熊本大学学術リポジトリ Kumamoto University Repository System

熊本大学学術リポジトリ
Kumamoto University Repository System
Title
ホメオボックス型転写因子 Dlx による Steroidogenic
acute regulatory protein (StAR) 遺伝子発現調節 : 先
天性疾患発症の分子機構解明に向けた解析
Author(s)
西田, 尚代
Citation
Issue date
2008-03-25
Type
Thesis or Dissertation
URL
http://hdl.handle.net/2298/9385
Right
↻ᮇኬᏕᏕనㄵᩝ
࣒࢛࣌࣍ࢴࢠࢪᆵ㌹෕ᅄᏄ Dlx ࡞ࡻࡾ Steroidogenic acute
regulatory protein (StAR)㐿ఎᏄⓆ⌟ㄢ⟿
̾඙ኮᛮ⑄ᝀⓆ⑍ࡡฦᏄᶭᵋよ᪺࡞ྡྷࡄࡒよᯊ̾
2007
↻ᮇኬᏕኬᏕ㝌ⷾᏕᩅ⫩㒂 ฦᏄᶭ⬗ⷾᏕᑍᨯ
ฦᏄᶭ⬗ⷾᏕㅦᗑ ⑋ឺ㐿ఎᏄよᯊᏕฦ㔕
こ⏛ ᑠ௥
Regulation of steroidogenic acute regulatory protein (StAR) gene
expression through the interaction between Dlx5 and GATA-4
for testicular steroidogenesis
- An approach toward the elucidation of molecular pathogenesis
of congenital anomalies-
Hisayo Nishida
1
Regulation of steroidogenic acute regulatory protein (StAR) gene
expression through the interaction between Dlx5 and GATA-4
for testicular steroidogenesis
- An approach toward the elucidation of molecular pathogenesis of congenital anomalies-
Hisayo Nishida
Androgen is essential for the control of the male organ development through binding to
the androgen receptor, resulting in masculinization of the external genitalia, Wolffian duct
derivatives and prostate. In human and mouse, any defects along the pathway of androgen
production cause the congenital disorders such as micropenis, hypospadias and
cryptorchidism.
Dlx homeobox genes are the mammalian homologs of Drosophila Distal-less (Dll).
Genetic lesions in the Dlx5 and Dlx6 (Dlx5/6) locus are associated with the human genetic
disorder Split Hand/Foot Malformations Type 1 (SHFM1) characterized by a profound
median cleft of the hands and/or feet. Intriguingly, several clinical reports described that
SHFM patients are occasionally associated with urogenital dysplasia such as micropenis,
hypospadias and small testis. Although Dlx5 is expressed in the mouse adult testis, their
possible function during testis differentiation has not been studied.
In this study I showed that Dlx5/6 are expressed in the mouse fetal Leydig cells during
testis development. During embryogenesis, Steroidogenic acute regulatory protein (StAR)
which is essential for testicular steroidogenesis is expressed in the fetal Leydig cells.
Overexpression of Dlx5 in the Leydig cell line (mLTC-1) induces the activation of the StAR
gene promoter. I also demonstrated that this activation depends on the interaction between
Dlx5 and GATA-4 that is another transcription factor essential for testicular steroidogenesis
by a combination of immunoprecipitation and transactivation domain analyses. Furthermore, I
showed that the double inactivation of Dlx5 and Dlx6 in the mouse embryos leads to
decreased testosterone level and abnormal masculinization phenotype. These results
suggested that Dlx5 and Dlx6 participate in the control of steroidogenesis during testis
development.
2
To clarify the pathogenesis of human congenital anomalies, the elucidation of androgen
signaling must be required. External genitalia are typical androgen responsive organs during
embryogenesis. To identify candidate genes of androgen target during the development of
genital tubercle (GT), external genitalia anlage, we previously performed DNA microarray
analysis. To examine the expression pattern of potential candidate genes from the DNA
microarray analysis, I performed gene expression analyses with real-time quantitative PCR
and section in situ hybridization. These analyses suggested that Cyp1b1, Fkbp51 and MafB
might be candidates for androgen target genes during GT masculinization.
In summary, Dlx5 can interact with GATA-4 and enhance the GATA-4-mediated StAR
promoter activity in mLTC-1. Furthermore, the embryonic mutant for Dlx5/6 genes showed
defects in testosterone production and male sexual differentiation. Altogether, these results
indicated that Dlx5/6 genes are involved in the proper testicular steroidogenesis and fetal
masculinization. Additionally, I identified the androgen target genes such as Cyp1b1, Fkbp51
and MafB during GT masculinization. These studies may open the way to analyze human
congenital birth defects.
3
␆ㄊ⾪
࣬ᮇㄵᩝ࡚ࡢ௧ୖࡡ␆ㄊࢅ౐⏕ࡌࡾࠊ
aa
: amino acid
AR
: androgen receptor
BCIP : 5-bromo,4-chrolo,3-indolyphosphate
bp
: base pair
C/EBP
: CCAAT/Enhancer-binding protein
cDNA
: complementary deoxyribonucleic acid
DAB
: 3,3’-diaminobenzidine tetra-hydrochloride
DEPC
: diethyl pyrocarbonate
Dhh
: Desert hedgehog
DIG
: dioxigenin
DMEM
: Dulbecco’s modified Eagle Medium
DNA
: deoxyribonucleic acid
ECL
: enhanced chemiluminescence
ER
: endoplasmic reticulum
FBS
: fetal bovine serum
GAPDH
:glyceraldehydes-3-phosphate dehydrogenase
HD
: homeodomain
HE
: hematoxylin and eosin staining
Hox
: homeobox transcriptional factor
HRP
: horseradish peroxidase
HSD
: hydroxysteroid dehydrogenase
IgG
: immunoglobulin G
lipoid CAH
: lipoid congenital adrenal hypoplasia
Insl3
: insulin-like factor 3
MIS
: Mullerian inhibiting substance (anti-Mullerian hormone, AMH)
mRNA
: messenger ribonucleic acid
NBT
: nitroblue tetrazolium
PAGE
: polyacrylamide gel electrophoresis
4
PBS
: phosphate buffered saline
PBST
: phosphate buffered saline with 1% Tween20
Pbx
: Pre-B cell luekaemia transcription factor
PCR
: polymerase chain reaction
PDEFRα
: platelet-derived growth factor receptor α
PFA
: paraformaldehyde
Ptc1
: patched 1
P450scc
: cytochrom p450 side chain cleavage enzyme
RNA
: ribonucleoic acid
RPMI
: Roswell Park Memorial Institute
RT-PCR
: reverse transcriptase-polymerase chain reaction
SDS
: sodium dodecyl sulfate
SHFM
: split hand/foot malformation
Tris
: Tris (hydroxymethly) aminomethane
WT
: wild type
5
ᮇㄵᩝࡢࠉᏕ⾙㞟ヽ࡞᥎㍍ࡈࡿࡒḗࡡㄵᩝࢅᇱ♇࡛ࡌࡾࡵࡡ࡚࠵ࡾࠊ
(1) Gene expression analyses on the embryonic external genitalia: identification of regulatory
genes passively involved in masculinization processes.
Congenital Anomalies, in press.
Hisayo Nishida*, Shinichi Miyagawa*, Daisuke Matsumaru, Yoshihiro Wada, Yoshihiko
Satoh, Yukiko Ogino, Shinji Fukuda, Taisen Iguchi, Gen Yamada.
*
These two authors contributed equally to this work.
(2) Positive regulation of steroidogenic acute regulatory protein (StAR) gene expression
through the interaction between Dlx and GATA-4 for testicular steroidogenesis.
Submitted.
Hisayo Nishida, Shinichi Miyagawa, Maxence Vieux-Rochas, Monica Morini, Yukiko
Ogino, Kentra Suzuki, Naomi Nakagata, Hueng-Sik Choi, Giovanni Levi and Gen
Yamada.
(3) External Genitalia Development: A Model System to Study Organogenesis.
Genetically Engineered Mice Handbook, 263-278, 2005 CRC Press, LLC, Boca Raton,
FL. Edited by John P. Sundberg and Tsutomu Ichiki
Kentaro Suzuki, Hisayo Nishida, Sho Ohta, Yoshihiko Satoh, Y. Xu, Y. Zhang, Yoshihiro
Wada, Yukiko Ogino, Naomi Nakagata, T. Ohba and Gen Yamada.
(4)ࠔဳ஘ິ∸አ⏍ṢჹࡡⓆ⏍࡛ฦ໩โᚒࠕ໩Ꮥ࡛⏍∸ Vol.42, No.10, 2004 P666-672.
こ⏛ᑠ௥ࠉ㕝ᮄᇻኯ㑳ࠉⲮ㔕⏜⣎Ꮔࠉᑹᮄ⚵├ࠉ⏛௥ᾀᚠࠉᒜ⏛″
6
┘ḗ
➠ୌ❮ ⥬ゕ
9
➠஦❮ ⤎ᯕ 13
➠ୌ⟿
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽ࡄࡾⓆ⌟よᯊ
➠஦⟿
Dlx5 ࡞ࡻࡾ StAR 㐿ఎᏄⓆ⌟โᚒ
➠୔⟿
Dlx5 ࡛ GATA-4 ࡡࢰࣤࣂࢠ㈻㛣┞பష⏕
18
➠ᄿ⟿
Dlx5 ࢰࣤࣂࢠ㈻ࡡ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ໩ࢺ࣒࢕ࣤࡡྜྷᏽ
21
➠஫⟿
Dlx5 and Dlx6 double knockout (Dlx5/6 DKO)࣏ࢗࢪࡡ⾪⌟ᆵよᯊ
22
➠ඵ⟿
࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄࡡⓆ⌟よᯊ 26
➠୔❮ ⩻ᐳ 29
13
16
➠ୌ⟿
Dlx5 ࡛ GATA-4 ࡞ࡻࡾࢰࣤࣂࢠ㈻㛣┞பష⏕ࡡᙲ๪ 29
➠஦⟿
Dlx5 ࡞ࡻࡾ StAR 㐿ఎᏄ㌹෕Ὡᛮ໩ᶭᵋ 30
➠୔⟿
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡ⫶ඡ⢥ᕛ࡞࠽ࡄࡾᶭ⬗ 31
➠ᄿ⟿
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࡛ࢼ࣭ࣖࣞࢪࢷࣞ࢕ࢺ࡛ࡡ㛭㏻ᛮ 31
➠஫⟿
࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄࡡአ⏍Ṣჹᙟᠺ㐛⛤࡞࠽ࡄࡾᶭ⬗ 32
➠ᄿ❮ ⥪ᣋ
35
➠஫❮ ᐁ㥺᪁Ἢ 36
➠ୌ⟿
ᐁ㥺ິ∸࠽ࡻࡦᇰ㣬⣵⬂
36
➠஦⟿
Plasmid DNA ࡡష⿿
36
➠୔⟿
Luciferase assay
37
➠ᄿ⟿
Western blotting Ἢ
37
➠஫⟿
ඞ␷ỷ㜾Ἢ
38
➠ඵ⟿
Mammalian two-hybrid assay
38
➠୏⟿
㐿ఎᏄⓆ⌟よᯊ
38
➠ୌ㡧
Section in situ hybridization
➠஦㡧
RNA ᢫ฝ࠽ࡻࡦ Real-time quantitative PCR
➠ඳ⟿
38
⤄⧂ᏕⓏよᯊ
39
40
7
➠ୌ㡧
ࣉ࣏ࢹ࢞ࢨ࢙࢛ࣝࣤ࣬ࢩࣤ᯹Ⰵ
40
➠஦㡧
ඞ␷⤄⧂᯹Ⰵ
40
ㅨ㎙
41
ཤ⩻ᩝ⊡
42
8
➠ୌ❮ ⥬ㄵ
࢓ࣤࢺࣞࢣࣤ(Androgen)ࡢဳ஘㢦࡞࠽࠷࡙⏠ᛮࡡ஦ḗᛮᚡࡡⓆ⌟࠽ࡻࡦ⥌ᣚ࡞ᚪ㡪
ࡡᅄᏄ࡚࠵ࡾࠊ࢓ࣤࢺࣞࢣࣤࡡୌࡗ࡚࠵ࡾࢷࢪࢹࢪࢷࣞࣤ(testosterone)ࡢ⢥ᕛ㛣㈻㡷
ᇡ࡞Ꮛᅹࡌࡾࣚ࢕ࢸ࢔ࢴࣃ⣵⬂(Leydig cells)࡚ྙᠺ࣬ฦἢࡈࡿࡾࠊ⾉ᾦ୯࡫ฦἢࡈࡿ
ࡒ ࢷ ࢪ ࢹ ࢪ ࢷ ࣞ ࣤࡢ አ ⏍Ṣ ჹ ࡷ๑ ❟ ⭚ ࡝࡜ ࡡ ࢓ ࣤࢺ ࣞ ࢣ ࣤᵾ Ⓩ ჹᏻ ࡞ ࠽࠷ ࡙
5α-reductase ࡡష⏕ࢅུࡄࠉࢩࣃࢺࣞࢷࢪࢹࢪࢷࣞࣤ(dihydrotestosterone, DHT)࡫࡛ን
ᥦࡈࡿࡾࠊDHT ࡢࢷࢪࢹࢪࢷࣞࣤࡻࡽࡵ࢓ࣤࢺࣞࢣࣤὩᛮ࠿ 2ࠤ3 ಶᙁ࠷ࡒࡴࠉᵾⓏ
ჹᏻ࡚ᙁࡂష⏕ࡌࡾࡆ࡛࠿ฝᮮࡾ(Fig. 1)ࠊ
Figure 1.ࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ⤊㊪
9
⫶⏍ᚃ᭿࡚ࡢࠉࡆࡿࡼࡡ࢓ࣤࢺࣞࢣࣤࡢ⏠ᛮ⏍ṢჹࡡⓆ⏍࡞㛭୙ࡊࠉአ⏍Ṣჹ(external
genitalia)ࠉ୯⭀⟮(Wolffian duct)ࠉ๑❟⭚(prostate)ࡡ㞕ᛮ໩(masculinization)ࢅㄇᑙࡌࡾࠊ
ࣃࢹ࠽ࡻࡦ࣏ࢗࢪ࡞࠽࠷࡙ࠉ࢓ࣤࢺࣞࢣࣤᶭ⬗ࡡ఩ୖࡢࠉᑺ㐠ୖ⿛(hypospadias) ࠉೳ
⏻⢥ᕛ(cryptorchism)࡝࡜ࡡἢᑺ⏍Ṣჹᙟᠺ␏ᖏࢅᘤࡀ㉫ࡆࡌ 1-3ࠊ
ᑺ㐠ୖ⿛ࡢᑺ㐠‹ࡡ㛚㙈࠿୘Ᏸධ࡝ࡒࡴࠉᑺ㐠࠿ஞ㢄඙❻࡞ᙟᠺࡈࡿ࡝࠷⑋ឺࢅᣞ
ࡌ(Fig. 2)ࠊ❟నᤴᑺ㝸ᐐࡷ⭴හᑏ⢥㝸ᐐ࡞ຊ࠻ࠉᖺᑛ᭿ࡡඡ❲࡫ࡡᚨ⌦Ⓩᙫ㡢ࡢኬࡀ
ࡂࠉྙె⑍࡛ࡊ࡙ೳ⏻⢥ᕛࡷ๑❟⭚ᑚᐄ࡝࡜ࡡ㞕ᛮ໩୘ධ࠿ぜࡼࡿࡾࠊᑺ㐠ୖ⿛ࡡ≁
ᚡ࡛ࡊ࡙ࠉฝ⏍⏠Ꮔ 250 ெ࡞ᑊࡊ 1 ౚ⛤ᗐ࡛㟸ᖏ࡞Ⓠ⑍㢎ᗐ࠿㧏࠷஥࠿ᣪࡅࡼࡿࠉࡱ
ࡒࠉᐓ᪐ᛮ࡞Ⓠ⑍ࡌࡾ஥ࡵሒ࿈ࡈࡿ࡙࠷ࡾ 4ࠊ㎾ᖳࠉ࢓࣒ࣛ࢜ࠉ࢕࢟ࣛࢪࠉࢸ࣏࣭ࣤ
ࢠࠉࢪ࣭ࢗ࢘ࢸࣤࠉ᪝ᮇ࡝࡜ࡡ඙㐅ㅎᅗ࡞࠽࠷࡙ᑺ㐠ୖ⿛ࡡⓆ⑍㢎ᗐࡡ୕᪴࠿ሒ࿈ࡈ
ࡿ࡙࠽ࡽࠉ⎌ሾỗ᯹∸㈻࡞ࡻࡾᙫ㡢࡝࡜ࡵ♟ြࡈࡿ࡙࠷ࡾ 5,6ࠊ
Figure 2. ᑺ㐠ୖ⿛
A ▦༰㸯ḿᖏ㒂న࡞న⨠ࡌࡾᑺ
㐠㛜ཾ㒂ࠊB, F ▦༰㸯␏ᖏ࡝㒂
న࡞న⨠ࡌࡾᑺ㐠㛜ཾ㒂ࠊᑺ㐠
㛜ཾ㒂࠿㝔ᄙ࡞㎾࠷࡮࡜አ⏍Ṣ
ჹࡢ༖㝔㝟ࡡᙟឺࢅ࡛ࡾࠊ
Enviro Health Prespect 109:1175-1183 (2001)
ೳ⏻⢥ᕛࡢ㝔ᄙහ࡞⢥ᕛ࠿りࡿ࡝࠷≟ឺࢅᣞࡊࠉ⏠ඡࡡ⏍Ṣჹࡡ␏ᖏ࡛ࡊ࡙᭩ࡵኣ
࠷⑄ᝀࡡୌࡗ࡚࠵ࡾࠊࣃࢹࡡሔྙࠉ⢥ᕛࡢ⫶⏍ 3 ࣧ᭮ࢅ㐛ࡁࡾ࡛⭙⭅හ࠾ࡼ㜾ୖࡊጙ
ࡴࠉ30-32 㐄ࡱ࡚࡞㝔ᄙහ࡞㜾ࡽ࡙ࡂࡾࠊೳ⏻⢥ᕛࡢࠉࡆࡡ⢥ᕛࡡ㜾ୖ㐛⛤࠿ḿᖏ࡞
⾔ࢂࡿࡍ࡞⭙⭅හ࡞⏻ࡱࡖࡒ≟ឺ࡚࠵ࡽࠉ᩺⏍ඡධమࡡ 3-5㸚࡞ヾࡴࡼࡿࠉ఩ฝ⏍మ
㔔ඡࡷ᪡⏐ඡ࡚ࡢࠉ30㸚࡛Ⓠ⑍㢎ᗐ࠿㧏ࡂ࡝ࡾࠊࡱࡒࠉୌ⯙Ⓩ࡞ᡥ⾙⒢Ἢ࡞ࡻࡽ἖⒢
ࢅ⾔࠹࠿ࠉ⾔ࢂ࡝࠷ሔྙࡢ୘ዲࢅᘤࡀ㉫ࡆࡊࠉࡈࡼ࡞⢥ᕛ⭐⑾ࡡⓆ⑍⋙࠿㣍㌅Ⓩ࡞୕
᪴ࡌࡾ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ 2,7ࠊ
ࡆࡡᵕ࡝⫴ᬊ࠾ࡼࠉ⫶⏍ᚃ᭿࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤᶭ⬗ࡡฦᏄᶭᵋࡡよᯊࡢᏕ⾙Ⓩ
࡝㔔さᛮ࡞ຊ࠻ࠉᚺ⏕༈Ꮥฦ㔕࡞࠽࠷࡙ࡵኬࡀ࡝ណ⩇ࢅࡵࡗ࡛࠷࠻ࡾࠊ
10
Dlx 㐿ఎᏄࡢ Drosophila Distal-less (Dll)ࡡဳ஘㢦࣓࣌ࣞࢡ࡚࠵ࡽࠉ௛ᒌ⫝ࡡ㐪న㒂ᙟ
ᠺ࡞㔔さ࡝ᙲ๪ࢅࡵࡗ࣒࢛࣌ࢺ࣒࢕ࣤᆵ㌹෕ᅄᏄ࡚࠵ࡾ 8ࠊࣃࢹ࠽ࡻࡦ࣏ࢗࢪ࡚ࡢࠉ
Dlx1 ࠾ࡼ Dlx6 ࡱ࡚ࡡ 6 ⛸㢦࠿Ꮛᅹࡊࠉ3 ࢠࣚࢪࢰ࣭(Dlx1/2, Dlx3/4, Dlx5/6)ࢅᙟᠺࡊ࡙
࠷ࡾࠊDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ(Dlx5/6)ࡢࠉ๑⬳(forebrain)ࠉ㪨ᘢ(branchial arches)ࠉ
ᄿ⫝(limb)ࡡჹᏻᙟᠺ࡞㛭୙ࡊ࡙࠷ࡾ஥࠿▩ࡼࡿ࡙࠷ࡾ 8-11ࠊ
Split hand/foot malformation (SHFM)ࡢ୯ኳ⿛ᡥ㊂ࢅఔ࠹⑍ುᛮᄿ⫝ᙟᠺ୘ධࢅ୹⑍
≟࡛ࡌࡾࣃࢹ඙ኮᛮ⑄ᝀ࡚࠵ࡽࠉ⌟ᅹࡱ࡚࡞ࠉࣃࢹ SHFM ࡞࠽࠷࡙ 5 ࡗࡡ㐿ఎᏄᗑࡡ
ን␏࠿ሒ࿈ࡈࡿ࡙࠷ࡾ 12ࠊSHFM type 1 (SHFM1)ࡢࠉᖏ᯹Ⰵమ 7q21.3-q22 ࡞న⨠ࡘࡄ
ࡼࡿ࡙࠽ࡽࠉࡆࡡ㡷ᇡࡢ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࢅྱࢆ࡚࠷ࡾ 13ࠊDlx5 㐿ఎᏄ
࠽ࡻࡦ Dlx6 㐿ఎᏄ㐿ఎᏄḖ᥾࣏ࢗࢪ(Dlx5 and Dlx6 double knockout mouse, Dlx5/6
DKO)ࡢᄿ⫝ᙟᠺ␏ᖏࢅ♟ࡊ
14,15
ࠉࡐࡡ⾪⌟ᆵࡢ SHFM ࡞㢦జࡊ࡙࠷ࡾ
13
ࠊࡆࡿࡼࡡ
▩ぜ࠾ࡼࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ SHFM1 ࡡཋᅄ㐿ఎᏄࡡୌࡗ࡚࠵ࡾ࡛⩻
࠻ࡼࡿ࡙࠷ࡾ 13ࠊ⮾࿝῕࠷஥࡞ࠉୌ㒂ࡡ SHFM ᝀ⩽࡚ࡢᄿ⫝ᙟᠺ␏ᖏ࡞ຊ࠻࡙ࠉᑚ㝔
ⱴ⑍ࠉᑺ㐠ୖ⿛ࠉ⢥ᕛⴆ⦨࡝࡜ࡡἢᑺ⏍Ṣჹᙟᠺ␏ᖏࢅెⓆࡌࡾ࡛࠷࠹ሒ࿈࠿࠵ࡾ
16-18
(Fig. 3)ࠊࡆࡿࡼࡡᙟឺᙟᠺ␏ᖏࡢ⫶ඡ᭿⢥ᕛ࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤྙᠺ఩ୖ࡞ࡻ
ࡽᘤࡀ㉫ࡆࡈࡿࡒ࡛᥆ᐳࡈࡿࡾ࠿ࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡ⢥ᕛ࡞࠽ࡄࡾᶭ
⬗ࡢධࡂฦ࠾ࡖ࡙࠷࡝࠾ࡖࡒࠊ
Figure 3. Split hand/foot malformation ࡞࠽ࡄࡾᄿ⫝ᙟᠺ␏ᖏ࠽ࡻࡦἢᑺ⏍ Ṣჹᙟᠺ୘ධ
Am J Med Genet A 135, 21-27 (2005).
Am J Med Genet A 140, 1366-1374 (2006)
11
Steroidogenic acute regulatory protein (StAR)ࡢࢤࣝࢪࢷ࣭ࣞࣜࡡ࣐ࢹࢤࣤࢺࣛ࢓አ⭯
࠾ࡼහ⭯࡫ࡡ㍲㏞ࢅㄢ⟿ࡌࡾᅄᏄ࡚࠵ࡽ(Fig. 1)ࠉࡆࡡ㐛⛤ࡢࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ
ࡡᚂ㏷ṹ㝭࡚࠵ࡾ 19,20ࠊ࣐ࢹࢤࣤࢺࣛ࢓හ⭯࡞㍲㏞ࡈࡿࡒࢤࣝࢪࢷ࣭ࣞࣜࡢ cytochrom
p450 side chain cleavage enzyme (P450scc)࡞ࡻࡽࣈࣝࢡࢾࢿࣞࣤ(pregnenolone)࡫ንᥦࡈ
ࡿࠉࡐࡡᚃࠉࡈࡱࡉࡱ࡝㓕⣪ష⏕ࢅུࡄ࡙ࢷࢪࢹࢪࢷࣞࣤ࡫࡛ንᥦࡈࡿࡾࠊࣃࢹ࡞࠽
࠷࡙ࠉStAR 㐿ఎᏄࡡን␏ࡢ඙ኮᛮ⑄ᝀ࡚࠵ࡾࣛ࣎࢕ࢺ㐛ᙟᠺ⑍(lipoid congenital
adrenal hypoplasia, lipoid CAH)ࡡཋᅄ࡛࡝ࡾࠊLipoid CAH ࡢ๧⭀࠽ࡻࡦ⏍Ṣ⭚࡚ࡡࢪ
ࢷ ࣞ ࢕ ࢺ ࣌ ࣜ ࣓ ࣤ ྙ ᠺ ఩ ୖ ࢅ ♟ ࡊ ࠉ ኣ ࡂ ࡢ ⏠ ᛮ ௫ ᛮ ༖ 㝔 㝟 (male
pseudohermaphoroditism, MPH)ࢅྙెࡌࡾ
21
ࠊࡈࡼ࡞ࠉStAR 㐿ఎᏄḖ᥾࣏ࢗࢪ(StAR
knockout mouse, StAR KO)ࡢࣃࢹࡡ lipoid CAH ࡛㢦జࡊࡒ⾪⌟ᆵࢅ࡛ࡾ 22,23ࠊࡆࡿࡼࡡ
▩ぜࡢࠉStAR ࠿ࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ࡞ᚪ㡪ࡡᅄᏄ࡚࠵ࡾ஥ࢅ♟ࡊ࡙࠷ࡾࠊ
StAR 㐿ఎᏄⓆ⌟ࡢᛮ⭚ๆ⃥࣓࣌ࣜࣤᨲฝ࣓࣌ࣜࣤ(gonadotropin releasing hormone,
GnRH) ࠉ 㯜 మ ᙟ ᠺ ࣌ ࣜ ࣓ ࣤ (luteinizing hormone, LH) ࠉ༵ ⬂ ๆ ⃥ ࣌ ࣓ࣜ ࣤ (follicle
stimulating hormone, FSH)ࠉ⏝≟⭚࣓࣌ࣜࣤ(thyroid hormone)ࠉቌṢᅄᏄ(growth factor)ࠉ
ࣈࣞࢪࢰࢡࣚࣤࢩࣤ(prostaglandins)࠽ࡻࡦࢪࢷࣞ࢕ࢺ➴࡞ࡻࡽᙫ㡢ࢅུࡄࡾ஥࠿▩ࡼ
ࡿ࡙࠷ࡾ
19,20
ࠊࡱࡒࠉStAR 㐿ఎᏄࡡ㌹෕โᚒࡢࠉStAR 㐿ఎᏄࡡ୕Ὦ㒼าࢅヾㆉࡌࡾ
steroidogenic factor 1(SF-1/Ad4BP)24-27, C/EBP25,28,29ࠉSp124,30ࠉCREB/CREM31,32ࠉyin yang
(YY1)33,34 ➴ࡡᵕࠍ࡝㌹෕ᅄᏄ⩄࡞ࡻࡖ࡙โᚒࡈࡿ࡙࠷ࡾ஥࠿ሒ࿈ࡈࡿ࡙࠷ࡾࠊ
Zinc-finger ᆵ㌹෕ᅄᏄ࡚࠵ࡾ GATA-4 ࡵ StAR 㐿ఎᏄࡡ㌹෕Ὡᛮ໩ᅄᏄ࡚࠵ࡽ 28-30ࠉ᭩
㎾ࡡሒ࿈࡚ࡢ GATA-4 ࠿⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂(fetal Leydig cells)ࡡ㐲ว࡝ฦ໩࡞⣵⬂
⮤ᚂⓏ࡞ᚪさ࡚࠵ࡾ஥࠿♟ࡈࡿ࡙࠷ࡾ 35ࠊ
ᮇ◂✪࡚ࡢࠉ⫶ඡ᭿⢥ᕛ࡞࠽࠷࡙ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࠿࢓ࣤࢺࣞࢣࣤྙ
ᠺ⣵⬂࡚࠵ࡾ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂࡚Ⓠ⌟ࡌࡾ஥ࢅ᪺ࡼ࠾࡞ࡊࡒࠊࡱࡒࠉDlx5 ࡢ
GATA-4 ࡛ࢰࣤࣂࢠ㈻㛣┞பష⏕ࡊࠉStAR 㐿ఎᏄࡡ㌹෕Ὡᛮࢅ୕᪴ࡈࡎࡾࡆ࡛ࢅぜฝ
ࡊࡒࠊࡱࡒࠉ࢓ࣤࢺࣞࢣࣤ౪ᏋⓏ࡝ᙟឺᙟᠺࢅ⾔࠹አ⏍Ṣჹ࡞Ἰ┘ࡊࠉDNA ࣏࢕ࢠ
ࣞ࢓ࣝ࢕よᯊ࡞ࡻࡽྜྷᏽࡈࡿࡒ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄࡡአ⏍Ṣჹᙟᠺ㐛⛤࡞
࠽ࡄࡾⓆ⌟よᯊࢅ⾔ࡖࡒࠊ
௧ୖࠉ྘❮࡚ᚋࡼࡿࡒ▩ぜࢅレ㏑ࡌࡾࠊ
12
➠஦❮ ⤎ᯕ
➠ୌ⟿ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽ࡄࡾⓆ⌟よᯊ
ࡆࡿࡱ࡚࡞ SHFM1 ࡡཋᅄ㐿ఎᏄࡡୌࡗ࡚࠵ࡾ Dlx5 㐿ఎᏄࡢ࣏ࢗࢪᠺమ⢥ᕛࢅྱࡴ
ᵕࠍ࡝ჹᏻ࡚Ⓠ⌟ࡊ࡙࠷ࡾ஥࠿ሒ࿈ࡈࡿ࡙࠷ࡾ 36,37ࠊࡊ࠾ࡊࠉ⢥ᕛࡡⓆ⏍ṹ㝭࡚ࡡⓆ
⌟ࣂࢰ࣭ࣤࡢ୘᪺࡚࠵ࡖࡒࠊ⫶ඡ⢥ᕛࡡḿᖏ࡝Ⓠ⏍࡛ࠉࡐࡆ࡚⾔ࢂࡿࡾ࢓ࣤࢺࣞࢣࣤ
⏐⏍ࡢࠉ⫶ඡࡡ㞕ᛮ໩࡞ᚪさ࡝࢕࣊ࣤࢹ࡚࠵ࡾࠊࡐࡆ࡚ࠉ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽ࡄࡾ
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄⓆ⌟ࡡ᭯↋ࢅㄢ࡬ࡾࡒࡴ࡞ࠉsemiquantitative RT-PCR
Ἢ࠽ࡻࡦ real-time quantitative PCR Ἢࢅ⏕࠷ࡒⓆ⌟よᯊࢅ⾔ࡖࡒࠊ⏍Ṣ⭚ࡡ⢥ᕛ࡫ࡡฦ
໩࠿᪺ࡼ࠾࡛࡝ࡾ⫶⏍ 12.5 ᪝࠾ࡼࠉฝ⏍├๑ࡡ⫶⏍ 18.5 ᪝ࡱ࡚ࡡ࣏ࢗࢪ⫶ඡ⢥ᕛ࠾
ࡼ᢫ฝࡊࡒ total RNA ࢅ⏕࠷࡙ semiquantitative RT-PCR ࢅ⾔ࡖࡒ࡛ࡆࢀࠉDlx5 㐿ఎᏄ
࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ⫶⏍ 12.5 ᪝࠾ࡼ⫶⏍ 18.5 ᪝࡞࠾ࡄ࡙࣏ࢗࢪ⫶ඡ⢥ᕛ࡚Ⓠ⌟ࡊ࡙
࠷ࡾࡆ࡛࠿᪺ࡼ࠾࡞࡝ࡖࡒ(Fig. 4A)ࠊࡈࡼ࡞ࠉreal-time quantitative PCR ࡞ࡻࡽࠉ୦㐿
ఎᏄࡢⓆ⏍࠿㐅ࡳ࡞ࡗࡿⓆ⌟㔖࠿ቌຊࡊ࡙࠷ࡾࡆ࡛࠿ࢂ࠾ࡖࡒ(Fig. 4B)ࠊ࣏ࢗࢪ⫶ඡ
⢥ᕛ࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤྙᠺࡢ⫶⏍ 13.5 ᪝࠾ࡼ⾔ࢂࡿ࡙࠽ࡽࠉ⫶⏍ 14.5 ᪝࠾ࡼࡐ
ࡡྙᠺ㔖࠿ቌຊࡌࡾ஥࠿▩ࡼࡿ࡙࠷ࡾ
38
ࠊ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽ࡄࡾ Dlx5 㐿ఎᏄ࠽ࡻ
ࡦ Dlx6 㐿ఎᏄࡡⓆ⌟୕᪴ࡢࠉ࢓ࣤࢺࣞࢣࣤྙᠺቌຊ࡛᫤㛣Ⓩ࡝ୌ⮬࠿ヾࡴࡼࡿࡒࠊ
Figure 4. Dlx5 and Dlx6 are expressed in fetal testis.
Quantification of Dlx5/6 mRNA expression in testis from 12.5 to 18.5 day postcoitum
(dpc), using semiquantitative RT-PCR (A) and Real-time quantitative PCR (B) analyses.
The expression of Dlx5/6 mRNA in 12.5 dpc testis was designated as the basal level (1.0).
13
ḗ࡞ࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄⓆ⌟⣵⬂ࢅྜྷᏽࡌࡾࡒࡴ࡞ section in situ
hybridization Ἢࢅ⏕࠷ࡒⓆ⌟よᯊࢅ⾔ࡖࡒࠊࡐࡡ⤎ᯕࠉ୦㐿ఎᏄࡢ⢥ᕛ㛣㈻࡞Ⓠ⌟ࡌ
ࡾࡆ࡛࠿ࢂ࠾ࡖࡒ(Fig. 5A)ࠊ⢥ᕛ㛣㈻࡞ࡢ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂(fetal Leydig cells)ࠉ
➵ᵕ⣵⬂(peritubular myoid cells)ࠉ⥲⥌ⰾ⣵⬂ᵕ⣵⬂(fibroblast like cells)࠿Ꮛᅹࡌࡾࠊ
⢥ᕛᙟᠺ㐛⛤࡞࠽࠷࡙ࠉP450scc ࡢ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂࣏࣭࣭࡛࢜ࡊ࡙⏕࠷ࡼࡿ࡙
࠷ࡾࠊSection in situ hybridization (Dlx5, Dlx6)࡛ඞ␷⤄⧂᯹Ⰵ(P450scc)ࡡ஦㔔᯹Ⰵࢅ⾔
ࡖࡒ࡛ࡆࢀࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ P450scc ࡛ྜྷୌ⣵⬂࡚භⓆ⌟ࡊ࡙࠷ࡾ
஥࠿ࢂ࠾ࡖࡒ(Fig. 5B)ࠊࡆࡡ⤎ᯕࡢࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࠿⫶ඡࣚ࢕ࢸ࢔
ࢴࣃ⣵⬂࡚Ⓠ⌟ࡊ࡙࠷ࡾ஥ࢅ♟ࡌࡵࡡ࡚࠵ࡾࠊ௧୕ࡡࡆ࡛࠾ࡼࠉDlx5 㐿ఎᏄ࠽ࡻࡦ
Dlx6 㐿ఎᏄࡢ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂࡞࠽࠷࡙Ⓠ⌟ࡊ࡙࠽ࡽࠉ⫶ඡ᭿࡚ࡡࢪࢷࣞ࢕ࢺ࣌
࣓ࣜࣤྙᠺ࡞㛭㏻ࡌࡾᶭ⬗ࢅ᭯ࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡒࠊ
14
Figure 5. Dlx5 and Dlx6 are expressed in the fetal Leydig cells.
