0013-7227/06/$15.

00/0 Endocrinology 147(1):432– 440

Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2005-0507

Long-Day Suppressed Expression of Type 2 Deiodinase

Gene in the Mediobasal Hypothalamus of the Saanen
Goat, a Short-Day Breeder: Implication for Seasonal
Window of Thyroid Hormone Action on Reproductive
Neuroendocrine Axis

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Shinobu Yasuo,* Nobuhiro Nakao,* Satoshi Ohkura, Masayuki Iigo, Satoko Hagiwara, Akemitsu Goto,
Hiroshi Ando, Takashi Yamamura, Miwa Watanabe, Tsuyoshi Watanabe, Sen-ichi Oda, Kei-ichiro Maeda,
Gerald A. Lincoln, Hiroaki Okamura, Shizufumi Ebihara, and Takashi Yoshimura
Division of Biomodeling (S.Y., N.N., T.Ya., M.W., T.W., S.E., T.Yo.), Laboratory of Animal Management and Resources
(S.-i.O.), Division of Applied Genetics and Physiology (K.-i.M.), Graduate School of Bioagricultural Sciences, Technical
Center (S.H., A.G., H.A.), and Institute for Advanced Research (T.Yo.), Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8601, Japan; Laboratory of Neuroendocrinology (S.O., H.O.), National Institute of Agrobiological Sciences, Ikenodai,
Tsukuba 305-8602, Japan; and Department of Applied Biological Chemistry (M.I.), Faculty of Agriculture, Utsunomiya
University, Mine-machi, Utsunomiya, Tochigi 321-8505, Japan; and Medical Research Council Human Reproductive
Sciences Unit (G.A.L.), University of Edinburgh, Chancellor’s Building, Edinburgh EH16 4SB, Scotland, United Kingdom

In most animals that live in temperate regions, reproduction
is under photoperiodic control. In long-day breeders such as
Japanese quail and Djungarian hamsters, type 2 deiodinase
(Dio2) plays an important role in the mediobasal hypothala-
mus, catalyzing the conversion of prohormone T4 to bioactive
T3 to regulate the photoperiodic response of the gonads. How-
ever, the molecular basis for seasonal reproduction in short-
day breeders remains unclear. Because thyroid hormones are
also known to be involved in short-day breeders, we examined
the effect of an artificial long-day stimulus on Dio2 expression
in the male Saanen goat (Capra hircus), a short-day breeder.
Dio2 expression was observed in the caudal continuation of
the arcuate nucleus, known as the target site for both mela-
tonin and T4 action. In addition, expression of Dio2 and T3
content in the mediobasal hypothalamus was suppressed by
artificial long-day conditions, which is the opposite of the
results of long-day breeders. Thyroid hormone action on the
development of neuroendocrine anestrus is known to be lim-
ited to a specific seasonal window. This long-day suppression
of Dio2 may provide a mechanism that accounts for the lack
of responsiveness to thyroxine during the mid to late anestrus.
(Endocrinology 147: 432– 440, 2006)

