࡧ8࡛ࡣ୧⩌㛫࡛ᕪࡣ࡞ࡗࡓࠋBF㸣᥎⛣ࢆ⩌ෆ࡛ẚ㍑ࡋࡓࡇࢁ
25
W1DF࡛ࡣDay 6ࡽ9࠾ࡼࡧDay 8ࡽ9ࡅ࡚᭷ពቑຍࡋࡓࡀ㸪W2DF࡛ࡣ᭷ព࡞ቑ ຍࡣࡳࡽࢀ࡞ࡗࡓࠋ
Day 8࠾ࡅࡿ⾑୰E2⃰ᗘࢆTable 2.2.♧ࡋࡓࠋ⾑୰E2⃰ᗘࡣW1DF࡛᭷ព㧗ࡗࡓࠋ
2㸫6㸫3㸬ヨ㦂3ࡢ⤖ᯝ LHࢧ࣮ࢪ᭚㟢๓࠾ࡅࡿ༸⬊ᾮ୰E2࠾ࡼࡧA4⃰ᗘࡣ㸪W1DF࡛᭷ព㧗ࡗࡓࡀ㸪P4 ⃰ᗘᕪࡣ࡞ࡗࡓ㸦Table 2.4.㸧ࠋLHࢧ࣮ࢪ᭚㟢ᚋ࠾ࡅࡿ㸪༸⬊ᾮ୰P4࠾ࡼࡧE2⃰ᗘ ᕪࡣ࡞ࡗࡓ㸦Table 2.5.㸧ࠋ LHrCYP19A1 mRNAⓎ⌧㔞ࡢ⤖ᯝࢆFig. 2.10.♧ࡋࡓࠋLHrࡣW1DF࡛W2DFẚ㍑ࡋ ࡚᭷ព㧗ࡗࡓࡀ㸪CYP19AⓎ⌧㔞ᕪࡣ࡞ࡗࡓࠋCOX-2㸪PGES࠾ࡼࡧPGFS mRNA Ⓨ⌧㔞୧⩌㛫࡛ᕪࡣ࡞ࡗࡓ㸦Fig. 2.11㸧ࠋiNOS࠾ࡼࡧeNOS mRNAⓎ⌧㔞ࢆFig. 2.12. ♧ࡋࡓࠋiNOS ᕪࡣ࡞ࡗࡓࡀ㸪eNOSࡣW1DF࡛㧗࠸ഴྥࢆ♧ࡋࡓࠋVEGF120㸪 VEGF164࠾ࡼࡧFGF-2 mRNAⓎ⌧㔞ࡢ⤖ᯝࢆFig. 2.13.♧ࡋࡓࠋW1DF࠾࠸࡚㸪VEGF120 ࠾ࡼࡧFGF-2࡛㧗࠸ഴྥࢆ♧ࡋ㸪VEGF164ࡣ᭷ព㧗ࡗࡓࠋStAR㸪P450-scc࠾ࡼࡧ3β-HSD mRNAⓎ⌧㔞ࡢ⤖ᯝࢆFig. 2.14.♧ࡋࡓࠋW1DF࠾࠸࡚StAR࡛㧗࠸ഴྥࢆ♧ࡋ㸪P450-scc ࠾ࡼࡧ3β-HSD mRNAࡣ᭷ព㧗ࡗࡓࠋ 2㸫6㸫4㸬ヨ㦂4ࡢ⤖ᯝ Day 9࠾ࡅࡿW1DFW2DFࡢᖹᆒ┤ᚄBF㸣ࡢ⤖ᯝࢆTable 2.6.♧ࡋࡓࠋW1DF࠾ ࠸࡚ᖹᆒ┤ᚄ࠾ࡼࡧBF%ࡣࡁࡗࡓࠋ㯤య᩿㠃✚᥎⛣ࢆFig. 2.16.♧ࡋࡓࠋ㯤య᩿㠃✚ ࡣDay 15㸪18࠾ࡼࡧ21࡛W1CL࡛W2CLẚ㍑ࡋ࡚᭷ព㧗ࡗࡓࠋ
26 㯤య⾑ὶ㠃✚᥎⛣ࢆFig. 2.17.♧ࡋࡓࠋ㯤య⾑ὶ㠃✚ࡣDay 12࠾ࡼࡧ15࡛W1CL࡛W2CL ẚ㍑ࡋ࡚᭷ព㧗ࡗࡓࠋ ⾑୰P4⃰ᗘ᥎⛣ࢆFig .2.18.♧ࡋࡓࠋ⾑୰P4⃰ᗘࡣヨ㦂ᮇ㛫ࢆ㏻ࡌ࡚W1CL࡛W2CL ẚ㍑ࡋ࡚᭷ព㧗ࡗࡓࠋ 2㸫7㸬⪃ᐹ ᮏ❶࡛ࡢヨ㦂1 ࠾ࡼࡧ 2 ࡽ㸪W1DF ࡣ W2DF ẚ㍑ࡋ࡚㸪ᖍ༸⬊ࡢᖹᆒ┤ᚄ㸪༸ ⬊ቨ⾑ὶ㸪LH ࢧ࣮ࢪ᭚㟢┤๓ࡢ⾑୰ E2⃰ᗘࡀ㧗ࡗࡓࠋࡲࡓ㸪ヨ㦂3 ࡽ㸪༸⬊ᾮ E2 ⃰ᗘ࠾ࡼࡧ༸⬊ᾮA4⃰ᗘ㸪㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿLHr㸪⾑⟶᪂⏕ᅉᏊ㸪ࢫࢸࣟࢻ ྜᡂ㓝⣲ࡢmRNA Ⓨ⌧㔞ࡀ W1DF ࡛㧗ࡗࡓࠋヨ㦂 4 ࡽ㸪W1DF ༸ᚋᙧᡂࡉࢀ ࡿ㯤యࡢ᩿㠃✚㸪⾑ὶ࠾ࡼࡧ⾑୰P4⃰ᗘ᥎⛣ࡀ㧗࠸ࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ ᯝࡽ㸪W1DF ࡣ W2DF ẚ㍑ࡋ࡚༸⬊ࡋ࡚ࡼࡾᶵ⬟ⓗάຊࡀ㧗࠸ࡇࡀ⪃࠼ࡽࢀ ࡿࠋࡲࡓ㸪ࡇࢀࡽࡢᕪ␗ࡀ⏕ࡌࡿཎᅉࡋ࡚㸪W1DF