(A) The expression of Dlx5 and Dlx6 in mouse testis at 16.5 dpc was detected by section
in situ hybridization analysis. The morphological feature of fetal Leydig cell with a round
nucleus was observed in the Dlx5/6 expressing cells. (B) Double-staining for Dlx5, Dlx6
(purple) and P450scc (red) in fetal testis. Note that the expression of Dlx5/6 was
overlapped with that of P450scc.
15
➠஦⟿ Dlx5 ࡞ࡻࡾ StAR 㐿ఎᏄⓆ⌟โᚒ
Dlx ࡝࡜ࡡ࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻࠿ DNA ࡛⤎ྙࡌࡾࡒࡴ࡞ࡢࠉDNA ୕࡞࣒࣌
࢛ࢺ࣒࢕ࣤ⤎ྙࢦ࢕ࢹ࡚࠵ࡾ TAAT 㒼า࠿ᚪさ࡚࠵ࡾ
39,40
ࠊFeledy ࡼࡢࠉDlx3 ࡢ
(A/C/G)TAATT(G/A)(C/G)㒼าࢅヾㆉࡊ࡙ DNA ࡛⤎ྙࡌࡾ࡛ሒ࿈ࡊ࡙࠷ࡾ
41
ࠊࡱࡒࠉ
chromatin immunoprecipitation assay ࠽ࡻࡦ luciferase assay ࡞ࡻࡾよᯊ࠾ࡼࠉDlx5 ࡢ
TAAT 㒼าࢅྱࡳ DNA 㡷ᇡ࡞⤎ྙࡊࠉୖὮ㐿ఎᏄࡡ㌹෕ㄢ⟿ࢅ⾔࠹஥࠿▩ࡼࡿ࡙࠷
ࡾ 42-44ࠊࡐࡆ࡚࣏ࢗࢪ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂࡞࠽ࡄࡾ Dlx5 㐿ఎᏄࡡᵾⓏ㐿ఎᏄࢅྜྷᏽ
ࡌࡾࡒࡴ࡞ࠉEnsembl Genome Browser ࢅ⏕࠷࡙࢓ࣤࢺࣞࢣࣤྙᠺ㛭㏻㐿ఎᏄ(Fig. 1)
ࡡ㌹෕㛜ጙⅤࡻࡽ୕Ὦ 4.5kbp 㡷ᇡ࡚ TAAT 㒼าࢅ᳠⣬ࡊࡒࠊࡐࡡ⤎ᯕࠉStAR 㐿ఎᏄ
ࡡࣈ࣓࣭ࣞࢰ࣭㡷ᇡ(Gene Bank Accession Number, AC122752)27 ࡞々ᩐࡡ TAAT 㒼า࠿
Ꮛᅹࡌࡾ஥࠿᪺ࡼ࠾࡛࡝ࡖࡒ(Fig. 6A)ࠊ
ḗ࡞ࠉDlx5 ࠿ࡆࡿࡼࡡ࣒࢛࣌ࢺ࣒࢕ࣤ⤎ྙࢦ࢕ࢹࢅྱࡳ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭
ࡡ㌹෕Ὡᛮ࡞㛭୙ࡌࡾ࠾ྫྷ࠾ࢅࠉ࣏ࢗࢪࣚ࢕ࢸ࢔ࢴࣃ⭐⑾⣵⬂(mouse Leydig tumor
cells, mLTC-1)45 ࢅ⏕࠷࡙ luciferase assay ࡞ࡻࡽ᳠ッࡊࡒࠊStAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭ࡡ
TAAT 㒼าࢅྱࡳ(-1514/+25)㡷ᇡࡢࠉDlx5 ࢅ㐛๨Ⓠ⌟ࡈࡎ࡙ࡵࠉࡐࡡࣈ࣓࣭ࣞࢰ࣭Ὡ
ᛮ࡞ን໩ࡢヾࡴࡼࡿ࡝࠾ࡖࡒࠊྜྷᵕ࡞ TAAT 㒼าࢅྱࡱ࡝࠷(-966/+25)㡷ᇡ࡞࠽࠷࡙
ࡵ Dlx5 㐛๨Ⓠ⌟ୖ࡚᭯ណ࡝ን໩ࡢヾࡴࡼࡿ࡝࠾ࡖࡒ(Fig. 6A)ࠊࡊࡒ࠿ࡖ࡙ࠉDlx5 ࡢ
༟≺࡚ࡢ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ࡞ᙫ㡢ࡊ࡝࠷஥࠿ࢂ࠾ࡖࡒࠊ
ୌ⯙࡞ࠉ࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻ࡢ DNA ⤎ྙ࡞ᑊࡌࡾ≁␏ᛮ࠿఩ࡂࠉ௙ࡡ㌹෕
ᅄᏄ࠽ࡻࡦ㌹෕භᙲᅄᏄ࡛々ྙమࢅᙟᠺࡊࠉࡐࡡ≁␏ᛮࢅ୕ࡅ࡙࠷ࡾࡆ࡛࠿▩ࡼࡿ࡙
࠷ࡾ 46,47ࠊࡐࡆ࡚ࠉ࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻࡛┞பష⏕ࡊ 48ࠉࡈࡼ࡞ StAR 㐿ఎᏄࡡ
㌹෕ࢅㄢ⟿ࡊ࡙࠷ࡾ 28-30GATA-4 ࡞Ἰ┘ࡊࡒࠊࡆࡿࡱ࡚ࡡሒ࿈࡞࠵ࡾࡻ࠹࡞ࠉGATA-4
ࡡࡲࢅ㐛๨Ⓠ⌟ࡈࡎࡾ࡛ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ࠿୕᪴ࡊࡒ (Fig. 6A)
28-30
ࠊࡆ
ࡡ GATA-4 ࡞ࡻࡾ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ໩࡞ᑊࡌࡾ Dlx5 ࡡᙫ㡢ࢅㄢ࡬ࡾࡒࡴ
࡞ࠉGATA-4 ࡛ Dlx5 ࢅභⓆ⌟ࡈࡎࠉluciferase assay ࢅ⾔ࡖࡒࠊࡐࡡ⤎ᯕࠉGATA-4 ࡡ
ࡲࢅ㐛๨Ⓠ⌟ࡈࡎࡒሔྙ࡞Ẓ࡬ࠉ୦⩽ࢅභⓆ⌟ࡈࡎࡾࡆ࡛࡚ StAR 㐿ఎᏄࡡࣈ࣓࣭ࣞ
ࢰ࣭Ὡᛮ࠿ࡈࡼ࡞ቌኬࡌࡾࡆ࡛࠿ࢂ࠾ࡖࡒ(Fig. 6A)ࠊ
Dlx5 ࡛ GATA-4 ࡡභⓆ⌟࡞ࡻࡾ㌹෕Ὡᛮ໩࠿ GATA-4 ⤎ྙࢦ࢕ࢹ࡞౪Ꮛࡌࡾ࠾࡜
࠹࠾ࢅㄢ࡬ࡾࡒࡴ࡞ࠉStAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭(-66/-61)㡷ᇡ࡞Ꮛᅹࡌࡾ GATA-4 ⤎ྙ
ࢦ࢕ࢹ࡞ን␏ࢅᑙථࡊ(TTATCTЍTTAagT)29ࠉྜྷᵕࡡᐁ㥺ࢅ⾔ࡖࡒࠊGATA-4 ⤎ྙࢦ
16
࢕ࢹࡡን␏࡞ࡻࡽࠉDlx5 ࠽ࡻࡦ GATA-4 භⓆ⌟ୖ࡚ࡡ㌹෕Ὡᛮ໩࠿΅ᘽࡊࡒ஥࠾ࡼ
(Fig. 6B)ࠉࡆࡡ㌹෕Ὡᛮ໩ࡢ GATA-4 ⤎ྙࢦ࢕ࢹࢅ௒ࡊ࡙࠷ࡾࡵࡡ࡚࠵ࡾ࡛⩻࠻ࡼࡿ
ࡒࠊ௧୕ࡡ⤎ᯕ࠾ࡼࠉDlx5 ࡢ GATA-4 Ꮛᅹୖ࡞࠽࠷࡙ GATA-4 ⤎ྙࢦ࢕ࢹࢅ௒ࡊ࡙
StAR 㐿ఎᏄࡡ㌹෕ㄢ⟿ࢅ⾔ࡖ࡙࠷ࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡒࠊ
Figure 6. Dlx5 enhances the GATA-4-mediated StAR promoter activity.
(A) Mouse StAR promoter (-1514/+25 bp and -966/+25 bp)/ luciferase constructs are
schematically illustrated in the upper panel. Homeodomain binding sites are indicated by
arrowheads. mLTC-1 cells were transfected with the above reporter constructs and
pRL-SV40, together with empty vector, Dlx5 and GATA-4 expression vector as indicated.
The activity of firefly luciferase was normalized with Renilla luciferase. (B) Nucleotide
substitutions were introduced at the GATA-4 binding site (TTATCT to TTAagT). mLCT-1
cells were transfected with wildtype (WT) or mutant StAR promoter/luciferase reporter
constructs together with a empty vector, Dlx5 and GATA-4 expression vector as indicated.
The asterisks indicate statistical significance by Student’s t-test; *, P<0.001.
17
➠୔⟿ Dlx5 ࡛ GATA-4 ࡡࢰࣤࣂࢠ㈻㛣┞பష⏕
Dlx5 ࡛ GATA-4 ࡞ࡻࡾ༝ㄢⓏ࡝ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭โᚒࢅࡻࡽレ⣵࡞ㄢ࡬ࡾ
ࡒࡴ࡞ࠉDlx5 ࡛ GATA-4 ࡡࢰࣤࣂࢠ㈻㛣┞பష⏕ࢅඞ␷ỷ㜾Ἢ࡞ࡻࡽ᳠ゞࡊࡒࠊDlx5
࠽ࡻࡦ GATA-4 ࢅ㐛๨Ⓠ⌟ࡈࡎࡒᚃ࡞ COS-7 ⣵⬂ࡡࢰࣤࣂࢠ㈻᢫ฝᾦࢅ⏕࠷࡙ඞ␷
ỷ㜾ࢅ⾔ࡖࡒࠊࡐࡡ⤎ᯕࠉDlx5 ࡱࡒࡢ GATA-4 ࡞ᑊࡌࡾᢘమࢅ⏕࠷ࡒ࠷ࡍࡿ࡞࠽࠷
࡙ࡵࠉ୦⩽ࡢࢰࣤࣂࢠ㈻々ྙమࢅᙟᠺࡌࡾ஥࠿᪺ࡼ࠾࡞࡝ࡖࡒ(Fig. 7A)ࠊࡈࡼ࡞ࡆࡡ
┞பష⏕ࢅࡻࡽレ⣵࡞ㄢ࡬ࡾࡒࡴ࡞ mLTC-1 ⣵⬂࡞࠽࠷࡙ Dlx5 ࢅ࣊࢕ࢹ࡛ࡊࡒ
mammalian two-hybrid assay ࢅ⾔ࡖࡒࠊDlx5-GAL4DBD ⼝ྙࢰࣤࣂࢠ㈻(Dlx5/pBIND)
࠽ࡻࡦ GATA-4-VP16 ⼝ྙࢰࣤࣂࢠ㈻(GATA4/pACT)ࢅ mLTC-1 ⣵⬂࡞࠽࠷࡙㐛๨Ⓠ
⌟ࡈࡎࠉ୦⩽ࡡ┞பష⏕ࢅࣜࢨࣆ࣭࢘ࣚࢭὩᛮࢅᣞᵾ࡞᳠ゞࡊࡒࠊࡐࡡ⤎ᯕࠉ
Dlx5/pBIND ࡱࡒࡢ GATA-4/pACT ༟≺Ⓠ⌟᫤࡞Ẓ࡬ࠉ୦Ⓠ⌟࣊ࢠࢰ࣭ࡡභⓆ⌟ୖ࡞
࠽࠷࡙᭯ណ࡝࣭ࣝ࣎ࢰ࣭Ὡᛮࡡ୕᪴࠿ヾࡴࡼࡿࡒ(Fig. 7B)ࠊࡆࡡ⤎ᯕ࠾ࡼࠉCOS-7 ⣵
⬂࡚ࡡඞ␷ỷ㜾࡚ᚋࡼࡿࡒ⤎ᯕ࡛ྜྷᵕ࡞ࠉmLTC-1 ⣵⬂࡞࠽࠷࡙ࡵ Dlx5 ࡢ GATA-4
࡛々ྙమࢅᙟᠺࡌࡾ஥࠿ࢂ࠾ࡖࡒࠊ
ࡈࡼ࡞ࠉGATA-4 ࡞ᑊࡌࡾ Dlx5 ࡡ⤎ྙ㡷ᇡࢅ᪺ࡼ࠾࡞ࡌࡾࡒࡴ࡞ࠉMyc ࢰࢡࢅ௛
ຊࡊࡒᵕࠍ࡝ Dlx5 ࣆࣚࢡ࣒ࣤࢹ࡛ GATA-4 ࡛ࡡ┞பష⏕ࢅඞ␷ỷ㜾࡞ࡻࡽよᯊࡊࡒ
(Fig. 8A)ࠊࡐࡡ⤎ᯕࠉDlx5ΔN (134-289 amino acid, aa)ࠉDlx5ΔC (1-195 aa)࠽ࡻࡦ Dlx5N
(1-134 aa)࠿ GATA-4 ࡛々ྙమࢅᙟᠺࡌࡾୌ᪁࡚ࠉDlx5C (196-289 aa)ࡢ GATA-4 ࡛┞
பష⏕ࡊ࡝࠾ࡖࡒ (Fig. 8B)ࠊ௙ࡡ᩷∞໩ࢰࣤࣂࢠ㈻࡛Ẓ࡬࡙ࠉDlx5HD (135-195 aa)
ࡡⓆ⌟㔖ࡢ఩࠾ࡖࡒ࠿ࠉࡆࡡ㡷ᇡࡵ GATA-4 ࡛ࡡ┞பష⏕࠿᳠ฝࡈࡿࡒ(Fig. 8B ྎࣂ
ࢾࣜ▦㢄)ࠊࡆࡿࡼࡡ⤎ᯕ࠾ࡼࠉDlx5 ࡢ N ᮆ❻㡷ᇡ(1-135 aa)ࡱࡒࡢ࣒࢛࣌ࢺ࣒࢕ࣤ㡷
ᇡ(135-195 aa)ࢅ௒ࡊ࡙ GATA-4 ࡛┞பష⏕ࡌࡾࡆ࡛࠿♟ြࡈࡿࡒࠊ
18
Figure 7. Protein-protein interaction between Dlx5 and GATA-4.
(A) COS-7 cells were transfected with Dlx5, GATA-4 expression vectors and empty
vector as indicated. After 24 h incubation, cell lysates were subjected to
immunoprecipitation assay. Immunocomplexes were analyzed by standerd SDS-PAGE
and Western blotting methods, using respective antibodies. (B) Mammalian two-hybrid
assay was performed using the expression vectors encoding Dlx5-GAL4-DBD
(Dlx5/pBIND) and/or GATA-4-VP16 (GATA-4/pACT). mLTC-1 cells were transfected
with the indicated constructs and assayed for luciferase activity at 24 h post-transfection.
19
Figure 8. Identification of interaction domains of Dlx5 for GATA-4.
(A) Schematic representation of various Dlx5 deletion mutants used to identify the
interaction domains. The results from B are presented schematically. The binding of the
various Dlx5 recombinants to GATA-4 are presented as follows: +, binding; -, no binding
(left palnel). (B) Series of plasmids that express Myc-tagged Dlx5 deletion mutants and
GATA-4 expression vector were transfected into COS-7cells and analyzed as previously
described. Two different exposure conditions were shown (left panel, short exposure; right
panel, long exposure; arrowheads indicate the Dlx5HD protein.