I N MOST ANIMALS living in temperate regions, repro-

ductive seasonality ensures the birth of young in spring
or summer, as is appropriate for survival. Species with a
short incubation or gestation period such as Japanese quail
and hamsters, or species with a gestation period of nearly 1
yr such as horses, are long-day breeders, and their fertile
period occurs in the springtime. On the other hand, species
with a gestation period around 5– 6 months such as sheep
and goats are short-day breeders, and their breeding take
places in autumn (1). In mammals, photoperiodic informa-
tion is translated into a daily cycle of melatonin secretion
from the pineal gland (2, 3). The duration of the night is
First Published Online September 29, 2005
* S.Y. and N.N. contributed equally to this work.
Abbreviations: AR, Androgen receptor; ARC, arcuate nucleus; cARC,
caudal continuation of the hypothalamic ARC; Dio2, type 2 deiodinase;
EC, ependymal cell layer lining the infralateral walls of the third ven-
tricle; ER, estrogen receptor; MBH, mediobasal hypothalamus; ME, me-
dian eminence; PMH, premammillary hypothalamic area; PMv, ventral
division of the premammillary nucleus; POA, preoptic area; TIS, tu-
beroinfundibular sulcus; TR, thyroid hormone receptor.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
reflected in the length of nocturnal melatonin secretion, and
melatonin regulates GnRH secretion from the hypothalamus.
An increase in melatonin secretion is associated with de-
creased GnRH secretion in long-day breeders and increased
GnRH secretion in short-day breeders, respectively. The
mechanism for the reversed effect of melatonin on GnRH
secretion between long- and short-day breeders has been a
profound mystery.
Melatonin is thought to act through the mediobasal hy-
pothalamus (MBH) in both long- and short-day breeders. In
Syrian hamsters, lesions of the MBH block the gonadal re-
sponse to short photoperiod (4, 5). In the ewe, melatonin
microimplants positioned in the MBH mimic a short-day
effect on LH secretion, whereas no effect is observed in other
hypothalamic areas (6, 7). Detailed analysis identified the
target site of melatonin as the premammillary hypothalamic
area (PMH). The PMH consists of three subdivisions: a cau-
dal continuation of the hypothalamic arcuate nucleus
(cARC), the ventral division of the premammillary nucleus,
and the ventral tuberomammillary nucleus, and the PMH
contains 125I-melatonin binding sites (8 –10). The mechanism
by which melatonin regulates GnRH secretion is also cur-
rently unknown.