Ⓨ⫱ᮇ㛫୰ࡢప࠸⾑୰ P4⃰ᗘక ࠺㧗࠸LH ࣃࣝࢫ㢖ᗘࡼࡿ༸⬊⣽⬊ࡢ่⃭ࡀ᥎ ࡉࢀࡓࠋ ヨ㦂1 ࡼࡾ㸪༸⬊ἼⓎ⌧㛤ጞࡽ 7 ᪥┠ࡲ࡛ࡢᖍ༸⬊ࡢⓎ⫱ࢆẚ㍑ࡍࡿ㸪༸⬊㑅 ᢤ㛤ጞᚋࡽ㸪Ⓨ⫱⋡ࡸᖍ༸⬊ࡢᖹᆒ┤ᚄࡣW1DF ࡛ࡁࡃ࡞ࡿࡇࡀ♧ࡉࢀࡓࠋࡲ ࡓ㸪W1DF ࠾ࡼࡧ W2DF ࡑࢀࡒࢀࡢⓎ⫱ᮇ㛫୰ࡢ⾑୰ P4⃰ᗘࢆẚ㍑ࡍࡿ㸪Day 0 ࡽ Day 6 ࡅ࡚ W1DF ࡛᭷ពపࡃ᥎⛣ࡍࡿࡇࡀ᫂ࡽࡉࢀࡓࠋ㐣ཤࡢ◊✲࠾࠸ ࡚㸪㯤యᙧᡂᮇࡣ㯤య㛤ⰼᮇẚ㍑ࡋ࡚㸪LH ࣃࣝࢫ㢖ᗘࡀ㧗ࡃ࡞ࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ㸦73㸧ࠋࡲࡓ㸪⭐ෆ␃⨨ᆺ P4〇ࢆ⏝࠸࡚㸪⾑୰P4⃰ᗘࢆ㧗ࡃࡍࡿLH ࣃࣝࢫ㢖 ᗘࡣపୗࡍࡿ୍᪉࡛㸪⾑୰P4⃰ᗘࢆపࡃ⥔ᣢࡍࡿLH ࣃࣝࢫ㢖ᗘࡣୖ᪼ࡍࡿࡇࡀ▱ ࡽࢀ࡚࠸ࡿ㸦15㸪52㸧ࠋࡲࡓ㸪Adams et al.㸦5㸧ࡣ㸪⮬↛Ⓨᚋࡢ༸☜ㄆ⭐ෆ␃⨨ ᆺP4〇ࢆᤄධࡋ㸪ࡑࡢᚋࡢ༸ᕢෆ࡛Ⓨ⫱ࡍࡿ➨1 ༸⬊Ἴࡢ༸⬊Ⓨ⫱ືែࢆほᐹࡋࡓ
27 ࡇࢁ㸪ᤄධࡋ࡞ࡗࡓ∵ẚᖍ༸⬊ࡢ᭱㐩ࡋࡓᖹᆒ┤ᚄࡀᑠࡉ࠸ࡇࢆሗ࿌ࡋ ࡚࠸ࡿࠋ༸⬊Ἴࡢ୰࡛༸⬊ࡀᖍ༸⬊ࡋ࡚㑅ᢤࡉࢀ㸪ࡑࡢᚋⓎ⫱ࢆ⥅⥆ࡍࡿࡓࡵࡣ㸪 㢛⢏ᒙ⣽⬊࡛LHr ࡢⓎ⌧ࡀᚲ㡲࡛࠶ࡿ㸦13㸧ࠋヨ㦂 1 ࡛ࡳࡽࢀࡓ㸪W1DF ࡢ W2DF ẚ ࡚ᖹᆒ┤ᚄࡀࡁࡃ᥎⛣ࡋࡓࡇࡽ㸪W1DF ࡢⓎ⫱ᮇ㛫୰ࡢప࠸⾑୰ P4⃰ᗘ᥎⛣ ㉳ᅉࡍࡿ㧗࠸LH ࣃࣝࢫ㢖ᗘࡼࡾᖍ༸⬊ࡢⓎ⫱ࡀಁࡉࢀࡓ᥎ ࡉࢀࡿࠋࡉࡽ㸪 ヨ㦂1 ࡛ࡣ Day 6 ༸⬊ቨ⾑ὶࢆほᐹࡋࡓࡇࢁ㸪W1DF ࡣ W2DF ẚ㍑ࡋ࡚㧗࠸ഴྥ ࢆ♧ࡋࡓࠋ༸⬊ࡢⓎ⫱㐣⛬࠾࠸࡚㸪༸⬊ቨ࡛ࡢ⾑⟶ᙧᡂࡣ㔜せ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚ ࠸ࡿ㸦95㸧ࠋ㐣ཤࡢ◊✲࠾࠸࡚㸪㑅ᢤᚋࡢᖍ༸⬊࡛ࡢ༸⬊ቨ⾑ὶࡀቑຍࡍࡿࡇࡀሗ ࿌ࡉࢀ࡚࠸ࡿ㸦42㸧ࠋࡲࡓ㸪㛢㙐๓ᚋࡢ༸⬊ቨ⾑ὶࢆẚ㍑ࡍࡿ㛢㙐ᚋ༸⬊ቨ⾑ὶࡀほ ᐹࡉࢀ࡞ࡃ࡞ࡿ࠸࠺ሗ࿌ࡶ࠶ࡿ㸦2㸧ࠋࡇࢀࡽࡢࡇࡽ㸪࣮࢝ࣛࢻࣉࣛࢆ⏝࠸ࡓ༸⬊ ቨ⾑ὶࡢホ౯ࡣ༸⬊ࡢάຊࡸᶵ⬟ࢆホ౯ࡍࡿࡢ᭷ຠ࡞ᣦᶆ࡞ࡿ⪃࠼ࡽࢀ㸪ᮏ◊✲ ࡛ࡳࡽࢀࡓW1DFW2DF࠾ࡅࡿ༸⬊ቨ⾑ὶࡢ㐪࠸ࡣ༸⬊ࡢᶵ⬟ࡢᕪࢆ♧ࡋ࡚࠸ࡿ ⪃࠼ࡽࢀࡿࠋ ᮏ◊✲ࡢ⤖ᯝ࠾࠸࡚㸪W1DF ࡛ࡣ W2DF ẚⓎ⫱⋡㸪ᖹᆒ┤ᚄ࠾ࡼ ࡧ༸⬊ቨ⾑ὶࡀ㧗ࡗࡓࡇࡽ㸪ᶵ⬟ᛶࡀ㧗࠸༸⬊࡛࠶ࡿࡇࡀ᥎ ࡉࢀࡓࠋ ヨ㦂2 ࡛ࡣ PGF2α࠾ࡼࡧGnRH ࢆ⏝࠸࡚㸪㯤య㏥⾜ㄏ㉳ᚋࡢ༸⬊ࡢᡂ⇍࠾ࡼࡧ༸ ⮳ࡿ㐣⛬ࡢW1DF W2DF ࡢᛶ㉁ࢆㄪ㸪ẚ㍑ࡋࡓࠋᖍ༸⬊ࡢᖹᆒ┤ᚄࡣ Day 6 ࡽ Day 9 ࡅ࡚ᮇ㛫ࢆ㏻ࡋ࡚ W1DF ࡛ࡁࡃ᥎⛣ࡋࡓࠋ༸⬊ቨ⾑ὶࡢኚࢆẚ㍑ࡋࡓ ࡇࢁ㸪W1DF ࠾࠸࡚ GnRH ᢞ๓ᚋ࡛᭷ព࡞ቑຍࡀほᐹࡉࢀࡓࡀ㸪W2DF ࠾࠸࡚ࡣ ᭷ព࡞ቑຍࡣほᐹࡉࢀࡎ㸪Day 9 ࠾࠸࡚ W1DF ࡛༸⬊ቨ⾑ὶࡀ W2DF ẚ᭷ព ࡁࡗࡓࠋ࢘ࢩ࡛ࡣLH ࢧ࣮ࢪࡽ࠾ࡼࡑ 27 㛫ᚋ༸ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦24㸧ࠋ ࡲࡓ࢘ࢩ࡛ࡣ㸪༸┤๓㸦LH ࢧ࣮ࢪ᭚㟢࠾ࡼࡑ 19㹼21 㛫ᚋ㸧ࡢ༸⬊ቨ⾑ὶࡀቑຍࡍ ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ 㸦85㸧ࠋ༸⬊ࡀ LH ࢧ࣮ࢪ᭚㟢ࡉࢀࡿ㸪༸⬊ෆ࡛ PG 㢮㸪 ࣄࢫࢱ࣑ࣥ㸪ࢥࣛࢤࢼ࣮ࢮ㸪࢟ࢽࣥ࠾ࡼࡧࣉࣛࢫ࣑ࣀ࣮ࢤࣥࢡࢳ࣮࣋ࢱ࡞ࡢ⏘⏕ࡀ
28 ஹ㐍ࡋ༸⬊⾑⟶⥙࡛ࡢ㏱㐣ᛶஹ㐍ࡸ༸⬊ቨࡢ⼥ゎ㸪⳼ⷧࡀ㉳ࡇࡿࡇ࡛༸ࡀ㐍⾜ࡋ㸪 ࡑࡢᛂࡣ⅖⌧㇟㢮ఝࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦25㸧ࠋࡑࡢࡓࡵ㸪LH ࢧ࣮ࢪ᭚ 㟢ᚋቑຍࡍࡿ༸⬊ቨ⾑ὶࡣ㸪ࡇࢀࡽࡢ⅖ᛶኚ㉳ᅉࡍࡿ⾑⟶ᶵ⬟ࡢኚࡼࡿ ⪃࠼ࡽࢀࡿࠋࡋࡋ࡞ࡀࡽ㸪ᮏヨ㦂࡛ࡣGnRH ᢞ 24 㛫ᚋ࠾ࡅࡿ༸⬊ቨ⾑ὶࡢ᭷ ព࡞ቑຍࡣW1DF ࡢࡳ࡛ࡋほᐹࡉࢀ࡞ࡗࡓࠋࡲࡓ㸪BF㸣࠾࠸࡚ࡶ Day 9 ࠾࠸࡚ W1DF ࡛㧗࠸ࡇࡽ㸪W1DF ࡢ⾑ὶࡢቑຍ࠾ࡼࡧ㧗್ࡢཎᅉࡋ࡚㸪༸⬊ቨෆ࡛ࡢ⾑ ⟶ࡢศᕸࡀࡼࡾᗈ⠊ᅖ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࡲࡓ㸪LH ࢧ࣮ࢪᑐࡍࡿᛂᛶࡀ 㧗࠸ࡇ࡛༸⬊ቨ࡛ࡢ⅖ᛶኚࡀࡼࡾࡁࡃ࡞ࡾ㸪⾑⟶ࡢ⾑ᾮ㑏ὶ㔞ࡀቑຍࡋ࡚࠸ ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋᮍ⤒⏘∵ࢆ⏝࠸࡚Ⓨㄏ㉳ࢆ⾜࡞ࡗࡓヨ㦂࠾࠸࡚㸪GnRH ᢞ 26 㛫ᚋࡢ༸⬊ቨ⾑ὶࡑࡢᐇࡋࡓேᕤᤵ⢭ᚋࡢཷ⫾ࡢ᭷↓ࢆẚ㍑ࡋࡓࡇ ࢁ㸪ዷፎࡋࡓ∵࡛༸⬊ቨ⾑ὶࡀ᭷ព㧗࠸ࡇࢆሗ࿌ࡋ࡚࠸ࡿ㸦84㸧ࠋࡇࡢ⌧㇟ࡣ㸪࣐࢘ ࢆ⏝࠸ࡓヨ㦂࠾࠸࡚ࡶྠᵝࡢ⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿ㸦87㸧ࠋࡲࡓ㸪Ⓨࢆㄏ㉳ࡋ GnRH ᢞ26 㛫ᚋࡢ༸๓༸⬊ࡽᅇࡋࡓ༸Ꮚᑐࡋ࡚㸪యእཷ⢭ࢆ⾜࠸ࡑࡢᚋࡢ⬇Ⓨ⫱ ࢆẚ㍑ࡋࡓ◊✲࡛ࡣ㸪༸Ꮚᅇࡢ༸๓༸⬊ࡢ༸⬊ቨ⾑ὶࡀࡁ࠸ሙྜ㸪⬇Ⓨ⫱ࡀⰋ ዲ࡛࠶ࡿࡇࡶ᫂ࡽࡉࢀ࡚࠸ࡿ㸦86㸧ࠋࡇࢀࡽࡢࡇࡽ㸪༸๓༸⬊ࡢ༸⬊ቨ⾑ὶ ࡣ㸪ࡑࡢ༸⬊ࡢάຊࡢ⛬ᗘࢆ♧ࡍࡇࡀ࡛ࡁࡿ⪃࠼ࡽࢀࡿࠋࡲࡓ㸪ᮏ◊✲ࡽ㸪GnRH ᢞࡢDay 8 ࠾ࡅࡿ⾑୰ E2⃰ᗘࡣW1DF ࡛㧗࠸ࡇࡀ♧ࡉࢀࡓࠋ㐣ཤࡢᐃேᕤᤵ ⢭ࢆ⏝࠸ࡓヨ㦂࠾࠸࡚㸪GnRH ᢞࡢ⾑୰ E2⃰ᗘࡑࡢᚋࡢཷ⫾ྍ⬟ᛶࡢ㛫ࡣ ṇࡢ┦㛵㛵ಀࡀᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦14㸧ࠋࡲࡓ㸪ᐃேᕤᤵ⢭Ⓨᚩೃࢆ ♧ࡋ࡚࠸ࡓ࢘ࢩࡣ㸪Ⓨᚩೃࢆ♧ࡋ࡚࠸࡞ࡗࡓ࢘ࢩẚ㍑ࡋ࡚ཷ⫾⋡ࡀ㧗࠸ࡇࡀሗ ࿌㸦70㸧ࡉࢀ࡚࠾ࡾ㸪༸๓ࡢ⾑୰ E2⃰ᗘࡣཷ⫾ᛶࡸ༸⬊ࡢάຊࢆ♧ࡍᣦᶆ࡞ࡿࠋࡇ ࢀࡽࡢࡇࡽ㸪W1DFࡣ༸⬊ቨ⾑ὶࡀࡁࡃ㸪༸┤๓ࡢ⾑୰E2⃰ᗘࡀ㧗࠸ࡇࡽ㸪 W2DF ẚάຊࡀ㧗࠸ࡇࡀ᥎ ࡉࢀࡿࠋ