20
➠ᄿ⟿ Dlx5 ࢰࣤࣂࢠ㈻ࡡ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ໩ࢺ࣒࢕ࣤࡡྜྷᏽ
Dlx5 ࡛ GATA-4 ࡞ࡻࡾ༝ㄢⓏ࡝ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭㌹෕Ὡᛮ࡞ᚪさ࡝ Dlx5
ࢰࣤࣂࢠ㈻ࢺ࣒࢕ࣤࢅ᪺ࡼ࠾࡞ࡌࡾࡒࡴ࡞ࠉmLTC-1 ⣵⬂࡞࠽࠷࡙ Dlx5 ࣆࣚࢡ࣒ࣤࢹ
ࢅ⏕࠷ࡒ luciferase assay ࢅ⾔ࡖࡒࠊDlx5ΔN ࠽ࡻࡦ Dlx5ΔC ࢅ㐛๨Ⓠ⌟ࡈࡎࡾ࡛ࠉධ㛏
Dlx5 ࡡሔྙ࡛ྜྷᵕ࡞ GATA-4 Ꮛᅹୖ࡚ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ࠿᭯ណ࡞୕᪴ࡊ
ࡒࠊୌ᪁ࠉDlx5N ࠽ࡻࡦ Dlx5C ࡚ࡢࠉStAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ࡞ᙫ㡢ࢅཀྵ࡯ࡈ
࡝࠾ࡖࡒࠊࡱࡒࠉDlx5HD ࡡ㐛๨Ⓠ⌟࡚ࡢࠉഷ࠾࡞ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭Ὡᛮ࠿୕
᪴ࡊࡒ(Fig.9)ࠊࡆࡡ⤎ᯕ࠾ࡼࠉGATA-4 Ꮛᅹୖ࡞࠽ࡄࡾ Dlx5 ࡞ࡻࡾ StAR 㐿ఎᏄࣈࣞ
࣓࣭ࢰ࣭Ὡᛮࡡ୕᪴࡞ࡢ Dlx5 ࡡ࣒࢛࣌ࢺ࣒࢕ࣤ㡷ᇡ࠿ᚪさ୘ྊḖ࡚࠵ࡾࡆ࡛࠿ฦ࠾
ࡖࡒࠊࡱࡒࠉDlx5 ࡡ N ᮆ❻࠽ࡻࡦ C ᮆ❻㡷ᇡࡢ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭ࡡ㌹෕ຝ⋙
ࢅโᚒࡌࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊ
Figure 9. Differential ability of Dlx5 mutant proteins for GATA-4-mediated StAR
promoter activation.
mLTC-1 cells were transiently transfected with StAR promoter/luciferase construct
(-966/+25 bp) and pRL-SV40, together with an empty vector, various Dlx5 deletion
mutants and GATA-4 expression vectors as indicated. The asterisks mean statistical
siginificance by Student’s t-test; *, P<0.01; **, P<0.001.
21
➠஫⟿ Dlx5 and Dlx6 double knockout (Dlx5/6 DKO)࣏ࢗࢪࡡ⾪⌟ᆵよᯊ
୕㏑ࡊࡒ in vitro よᯊ࠾ࡼࠉDlx5 ࡢ GATA-4 ࡛ࡡࢰࣤࣂࢠ㈻㛣┞பష⏕࡞ࡻࡽ StAR
㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭ࡡὩᛮ໩ࢅ⾔࠹ྊ⬗ᛮ࠿♟ြࡈࡿࡒࡆ࡛࠾ࡼࠉDlx5/6 DKO ࣏ࢗ
ࢪ⢥ᕛࡡ⾪⌟ᆵよᯊࢅ⾔ࡖࡒࠊDlx5/6 DKO ࣏ࢗࢪࡢ㢄⵱㦭ᙟᠺ୘ධ࠽ࡻࡦ㢙ᙟᠺ୘
ධ࡞ࡻࡽฝ⏍├ᚃ࡞Ṓஷࡌࡾࡒࡴ
13
ࠉよᯊ࡞ࡢ⫶ඡ࣏ࢗࢪࢅ⏕࠷ࡒࠊDlx5/6 DKO ࣏
ࢗࢪ࠽ࡻࡦࢤࣤࢹ࣭ࣞࣜࡡ⫶ඡ⢥ᕛࢅ HE ࡚᯹Ⰵࡊ࡙⤄⧂ᏕⓏよᯊࢅ⾔ࡖࡒ࡛ࡆࢀࠉ
⾉⟮࠽ࡻࡦ⢥⣵⟮ᙟᠺࡢḿᖏ࡞⾔ࢂࡿ࡙࠷ࡾࡆ࡛࠿ฦ࠾ࡖࡒ(Fig. 10a,b)ࠊḗ࡞ࠉ⏍Ṣ
⣵ ⬂ ࡡ ࣏ ࣭࢜ ࣭࡚࠵ࡾ TRA9849 ࠽ࡻࡦ ࢬ ࣜࢹࣛ⣵⬂ ࣏ ࣭࣭࡚࢜࠵ ࡾ Mullerian
inhibiting substrate (MIS)50 ࡡⓆ⌟ࢅඞ␷⤄⧂᯹Ⰵ࡞ࡻࡽよᯊࡊࠉྜྷࡋࡂࢬࣜࢹࣛ⣵⬂࡚
Ⓠ⌟ࡌࡾ Desert hedgehog (Dhh)51ࠉ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂ࢅྱࡳ㛣㈻㡷ᇡ࡞Ⓠ⌟ࡌࡾ
Patched1 㐿ఎᏄ(Ptc1)51ࠉplatelet-derived growth factor receptorα㐿ఎᏄ(PDGFRα)52ࠉ⫶
ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂ࢅ㝎ࡂ㛣㈻㡷ᇡ࡚Ⓠ⌟ࡌࡾ Arx 㐿ఎᏄ
53
ࡡⓆ⌟ࢅ section in situ
hybridization ࡞ࡻࡽよᯊࡊࡒࠊࡐࡡ⤎ᯕࠉDlx5/6 DKO ࣏ࢗࢪ⫶ඡ⢥ᕛࡢࢤࣤࢹ࣭ࣞࣜ
⩄࡛ྜྷᵕ࡞ࡆࡿࡼࡡ࣏࣭࣭࢜ᅄᏄࢅⓆ⌟ࡊ࡙࠷ࡾ஥࠿ฦ࠾ࡖࡒ(Fig. 10c-n)ࠊࡊࡒ࠿ࡖ
࡙ࠉDlx5/6 DKO ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽࠷࡙ࠉ⏍Ṣ⣵⬂ࠉࢬࣜࢹࣛ⣵⬂ࠉ⫶ඡࣚ࢕ࢸ࢔
ࢴࣃ⣵⬂ࡡⓆ⏍ࡢḿᖏ࡞⾔ࢂࡿ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡒࠊInsulin-like factor 3 (Insl3)ࡢᠺ⇅
ࡊࡒ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂ࡡ࣏࣭࣭࢜㐿ఎᏄ࡚࠵ࡾ 54,55ࠊInsl3 㐿ఎᏄࡵ Dlx5/6 DKO
࣏ࢗࢪ⫶ඡ⢥ᕛ࡚ḿᖏ࡞Ⓠ⌟ࡊ࡙࠷ࡒࡆ࡛࠾ࡼ(Fig. 11)ࠉ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂ࡡฦ
໩㐛⛤ࡵḿᖏ࡞⾔ࢂࡿ࡙࠷ࡾ࡛♟ြࡈࡿࡒࠊୌ᪁࡚ࠉsection in situ hybridization ࡞ࡻ
ࡾⓆ⌟よᯊ࠾ࡼࠉDlx5/6 DKO ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞࠽࠷࡙ୌ㒂ࡡ಴మ(⣑ 40%)࡞࠽࠷࡙
StAR 㐿ఎᏄࡡⓆ⌟఩ୖ࠿ヾࡴࡼࡿࡒ(Fig.11)ࠊࡆࡿࡼࡡ⤎ᯕ࠾ࡼࠉDlx5 㐿ఎᏄ࠽ࡻࡦ
Dlx6 㐿ఎᏄࡢ StAR 㐿ఎᏄⓆ⌟࡞ᙫ㡢ࢅ୙࠻ࡾୌ᪁ࠉ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂ࡡⓆ⏍࣬
ฦ໩࡞ࡢ├᥃㛭୙ࡊ࡝࠷ࡆ࡛ࢅ♟ြࡊࡒࠊ
22
Figure 10. Histogical and marker analyses of Dlx5/6 DKO embryonic testes.
Control testes (a, c, e, g, i, k, m), Dlx5/6 DKO testes (b, d, f, h, j, l,n) at E16.5. HE
staining (a, b). Sertoli cell markers, Dhh (c, d) and AMH (e, f). Germ cell marker, TRA98
(G,h). Fetal Leydig cell markers, Ptc1 (i, j), PDGFRα (k, l). Arx is expressed in interstitial
region except for fetal Leydig cells (m, n). Section in situ hybridization analyses (c, d,
g-n). immunohistochemical analysis (e-h). Basically both control and DKO testis display
similar histogenesis and marker expressions.
23
Figure 11. Comparison of insulin-like factor 3 (Insl3) and StAR expression in testis
of Control (A, C) and Dlx5/6 DKO (B, D) mice at E18.5 by section in situ
hybridization. Approximately, 40% of Dlx5/6 DKO embryos displayed lower degree
of StAR expression in their testes.
24
Dlx5/6 DKO ࣏ࢗࢪ⫶ඡࡡ⢥ᕛྱ᭯୯ࢷࢪࢹࢪࢷࣞࣤࢅῼᏽࡊࡒ࡛ࡆࢀࠉDlx5/6
DKO ࣏ࢗࢪ⫶ඡ࡚ࡢࢤࣤࢹ࣭ࣞࣜ࡞Ẓ࡬᭯ណ࡞఩ୖࡊ࡙࠷ࡒ(Fig. 12A)ࠊࡱࡒࠉ⾉ΰ
୯ࢷࢪࢹࢪࢷ⃨ࣞࣤᗐ࡞࠽࠷࡙ࡵ Dlx5/6 DKO ࣏ࢗࢪ⫶ඡ࡚ࡢࢤࣤࢹ࣭ࣞࣜ࡞Ẓ࡬఩
ୖലྡྷ࠿ふᐳࡈࡿࡒ(Fig. 12B)ࠊࡈࡼ࡞ࠉ㞕ᛮ໩ࡡୌࡗࡡᣞᵾ࡛ࡊ࡙⏕࠷ࡼࡿ࡙࠷ࡾ
anogenital distance (AGD)ࡵ Dlx5/6 DKO ࣏ࢗࢪ⫶ඡ࡞࠽࠷࡙᭯ណ࡞఩ୖࡊ࡙࠷ࡒ(Fig.
12C)ࠊࡆࡿࡼࡡ⤎ᯕ࠾ࡼࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ⫶ඡ᭿⢥ᕛ࡞࠽ࡄࡾࢷࢪ
ࢹࢪࢷࣞࣤྙᠺ࡞ᚪさ࡚࠵ࡽࠉ⫶ඡࡡ㞕ᛮ໩࡞ᐞ୙ࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡒࠊ
Figure 12. Abnormal masculinization in Dlx5/6 DKO embryos.
(A) Measurement of intratesticular testosterone by CLIA method. An asterisk means
statistical significance by Student’s t-test (*, P<0.01) (B) Measurement of serum
testosterone by LC-MS/MS method. (C) The distance between the anus and genitals of
mice (i.e., the anogenital distance; AGD). The AGD is one of the frequently utilized
androgen dependent markers in teratology and a reliable parameter of external
masculinization in mice.
25
➠ඵ⟿ ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄࡡⓆ⌟よᯊ
ᮇ◂✪࠾ࡼ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࡞ࡻࡾ⫶ඡ⢥ᕛ࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤྙ
ᠺㄢ⟿ᶭᵋࡡୌ❻࠿᪺ࡼ࠾࡞࡝ࡖࡒࠊࡊ࠾ࡊࠉ࢓ࣤࢺࣞࢣࣤᶭ⬗୘ධ࡞ࡻࡾἢᑺ⏍Ṣ
ჹᙟᠺ␏ᖏࡡ⌦よ࡞ࡢࠉ㞕ᛮ⏍Ṣჹᏻ࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤࢨࢡࢻࣜࡡฦᏄᶭᵋよ᪺
࠿ᚪさ୘ྊḖ࡚࠵ࡾࠊࡐࡆ࡚ࠉ⫶ඡ᭿࡞࠽ࡄࡾ㞕ᛮ໩ࡡฦᏄᶭᵋࢅ᪺ࡼ࠾࡞ࡌࡾࡒࡴ
࡞ࠉ࢓ࣤࢺࣞࢣࣤᵾⓏ㐿ఎᏄࡡⓆ⌟よᯊࢅ⾔ࡖࡒࠊ
አ⏍Ṣჹࡢ࢓ࣤࢺࣞࢣࣤࡡష⏕ࢅུࡄ࡙㞕ᛮ໩ࡌࡾჹᏻࡡୌࡗ࡚࠵ࡾࠊ⫶⏍ 14.5
᪝࠾ࡼ⢥ᕛ࡚ࡡ࢓ࣤࢺࣞࢣࣤྙᠺ࠿୕᪴ࡌࡾ࠿ࠉአ⏍Ṣჹ࡚ࡢ⫶⏍ 16.5 ᪝࠾ࡼᙟឺⓏ
࡝㞜㞕ᕣ࠿ヾࡴࡼࡿࡾ 56ࠊ㞜ࡡአ⏍Ṣჹ࡛Ẓ࡬ࠉ㞕࡚ࡢⓆ㐡ࡊࡒໜ⓮ࠉᑺ㐠ࣃࢱࡡ⼝
ྙࠉⓉ⭯࡞しࢂࡿࡒஞ㢄࡝࡜㞕≁᭯ࡡᙟឺ࠿ふᐳࡈࡿࡾ(Fig.13)ࠊ
Figure13. Histology of external genitalia
Transverse sections of male (B) and female (C) genital tubercle (GT), external genital
anlage, at 18.5 dpc. In male, the fusion of the urethral folds in ventral midline of GT is
prominent and the glands lamellae completely encircles the shaft (glans penis). L: glans
lamellae, PP: prepuce, U: urethra,
UF: urethral fold.
26
አ⏍Ṣჹᙟᠺ㐛⛤࡞࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤᵾⓏ㐿ఎᏄࢅྜྷᏽࡌࡾࡒࡴ࡞ࠉᙔ◂✪ᐄ࡚
DNA ࣏࢕ࢠࣞ࢓ࣝ࢕よᯊ࠿⾔ࢂࡿࠉ々ᩐࡡು⿭㐿ఎᏄ࠿ᚋࡼࡿ࡙࠷ࡒ(unpublished
data)ࠊᮇ◂✪࡚ࡢࠉࡐࡿࡼು⿭㐿ఎᏄࡡ୯࠾ࡼࠉࢲࢹࢠ࣭࣑ࣞ P450 ฦᏄ⛸ࡡୌࡗ࡚
࠵ࡾ Cyp1b1 㐿ఎᏄࠉ࢓ࣤࢺࣞࢣུࣤᐖమ㸝androgen receptor, AR㸞ࡡࢨ࡛ࣔ࣋ࣞࣤࡊ
࡙ᶭ⬗ࡌࡾ Fkbp51 㐿ఎᏄࠉbasic leucine zipper ᆵ㌹෕ᅄᏄ࡚࠵ࡾ MafB 㐿ఎᏄࡡⓆ⌟
ࣂࢰ࣭ࣤࢅ real-time quantitative PCR Ἢ࠽ࡻࡦ section in situ hybridization Ἢࢅ⏕࠷࡙よ
ᯊࡊࡒࠊ
㞜㞕ࡡᙟឺን໩࠿ふᐳࡈࡿࡾ௧๑ࡡ⫶⏍ 15.5 ᪝ࡡአ⏍Ṣჹ࠾ࡼᚋࡼࡿࡒ cDNA ࢅ
⏕࠷࡙ real-time quantitative PCR ࡞ࡻࡽ Cyp1b1 㐿ఎᏄࠉFkbp51 㐿ఎᏄࠉMafB 㐿ఎᏄ
ࡡⓆ⌟ࢅ㞜㞕࡚Ẓ㍉ࡊࡒ࡛ࡆࢀࠉࡆࡿࡼࡡ㐿ఎᏄࡢ DNA ࣏࢕ࢠࣞ࢓ࣝ࢕࡚ᚋࡼࡿࡒ
⤎ᯕ࡛ྜྷᵕ࡞ࠉ㞜࡞Ẓ࡬㞕࡚ᙁࡂⓆ⌟ࡊ࡙࠷ࡾࡆ࡛࠿ࢂ࠾ࡖࡒ(Fig. 14A)ࠊḗ࡞ࠉࡆࡿ
ࡼࡡ㐿ఎᏄࡡⓆ⌟㡷ᇡࢅ᪺ࡼ࠾࡞ࡌࡾࡒࡴ࡞ࠉsection in situ hybridization ࢅ⏕࠷ࡒⓆ
⌟よᯊࢅ⾔ࡖࡒࠊCyp1b1 㐿ఎᏄ࠽ࡻࡦ Fkbp51 㐿ఎᏄࡢአ⏍Ṣჹ㎾న㒂ࡡ㛣ⴝ㡷ᇡ࡚
Ⓠ⌟ࡊ࡙࠽ࡽࠉ㞜࡞Ẓ࡬㞕࡚Ⓠ⌟࠿ᙁ࠷ലྡྷ࠿ヾࡴࡼࡿࡒ(Fig. 14B-E)ࠊ࢓ࣤࢺࣞࢣࣤ
ࡡ AR ࡫ࡡ⤎ྙࢅ㜴ᐐࡌࡾࣆࣜࢰ࣐ࢺࢅዲፈ࣏ࢗࢪ࡞ᢖ୙ࡌࡾ࡛ࠉ㞕⫶ඡ࣏ࢗࢪࡡአ
⏍Ṣჹ㎾న㒂࡚ᑺ㐠ୖ⿛࠿ㄇᑙࡈࡿࡾ 56ࠊࡆࡡࡆ࡛࠾ࡼࠉአ⏍Ṣჹ㎾న㒂ࡢ࢓ࣤࢺࣞ
ࢣࣤ࠿ᙁࡂష⏕ࡊ࡙࠷ࡾ㡷ᇡ࡚࠵ࡽࠉCyp1b1 㐿ఎᏄࡷ Fkbp51 㐿ఎᏄ࠿࢓ࣤࢺࣞࢣࣤ
࡞ᚺ➽ࡊ࡙Ⓠ⌟ࡊ࡙࠷ࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊ
MafB 㐿ఎᏄࡢᑺ㐠୦ഁ㛣ⴝ࡚Ⓠ⌟ࡊ࡙࠽ࡽࠉ㞜࡞Ẓ࡬㞕࡚㟸ᖏ࡞ᙁࡂⓆ⌟ࡊ࡙࠷
ࡒ(Fig. 14F,G)ࠊᑺ㐠୦ഁ㛣ⴝ࡚ࡢ㞕≁᭯ࡡᙟឺᙟᠺ࡚࠵ࡾᑺ㐠ࣃࢱࡡ⼝ྙ࠿ふᐳࡈࡿ
ࡾࡆ࡛࠾ࡼࠉࡆࡡ㡷ᇡࡢ࢓ࣤࢺࣞࢣࣤష⏕ࢅᙁࡂུࡄࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊࡱࡒࠉ
࣏ࢗࢪአ⏍Ṣჹᙟᠺ㐛⛤࡞࠽࠷࡙ AR ࡢࡆࡡ㡷ᇡ࡚ᙁࡂⓆ⌟ࡊ࡙࠷ࡾ(data not shown)ࠊ
ࡆࡿࡼࡡࡆ࡛࠾ࡼࠉ࢓ࣤࢺࣞࢣࣤᚺ➽㐿ఎᏄ࡚࠵ࡾ MafB 㐿ఎᏄ࠿ᑺ㐠୦ഁ㛣ⴝ࡞࠽
࠷࡙ᑺ㐠ࣃࢱࡡ⼝ྙ࡞㛭୙ࡌࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊ
27
Figure 14. The comparison of Cyp1b1, Fkbp5 and MafB expression between male and
female GT.