432
Yasuo et al. • Long-Day Suppressed Expression of Dio2
Using Japanese quail, an excellent animal model for study-
ing photoperiodism, we found that expression of type 2
iodothyronine deiodinase (Dio2) was induced by long-day
conditions in the MBH. Dio2 catalyzes the conversion of
prohormoneT4 to bioactive T3 and controls local thyroid hor-
mone concentration. Thyroid hormone contents in the MBH
were about 10-fold higher in quail kept under long-day con-
ditions than in those under short-day conditions, and intra-
cerebroventricular infusion of T3 mimicked photoperiodic
testicular growth under short days. These results suggested
that light-induced conversion of T4 to T3 in the MBH is critical
for the photoperiodic response of gonads in birds (11). Be-
cause thyroid hormones are known to be essential for the
maintenance of seasonal reproductive changes in both birds
and mammals, this mechanism also appears to be conserved
in mammals (12, 13). Indeed, similar to quail, up-regulation
of Dio2 expression under long-day conditions was observed
in the ependymal cell layer lining the infralateral walls of the
third ventricle (EC), the cell-clear zone overlying the tu-
beroinfundibular sulcus (TIS), and the arcuate nucleus (ARC)
of Djungarian hamster hypothalamus, and its expression was
suppressed by melatonin administration (14). In ewe, thyroid
hormone is known to be involved in the transition to anestrus
(15), and more recently, it was reported that microimplan-
tation of thyroid hormone in the PMH of thyroidectomized
ewes could inhibit the secretion of LH, suggesting that the
PMH is the target site for thyroid hormone action (16). In the
present study, therefore, we examined the effect of long days
on Dio2 expression in the MBH of the Saanen goat (Capra
hircus), a short-day breeder.
In ewes, thyroid hormone is known to be involved in
changes in the responsiveness of the GnRH axis to estrogen-
negative feedback at the transition to anestrus (17, 18). It has
also been reported that thyroid hormone can affect the ex-
pression of the estrogen receptor (ER) gene (19, 20) and
estrogen-sensitive gene expression (21) via estrogen and thy-
roid hormone receptor (TR) interactions. Although LH se-
cretion is also under the negative feedback regulation of
testicular steroids in males, the extent to which of these
FIG. 1. Schematic drawing of the seasonal
changes in plasma prolactin and testicular vol-
ume of goats (A) and photoperiodic treatments
and experimental design (B). A, The changes
in plasma prolactin and testicular volume are
based on reports of Alpine and Saanen goats
and Soay rams (23–25). B, Saanen goats were
born around April 2003 (hatched area) and
raised under natural photoperiods (latitude
35° 8⬘N). In November 2003, half of the goats
were transferred to artificial long-day condi-
tions (LD16:8).
Endocrinology, January 2006, 147(1):432– 440 433
actions of testicular steroids result from the direct action of
testosterone or its metabolite estradiol is unclear (22). There-
fore, we further examined the expression of TR␣, TR␤, ER␣,
and androgen receptor (AR) in the goat MBH and the effect
of long-day stimulus on the expression of these genes. In the
Saanen goat, prolactin secretion increases during spring and
summer, whereas plasma LH and testosterone increases dur-
ing autumn and winter (23–25) (Fig. 1A). We monitored the
testicular size and plasma prolactin concentration during the
experiment to determine the effect of long-day stimulus and
physiological condition of goats.
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Materials and Methods
Animals and housing
Male Saanen goats, approximately 4 months old, were purchased
from a local dealer and kept in the Experimental Station of Highland
Animal Production of Nagoya University (latitude 35° 8⬘N) under the
influence of natural changes in photoperiod and temperature. Food and
water were available ad libitum. The experimental schedule and lighting
conditions are shown in Fig. 1B. Because maximum testicular regression
occurs after approximately 16 wk in Soay rams (25), we exposed animals
to an artificial long-day stimulus for 4 months. Eight goats were ran-
domly divided into two groups in November 2003, and four goats were
transferred to the artificial long-day conditions (16 h light, 8 h dark). The
other goats were kept under natural daylengths. The measurement of
testicular length was conducted once a month from September 2003 to
February 2004. In March 2004, goats were killed by an overdose of
pentobarbital sodium. The brains were rapidly removed and a block of
hypothalamus (Fig. 2) was frozen on dry ice. The hypothalamic block
was stored at ⫺80 C until in situ hybridization. Animals were treated in
accordance with the guidelines of Nagoya University.
Quantification of prolactin
Plasma samples were collected once a month from November 2003
to March 2004. Prolactin concentrations in blood plasma samples
were measured using a routine RIA validated for goat plasma (26).
The assay used a rabbit antisera (ASM R50) and a purified ovine
prolactin standard (NIH-PRL-S13). Intraassay coefficient of variation
was less than 10%. The lower limit of sensitivity was 0.5 ng NIH-
PRL-S13/ml plasma.
434 Endocrinology, January 2006, 147(1):432– 440