29 ヨ㦂3 ࡛ࡣ㸪ヨ㦂 2 ࡛ᚓࡽࢀࡓ㸪W1DF ࡢᛶ㉁ࢆࡉࡽヲ⣽ẚ㍑ࡍࡿࡓࡵ㸪LH ࢧ ࣮ࢪ᭚㟢๓ᚋࡢ༸⬊ᾮᛶࢫࢸࣟࢻ࣍ࣝࣔࣥࡸ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿ mRNA Ⓨ⌧ 㔞ࢆẚ㍑ࡋࡓࠋ LH ࢧ࣮ࢪ᭚㟢๓ࡢᡂ⇍༸⬊ෆ࡛㸪ࢫࢸࣟࢻ࣍ࣝࣔࣥྜᡂ㐣⛬ࡣ㸪༸⬊ቨࡢෆ༸⬊ ⭷⣽⬊࠾࠸࡚㸪ࢫࢸࣟࢻ࣍ࣝࣔࣥࡢᇶ㉁࡞ࡿࢥࣞࢫࢸ࣮ࣟࣝࡀ⣽⬊ෆྲྀࡾ㎸ࡲ ࢀࡿࡇࡽጞࡲࡾ㸪ࡇࡢࢥࣞࢫࢸ࣮ࣟࣝࡣ StAR ࡼࡾ࣑ࢺࢥࣥࢻࣜෆྲྀࡾ㎸ࡲ ࢀ㸪⥆࠸࡚࣑ࢺࢥࣥࢻࣜෆ⭷ୖᏑᅾࡍࡿP450-scc ࡼࡾࢥࣞࢫࢸ࣮ࣟࣝࡽࣉࣞࢢ ࢿࣀࣟࣥኚࡉࢀ㸪ࡑࡢᚋ㠃ᑠ⬊యୖᏑᅾࡋ࡚࠸ࡿ 3β-HSD ࡼࡾࣉࣞࢢࢿࣀࣟ ࣥࡽP4ኚࡉࢀࡿ㸦22㸧ࠋP4ࡣC17 Ỉ㓟㓝⣲άᛶࡼࡾ 17α̺ࣄࢻࣟ࢟ࢩࣉࣟ ࢪ࢙ࢫࢸࣟࣥኚࡉࢀ㸪C17-20 ഃ㙐ษ᩿㓝⣲άᛶࡼࡾ A4ኚࡉࢀࡿ㸦31㸪59㸧ࠋ ࡉࡽ A4ࡣ༸⬊⭷⣽⬊ࡽ㢛⢏ᒙ⣽⬊⛣⾜ࡋࡑࡇ࡛࣐ࣟࢱ࣮ࢮ㸦CYP19A1㸧ࡼ ࡾE2ኚࡉࢀࡿ㸦12㸧ࡇ࡛༸⬊ෆ࡛ࢫࢸࣟࢻࡀ⏘⏕ࡉࢀ࡚࠸ࡿࠋᮏヨ㦂࠾࠸࡚㸪 ༸⬊ᾮ୰E2࠾ࡼࡧA4⃰ᗘࡣW1DF ࡛㧗ࡗࡓࡀ㸪CYP19A1 mRNA Ⓨ⌧㔞࠾࠸࡚ࡣ ୧⩌ᕪࡣ࡞ࡗࡓࠋ㐣ཤ⾜ࢃࢀࡓ◊✲࠾࠸࡚㸪༸⬊ෆ࡛ࡢ E2⏘⏕ࡢไ㝈せᅉࡣ㸪 ࣐ࣟࢱ࣮ࢮάᛶ࡛ࡣ࡞ࡃ㸪ෆ༸⬊⭷࡛⏘⏕ࡉࢀࡿA4ࡼࡿࡇࢁࡀࡁ࠸࠸࠺ሗ࿌ ࡀ࠶ࡿ㸦9㸧ࠋࡇࢀࡽࡢࡇࡽ㸪W1DF ࠾ࡅࡿ༸⬊ᾮ E2⃰ᗘࡢ㧗್ࡣW1DF ࡢෆ༸ ⬊⭷࡛ࡢ㧗࠸A4⏘⏕⬟ࡼࡿࡶࡢ࡛࠶ࡿ᥎ ࡉࢀࡿࠋLH ࢧ࣮ࢪ᭚㟢๓ࡢ㢛⢏ᒙ⣽⬊ ࡛Ⓨ⌧ࡋ࡚࠸ࡿLHr mRNA Ⓨ⌧㔞ࢆẚ㍑ࡍࡿ㸪W1DF ࡀ㧗࠸ࡇࡀ♧ࡉࢀࡓࠋ࢘ࢩࡢ 㢛⢏ᒙ⣽⬊࠾࠸࡚㸪LH ࡣ LHr mRNA Ⓨ⌧㔞ࢆቑຍࡉࡏࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸦56㸧㸪 ヨ㦂1 ࠾࠸࡚ W1DF Ⓨ⫱ᮇ㛫୰ࡢ⾑୰ P4⃰ᗘࡀపࡗࡓࡇࡽ㸪W1DF Ⓨ⫱ᮇ㛫୰ ࡢ㧗࠸LHࣃࣝࢫ㢖ᗘࡀW1DF㢛⢏ᒙ⣽⬊୰࠾ࡅࡿLHrⓎ⌧㔞ࢆ㧗ࡵࡓ⪃࠼ࡽࢀࡿࠋ ༸⬊ࡀ LH ࢧ࣮ࢪᛂࡋ࡚༸࡛ࡁࡿࡼ࠺࡞ࡿࡓࡵࡣ㸪㢛⢏ᒙ⣽⬊࠾ࡅࡿ LHr Ⓨ⌧ࡀᚲ㡲࡛࠶ࡿࡇࡽ㸪W1DF ࡢ㢛⢏ᒙ⣽⬊ࡣ LH ࢧ࣮ࢪࢆཷࡅ࡚༸ྥ࠺
30 ᛂᛶࡀࡼࡾ㧗࠸ࡇࡀ᥎ ࡉࢀ㸪ヨ㦂2 ࠾ࡅࡿ Day 9 ࡛ࡢ W1DF ࡢ༸⬊ቨ⾑ὶࡢ㧗್ ࡣ㸪W1DF ࡢ LH ࢧ࣮ࢪᑐࡍࡿ༸๓ࡢ༸⬊ቨ⾑ὶቑຍ࠾ࡅࡿᛂᛶࡢ㧗ࡉࢆᫎ ࡋ࡚࠸ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡿࠋ ༸⬊ࡀ LH ࢧ࣮ࢪ᭚㟢ࡉࢀࡿ㸪༸⬊ࢆᵓᡂࡍࡿ⣽⬊࡛࠶ࡿ㢛⢏ᒙ⣽⬊ࡸ༸⬊⭷⣽ ⬊ࡣ㯤య⣽⬊ኚࡍࡿࡇ࡛㸪ࢫࢸࣟࢻྜᡂࡣ E2ྜᡂࡽ P4ྜᡂᛴ㏿⛣ ⾜ࡋ࡚࠸ࡃ㸦89㸧ࠋࡲࡓ㸪LH ࢧ࣮ࢪ᭚㟢ᚋࡢ༸⬊ᾮ୰࠾࠸࡚㸪PG 㢮ࡢ⃰ᗘࡀᛴ⃭ ୖ᪼ࡋ࡚࠸ࡃ㸦1㸪3㸧ࠋPG ࡣᒁᡤ࠾ࡅࡿ⾑⟶సືᛶస⏝㸦21㸪98㸧ࡸ⾑⟶㏱㐣ᛶస⏝ 㸦26㸧ࢆᣢࡘࡇࡀ▱ࡽࢀ࡚࠾ࡾ㸪༸┤๓ࡢ༸⬊య✚ቑຍ㸦61㸧ࡑࡢᚋࡢ༸⬊◚ 㸦26㸧㛵ࢃࡿࡉࢀ࡚࠸ࡿࠋࡉࡽ㸪༸┤๓࠾ࡅࡿ PGE2ྜᡂࢆᢚไࡍࡿ㸪༸ ⬊ࡢ◚ࢆᢚไࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦71㸧ࠋ࢘ࢩ࠾࠸࡚㏥⾜㛤ጞࡢ㯤య࠾ࡅࡿ ⾑ὶࡢቑຍࡣ⾑⟶ᣑᙇᅉᏊ࡛࠶ࡿ୍㓟❅⣲㸦NO㸧ࡀ㛵ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ 㸦83㸧㸪࣐࢘ࢫ࠾࠸࡚༸⬊ෆ࡛ࡶⓎ⌧ࡀ☜ㄆࡉࢀ࡚࠸ࡿ㸦104㸧ࠋVEGF ࡸ FGF ࡣ༸⬊ Ⓨ⫱㸦82㸧ࡸ㯤యᙧᡂ㸦105㸧࠾ࡅࡿ⾑⟶᪂⏕㔜せ࡞ᙺࢆᯝࡓࡍࡇࡀ▱ࡽࢀ࡚࠾ ࡾ㸪ࡇࢀࡽࡢᅉᏊࡢⓎ⌧㔞ࢆẚ㍑ࡍࡿࡇ࡛LH ࢧ࣮ࢪ๓ᚋ༸⮳ࡿ㐣⛬࠾ࡅࡿ ༸⬊ᶵ⬟ࢆホ౯࡛ࡁࡿ⪃࠼ࡓࠋ ᮏヨ㦂࡛ࡣ㸪COX-2㸪PGES ࠾ࡼࡧ PGFS mRNA Ⓨ⌧㔞᭷ព࡞ᕪࡀ࡞ࡗࡓࡇ ࡽ㸪༸㐣⛬ᚲ㡲࡞ࡇࢀࡽࡢᅉᏊࡢⓎ⌧㔞ཬࡰࡍ㸪LH ࢧ࣮ࢪᑐࡍࡿᛂᛶࡀ W1DF W2DF ࡢ㛫࡛ᕪ␗ࡣ࡞࠸ࡇࢆ♧၀ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋࡘࡲࡾ㸪ࡇࢀࡽ ࡢᅉᏊࡢⓎ⌧㔞ࡢ㐪࠸ࡀ㸪༸⬊ቨ⾑ὶࡢᕪ␗ࢆࡶࡓࡽࡋࡓཎᅉ࡛࠶ࡿࡣ⪃࠼ࡽࢀ࡞࠸ࠋ ୍᪉࡛㸪eNOS mRNA Ⓨ⌧㔞ࡣ W1DF ࡛㧗࠸ഴྥࢆ♧ࡋࡓࡇࡽ㸪༸⬊ࡢ⾑⟶ᣑᙇస ⏝W1DF W2DF 㛫࡛ᕪࡀ⏕ࡌ࡚࠸ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋࡋࡋ࡞ࡀࡽ㸪༸⬊ቨࡢ ⾑⟶⥙ࡣෆ༸⬊⭷ศᕸࡋ࡚࠾ࡾ㸪㢛⢏ᒙ⣽⬊ࡣ⾑⟶ࡀධࡋ࡚࠸࡞࠸㸦29㸧ࡇ ࡽ㸪ෆ༸⬊⭷࡛ࡢ⾑⟶⥙ࡢศᕸࡸෆ༸⬊⭷⣽⬊࡛ࡢiNOS ࠾ࡼࡧ eNOS ࡞ࡢ⾑⟶సື
31 ᛶᅉᏊࡢⓎ⌧㔞ࢆẚ㍑ࡍࡿࡇ࡛㸪༸⬊ቨ⾑ὶࡢᕪ␗ࢆࡶࡓࡽࡋࡓཎᅉࡘ࠸࡚㏣ཬࡋ ࡚࠸ࡃᚲせࡀ࠶ࡿ⪃࠼ࡿࠋ P4⏘⏕㛵ࢃࡿࢫࢸࣟࢻྜᡂ㓝⣲࡛࠶ࡿStAR㸪P450-scc ࠾ࡼࡧ 3β-HSD mRNA Ⓨ⌧ 㔞ࢆW1DF W2DF 㛫࡛ẚ㍑ࡋࡓࡇࢁ㸪P450-scc ࠾ࡼࡧ 3β-HSD mRNA ࡀ W1DF ࡛ 㧗ࡃⓎ⌧ࡋ࡚࠸ࡓࠋࡲࡓ㸪⾑⟶ᙧᡂᅉᏊ࡛࠶ࡿVEGF120㸪164 ࠾ࡼࡧ FGF-2 mRNA Ⓨ⌧ 㔞࠾࠸࡚ࡶ㸪VEGF164 mRNA Ⓨ⌧㔞ࡀ W1DF ࡛㧗ࡃⓎ⌧ࡋ࡚࠸ࡓࠋ㢛⢏ᒙ⣽⬊ᑐ ࡋ࡚㸪LH ࡣ VEGF㸦45㸧࠾ࡼࡧ P450-scc 㸦106㸧ࡢⓎ⌧ࢆୖ᪼ࡉࡏࡿሗ࿌ࡉࢀ࡚࠾ࡾ㸪 W1DF ࡢ LH ࢧ࣮ࢪ᭚㟢๓ࡢ㢛⢏ᒙ⣽⬊࡛ LHr mRNA ࡢⓎ⌧ࡀ㧗ࡗࡓࡇࡽ㸪ࡇࢀ ࡽࡢᅉᏊࡢⓎ⌧㔞ࡀୖ᪼ࡋࡓ᥎ ࡉࢀࡿࠋࡇࡢࡼ࠺ࢫࢸࣟࢻྜᡂ㓝⣲࠾ࡼࡧ⾑⟶ ᪂⏕ᅉᏊࡢⓎ⌧ࡀW1DF ࡛㧗ࡃ࡞ࡗ࡚࠸ࡓࡇࡽ㸪༸ᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែࡸ ᶵ⬟ࡀW1CL ࠾࠸࡚㧗࠸ࡇࡀ᥎ ࡉࢀࡓࠋ ヨ㦂4 ࠾࠸࡚㸪W1DF ༸ᚋᙧᡂࡉࢀࡿ W1CL ࡢ㯤య᩿㠃✚㸪㯤య⾑ὶ㠃✚࠾ࡼ ࡧ⾑୰P4⃰ᗘࡣ㧗ࡗࡓࠋࡇࢀࡲ࡛㸪༸๓༸⬊VEGF ࡢࣥࢱࢦࢽࢫࢺࢆᢞࡋࡓ ሙྜ㸪ࡑࡢᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែࡸᶵ⬟ࢆపୗࡉࡏࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦46㸧ࠋ ࡲࡓ㸪FGFR1 ᑐࡍࡿࣥࣄࣅࢱ࣮ࢆᇵ㣴ࡋࡓ㯤య⣽⬊ῧຍࡍࡿ⾑⟶ෆ⓶⣽⬊ࡢࢿ ࢵࢺ࣮࣡ࢡࡸ⾑⟶ഃᯞࢆⴭࡋࡃῶᑡࡉࡏࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦101㸧ࠋࡇࢀࡽࡢࡇ ࡽ㸪W1DF ࠾ࡅࡿ⾑⟶ᙧᡂᅉᏊࡢ㧗࠸Ⓨ⌧ࡼࡾ㸪㯤యᙧᡂึᮇࡢ⾑⟶ᙧᡂࡢ⛬ᗘࡀ W1CL ࡛ࡁࡃ࡞ࡗࡓ⪃࠼ࡽࢀࡿࠋࡇࢀࡲ࡛㸪༸๓༸⬊ࡢ┤ᚄࡀࡁ࠸ሙྜ㸪ࡑࡢ ᚋᙧᡂࡉࢀࡿ㯤యࡢ┤ᚄࡶࡁࡃ࡞ࡿ࠸࠺ሗ࿌ࡀ࠶ࡿ㸦97㸧ࠋᮏヨ㦂࠾࠸࡚ࡶ༸ ๓༸⬊ࡣW1DF ࡛ࡁࡃ㸪ࡑࡢᚋࡢ㯤య᩿㠃✚ࡀࡁ࠸ࡇࡽ㸪㐣ཤࡢሗ࿌୍⮴ࡍ ࡿࠋࡉࡽ㸪W1CL ࡛ࡣ㯤యᙧᡂึᮇ࡛ࡢ⾑⟶ᙧᡂࡢ⛬ᗘࡀࡁࡗࡓࡇࡽ㸪⾑ᾮ 㑏ὶ㔞ࡀቑຍࡋ㸪㯤యࡢⓎ⫱ࡀࡉࡽಁࡉࢀ࡚ࡁࡃⓎ⫱ࡋࡓ⪃࠼ࡽࢀࡿࠋ㐣ཤࡢሗ ࿌࠾࠸࡚㸪㯤యࡢ┤ᚄ⾑୰P4⃰ᗘࡣṇࡢ┦㛵㛵ಀࡀᏑᅾࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ
32 㸦58㸧ࠋᮏヨ㦂࠾࠸࡚ࡶ㸪⾑୰ P4⃰ᗘࡀ㧗ࡃ᥎⛣ࡋࡓཎᅉ㸪W1CL ࡢࢧࢬࡀࡁ ࠸ࡇࡶ⪃࠼ࡽࢀࡿࡀ㸪㯤యᙧᡂึᮇ⾑ὶ㔞ࡀከ࠸ࡇ࡛㯤య⣽⬊࡛ࡢP4⏘⏕ࡢஹ㐍 ࡸ㸪⏘⏕ࡉࢀࡓ P4ࢆࡼࡾຠ⋡Ⰻࡃ㌟ᚠ⎔ᨺฟ࡛ࡁࡓࡇࡀ㧗࠸⾑୰ P4⃰ᗘࢆࡶࡓ ࡽࡋࡓࡢ࡛ࡣ࡞࠸᥎ ࡉࢀࡿࠋࡲࡓ㸪ヨ㦂3 ࡽ P4ྜᡂ㓝⣲ࡢmRNA Ⓨ⌧㔞ࡀ㧗 ࡗࡓࡇࡽ㸪㯤య⣽⬊࡛ࡢࢫࢸࣟࢻྜᡂ⬟ࡢᶵ⬟ࡢ㧗࠸ࡇࡶせᅉࡢ୍ࡘ࡞ࡿ ⪃࠼ࡽࢀࡿࠋࡇࢀࡽࡢࡇࡽ㸪W1DF ࡣ༸⬊ࡢᶵ⬟ࡔࡅ࡛࡞ࡃ㸪ࡑࡢᚋᙧᡂࡉࢀ ࡿ㯤యࡢάຊࡶ㧗࠸ࡇࡀ♧ࡉࢀࡓࠋࡲࡓ㸪㯤యᶵ⬟ࡣ༸┤๓ࡢ༸⬊ᶵ⬟ࡁࡃᙳ 㡪ࢆཷࡅࡿࡇࡀ♧၀ࡉࢀࡓࠋ ௨ୖࡢࡇࡽ㸪W1DF ࡢᛶ㉁ࢆ᫂ࡽࡍࡿ┠ⓗ࡛ W2DF ༸⬊Ⓨ⫱ືែ㸪༸ ⮳ࡿࡲ࡛ࡢ༸⬊ቨ⾑ὶࡸ⾑୰E2⃰ᗘ㸪LH ࢧ࣮ࢪ᭚㟢๓ᚋ࠾ࡅࡿ༸⬊ᾮᛶࢫࢸࣟࢻ ⃰ᗘ㸪㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿ LHr㸪ࢫࢸࣟࢻྜᡂ㓝⣲㸪⾑⟶సືᛶᅉᏊ࠾ࡼࡧ⾑ ⟶ᙧᡂᅉᏊࡢmRNA Ⓨ⌧㔞㸪ࡉࡽࡣ༸ᚋᙧᡂࡉࢀࡿ㯤యࡢᙧែ࠾ࡼࡧᶵ⬟ࢆẚ㍑ ࡋࡓࡇࢁ㸪W1DF