(A) The amounts of Cyp1b1, Fkbp51 and MafB mRNA in male and female GT at 15.5
dpc were quantified by real-time quantitative PCR analyses. Relative RNA expression
levels for each sample were standardized by comparing the L8 mRNA levels. The
error bars represent the standard deviation. The expression patterns of Cyp1b1 (B, C),
Fkbp51 (D, E) and MafB (F, G) in male (B, D, F) and female (C, E, G) GT at 15.5
dpc.
28
➠୔❮ ⩻ᐳ
ࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤࡢ⫶ඡⓆ⏍㐛⛤࡚ࡡ⤄⧂ࡡฦ໩ࡓࡄ࡚࡝ࡂࠉᠺమ࡞࠽ࡄࡾ⏍Ṣ
ᶭ⬗ࡡⓆ㐡࠽ࡻࡦᜇᖏᛮࡡ⥌ᣚ࡞㔔さ࡝ᙲ๪ࢅᯕࡒࡌࠊࡐࡡ୯࡚ࡵ࢓ࣤࢺࣞࢣࣤࡢࠉ
㞕ࡡ⏍Ṣᶭ⬗ࡡⓆ⌟࣬⥌ᣚ࡞ᚪ㡪ࡡᅄᏄ࡚࠵ࡾࠊࡊࡒ࠿ࡖ࡙ࠉࢪࢷࣞ࢕ࢺྙᠺᶭᵋࡡ
よ᪺ࡢࠉࣃࢹ඙ኮᛮ⑄ᝀࡡⓆ⑍ᶭᵋࡡ⌦よࡓࡄ࡚࡝ࡂࠉᠺమ࡞࠽ࡄࡾᵕࠍ࡝හฦἢ␏
ᖏࡡよᯊ࡞ࡵኬࡀࡂ㈁⊡ࡌࡾࠊ
ᮇ◂✪࠾ࡼࠉ࣒࢛࣌࣍ࢴࢠࢪᆵ㌹෕ᅄᏄ Dlx5 ࠿ Zinc finger ᆵ㌹෕ᅄᏄ GATA-4 ࡛
々ྙమࢅᙟᠺࡊࠉࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺࡡᚂ㏷ṹ㝭ࢅㄢ⟿ࡌࡾ Steroidogenic acute
regulatory protein (StAR)㐿ఎᏄࡡࣈ࣓࣭ࣞࢰ࣭Ὡᛮࢅಀ㐅ࡈࡎࡾࡆ࡛ࢅ᪺ࡼ࠾࡞࡝ࡖ
ࡒࠊࡈࡼ࡞ࠉDlx5 and Dlx6 double knockout mouse (Dlx5/6 DKO)ࢅ⏕࠷ࡒよᯊ࡞ࡻࡽࠉ
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ⫶ඡ᭿⢥ᕛ࡞࠽࠷࡙ StAR 㐿ఎᏄⓆ⌟࠽ࡻࡦࢷࢪࢹࢪ
ࢷࣞࣤྙᠺࢅಀ㐅ࡌࡾྊ⬗ᛮࢅ᪺ࡼ࠾࡞ࡊࡒࠊࡱࡒࠉࡆࡿࡼࡡ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6
㐿ఎᏄ࠿᭯ࡌࡾᶭ⬗ࡢࠉ⫶ඡࡡ㞕ᛮ໩࡞ᐞ୙ࡌࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊ
➠ୌ⟿ Dlx5 ࡛ GATA-4 ࡞ࡻࡾࢰࣤࣂࢠ㈻㛣┞பష⏕ࡡᙲ๪
࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻ࡡ DNA ⤎ྙ㡷ᇡࡡ≁ᏽࠉ㌹෕Ὡᛮࠉ⣵⬂හᑻᅹ࡝࡜ࡢࠉ
ୌ⯙࡞ࢰࣤࣂࢠ㈻㛣┞பష⏕࡞ࡻࡖ࡙โᚒࡈࡿ࡙࠷ࡾ
46,47
ࠊHox 㐿ఎᏄ⩄࡞ࡢ Pbxࠉ
MeisࠉPrep ࡝࡜々ᩐࡡ㌹෕භᙲᅄᏄ࠿ᏋᅹࡌࡾࠊPBX1 ࡢ HoxA9 ࡛ࢰࣤࣂࢠ㈻㛣┞ப
ష⏕ࡊࠉHox ࡡ DNA ⤎ྙ㐽ᢝᛮ(DNA-binding specificity)ࢅን໩ࡈࡎ 57ࠉࡐࡡ㌹෕Ὡᛮ
ࢅㄢ⟿ࡌࡾ 58ࠊࢨࣘࢗࢩࣘࢗࣁ࢙࡚ࡢ࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻࡚࠵ࡾ Extradentricle
(EXD)ࡡᰶහ⛛ິ࡞ࠉྜྷࡋࡂ࣒࢛࣌ࢺ࣒࢕ࣤࢰࣤࣂࢠ㈻࡚࠵ࡾ homothorax (HTH)࡛ࡡ
ࢰࣤࣂࢠ㈻㛣┞பష⏕࠿ᚪさ࡚࠵ࡾ 59ࠊ
Dlx5 ࡞࠽࠷࡙ࡵ々ᩐࡡභᙲᅄᏄ࠿ሒ࿈ࡈࡿ࡙࠷ࡾࠊYeast two-hybrid assay ࡞ࡻࡽྜྷ
ᏽࡈࡿࡒ Dlxin-1 ࡛ GRIP1b ࡢ Dlx5 ࡡ㌹෕භᙲᅄᏄ࡛ࡊ࡙ᶭ⬗ࡌࡾ
60,61
ࠊDlxin-1 ࡢ
necdin/melanoma-associated antigen (MAGE) family ࡞ᒌࡊ࡙࠽ࡽࠉ⬳ࠉ㦭࠽ࡻࡦ⢥ᕛ࡝
࡜ࡡ Dlx5 ࠿Ⓠ⌟ࡊࠉᶭ⬗ࡌࡾჹᏻ࡚Ⓠ⌟ࡊ࡙࠷ࡾ 60ࠊࡱࡒࠉGRIP1b ࡢ GRIP1(glutamate
receptor interacting protein 1)ࡡ alternative splicing form ࡚࠵ࡾ 61ࠊGRIP1b ࡢ 5 ࡗࡡ PDZ
ࢺ࣒࢕ࣤ㸝PSD-95ࠉDlgࠉZO-1 ࡞භ㏳ࡡࢺ࣒࢕࡚ࣤ࠵ࡽࠉࡆࡿࡼࡡ㢄ᩝᏊࢅ࡛ࡖ࡙ྞ
௛ࡄࡼࡿࡒ㸞ࢅᣚࡔࠉࡆࡡ PDZ ࢺ࣒࢕ࣤࢅ௒ࡊ࡙ Dlx5 ࡛⤎ྙࡌࡾ 61ࠊDlxin-1 ࡛ GRIP1b
ࡢ࠷ࡍࡿࡵ Dlx5 ࡡ N ᮆ❻㡷ᇡ࡛⤎ྙࡊࠉDlx5 ࡡ㌹෕Ὡᛮࢅቌᖕࡌࡾ 60,61ࠊࡈࡼ࡞ࠉ
29
Dlx5 ࡢ Drosophila muscle segment homeobox (msh)㐿ఎᏄࡡဳ஘㢦࣓࣌ࣞࢡ࡚࠵ࡾ Msx1
࠽ࡻࡦ Msx2 ࡛ப࠷ࡡ࣒࢛࣌ࢺ࣒࢕ࣤ㡷ᇡࢅ௒ࡊ࡙ࢰࣤࣂࢠ㈻㛣┞பష⏕ࡌࡾ
62
ࠊ
Dlx5 ࡢ Msx1 ࡱࡒࡢ Msx2 ࡛々ྙమࢅᙟᠺࡌࡾࡆ࡛࡞ࡻࡖ࡙ DNA ⤎ྙ࠿㜴ᐐࡈࡿࠉ
ୖὮ㐿ఎᏄ࡞ᑊࡌࡾ㌹෕Ὡᛮ࠿఩ୖࡌࡾ 62ࠊࡆࡡࡻ࠹࡞ࠉDlx5 ࡢᵕࠍ࡝ࢰࣤࣂࢠ㈻࡛
┞பష⏕ࡌࡾࡆ࡛࡞ࡻࡽ⮤㌗࠿ᣚࡗᶭ⬗࠽ࡻࡦ௙ࡡࢰࣤࣂࢠ㈻ࡡᶭ⬗ࢅโᚒࡌࡾࠊ
ᮇ◂✪࡚ࡢࠉDlx5 ࡢ N ᮆ❻㡷ᇡࡱࡒࡢ࣒࢛࣌ࢺ࣒࢕ࣤ㡷ᇡࢅ௒ࡊ࡙ GATA-4 ࡛ࢰ
ࣤࣂࢠ㈻㛣┞பష⏕ࡌࡾࡆ࡛࠿᩺ࡒ࡞ฦ࠾ࡖࡒࠊࡈࡼ࡞ Dlx5 ࡢ GATA-4 ࡛ࡡභⓆ⌟
ୖ࡞࠽࠷࡙ StAR 㐿ఎᏄࡡࣈ࣓࣭ࣞࢰ࣭Ὡᛮࢅ୕᪴ࡈࡎࡾ஥࠿᪺ࡼ࠾࡞࡝ࡖࡒࠊDlx
࠿ DNA ࡛⤎ྙࡌࡾࡒࡴ࡞ࡢ࣒࢛࣌ࢺ࣒࢕ࣤ⤎ྙࢦ࢕ࢹ࠿ᚪさ࡚࠵ࡾࡆ࡛࠿▩ࡼࡿ࡙
࠽ࡽ 39,40ࠉ☔࠾࡞ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭୕࡞ࡢ඼ᆵⓏ࡝࣒࢛࣌ࢺ࣒࢕ࣤ⤎ྙ㒼า࠿
Ꮛᅹࡊ࡙࠷ࡒࠊࡊ࠾ࡊࠉDlx5 ༟≺ࡡ㐛๨Ⓠ⌟࡚ࡢࠉStAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭ࡢὩᛮ
໩ࡊ࡝࠾ࡖࡒࠊ⮾࿝῕࠷஥࡞ࠉDlx5 ࡛ GATA-4 ࡡ༝ㄢⓏ࡝ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭
ࡡὩᛮ໩ࡢ࣒࢛࣌ࢺ࣒࢕ࣤ⤎ྙࢦ࢕ࢹ࡚ࡢ࡝ࡂࠉGATA-4 ⤎ྙࢦ࢕ࢹࢅ௒ࡊ࡙࠷ࡾ஥
࠿ฦ࠾ࡖࡒࠊ௧୕ࡡࡆ࡛࠾ࡼࠉᮇ◂✪࡚ᚋࡼࡿࡒ⤎ᯕࡢ StAR 㐿ఎᏄ㌹෕โᚒ࡞࠽࠷
࡙ Dlx5 ࠿ GATA-4 ࡡ㌹෕භᙲᅄᏄ࡛ࡊ࡙ᶭ⬗ࡌࡾྊ⬗ᛮࢅ♟ࡌࡵࡡ࡚࠵ࡾࠊ
➠஦⟿ Dlx5 ࡞ࡻࡾ StAR 㐿ఎᏄ㌹෕Ὡᛮ໩ᶭᵋ
Dlx5 ࡢ࡜ࡡࡻ࠹࡞ࡊ࡙ StAR 㐿ఎᏄࡡ㌹෕Ὡᛮࢅಀ㐅ࡌࡾࡡࡓࢀ࠹࠾ࠊDlx5 ࢰࣤ
ࣂࢠ㈻ࡢ࣒࢛࣌ࢺ࣒࢕ࣤ㡷ᇡࡡ୦ഁ࡞㸧ࡗࡡࣈࣞࣛࣤࣛࢴࢲ㡷ᇡࢅᣚࡖ࡙࠷ࡾࠊୌ⯙
Ⓩ࡞ࠉࣈࣞࣛࣤࣛࢴࢲ㡷ᇡࡢ㌹෕Ὡᛮ࡞ᑊࡊ࡙ಀ㐅Ⓩ࡞഼ࡂ஥࠿▩ࡼࡿ࡙࠷ࡾ 63ࠊᐁ
㝷ࠉDlx5 ࡡ N ᮆ❻㡷ᇡࡢ㌹෕Ὡᛮ໩ࢺ࣒࢕࡛ࣤࡊ࡙ᶭ⬗ࡌࡾ
60
ࠊᮇ◂✪࡞࠽࠷࡙ࠉ
Dlx5 ࣆࣚࢡ࣒ࣤࢹࢅ⏕࠷ࡒ luciferase assay ࠾ࡼࠉDlx5 ࡡ N ᮆ❻㡷ᇡ࠽ࡻࡦ C ᮆ❻㡷
ᇡࡢ GATA-4 භⓆ⌟ୖ࡞࠽࠷࡙ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭ࡡ㌹෕Ὡᛮ໩ࢺ࣒࢕࡛ࣤࡊ
࡙ᶭ⬗ࡊᚋࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖࡒࠊࣃࢪࢹࣤ࢓ࢬࢲࣜ໩㓕⣪࡚࠵ࡾ p300 ࡢ GATA-4
ࡡ㌹෕භᙲὩᛮ໩ᅄᏄ࡛ࡊ࡙ᶭ⬗ࡊ
64
ࠉGATA-4 ࡡ StAR 㐿ఎᏄ࡞ᑊࡌࡾ㌹෕Ὡᛮࢅ
ቌᖕࡌࡾ 30ࠊࡈࡼ࡞ࠉࣈࣞࣛࣤࣛࢴࢲ㡷ᇡࡢ p300 ࡷᇱᮇ㌹෕ᅄᏄࡡୌࡗ࡚࠵ࡾ TFIID
࡛┞பష⏕ࡌࡾ஥࠿▩ࡼࡿ࡙࠷ࡾ
65,66
ࠊࡆࡿࡼࡡ▩ぜ࠾ࡼࠉDlx5 ࡡ N ᮆ❻࠽ࡻࡦ C
ᮆ❻㡷ᇡ࡞Ꮛᅹࡌࡾࣈࣞࣛࣤࣛࢴࢲ㡷ᇡ࠿ p300ࠉTFIID ࡝࡜ࢅࣛࢠ࣭ࣜࢹࡌࡾࡆ࡛࡞
ࡻࡽ㌹෕Ὡᛮ໩ࢺ࣒࢕࡛ࣤࡊ࡙ᶭ⬗ࡊᚋࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ
30
➠୔⟿ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡ⫶ඡ⢥ᕛ࡞࠽ࡄࡾᶭ⬗
ࡆࡡࡻ࠹࡞ Dlx5 ࡢ GATA-4 ࡛࡛ࡵ࡞ StAR 㐿ఎᏄࡡⓆ⌟ㄢ⟿࡞㛭୙ࡌࡾࡆ࡛࠿♟ြ
ࡈࡿࡒࠊ๑㏑ࡡࡻ࠹࡞ StAR ࡢࢪࢷࣞ࢕ࢺྙᠺ࡞ᚪ㡪࡚࠵ࡽࠉStAR 㐿ఎᏄን␏࡞ࡻࡽ