FIG. 2. Schematic drawing of the hypothalamic block dissected
(dashed box) and slices used in the T3 and T4 RIA. PVN, Paraven-
tricular nucleus; MB, mammillary body; PT, pars tuberalis; PD, pars
distalis; OC, optic chiasm; IIIV, third ventricle.
In situ hybridization
In situ hybridization was carried out according to Yoshimura et al. (27).
Antisense 45-oligomer oligonucleotide probes for goat Dio2 (anti-
sense: 5⬘-tgcttgaggagaatgaccgagtcatacagcgccaggaagaggcag-3⬘) (Gen-
Bank accession no. AB201476), ovine TR␣ (5⬘-gtactgctctcctctgggtct-
gacccacactccaccttgcttggc-3⬘), ovine TR␤ (5⬘-acacaggcaagccctgggcgat-
ctgaagacatcagcaggacggcc-3⬘), goat ER␣ (5⬘-ccatgcccacttcatagcattcgc-
gtagccggcagtcctggcaac-3⬘), and goat AR (5⬘-aaggaccgccagcccatggca-
aacaccataagccccatccaggag-3⬘) were labeled with [33P]deoxy-ATP (NEN
Life Science Products, Boston, MA) using terminal deoxyribonucleot-
idyl transferase (Life Technologies, Inc., Frederick, MD). Coronal sect-
ions (20 ␮m thickness) of the MBH were prepared using a Cryostat.
Hybridization was carried out overnight at 42 C. Two high-stringency
posthybridization washes were performed at 55 C. For negative cont-
rols, sense probes were labeled and hybridized in the same way. The
sections were air-dried and apposed to Biomax-MR film (Kodak, Ro-
chester, NY) for 2 wk. 14C standards (American Radiolabeled Chemic-
als, St. Louis, MO) were included in each cassette, and the relative OD
was measured using a computed image-analyzing system (MCID, Im-
aging Research, St. Catherines, Ontario, Canada) and converted into the
radioactive value (nanocuries) using the 14C standard measurements.
Data were normalized by subtracting the value at the ventroanterior
nucleus of the thalamus, which is located in the same section and does
not exhibit a hybridization signal. After exposure to x-ray film, each
Yasuo et al. • Long-Day Suppressed Expression of Dio2
slide was dipped in type NTB2 autoradiography emulsion (Kodak)
diluted twice with sterile distilled water and developed after 6 wk of
exposure at 4 C. After development, sections were counterstained with
0.5% cresyl violet and observed with photomicroscope.
Quantification of T3 and T4
When slices for in situ hybridization were prepared, slices (200 ␮m
thickness) of MBH were collected every 500 ␮m, and 13 slices per animal
were pooled for the measurement of T3 and T4 concentration in the MBH
(Fig. 2). Thyroid hormones in the MBH were extracted with ethanol and
measured by RIA as described (28, 29). The extraction rates determined
by the use of [125I]T3 or [125I]T4 (⬃10,000 cpm/sample) were 90.6 ⫾ 2.4
and 91.4 ⫾ 1.7% (mean ⫾ sem, n ⫽ 6) for T3 and T4, respectively. The
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inhibition curves for the brain extracts were parallel to the curves for
authentic T3 and T4 standards. The relationship between the quantity of
T3 or T4 added and the quantity recovered was analyzed using linear
regression analysis. Significant correlations were obtained between
amounts of T3 or T4 added and recovered (T3: Y ⫽ 1.14␹ ⫺0.04, r ⫽ 0.997,
P ⬍ 0.001; T4: Y ⫽ 1.21␹ ⫺0.02, r ⫽ 0.997, P ⬍ 0.001). Intra- and interassay
coefficients of variation were 3.7 and 9.2% for T3 and 6.9 and 10.0% for
T4, respectively. The lower limits of sensitivity were 6.3 pg/tube (cor-
responding to 68.8 pg/sample) for T3, and 2.9 pg/tube (corresponding
to 31.7 pg/sample) for T4, respectively.
Results
Effect of artificial long days on testicular size and
prolactin secretion
Goats were born around April 2003 and males were fertile
with fully functional testes by the first autumn in our latitude
(35° 8⬘N). Therefore, in the present study, we examined the
inhibitory effect of artificial long photoperiods on the repro-
ductive axis in the first winter (Fig. 1B). In goats kept under
natural daylength, the testicular length did not change
throughout the experiment [one-way ANOVA, F(5,18) ⫽
0.29, P ⬎ 0.05], whereas that of goats kept under artificial long
days significantly decreased from January 2004 [one-way
ANOVA, F(5,18) ⫽ 4.33, P ⬍ 0.05] (Fig. 3A). When the an-
imals were killed in March 2004, the paired testes weight of
goats kept under artificial long days was significantly less
(⬃70%) than that of goats kept under natural daylength
(Mann-Whitney U test, P ⬍ 0.05) (Fig. 3B). In goats kept
under natural daylength, plasma prolactin increased from
February 2004 [one-way ANOVA, F(4,15) ⫽ 11.950, P ⬍ 0.01].
In contrast, in goats kept under artificial long days, plasma
prolactin increased in advance [one-way ANOVA, F(4,15) ⫽
6.310, P ⬍ 0.01] (Fig. 3C).
Distribution of Dio2 mRNA in the MBH
We examined Dio2 mRNA expression in the MBH of
Saanen goats. From the rostral to the middle region of the
MBH, Dio2 mRNA expression was observed in the external
zone of the median eminence (ME), especially in the outer-
most zone (external edge) of the ME, but not in the internal
layer (Fig. 4, A and B). In the middle region of the MBH, Dio2
mRNA expression was also observed in the cell-clear zone
overlying the TIS and EC, which is consistent with previous
studies using the Djungarian hamster (14). From the middle
to the caudal region of the MBH, cARC was labeled with a
Dio2 mRNA probe (Fig. 4, C and D) (10). No signal was
detected in these regions by sense probes (Fig. 4, A–D).