ࡣࢧࢬࡸᶵ⬟ᛶࡀ㧗ࡃ㸪άຊࡢ㧗࠸༸⬊࡛࠶ࡿࡇࡀ᫂ࡽ࡞ ࡗࡓࠋ 2㸫8㸬ᑠᣓ 㯤యᙧᡂᮇⓎ⫱ࡍࡿW1DFࡣ㸪㯤య㛤ⰼᮇⓎ⫱ࡍࡿW2DFẚ༸⬊ࡢⓎ⫱㸪༸⬊ ቨ⾑ὶ㸪༸๓༸⬊ࡢ㢛⢏ᒙ⣽⬊࡛Ⓨ⌧ࡋ࡚࠸ࡿLHr㸪⾑⟶᪂⏕ᅉᏊ࠾ࡼࡧࢫࢸࣟࢻྜ ᡂ㓝⣲ࡢmRNAⓎ⌧㔞㸪ࢫࢸࣟࢻྜᡂ⬟㸪㯤యᙧᡂ࠾ࡼࡧ㯤యᶵ⬟ࡀ㧗ࡃ㸪άຊࡀ㧗࠸ ࡇࡀ♧၀ࡉࢀࡓࠋࡇࢀࡽࡢW1DFࡢᛶ㉁ࡣ㸪W1DFⓎ⫱ᮇ㛫୰ࡢ⾑୰P4⃰ᗘࡢప࠸᥎⛣ ࡼࡿࡶࡢ᥎ ࡉࢀࡓࠋ
Fig. 2.1.
Schematic diagram of experimental model of W1DF and W2DF in Experiment 1. In the first follicular wave, the day of spontaneous ovulation was defined as Day 0. In the second follicular wave, the day of spontaneous ovulation was defined as Day 7. Ovulation of the first-wave dominant follicle (DF) was induced by GnRH treatment on Day 2 to induce a new follicular wave, and induced ovulation was confirmed 36 h after GnRH treatment on Day 0. W1DF; DF in the first follicular wave. W2DF;DF in the second follicular wave. B-mode US = trans-rectal ultrasonography. Color Doppler US = trans-rectal color Doppler ultrasonography.
W1DF
B-mode US Color Doppler US
Spontaneous
ovulation Induced ovulation B-mode US Color Doppler US
W2DF
Day0 Day1 Day2 Day3 Day4 Day5 Day6
Day0 Day1 Day2 Day3 Day4 Day5 Day6 Spontaneous ovulation Day2 Day7 GnRH 33
Fig. 2.2.
Measuring method of blood flow area (BFA). Image of (A) is dominant follicle with blood flow signals in the follicular wall using color Doppler ultrasonography. The area of blood flow signals which is enclosed by white line is measured as BFA (image B).