Ⓠ⑍ࡌࡾ lipoid CAH ࡞࠽࠷࡙⏠ᛮ௫ᛮ༖㝔㝟ࡡ⑍≟࠿ヾࡴࡼࡿࡾ 21ࠊᐁ㝷࡞ Dlx5 ࠿
ࢪࢷࣞ࢕ࢺྙᠺ࡞㛭୙ࡊ࡙࠷ࡾ࠾ㄢ࡬ࡾࡒࡴ࡞ࠉDlx5/6 DKO ࣏ࢗࢪ⫶ඡ⢥ᕛࡡ⾪⌟
ᆵࢅよᯊࡊࡒࠊࡐࡡ⤎ᯕࠉ⑍≟࡞಴మᕣ࠿࠵ࡾࡵࡡࡡ Dlx5/6 DKO ࣏ࢗࢪ⫶ඡ⢥ᕛ࡞
࠽࠷࡙☔࠾࡞ StAR 㐿ఎᏄࡡⓆ⌟࠿఩ୖࡌࡾലྡྷ࠿☔ヾࡈࡿࡒࠊࡱࡒࠉDlx5/6 DKO ࣏
ࢗࢪ⫶ඡ࡚ࡢࠉ⢥ᕛྱ᭯ࢷࢪࢹࢪࢷ⃨ࣞࣤᗐࡡ఩ୖ࡛࡛ࡵ࡞ࠉAGD ఩ୖ࡛࠷ࡖࡒ㞕
ᛮ໩␏ᖏ࠿ふᐳࡈࡿࡒࠊ௧୕ࡡࡆ࡛࠾ࡼࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ⫶ඡ⢥ᕛ
Ⓠ⏍㐛⛤࡞࠽࠷࡙ࢷࢪࢹࢪࢷࣞࣤྙᠺ࡞㛭୙ࡌࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖࡒࠊ
Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ⫶ඡ⢥ᕛࡓࡄ࡚࡝ࡂࠉᠺమ⢥ᕛ࡞࠽࠷࡙ࡵⓆ⌟ࡊ
࡙࠷ࡾ 36,37ࠊࡱࡒᮇ◂✪࡞࠽࠷࡙ࠉ୦㐿ఎᏄࡢᠺమ༵ᕛ࡚ࡵⓆ⌟ࡊ࡙࠷ࡒ࠿ࠉ⫶ඡ༵
ᕛ࡚ࡢࡐࡿࡼࡡⓆ⌟ࡢヾࡴࡼࡿ࡝࠾ࡖࡒ(data not shown)ࠊ࣏ࢗࢪ࡚ࡢ⫶ඡ༵ᕛࡢࢪࢷ
ࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺࢅ⾔ࢂ࡝࠷ࡆ࡛࠿▩ࡼࡿ࡙࠷ࡾࠊࡆࡿࡼࡡ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6
㐿ఎᏄࡡⓆ⌟ࣂࢰ࣭ࣤࡢࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ࡛᫤㛣✭㛣Ⓩ࡝ୌ⮬࠿ヾࡴࡼࡿࡾ
஥࠾ࡼࠉ୦㐿ఎᏄࡢᠺమ⢥ᕛ࠽ࡻࡦᠺమ༵ᕛ࡞࠽࠷࡙ࡵࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ࡞㛭
୙ࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ
➠ᄿ⟿ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࡛ࢼ࣭ࣖࣞࢪࢷࣞ࢕ࢺ࡛ࡡ㛭㏻ᛮ
⬳♼⤊⣌ࡢᮆᲀහฦἢ⭚࠿ྙᠺࡌࡾࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤࡡᵾⓏჹᏻ࡛ࡊ࡙ᤂ࠻ࡼ
ࡿ࡙ࡀࡒࠊࡊ࠾ࡊࠉ㎾ᖳࠉ⬳♼⤊࠿≺⮤࡞ࢤࣝࢪࢷ࣭ࣞࣜࢅࡵ࡛࡞ࢪࢷࣞ࢕ࢺ࣓࣌ࣜ
ࣤࢅྙᠺࡊ࡙࠷ࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖ࡙ࡀࡒ 67,68ࠊ⬳♼⤊⣌࠿ྙᠺࡌࡾࡆࡡ᩺ࡊ࠷ᴣ
ᛍࡡ⬳ฦᏄࡢࠉᢧᾐහฦἢ⭚࠿ࡗࡂࡾᚉᮮࡡཿ඼Ⓩࢪࢷࣞ࢕ࢺ(classical steroid)࡛༇ื
ࡊ࡙ࠉࢼ࣭ࣖࣞࢪࢷࣞ࢕ࢺ㸝neurosteroid㸞࡛ྞ௛ࡄࡼࡿࡒࠊࢼ࣭ࣖࣞࢪࢷࣞ࢕ࢺࡢ♼
⤊ࡡⓆ⏍ࡷᶭ⬗࡞ኬࡀ࡝ᙫ㡢ࢅ୙࠻ࡾࡆ࡛࠿▩ࡼࡿ࡙࠷ࡾ 67,68ࠊ᭩㎾ࡡሒ࿈࠾ࡼࠉStAR
㐿ఎᏄ࠿୯ᯙ♼⤊࠽ࡻࡦᮆᲀ♼⤊࡞࠽࠷࡙Ⓠ⌟ࡊ࡙࠷ࡾࡆ࡛࠿᪺ࡼ࠾࡞࡝ࡖࡒࠊDlx5
㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ๑⬳ᙟᠺ࡞ᚪさ࡝ᅄᏄ࡚࠵ࡽ
⓮㈻࡝࡜ࡡ StAR 㐿ఎᏄⓆ⌟㡷ᇡ࡞࠽࠷࡙Ⓠ⌟ࡊ࡙࠷ࡾ
69
ࠉっᗃୖ㒂ࠉႡ⌣ࠉኬ⬳
70-73
ࠊࡆࡿࡼࡡ஥ᐁࡢࠉDlx5
㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࠿⬳ࡡࢪࢷࣞ࢕ࢺྙᠺ⣵⬂࡚Ⓠ⌟ࡊ࡙࠷ࡾྊ⬗ᛮࢅ♟ြࡌ
31
ࡾࡵࡡ࡚࠵ࡾࠊࡊࡒ࠿ࡖ࡙ࠉDlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄ࠿⬳♼⤊⣌࡞࠽࠷࡙ࡵࢪ
ࢷࣞ࢕ࢺྙᠺ࡞㛭୙ࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ
➠஫⟿ ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄࡡአ⏍Ṣჹᙟᠺ㐛⛤࡞࠽ࡄࡾᶭ⬗
࢓ࣤࢺࣞࢣࣤࡢ㞕ᛮ⏍Ṣჹᙟᠺ࡞ᚪ㡪ࡡᅄᏄ࡚࠵ࡽࠉᠺమ࡞࠽࠷࡙ࡵ⏠ᛮ⏍⌦ᶭ⬗
ࡡ⥌ᣚࢅᢰ࠹㟸ᖏ࡞ኣᵕ࡝ᙲ๪ࢅᣚࡗࢪࢷࣞ࢕ࢺ࣓࡚࣌ࣜࣤ࠵ࡾࠊ᭩㎾ࡡሒ࿈࡚ࡢࠉ
࢓ࣤࢺࣞࢣࣤ࠿ ES ⣵⬂࠾ࡼᚨ➵࡫ࡡฦ໩ㄇᑙࢅ⾔࠹ྊ⬗ᛮ࠿♟ြࡈࡿ࡙࠷ࡾ
74
ࠊࡱ
ࡒࠉchromatin immunoprecipitation together with microarray (Chip-chip)Ἢ࡞ࡻࡾよᯊ࠾ࡼ
ᰶහ㌹෕ᅄᏄ࡚࠵ࡾ AR ࡡᵾⓏ㐿ఎᏄࡢኣ⛸ኣᵕ࡚࠵ࡽࠉ々㞟࡝ࢨࢡࢻࣜ࢜ࢪࢢ࣭ࢺ
ࢅᣚࡗࡆ࡛࠿᪺ࡼ࠾࡞࡝ࡖ࡙࠷ࡾ࠿ 75ࠉ࢓ࣤࢺࣞࢣࣤࢨࢡࢻࣜධㇲよ᪺࡞ࡢ⮫ࡖ࡙࠷
࡝࠷ࠊ
AR 㐿ఎᏄን␏࣏ࢗࢪ(testicular feminization, Tfm)ࡷ AR 㐿ఎᏄḖ᥾࣏ࢗࢪ(AR KO)࡞
ࡻࡾよᯊ࠾ࡼࠉአ⏍Ṣჹᙟᠺ࡞࠽࠷࡙ࡵ࢓ࣤࢺࣞࢣࣤ࠿㞕ᛮ໩ᅄᏄ࡛ࡊ࡙ᶭ⬗ࡌࡾࡆ
࡛ࡢ᪺ࡼ࠾࡚࠵ࡾ 76ࠊᮇ◂✪࡚ࡢ⫶ඡ᭿㞕ᛮ໩ᶭᗆࢅ᪺ࡼ࠾࡞ࡌࡾࡆ࡛ࢅᛍ㢄࡞ࠉࡐ
ࡡ㞕ᛮ໩㐛⛤ࡡ◂✪࠿ࡵࡖ࡛ࡵ㐅ࢆ࡚࠷ࡾአ⏍Ṣჹࢅ࣓ࢸ࡛ࣜࡊ࡙ࠉ࢓ࣤࢺࣞࢣࣤᵾ
Ⓩ㐿ఎᏄࡡⓆ⌟よᯊࢅ࠽ࡆ࡝ࡖࡒࠊࡐࡡ⤎ᯕࠉ3 ࡗࡡ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄ
(Cyp1b1 㐿ఎᏄࠉFkbp51 㐿ఎᏄ࠽ࡻࡦ MafB 㐿ఎᏄ) ࠿᪺ࡼ࠾࡛࡝ࡽࠉአ⏍Ṣჹᙟᠺ
㐛⛤࡞࠽ࡄࡾࡐࡿࡼࡡᶭ⬗ࢅ௧ୖ࡞⩻ᐳࡊࡒࠊ
ⷾ∸௥ㅨ㓕⣪ࡡୌࡗ࡚࠵ࡾ Cyp1b1 㐿ఎᏄࡢࠉ஘⒬ࠉ༵ᕛ⒬࡝࡜ኣࡂࡡ⭐⑾⤄⧂࡚
ࡐࡡⓆ⌟࠿ㄇᑙࡈࡿ࡙࠽ࡽࠉ≁࡞๑❟⭚⒬࡚ࡢࡐࡡ㐿ఎᏄኣᆵ㸝single nucleotide
polymorphisms, SNP㸞࡞ࡻࡽ⑋≟࠿ኬࡀࡂ␏࡝ࡾࡆ࡛࠿ሒ࿈ࡈࡿ࡙࠷ࡾ
77,78
ࠊࡱࡒࠉ
ⷾ๠ࡓࡄ࡚ࡢ࡝ࡂࠉ࢙ࢪࢹࣞࢣࣤࡷࢷࢪࢹࢪࢷࣞࣤࡡ௥ㅨ࡞ࡵ㛭୙ࡊ࡙࠷ࡾ 78ࠊ࣏ࢗ
ࢪአ⏍Ṣჹᙟᠺ㐛⛤࡞࠽࠷࡙ࠉCyp1b1 㐿ఎᏄࡢ㞕አ⏍Ṣჹ㎾న㒂ࡡ㛣ⴝ㡷ᇡ࡚Ⓠ⌟
࠿ᙁࡂヾࡴࡼࡿࡒࠊ࢓ࣤࢺࣞࢣࣤᚺ➽㐿ఎᏄ࡚࠵ࡾ Cyp1b1 㐿ఎᏄࡢࢷࢪࢹࢪࢷࣞࣤ
ࡡ௥ㅨࢅ⾔࠹ࡆ࡛࠾ࡼࠉ࢓ࣤࢺࣞࢣࣤࠉCYP1B1 ࡡ୦⩽࡞ࡢㇿࡡࣆ࢔࣭ࢺࣁࢴࢠ࠿Ꮛ
ᅹࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ
ࡱࡒࠉCyp1b1 㐿ఎᏄࡢ࢓ࣛࣜࣀ࢕ࢺུ࣭ࣞ࢜࣍ࣤᐖమ(aryl hydrocarbon receptor,
AhR)ࡡᵾⓏ㐿ఎᏄ࡚ࡵ࠵ࡾ
79
ࠊࢱ࢕࢛࢞ࢨࣤࡢ AhR ࢅ௒ࡊ࡙⏍మẐᛮࢅⓆ⌟ࡌࡾࠊ
አ⏍Ṣჹᙟᠺ㐛⛤࡞࠽࠷࡙ࠉAhR 㐿ఎᏄⓆ⌟ࡢአ⏍Ṣჹ㛣ⴝ㡷ᇡ࡚Ⓠ⌟ࡊ࡙࠽ࡽࠉዲ
32
ፈ࣏ࢗࢪࢅ⏕࠷ࡒ TCDD ᢖ୙ᐁ㥺࡞ࡻࡽᑺ㐠ୖ⿛ᵕࡡ⑍≟࠿ㄇᑙࡈࡿࡾ 80,81ࠊᮇ◂✪
࡚ Cyp1b1 㐿ఎᏄࡢ࢓ࣤࢺࣞࢣࣤ࡞ᚺ➽ࡊㄇᑙࡈࡿࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࡆ࡛࠾ࡼࠉ
Cyp1b1 㐿ఎᏄⓆ⌟ㄢ⟿࡞ࡢ AR ࡛ AhR ࡡࢠࣞࢪࢹ࣭ࢠ࠿Ꮛᅹࡌࡾ௫ㄕ࠿⩻࠻ࡼࡿࡾࠊ
ࡵࡊࠉࡆࡡ௫ㄕ࠿ḿࡊ࠷ࡵࡡ࡚࠵ࡿࡣࠉࢱ࢕࢛࢞ࢨࣤ࡞ࡻࡽㄇᑙࡈࡿࡾᑺ㐠ୖ⿛ࡢ
Cyp1b1 㐿ఎᏄⓆ⌟ㄇᑙ࠿㐛๨࡞࡝ࡽࠉࡐࡿࢅུࡄ࡙ࠉష⏕ࡌࡾ࡬ࡀ࢓ࣤࢺࣞࢣࣤ࠿
௥ㅨࡈࡿࠉࡐࡡ⤎ᯕࣆࣜࢰ࣐ࢺᢖ୙࡛ྜྷᵕ࡞࢓ࣤࢺࣞࢣࣤᶭ⬗࠿఩ୖࡌࡾࡆ࡛࡞ࡻࡽ
ᑺ㐠ᙟᠺ␏ᖏࢅᘤࡀ㉫ࡆࡌྊ⬗ᛮ࠿᥆ᐳࡈࡿࡾࠊ
๑❟⭚⒬⣵⬂ᰬࢅ⏕࠷ࡒよᯊ࠾ࡼࠉAR ࡢ├᥃Ⓩ࡞ Fkbp51 㐿ఎᏄࡡⓆ⌟ㄢ⟿ࢅ⾔࠹
ࡆ࡛࠿᪺ࡼ࠾࡚࠵ࡾࠊࡈࡼ࡞ࠉFebbo ࡼࡢࠉAR ࡛ FKBP51 ࡢࢰࣤࣂࢠ㈻㛣┞பష⏕
ࢅᣚࡔࠉFkbp51 㐿ఎᏄࡡ㐛๨Ⓠ⌟ୖ࡚ࡢࠉAR ࡡ㌹෕Ὡᛮ࠿୕᪴ࡌࡾ࡛ሒ࿈ࡊ࡙࠷ࡾ
82
ࠊࡆࡿࡼࡡ▩ぜ࠾ࡼࠉFKBP51 ࡢ࢓ࣤࢺࣞࢣࣤࢨࢡࢻࣜ࡞ᙫ㡢ࢅ୙࠻ᚋࡾᅄᏄ࡚࠵
ࡾ࡛஢᝷ࡈࡿࡾ࠿ࠉFkbp51 㐿ఎᏄḖ᥾࣏ࢗࢪ(Fkbp51 KO)࡚ࡢἢᑺ⏍Ṣჹᙟᠺ࡞㢟ⴥ
࡝␏ᖏࡢヾࡴࡼࡿࡍࠉዲᏈᛮࡵḿᖏ࡚࠵ࡖࡒ 83ࠊFKBP51 ࡛ྜྷࡋࣆ࢒࣐࣭࡚ࣛ࠵ࡽࠉ
AR ࡡࢨ࡛ࣔ࣋ࣞࣤࡊ࡙ᶭ⬗ࡌࡾ Fkbp52 ࡡ㐿ఎᏄḖ᥾࣏ࢗࢪࡢࠉᑺ㐠ୖ⿛ᵕࡡᙟឺ␏
ᖏ࠽ࡻࡦ๑❟⭚ᙟᠺ୘ධࢅ♟ࡌ 83ࠊࡈࡼ࡞ FKBP52 Ḗ᥾ୖ࡚ࡢࠉAR ࡡ㌹෕Ὡᛮ࠿ⴥ
ࡊࡂ఩ୖࡌࡾࡆ࡛࠾ࡼࠉFKBP52 ࡢ AR ࡡ㌹෕Ὡᛮ࡞ᙫ㡢ࢅ୙࠻ࡾࡆ࡛࠿᪺ࡼ࠾࡞࡝
ࡖ࡙࠷ࡾ 83ࠊ௧୕ࡡࡆ࡛࠾ࡼࠉFkbp51 㐿ఎᏄࡢ࢓ࣤࢺࣞࢣࣤᚺ➽㐿ఎᏄ࡚࠵ࡾ࠿ࠉ㞕
አ⏍Ṣჹᙟᠺ࡞ࡢ├᥃Ⓩ࡞ࡢ㛭୙ࡊ࡝࠷ࡆ࡛࠿⩻࠻ࡼࡿࡾࠊࡊ࠾ࡊࠉFkbp51 㐿ఎᏄ