Yasuo et al. • Long-Day Suppressed Expression of Dio2
FIG. 3. Effect of artificial long days on testicular size and prolactin
secretion. A, Effect of artificial long days on testicular length from
September 2003 to February 2004. Significant change was observed
under artificial long days (open circle) (one-way ANOVA, P ⬍ 0.05),
whereas no change was detected under natural photoperiod (solid
circle). Asterisks indicate significant differences between the two
groups (Mann-Whitney U test, P ⬍ 0.05). B, Final paired testes weight
in March 2004, when goats were killed. A significant difference was
detected between the two groups (Mann-Whitney U test, P ⬍ 0.05).
Each value is the mean ⫾ SEM (n ⫽ 4). C, Effect of artificial long days
on prolactin secretion. Plasma samples were collected from November
2003 to March 2004. A significant difference between the two groups
was observed in January 2004 (asterisk, Mann-Whitney U test, P ⬍
0.05). Each value is the mean ⫾ SEM (n ⫽ 4).
Photoperiodic regulation of Dio2 expression in the MBH
We compared Dio2 mRNA levels between goats kept un-
der natural daylength and artificial long days. In the external
zone of the ME, the signal was stronger under artificial long
days than under natural daylength (Mann-Whitney U test,
P ⬍ 0.05) (Fig. 5A). In contrast, a significant decrease in Dio2
mRNA expression under artificial long days was observed in
the TIS, EC, and cARC (Mann-Whitney U test, P ⬍ 0.05) (Fig.
5, B and C).
T3 and T4 content in the MBH
To assess the functional significance of Dio2 in the MBH,
we measured T3 and T4 content in the MBH under natural
daylength and artificial long days. Due to the difficulty in
isolating each brain region because of overlap in some re-
gions, we measured T3 and T4 content of pooled brain slices
obtained from the whole MBH (Fig. 2). Although T4 con-
centrations did not differ between natural daylength and
Endocrinology, January 2006, 147(1):432– 440 435
artificial long days (Mann-Whitney U test, P ⬎ 0.05), changes
in T3 concentration were suppressed in the MBH of goats
kept under artificial long days (Table 1).
Distribution of TR␣, TR␤, ER␣, and AR mRNA and effect
of photoperiod on their expression
Thyroid hormone actions are mediated by TRs. Therefore,
we examined TR␣ and TR␤ expression in the goat MBH.
Expression of TR␣ was observed widely around the MBH
including the cARC (30, 31) (Fig. 6A), whereas TR␤ expres-
sion was restricted to the cARC (Fig. 6B). Expression of these
genes was not different between natural daylength and ar-
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tificial long days (data not shown). We next examined the
expression of ER␣ and AR in the goat MBH. ER␣ expression
was observed in the cARC, whereas AR expression was
observed not only in the cARC but also in the ventral division
of the premammillary nucleus (PMv) (Fig. 7, A and B). Ex-
pression levels of ER␣ and AR were not different between
natural daylength and artificial long-day conditions (Fig. 7,
E and F). No signals were detected by sense probes for TR␣,
TR␤, ER␣, and AR (Figs. 6, C and D, and 7, C and D).
Discussion
In the present study, long-day stimulus decreased testic-
ular weight of goats to approximately 70% of that of control
goats. This result was consistent with a previous report using
Alpine and Saanen goats (32), confirming that the male
Saanen goat is a useful animal model for studying seasonal
reproduction. Plasma prolactin of goats kept under natural
daylength started to increase from February, in accordance
with a previous report (25), whereas that of goats kept under
artificial long days increased in advance and a high level was
maintained until brains were collected. These results suggest
that brain collected for gene expression analysis from goats
kept under natural daylength represents spring status,
whereas that of goats kept under artificial long days mimics
the summer status.
Dio2 expression was widely distributed in the goat MBH
including the ME, the TIS, and the cARC. Among these
regions, cARC is particularly noteworthy, because the T4
microimplants that were placed close to or within the pos-
terior portion of the ARC or slightly caudal to it in thyroid-
ectomized ewes allowed the ewes to enter neuroendocrine
anestrus (16). Because T4 is a prohormone and has relatively
low biological activity, Dio2 expressed in the cARC seems to
generate bioactive T3 locally. Although it has also been re-
ported that micropimplants of T4 in the ventromedial POA
(preoptic area) were also effective in two of six ewes exam-
ined (16), our preliminary experiment failed to detect Dio2
expression in the goat ventromedial POA (data not shown).
In mammals, photoperiodic information is translated into
a daily cycle of melatonin secretion from the pineal gland (2,
3). It is also of interest to note that 1) melatonin microim-
plants positioned in the MBH mimic a short-day effect on LH
secretion (6, 7), 2) expression of melatonin 1A receptor
mRNA shows day-night variation in the PMH including the
cARC (33), and 3) the PMH is known to contain a melatonin
binding site (8). In addition, lesion of the cARC disrupts the
seasonal cycle of FSH secretion, and alters both the ampli-
436 Endocrinology, January 2006, 147(1):432– 440 Yasuo et al. • Long-Day Suppressed Expression of Dio2