(A)
(B)
W1DF W2DF
Fig. 2.3.
Comparative changes of plasma P4 concentration of W1DF and
W2DF from Day 0 to 6. Day 0 = The day of follicular wave emergence. W1DF; Dominant follicle (DF) in the first follicular wave. W2DF;DF in the second follicular wave. Effect of group (G) (P < 0.001), day (D) (P < 0.001) and interaction between group and day (G,D) (NS). Values are mean r SEM of each point.
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Day0 Day1 Day2 Day3 Day4 Day5 Day6
Pla
sma P4 concen
tration
of P4(ng/ml)
Days from follicular wave emergence G䠖P < 0.001 D䠖P < 0.001 G,D䠖NS
Plasma P
4c
oncen
tr
at
ion
(ng
/m
l)
35Fig. 2.4.
Comparative changes of dominant follicle (DF) of W1DF and W2DF from Day 3 to 6. Day 0 = The day of follicular wave emergence. W1DF; DF in the first follicular wave. W2DF; DF in the second follicular wave. Effect of group (G) (P < 0.01), day (D) (P < 0.01) and interaction between group and day (G,D) (P < 0.01). The asterisk denotes difference between each point, : P < 0.05. Values are mean r SEM of each point.㻌 9 10 11 12 13 14 15 16 17
Day3 Day4 Day5 Day6
Dominant follicle diameter (mm)
Days from follicular wave emergence
G䠖P < 0.01 D䠖P < 0.01 G,D䠖P < 0.01 W1DF W2DF