ࡢ࢓ࣤࢺࣞࢣࣤ࡞౪ᏋࡊࡒⓆ⌟ㄇᑙࢅུࡄࡾࡆ࡛࠾ࡼࠉ࢓ࣤࢺࣞࢣࣤᶭ⬗ࡡ࣭ࣝ࣎ࢰ
࣭㐿ఎᏄ࡛ࡊ࡙᭯ຝ࡚࠵ࡾ⩻࠻ࡼࡿࡾࠊࡆࡡ஥ᐁࡢࠉFkbp51 㐿ఎᏄ࠿௑ᚃࡡ࢓ࣤࢺ
ࣞࢣࣤ࡞ࡻࡾ㞕አ⏍ṢჹᙟᠺࡡฦᏄ࣒࢜ࢼࢫ࣑ࢅよᯊࡌࡾ୕࡚㔔さ࡝ᅄᏄ࡛࡝ࡽᚋ
ࡾࡆ࡛ࢅ♟ࡊ࡙࠷ࡾࠊ
MafB 㐿ఎᏄࡢᚃ⬳ࡷහ⪝ࡡᙟᠺ࡞㔔さ࡝ᅄᏄ࡚࠵ࡾ 84ࠊ⮾࿝῕࠷ࡆ࡛࡞ࠉMafB ࡢ
Fos ࡛ࣉࢷࣞࢱ࢕࣏࣭ࢅᙟᠺࡌࡾ 85ࠊFos ࡢ activator protein (AP-1)ࡡ୹࡝ᵋᠺᅄᏄࡡୌ
ࡗ࡚࠵ࡽࠉAP-1 々ྙమࡢ FGF ࡷ Wnt ࡝࡜ࡡቌṢᅄᏄࡡ㔔さ࡝ୖὮᅄᏄ࡚࠵ࡾࠊFgf10
㐿ఎᏄࡢᑺ㐠୦ഁ㛣ⴝ࡚Ⓠ⌟ࡊ࡙࠽ࡽࠉࡐࡡ㐿ఎᏄḖ᥾࣏ࢗࢪࢅ⏕࠷ࡒᶭ⬗よᯊ࠾ࡼ
Fgf10 㐿ఎᏄࡢᑺ㐠ᙟᠺ࡞㛭୙ࡊ࡙࠷ࡾࡆ࡛࠿ࢂ࠾ࡖ࡙࠷ࡾ 86ࠊࡈࡼ࡞ࠉFGF10 ࡡུ
ᐖ మ ࡚ ࠵ ࡾ Fgf receptor IIIb (FgfrIIIb) 㐿ఎᏄࡵᑺ㐠୦ഁ㛣ⴝ࡞Ⓠ⌟ࡊ࡙࠽ࡽ ࠉ
FGF-FGFRIIIb ࡡ┞பష⏕ࡢᑺ㐠ᙟᠺ࡞ᚪ㡪࡚࠵ࡾ 87,88ࠊWnt5a 㐿ఎᏄࡢአ⏍Ṣჹ㛣ⴝ
33
㡷ᇡ࡞Ⓠ⌟ࡊ࡙࠽ࡽࠉWnt5a 㐿ఎᏄḖ᥾࣏ࢗࢪࡢአ⏍Ṣჹᙟᠺ୘ධࢅ࿆ࡌࡾ 89ࠊࡆࡡ
ࡻ࠹࡞ࠉFGFࠉWnt ࡝࡜ࡡቌṢᅄᏄ⩄࠿አ⏍Ṣჹᙟᠺ㐛⛤࡞ᚪさ࡚࠵ࡾ஥ࡢ᪺ࡼ࠾࡚
࠵ࡾ࠿ࠉࡆࡿࡼ࠿࢓ࣤࢺࣞࢣࣤ౪ᏋⓏ࡝አ⏍Ṣჹᙟᠺ࡞࠽࠷࡙࡜ࡡࡻ࠹࡝ᙲ๪ࢅᣚࡗ
ࡡ࠾ࡢᮅࡓ୘᪺࡚࠵ࡾࠊ
ᮇ◂✪࡞ࡻࡽࠉMafB 㐿ఎᏄ࠿㞕አ⏍Ṣჹᑺ㐠୦ഁ㛣ⴝ࡚ᙁࡂⓆ⌟ࡌࡾࡆ࡛࠿᪺ࡼ
࠾࡞࡝ࡖࡒࠊFgf 㐿ఎᏄࠉWnt 㐿ఎᏄࡵྜྷ㡷ᇡ࡚Ⓠ⌟ࡌࡾࡆ࡛࠾ࡼࠉMafB ࡢࡆࡿࡼࡡ
ቌṢᅄᏄ⩄ࡡୖὮᅄᏄ࡚࠵ࡾ AP-1 ࡡ㌹෕Ὡᛮࢅಞ㣥ࡊࠉࡐࡿ࡞ࡻࡖ࡙㞕አ⏍Ṣჹ≁
᭯ࡡᙟឺᙟᠺࢅㄇᑙࡌࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ
34
➠ᄿ❮ ⥪ᣋ
ࣃࢹ⏠ඡ࡞࠽ࡄࡾᛮฦ໩␏ᖏ⑍ࡢࠉ⏍Ṣ⭚ᙟᠺ୘ධࠉᛮ࣓࣌ࣜࣤྙᠺ୘ධࠉ࢓ࣤࢺ
ࣞࢣࣤ୘ᚺ⑍࡝࡜ኣᒪ࡞ࢂࡒࡾࠊࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺ␏ᖏ࡞ࡻࡾᛮฦ໩␏ᖏ⑍ࡡ
ୌࡗ࡞ࣛ࣎࢕ࢺ㐛ᙟᠺ⑍࠿࠵ࡽࠉ࢓ࣤࢺࣞࢣࣤࢅࡢࡋࡴ࡛ࡌࡾᛮ࣓࣌ࣜࣤࡡྙᠺ୘ධ
࡛࡛ࡵ࡞ࠉ⏠ඡ࡚ࡢ⏠ᛮ௫ᛮ༖㝔㝟ࢅ࿆ࡌࡾࠊᮇ⑍ࡢ᪝ᮇெ࡞Ẓ㍉ⓏኣࡂⓆ⑍ࡊࠉࡐ
ࡡ࡮࡛ࢆ࡜࠿ StAR 㐿ఎᏄን␏࡞ࡻࡾࡵࡡ࡚࠵ࡾ࠿ࠉStAR 㐿ఎᏄࡡን␏࠿ྜྷᏽࡈࡿ࡝
࠷ౚࡵヾࡴࡼࡿ࡙࠷ࡾࠊ
ᮇ◂✪࡞࠽ࡄࡾࣃࢹ඙ኮᛮ⑄ᝀ SHFM1 ࡡཋᅄ㐿ఎᏄ࡚࠵ࡾ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6
㐿ఎᏄࡡᶭ⬗よᯊ࡞ࡻࡽࠉDlx5 ࠿ GATA-4 ࡛々ྙమࢅᙟᠺࡊࠉStAR 㐿ఎᏄࣈ࣓࣭ࣞ
ࢰ࣭Ὡᛮࢅ୕᪴ࡈࡎࡾࡆ࡛࠿♟ြࡈࡿࡒࠊࡈࡼ࡞ Dlx5/6 DKO ࣏ࢗࢪࡡ⾪⌟ᆵよᯊ࡞
ࡻࡽࠉ⫶ඡࣚ࢕ࢸ࢔ࢴࣃ⣵⬂࡚Ⓠ⌟ࡌࡾ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡢ࢓ࣤࢺࣞࢣ
ࣤྙᠺࢅಀ㐅ࡊࠉࡐࡿ࡞ࡻࡽ⫶ඡࡡ㞕ᛮ໩ࢅಀࡌྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊࡱࡒࠉDlx5
㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡⓆ⌟ࡢ⫶ඡ⢥ᕛࡓࡄ࡚࡝ࡂࠉᠺమ⢥ᕛࠉᠺమ༵ᕛࠉ⬳࡝
࡜ࡡ௙ࡡࢪࢷࣞ࢕ࢺ࣓࣌ࣜࣤྙᠺჹᏻ࡚ࡵヾࡴࡼࡿࡾࡆ࡛࠾ࡼࠉ௑ᚃࡆࡿࡼࡡჹᏻ࡞
࠽ࡄࡾ୦㐿ఎᏄࡡᙲ๪࡞ࡗ࠷࡙ࡵἸ┘ࡊ࡙࠷ࡾࠊ
࢓ࣤࢺࣞࢣࣤ࠿⏠ᛮ⏍Ṣჹᙟᠺ࡞㟸ᖏ࡞㔔さ࡝ᙲ๪ࢅⁿࡋ࡙࠷ࡾࡆ࡛ࡢ Tfm ࡷ AR
KO ࡡよᯊ࠾ࡼ᪺ࡼ࠾࡚࠵ࡾࠊ⫶ඡ⢥ᕛ࡞࠽ࡄࡾ Dlx5 㐿ఎᏄ࠽ࡻࡦ Dlx6 㐿ఎᏄࡡᶭ
⬗よᯊ࠾ࡼࠉ୦㐿ఎᏄ࠿࢓ࣤࢺࣞࢣࣤྙᠺ࡞ᐞ୙ࡌࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒ࠿ࠉࣃࢹ඙
ኮᛮ⑄ᝀ࡚࠵ࡾᑺ㐠ୖ⿛ࡷೳ⏻⢥ᕛࡡฦᏄ⑋ឺࢅ⌦よࡌࡾࡒࡴ࡞ࡢࠉ㞕ᛮ⏍Ṣჹᏻ࡞
࠽ࡄࡾ࢓ࣤࢺࣞࢣࣤࢨࢡࢻࣜࡡฦᏄᶭᵋࡡよ᪺࠿ᚪ㡪࡚࠵ࡾࠊࡆࡿࢅུࡄ࡙ࠉᮇ◂✪
࡚ࡢ Cyp1b1 㐿ఎᏄࠉFkbp51 㐿ఎᏄࠉMafB 㐿ఎᏄ࡝࡜ࡡ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎ
Ꮔ⩄ࡡⓆ⌟よᯊࢅ⾔࠷ࠉࡐࡿࡼࡡአ⏍Ṣჹᙟᠺ᭿࡞࠽ࡄࡾ஢᝷ࡈࡿࡾᶭ⬗ࢅ㏑࡬ࡒࠊ
௑ᚃࠉࡆࡿࡼࡡ࢓ࣤࢺࣞࢣࣤᵾⓏು⿭㐿ఎᏄ࠿አ⏍Ṣჹᙟᠺ࡚࡜ࡡࡻ࠹࡝ᙲ๪ࢅࡌࡾ
ࡡ࠾ࢅよᯊࡌࡾࡆ࡛࡚ࠉ࢓ࣤࢺࣞࢣࣤࢨࢡࢻࣜࡡฦᏄᶭᵋࡡ⌦よࢅ῕ࡴࡾࡆ࡛࠿࡚ࡀ
ࡾ࡛⩻࠻࡙࠷ࡾࠊ
1990 ᖳ࡞ᛮỬᏽ㐿ఎᏄ SRY ࠿ྜྷᏽࡈࡿ࡙௧㜾ࠉฦᏄ⏍∸Ꮥࡡ㐅ᒈ࡞ࡻࡽᵕࠍ࡝ᛮ
ฦ໩㐿ఎᏄ࠿ḗࠍ࡞ྜྷᏽࡈࡿࠉᛮࡡฦ໩ᶭᵋࡡ⌦よ࠿᛬㏷࡞῕ࡱࡖ࡙࠷ࡾࠊࡊ࠾ࡊࠉ
ࡱࡓࡱࡓᮅよỬࡡၡ㢗࠿ኣࡂṟࡖ࡙࠷ࡾࡆ࡛ࡵ஥ᐁ࡚࠵ࡾࠊᮇ◂✪ࡡⓆᒈ࠿௑ᚃࡡᛮ
ฦ໩␏ᖏ⑍ࡡฦᏄ⑋ឺࡡࡻࡽⰃ࠷⌦よ࡞⦽࠿ࡾࡆ࡛ࢅ᭿ᙽࡊ࡙࠷ࡾࠊ
35
➠஫❮ ᐁ㥺᪁Ἢ
➠ୌ⟿ ᐁ㥺ິ∸࠽ࡻࡦᇰ㣬⣵⬂
ᮇ◂✪࡚⾔ࢂࡿࡒິ∸ᐁ㥺ࡢࠉ↻ᮇኬᏕິ∸ᐁ㥺ᣞ㔢࡞ᇱࡘࡀ⾔ࡖࡒࠊᮇ◂✪࡚⏕
࠷ࡒ Dlx5 and Dlx6 double knockout (Dlx5/6 DKO)࣏ࢗࢪࡢࠉMerlo ࡼ 13 ࡞ࡻࡽሒ࿈ࡈࡿ
࡙࠷ࡾࡵࡡ࡚࠵ࡾࠊ࣏ࢗࢪࣚ࢕ࢸ࢔ࢴࣃ⭐⑾⣵⬂(mouse Leydig tumor cells, mLTC-145)
ࡢࠉ10% FBS (Invitrogen Life Technologies, Carlsbad, CA)࠽ࡻࡦ penicillin-streptomycin
(Invitrogen)ࢅྱࡳ RPMI1640 ᇰᆀ(Invitrogen)୯࡚ᇰ㣬ࡊࡒࠊCOS-7 ⣵⬂ࡢࠉ10% FBS
࠽ࡻࡦ penicillin-streptomycin ࢅྱࡳ DMEM ᇰᆀ(Invitrogen)୯࡚ᇰ㣬ࡊࡒࠊ
➠஦⟿ Plasmid DNA ࡡష⿿
C57BL/6J ࣏ࢗࢪࡡࢣࢿ࣑ DNA ࢅ⏕࠷࡙ PCR ࡞ࡻࡽ StAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭㡷ᇡ
(-1514/+25 bp ࠽ࡻࡦ-966/+25 bp)ࢅቌᖕࡊࠉpGL3 Basic vector (Promega, Madison, WI)
࡞⤄ࡲ㎲ࢆࡓࠊStAR 㐿ఎᏄࣈ࣓࣭ࣞࢰ࣭࡞࠵ࡾ GATA-4 ⤎ྙࢦ࢕ࢹ࡫ࡡን␏ᑙථࡢࠉ
㐛ཡ࡞ሒ࿈ࡈࡿ࡙࠷ࡾ᝗ሒ 29 ࡞ᇱࡘ࠷࡙⾔ࡖࡒࠊRT-PCR ࡞ࡻࡽᚋࡼࡿࡒ GATA-4 ࡡ
cDNA ࡢࠉpCAGGS vector ࠽ࡻࡦ pCMV-TnT vector (Promega)࡞⤄ࡲ㎲ࢆࡓࠊDlx5
deletion mutants ࡢ PCR ࡞ࡻࡽష⿿ࡊࠉpCMV-Myc vector (Clontech Laboratories, Palo
Alto, CA)ࡱࡒࡢ pCMV-TnT vector ࡞⤄ࡲ㎲ࢆࡓࠊ౐⏕ࡊࡒ primer ࡢ௧ୖࡡ㏳ࡽ࡚࠵ࡾࠊ
-1514/+25 bp fragment F, 5’-AGT CAT CTC TCC AGC CCA ACA CGT-3̓
-966/+25 bp fragment F, 5’-ACC ACA GGG ATC ACA TAC CTG CA-3̓
-1514/+25 bp and -966/+25 bp fragments R, 5’-TCA AGG TCC TGA GTC CTG CAG CT-3̓
GATA-4 F, 5’-TCA GAG CTT GGG GCG ATG TAC CAA-3̓
GATA-4 R, 5’- TAC GCG GTG ATT ATG TCC CCA TGA CT-3̓
Dlx5ΔΝ and Dlx5HD F, 5’-TGA ATT CGG ATG AAA CCA AAG AAA GTT CGT AAA
CCC-3̓
Dlx5ΔC and Dlx5N F, 5’-TGA ATT CGG ATG ACA GGA GTG TTT GAC AGA-3̓
Dlx5C F, 5’-TGA ATT CGG ATG AAA AAC GGG GAG ATG-3̓
Dlx5ΔN and Dlx5C R, 5’-TAA CTC GAG CAA GAG AAA GTA GCC-3̓
36
Dlx5ΔC and Dlx5HD R, 5’-ATG GTA CCG ATCT TCT TGA TCT TGG ATC TTT TG-3̓
Dlx5N R, 5’-ATG GTA CCT GGT TTA CCA TTC ACC ATC CT-3̓.