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FIG. 4. Distribution of Dio2 mRNA in the Saanen goat MBH. Rostral(A), middle (C), and caudal (D) parts of the MBH are shown. The
magnification of the ME (dashed squares of panel A) are also shown in B. In each region of the MBH, a dark-field photomicrograph showing
the distribution of Dio2 mRNA, sense controls, a light-field photomicrograph of a serial section stained by cresyl violet, and the schematic
drawings are shown (from left to right). The square in each schematic drawing shows the site where dark-field and light-field photomicrographs
were taken. Sections of goat under artificial long days are used in panels A and B), whereas those under natural daylength are used in panels
C and D). Dio2 mRNA was observed in the external zone of the ME (ez, large arrowheads), the cell-clear zone overlying the TIS (small
arrowheads), the EC (small arrows), and cARC (large arrows). Note that especially dense signals are observed in the outermost edge of the
external zone of the ME. Iz, Internal zone of the ME; PT, pars tuberalis; TMv, the ventral tuberomammillary nucleus; Fx, fornix; IIIV, third
ventricle.

tude and the timing of photoperiod-induced testicular
growth in the Soay ram (34). These results suggest that the
cARC is the integration center for melatonin signaling. Be-
cause Dio2 expression is regulated by melatonin in the MBH
of the Djungarian hamster (14), Dio2 expressed in the goat
cARC may also be regulated by melatonin.
In Japanese quail, photoperiodic regulation of Dio2 ex-
pression and thyroid hormone content was observed in the
MBH (11). Likewise, in the present study, we have demon-
strated photoperiodic regulation of Dio2 expression and T3
content in the goat MBH. However, the effect of long-day
stimulus was different among different brain regions. That is,
long-day stimulus suppressed Dio2 expression in the EC, the
TIS, and the cARC, whereas it increased expression in the
external layer of the ME. When we measured thyroid hor-
mone content in the MBH slices, T3 was suppressed by ar-
tificial long days. The suppressed T3 content under long-day
conditions reflects the expression pattern of Dio2 in the EC,
TIS, and the cARC, but not in the external zone of the ME.
In Japanese quail, although strong expression of Dio2 was
Yasuo et al. • Long-Day Suppressed Expression of Dio2 Endocrinology, January 2006, 147(1):432– 440 437

TABLE 1. T3 and T4 concentration in the MBH

Photoperiodic treatment T3 (pg/brain area) T4 (pg/brain area)

Natural daylength 180.7 ⫾ 40.7 160.5 ⫾ 17.3
Artificial long days ND 154.1 ⫾ 23.6
Not detected (ND) ⬍ 68.8 pg (T3); each value is the mean ⫾ SEM (n ⫽ 4).

also observed in the ME, its expression was restricted to the

inner and middle zones of the ME, and only minor expres-
sion was observed in the external zone. Because there are no
cell bodies in the external layer of the ME, Dio2 mRNA
appears to originate from cell bodies localized in other re-
gions and be transported to the external layer of the ME. Dio2

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protein is an endoplasmic reticulum-resident protein with a
short half-life (35, 36). The short half-life of Dio2 protein
results from the fact that Dio2 is a substrate for endoplasmic
reticulum-associated degradation and selectively targeted
for ubiquitination and subsequent proteasomal degradation
(37–39). Therefore, Dio2 protein must be associated with the
endoplasmic reticulum to exert an effect. However, endo-
plasmic reticulum cannot be observed in the external layer of
the ME and mRNA observed in the external layer of the ME,
does not appear to have a functional role. In addition, TR
mRNA, as well as AR and ER mRNA, is not expressed in the
ME, whereas all are expressed in the cARC. Taken together,
these observations suggest that Dio2 expressed in the cARC
appears to have a critical effect on the reproductive neu-
roendocrine axis in goats.
In the ewe, thyroid hormone action on the suppression of
seasonal activity is known to be limited to a specific seasonal
window (40, 41). From the mid to late anestrus season (i.e.
summer), thyroidectomized ewes cannot enter anestrus in
response to T4 treatment. Although the mechanism of this
seasonal window has been a mystery, our finding of long-
day suppression of Dio2 in the MBH seems to provide a
potential answer. That is, we suggest that under long days
(mid to late anestrus season), the lack of Dio2 protein due to
long-day suppressed Dio2 expression means that T4 cannot