➠୔⟿ Luciferase assay
ࢹࣚࣤࢪࣆ࢘ࢠࢨࣘࣤࡡ๑᪝࡞ࠉmLTC-1 ⣵⬂ࢅ 105 cells/well ࡡ⣵⬂ᐠᗐ࡚ 24 ࢗ࢘
ࣜࣈ࣭ࣝࢹ࡞᧓࠷ࡒࠊ⤽௥࠾ࡼ 24 ᫤㛣ᚃࠉFuGENE HD (Roche, Basel, Switzerland)ࢅ
⏕࠷࡙ࠉ┘Ⓩ㐿ఎᏄࢅᑙථࡊࡒࠊ྘ࠍࡡ㐿ఎᏄⓆ⌟࣊ࢠࢰ࣭(400 ng)ࠉLuciferase reporter
plasmids(200 ng)ࠉpRL-SV40 plasmid (80 ng)ࡡ᮪௲࡚ࠉtriplicate ࡚ᐁ㥺ࢅ⾔ࡖࡒࠊRenilla
luciferase ࢅࢤ࣭ࢺࡌࡾ pRL-SV40 plasmid ࡢහ㒂ᵾ‵࡛ࡊ࡙⏕࠷ࡒࠊ㐿ఎᏄᑙථᚃࠉ
24 ᫤㛣ᇰ㣬ࡊࡒ⣵⬂ࢅᅂ཭ࡊࠉ⣵⬂᢫ฝᾦࢅ⏕࠷࡙ Dual-Luciferase Reporter Assay
System (Promega)࡞ࡻࡽࠉluciferase Ὡᛮࢅῼᏽࡊࡒࠊ
➠ᄿ⟿ Western blotting Ἢ
ᇰ㣬⣵⬂ࢅSampling buffer (50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 10%
β-mercaptoethanol)࡞ࡻࡽ⁈よࡊࠉ㉰㡚ἴ◒◃ᶭ࡞ࡻࡽ◒◃ࡊࡒᚃ95Υ࡚5ฦ㛣ຊ⇍ࡊ
ࡒࡵࡡࢅモᩩ࡛ࡊࡒࠊ12% SDS-PAGE࡞ࡻࡽฦ㞫ࡊࡒࢰࣤࣂࢠ㈻ࢅࠉPVDF⭯࡞㌹෕
(40mA, 2᫤㛣)ࡊࡒࠊ㌹෕ᚃࡡPVDF⭯ࢅ5% ࢪ࣑࣐࢞ࣜࢠ/TBST (140 mM NaCl, 2.7
mM KCl, 0.1% Tween20, 25 mM Tris-HCl pH7.5)⁈ᾦ࡚ࣇࢴࣞ࢞ࣤࢡฌ⌦(ᐄῺࠉ2᫤㛣)
ࡊࠉTBST࡚ὑὯᚃࠉୌḗᢘమཬᚺࢅ4Υ࡚ୌᬄ⾔ࡖࡒࠊTBST࡚ὑὯᚃࠉ஦ḗᢘమཬ
ᚺ ࢅ ᐄ Ὼ ࡚1᫤㛣⾔ࡖ ࡒ ᚃࠉ PVDF ⭯ࢅTBST ࡚ὑὯࡊࠉ ECL kit (GE Healthcare,
Buckinghamshire, England)࡞ࡻࡽᢘమཬᚺࢅ᳠ฝࡊࡒࠊ౐⏕ࡊࡒᢘమࢅ௧ୖ࡞♟ࡌࠊ
Anti-Myc Tag mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY)
GATA-4(G-4) mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
Dlx5 (Y-20) goat polyclonal antibody (Santa Cruz)
HRP-Goat anti-Mouse IgG+A+M (H+L) (Zymed, South San Francisco, CA)
HRP-Rabbit anti-Goat IgG (H+L) (Zymed)
Mouse TrueBlot: HRP anti-mouse IgG (eBioscience, San Diego, CA)
37
➠஫⟿ ඞ␷ỷ㜾Ἢ
๑᪝࡞ COS-7 ⣵⬂ࢅ 4.5x105/60mm ࢸ࢔ࢴࢨࣖࡡᐠᗐ࡚᧓࠷ࡒࠊ྘ࠍࡡ㐿ఎᏄⓆ⌟
࣊ࢠࢰ࣭ࢅ TransFast (Promega)ࢅ⏕࠷࡙ COS-7 ⣵⬂࡞ᑙථࡊࡒࠊ24 ᫤㛣ᇰ㣬ᚃࠉ
Complete mini protease inhibitor cocktail (Roche)ࢅྱࡳ Lysis buffer (150 mM NaCl, 0.5%
NP40, 10 mM Tris pH7.5)࡚⣵⬂ࢅ⁈よࡊࠉ㉰㡚ἴ◒◃ᶭ࡞ࡻࡽ◒◃ࡊࡒᚃࠉ㐪ᚨ᧧ష
࡞ࡻࡽ⣵⬂ምࢅཱིࡽ㝎࠷ࡒࡵࡡࢅ⣵⬂⁈ฝᾦ࡛ࡊࡒࠊࡆࡡ⣵⬂⁈ฝᾦ࡞ୌḗᢘమࢅຊ
࠻ࠉ4Υ࡚ 1 ᫤㛣㟀┖ࡊࡒᚃࠉprotein G sepharose (GE Healthcare)ࢅຊ࠻ࠉ4Υ࡚ 1 ᫤㛣
㟀┖ࡊࡒࠊLysis Buffer ࡚ὑὯᚃࠉඞ␷々ྙమࢅ sampling buffer ࡞⁈よࡈࡎࡒࠊᚋࡼ
ࡿࡒモᩩࢅ⏕࠷࡙ SDS-PAGE ࠽ࡻࡦ Western Blotting ࢅ⾔࠷ࠉ┘Ⓩࢰࣤࣂࢠ㈻ࢅ᳠ฝ
ࡊࡒࠊඞ␷ỷ㜾࡞౐⏕ࡊࡒᢘమࡢ௧ୖ࡞♟ࡌࠊ
GATA-4 (G-4) mouse monoclonal antibody (Santa Cruz)
Dlx5 (C-20) goat polyclonal antibody (Santa Cruz)
➠ඵ⟿ mammalian two-hybrid assay
Dlx5 ࠽ࡻࡦ GATA-4 ࡡ cDNA ࢅ pACT vector, pBIND vector (Promega)࡫⤄ࡲ㎲ࢆࡓ
ࡵࡡࢅⓆ⌟࣊ࢠࢰ࣭࡛ࡊࡒࠊmLTC-1 ⣵⬂ࢅ 105cells/well ࡡᐠᗐ࡚ 24 ࢗ࢘ࣜࣈ࣭ࣝࢹ
࡞᧓ࡀ 24 ᫤㛣ᇰ㣬ࡊࡒᚃࠉ྘ࠍࡡⓆ⌟࣊ࢠࢰ࣭ࢅ FuGENE HD (Roche)ࢅ⏕࠷࡙㐿ఎ
Ꮔᑙථࡊࡒࠊ24 ᫤㛣ᇰ㣬ᚃࠉ⣵⬂ࢅᅂ཭ࡊࠉDual-Luciferase Reporter Assay System
(Promega)࡞ࡻࡽࠉluciferase Ὡᛮࢅῼᏽࡊࡒࠊ
➠୏⟿ 㐿ఎᏄⓆ⌟よᯊ
➠ୌ㡧 section in situ hybridization
ዲፈ࣏ࢗࢪࡻࡽᦤฝࡊࡒ⫶ඡࢅ4% PFA/PBS୯࡚ୌᬄ4Υ࡞࡙ᅖᏽࡊࡒᚃࠉ࣒ࢰࢿ࣭
ࣜ/PBSTᾦ࡞࡙ṹ㝭Ⓩ࡞⬲Ềࢅ⾔ࡖࡒࡵࡡࢅモᩩ࡛ࡊࡒࠊᚋࡼࡿࡒモᩩࢅ࢞ࢨࣝࣤ࡞
⨠ᥦᚃࠉࣂࣚࣆ࢔ࣤ࡞ໜᇔࡊࠉ8µmࡡว∞ࢅష⿿ࡊࡒࠊࢪࣚ࢕ࢺ࢝ࣚࢪ࡞ᏽ╌ࡈࡎࡒ
モᩩࢅ࢞ࢨࣝࣤ࡞ࡻࡽ⬲ࣂࣚࣆ࢔ࣤฌ⌦ࢅࡊࠉ࣒ࢰࢿ࣭ࣜ/H2Oᾦ࡞࡙ṹ㝭Ⓩ࡞්Ề࿰
38
ࡊࡒࠊ1µg/ml Proteinase Kฌ⌦ᚃࠉ4% PFA/PBSࢅ⏕࠷࡙ว∞ࢅ්ᅖᏽࡊࡒࠊPBST࡚ὑ
ὯᚃࠉDIGᵾㆉࡈࡿࡒ┘Ⓩ㐿ఎᏄRNA probe࡞ࡻࡽhybridizationཬᚺࢅ65Υ࡚ୌᬄ⾔ࡖ
ࡒࠊ5x SSC࠽ࡻࡦTBSTὑὯᚃࠉblocking solution (10% Blocking Reagent (Roche) in 100
mM maleate buffer pH 7.5, 25% heated FBS in TBST)ࢅ⏕࠷࡙ࣇࣞࢴ࢞ࣤࢡฌ⌦ࢅ⾔࠷ࠉ
anti-DIG antibody (Roche)ࢅ⏕࠷࡙4Υ࡞࡙ୌᬄᢘమཬᚺࢅ⾔ࡖࡒࠊTBSTࠉNTMT (100
mM NaCl, 50 mM MgCl2, 0.1% Tween20, 100 mM Tris-HCl pH 9.5)࡚ὑὯᚃࠉᇱ㈻ᾦࢅຊ
࠻㐵ක≟ឺ࡚㓕⣪ཬᚺࢅ⾔ࡖࡒࠊ4% PFA /PBS࡚㓕⣪ཬᚺࢅೳḾࡈࡎࠉPBSTὑὯᚃ࡞
ࢡࣛࢬ࣭ࣞࣜࢅ⏕࠷࡙⤄⧂ว∞ࢅᑌථࡊࠉ㢟᚜㙶ふᐳࢅ⾔ࡖࡒࠊ
➠஦㡧 RNA ᢫ฝ࠽ࡻࡦ Real-time quantitative PCR
࣏ࢗࢪ⫶ඡࡻࡽ⢥ᕛࢅᦤฝࡊࠉISOGEN (Nippongene, Toyama, Japan)ࢅ⏕࠷࡙ total
RNA ࢅ᢫ฝࡊࡒࠊᚋࡼࡿࡒ total RNA ࠾ࡼࠉࣚࣤࢱ࣑ࣈࣚ࢕࣏࣭ࠉSuperScript III ㏣
㌹෕㓕⣪(Invitrogen)ࢅ⏕࠷࡙ cDNA ࢅష⿿ࡊࡒࠊReal-time PCR ࡢ SYBR Green Master
Mix ࢅ⏕࠷࡙ 7500 real-time PCR System (Applied Biosystems, Foster City, CA)࡞ࡻࡽ⾔
ࡖࡒࠊPCR ཬᚺࡡ᮪௲ࡢᖏἪ࡞ࡊࡒ࠿ࡖࡒࠊ⏕࠷ࡒࣈࣚ࢕࣏࣭ࡢ௧ୖࡡ㏳ࡽ࡚࠵ࡾࠊ
Dlx5 F, 5’-AGC TAC CTG GAG AAC TCG GCT T-3̓
Dlx5 R, 5’-GAT TGA GCT GGC TGC GCT-3̓
Dlx6 F, 5’-GTA TGC CTC CCA ACA GCT ACA TG-3̓
Dlx6 R, 5’-GTG TCC TGG TGT GGT GAG GAA TA-3̓
StAR F, 5’-GGA GAT GCC GGA GCA GAG T-3̓
StAR R, 5’-GCC AGT GGA TGA AGC ACC AT-3̓
Cyp1b1 F, 5’-TTG ACC CCA TAG GAA ACT GC-3̓
Cyp1b1 R, 5’-GCT GTC TCT TGG TAG GAG GA-3̓
Fkbp51 F, 5’-GCT GGC AAA CAA CAC GAG AG-3'
Fkbp51 R, 5’-GAG GAG GGC CGA GTT CAT T-3'
MafB F, 5’-AGG CCG CGA GGC TTA TTC-3'
MafB R, 5’-CTC ACA AAG TTC TCA GAG CCA GAA-3'
GAPDH F, 5’-AAC GAC CCC TTC ATT GAC CTC-3̓
GAPDH R, 5’-CCT TGA CTG TGC CGT TGA ATT-3̓.
39
L8 F: 5’-ACA GAG CCG TTG TTG GTG TTG-3'
L8 R: 5’-CAG CAG TTC CTC TTT GCC TTG T-3'
➠ඳ⟿ ⤄⧂ᏕⓏよᯊ
➠ୌ㡧 ࣉ࣏ࢹ࢞ࢨ࢙࢛ࣝࣤ࣬ࢩࣤ᯹Ⰵ(Hematoxylin and Eosin staining)
࣏ࢗࢪ⫶ඡࢅ 4%ࣂࣚࣆ࢚࣑ࣜ࢓ࣜࢸࣃࢺ/PBS ᾦ୯࡚ୌᬄ 4Υ࡞࡙ᅖᏽࡊࡒᚃࠉ࣒
ࢰࢿ࣭ࣜ/PBST ᾦ࡞࡙ṹ㝭Ⓩ࡞⬲Ềࢅ⾔ࡖࡒࡵࡡࢅモᩩ࡛ࡊࡒࠊᚋࡼࡿࡒモᩩࢅ࢞ࢨ
ࣝࣤ࡞⨠ᥦᚃࠉࣂࣚࣆ࢔ࣤ࡞ໜᇔࡊࠉ8µm ࡡว∞ࢅష⿿ࡊࡒࠊ࢞ࢨࣝࣤࢅ⏕࠷࡙⬲ࣂ
ࣚࣆ࢔ࣤࡊࠉ࣒ࢰࢿ࣭ࣜ/H2O ᾦ࡞࡙ṹ㝭Ⓩ࡞්Ề࿰ࡊࡒᚃࠉࣉ࣏ࢹ࢞ࢨ࡚ࣝࣤ 10 ฦ
᯹Ⰵࡊࠉ්ᗐ࣒ࢰࢿ࣭ࣜ/H2O ᾦ࡞࡙ṹ㝭Ⓩ࡞⬲Ềࡊࠉ࢙࢛ࢩࣤ᯹Ⰵࢅ⾔ࡖࡒࠊ࢛࢕
࢞ࢴࢹࢅ⏕࠷࡙ᑌථࡊࠉ㢟᚜㙶ふᐳࢅ⾔ࡖࡒࠊ
➠஦㡧 ඞ␷⤄⧂᯹Ⰵ
8µm ࡡࣂࣚࣆ࢔ࣤว∞ࢅష⿿ࡊࡒࠊ࢞ࢨࣝࣤࢅ⏕࠷࡙⬲ࣂࣚࣆ࢔ࣤࡊࠉ࣒ࢰࢿ࣭ࣜ
/H2O ᾦ࡞࡙ṹ㝭Ⓩ࡞්Ề࿰ࡊࡒᚃࠉᢘཋ㈹Ὡ໩㸝10mM ࡡࢠ࢙ࣤ㓗⁈ᾦ୯࡞࡙࣏࢕ࢠ
࣭ࣞࢗ࢘ࣇฌ⌦㸞ࡊࠉ2% FBS ࡞࡙ࣇࣞࢴ࢞ࣤࢡฌ⌦ࢅ⾔ࡖࡒࠊࡐࡡᚃࠉanti-MIS ᢘ
మ(Santa Cruz Biochemistory)ࢅ⏕࠷࡙ୌḗᢘమཬᚺࢅ⾔ࡖࡒࠊPBS ὑὯᚃࠉHRP-Rabbit
anti-Goat IgG (H+L)(Zymed)࠽ࡻࡦ࡞ࡻࡾ஦ḗᢘమཬᚺࢅ⾔࠷ࠉDAB ࢅᇱ㈻ᾦ࡛ࡊࠉ㓕
⣪ཬᚺࢅ⾔ࡖࡒࠊanti-Rat cytochrom p450 side chain cleavage enzyme ᢘమ(American
Research products, Belmont, MA)ࢅ⏕࠷࡙ୌḗᢘమཬᚺࢅ⾔ࡖࡒ᫤ࡢࠉAlexa Fluor 546
goat anti-rabbit IgG (H+L)(Invitrogen)࡞ࡻࡽ஦ḗᢘమཬᚺࢅ⾔࠷ࠉࡐࡡᚃࠉ⺧ක㢟᚜㙶
ୖ࡚ふᐳࡊࡒࠊ
40
ㅨ㎙
ᮇ◂✪ࡢ↻ᮇኬᏕ⏍࿤㈠″◂✪࣬ᨥᥴࢬࣤࢰ࣭ᢇ⾙㛜Ⓠฦ㔕࡞࠽࠷࡙ᒜ⏛″ᩅ᤭ࡡ
ᚒᣞᑙࡡࡵ࡛⾔ࢂࡿࡒࡵࡡ࡚ࡌࠊᒜ⏛″ᩅ᤭ࡻࡽ⤂ጙᚒᣞᑙࠉᚒ㠬᧙ࢅ㈱ࡽࡱࡊࡒ஥
ࢅ῕ࡂវㅨ⏞ࡊ୕ࡅࡱࡌࠊ
↻ᮇኬᏕ⏍࿤㈠″◂✪ᨥᥴࢬࣤࢰ࣭ᢇ⾙㛜Ⓠฦ㔕ࠉⲮ㔕⏜⣎Ꮔ༡ኃࠉᐋᕖಘୌ༡ኃ࡞
ࡢ᪝ࠍࡡᐁ㥺ᡥἪ࡝ࡼࡦ࡞ㄵᩝషᠺ࡞㛭ࡊ࡙⤂ጙ᭯─࡝ᚒຐゕ࡛ᚒᣞᑙࢅ㡤ࡀࡱࡊ
ࡒࠊ῕ࡂវㅨࡡណࢅ⾪ࡊࡱࡌࠊ
↻ᮇኬᏕ⏍࿤㈠″◂✪ᨥᥴࢬࣤࢰ࣭ᢇ⾙㛜Ⓠฦ㔕ࡡຊ⸠Ὂᩅ༡ኃࠉኯ⏛ᑑ༡ኃࠉ୕ᮟ
⨶౅༡ኃࠉ㕝ᮄᇻኯ㑳༡ኃࠉཋཾ❫᦮༡ኃࠉఫ⸠⩇ᙢ༡ኃࠉᑹᮄ⚵├༡ኃࠉ♼ᕖ┷⨶
ಞኃࠉ໪ᕖᚷಕẮࠉ⸠ᕖఫ࿰ᏄẮ࡞ࡢኣࡂࡡᚒ༝ງ࡛ᚒᣞᑙࢅ㡤ࡀࡱࡊࡒࠊᚨ࠾ࡼវ
ㅨ⮬ࡊࡱࡌࠊ
↻ᮇኬᏕኬᏕ㝌⑋ឺ㐿ఎᏄよᯊᏕㅦᗑࡡ Ms. Mylah villacorteࠉᮟᔪல⣎Ꮥኃࠉኬ᲻ᬏ
ᏄᏕኃࠉ஬୕ዄ᭮Ꮥኃࠉ୔ཋ೸ୌ㑳Ꮥኃࠉᮿ୷ኬ㍔Ꮥኃࠉఫ⸠ួ♰Ꮥኃࠉ஬୕ᬓᜠᏕ
ኃࠉ୯⏛⩟ᏄẮࠉ⏛୯ୌᶖẮ࡞ࡢ᪝እኣኬ࡝ࡾᚒ༝ງࢅ㡤ࡀࡱࡊࡒࠊᚨ࠾ࡼវㅨ⮬ࡊ
ࡱࡌࠊ
ࡱࡒࠉ௧ୖࡡ඙⏍᪁࡞ࡢᮇ◂✪ࢅ㐑⾔ࡊ࡙࠷ࡂ୕࡚ࠉᐁ㥺࡞ᚪさ࡝ᮞᩩࡡฦ୙ࠉᚒຐ
ゕࠉᚒ༝ງࢅ㡤ࡀࡱࡊࡒࠊវㅨ⏞ࡊ୕ࡅࡱࡌࠊ
CNRS UMR5166-VMNHN Evolution des Regulations Endocriniennes
Giovanni Levi ༡ኃ
Hormone Research Center, Chonnam National University
Hueng-Sik Choi ༡ኃ
↻ᮇኬᏕⓆ⏍༈Ꮥ◂✪ࢬࣤࢰ࣭㌹෕โᚒฦ㔕 ⏛㈙ူஒ ༡ኃ
⚗⏛ಘ἖ ༡ኃ
᭩ᚃ࡞ࠉ⌟ᅹࡱ࡚ᖏ࡞ᝨࡊࡲ࡝࠷ᨥᥴ࡛ᐰኬ࡝⢥♼࡚⚶ࢅぜᏬࡽ⤾ࡄ࡙ୖࡈࡖࡒ∏࡛
ẍࠉࡱࡒ⢥♼㟻࠾ࡼᙁࡂᨥ࠻࡙ࡂࡿࡒ጗࡞ᚨ࠾ࡼវㅨ⮬ࡊࡱࡌࠊ
41
ཤ⩻ᩝ⊡
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