FIG. 5. Photoperiodic regulation of Dio2 mRNA in several MBH re-

gions. A, Rostral; B, middle; C, caudal parts of the MBH are shown.
In each part of the MBH, representative autoradiograms for Dio2
expression under natural photoperiod and artificial long-day condi-
tions, and a schematic drawing are shown. Each graph shows the
relative OD of Dio2 mRNA level in the external zone of the ME (ez,
large arrowheads), the cell-clear zone overlying the TIS (small ar-
rowheads), the EC (small arrows), and cARC (large arrows), respec-
tively. Solid bars, Natural daylength; open bars, artificial long days.
Asterisks shows the significant difference detected by Mann-Whitney
U test (P ⬍ 0.05). Each value is the mean ⫾ SEM (n ⫽ 4). Iz, Internal
zone of the ME; PT, pars tuberalis; TMv, the ventral tuberomammil- FIG. 6. Distribution of TR␣ (A) and TR␤ (B) mRNA in the cARC of
lary nucleus; Fx, fornix; IIIV, third ventricle. goat. Sense controls of TR␣ (C) and TR␤ (D) are also shown. Expres-
sion of these genes was not different between natural photoperiods
and artificial long days (data not shown).
438 Endocrinology, January 2006, 147(1):432– 440
FIG. 7. Distribution of ER␣ (A) and AR (B) mRNA in the cARC and
PMv of goats. Sense controls of ER␣ (C) and AR (D) are also shown.
Graphs show the ER␣ (E) and AR (F) mRNA level in the cARC under
natural daylength (solid bars) and the artificial long days (open bars).
No significant difference was observed (Mann-Whitney U test, P ⬎
0.05). Each value is the mean ⫾ SEM (n ⫽ 4).
be converted to the bioactive form T3, which results in a lack
of responsiveness to T4.
The mechanism by which melatonin regulates secretion of
GnRH secretion is currently unknown and the opposite effect
of melatonin between long-day and short-day breeders has
been a profound mystery. The suppression of Dio2 expres-
sion under long days observed in the present study is the
opposite of the results for long-day breeders (11, 14). The
present study may be the first demonstration of a reversed
event that may be causally involved in inducing a different
neuroendocrine response to a long-day melatonin signal
within the brain of long- and short-day breeders. This event
may also be found to provide the key to the switching mech-
anism for long-day breeders and short-day breeders in a
future study. To test this hypothesis, it is important to ex-
amine the detailed annual expression profiles of Dio2 in the
MBH and the effect of melatonin administration on Dio2
expression in this region.
The mechanism by which thyroid hormone regulates the
reproductive neuroendocrine axis is of particular interest.
Because thyroid hormone actions are mediated by TRs, we
examined expression of TRs. Expression of TR␣ was ob-
served widely around the MBH, whereas that of TR␤ was
restricted to cARC. Because TR␣ is colocalized in 47% of
GnRH neurons in the ewe MBH, thyroid hormone is thought
to act on the GnRH system directly in the MBH (30, 31).
Although little attention has been given to TR␤ in the MBH,
Yasuo et al. • Long-Day Suppressed Expression of Dio2
TR␤ in the cARC may also have a potential role in the reg-
ulation of the GnRH system.
In sheep, thyroid hormones are known to be involved in
both changes in the responsiveness of the GnRH axis to
estrogen-negative feedback at the transition to anestrus, and
in the steroid-independent cycles in LH pulse frequency (17,
18). Although the underlying mechanism for these actions of
thyroid hormones remains to be elucidated, the MBH, es-
pecially the cARC, appears to be very important because the
cARC is known to contain ERs (10, 42) and estrogen implan-
tation in the ARC results in the suppression of LH secretion
in the ram (43). Therefore, it is possible that thyroid hormone
regulates the responsiveness of the GnRH system to estra-
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diol-negative feedback in the cARC. Indeed, the influence of
thyroid hormone on estrogen actions has been convincingly
demonstrated (21). TRs and ERs are members of the nuclear
receptor superfamily and have a modular protein structure
with high homology in the central binding domain. Further-
more, thyroid hormone can affect the expression of the ER
gene (19, 20) and estrogen-sensitive gene expression (21) via
estrogen and TR interactions. Because it is not clear whether
the action of testicular steroid is mediated by testosterone
itself or its metabolite estradiol in male (22), we further ex-
amined the expression of ER␣ and AR in the goat MBH. As
previously reported, expression of ER␣ and AR was ob-
served in the cARC (42, 44). Because thyroid hormone can
affect steroid hormone action through receptor interactions,
overlapping of TR␣, TR␤, ER␣, and AR mRNA in the cARC
is noteworthy. Although we compared the expression level
of both genes, their expression was not altered by photope-
riodic treatment. Therefore, it seems that the effect of T3 on
estradiol-negative feedback is not directly mediated by ER␣
or AR gene expression, and it may be possible that thyroid
hormone affects the responsiveness to estradiol-negative
feedback through estrogen-sensitive genes. It is also possible
that thyroid hormone action is mediated by nuclear receptor
coregulators, which serve to enhance nuclear receptor-me-
diated transcription primarily by binding to the ligand-ac-
tivated receptors to remodel the local chromatin structure
(45, 46). This possibility is supported by the fact that the
mRNA expression of some nuclear receptor coregulators is
affected by thyroid hormones in rat pituitary cells (47), in the
rat brain (48), and in Xenopus laevis tissues during metamor-
phosis (49). It would be interesting, therefore, to examine
seasonal changes in the expression of nuclear receptor co-
regulators in the goat MBH in the future.
Thyroid hormones are also known to have essential roles
in the development and plasticity of the central nervous
system (50), and seasonal plasticity in the GnRH systems has
been reported. In Japanese quail, expression of TRs is ob-
served in the ME and the ensheathment of GnRH terminals
by glial processes was increased under nonbreeding short
days in the ME (51). In sheep, GnRH neurons in the POA in
anestrus ewes appear to be surrounded by higher glial pro-
cesses than those in breeding season ewes (52). Although
both studies report changes in the morphological interaction
between GnRH neurons and glia, the two studies focus on
completely different locations (i.e. encasement of GnRH
nerve terminals vs. synaptic input to GnRH cell bodies) and
whether these two phenomena are related remains to be
Yasuo et al. • Long-Day Suppressed Expression of Dio2
determined. In birds, melatonin is not required for seasonal
reproduction (53) and light information seems to reach
GnRH neurons directly because direct innervation of GnRH
neurons by encephalic photoreceptor cells has been reported
(54). In contrast, in mammals, the eye is the only organ to
receive light information and melatonin is essential (2).
Viewed in this light, although the fundamental mechanism
for seasonal reproduction appears to be conserved in birds
and mammals (14, 55), the existence of different regulatory
mechanisms among birds and mammals is not surprising,
particularly at the level of transmission of light information.
In sheep, it is thought that melatonin acts in the PMH in-
cluding the cARC and the signal is then brought to the GnRH
neurons through interneurons (56). In addition, ER␣-ex-
pressing neural populations in the cARC project to the rostral
preoptic area and diagonal band of Broca where the majority
of the GnRH perikarya are found in the ewe (57, 58). There-
fore, it would be interesting to examine the morphological
plasticity of cells expressing the melatonin receptor (33), ER␣,
or interneurons in the cARC.
In conclusion, we found expression of Dio2 in the cARC,
which is the target site for both melatonin and thyroxine. In
addition, Dio2 expression and T3 content were suppressed by
the artificial long-day conditions. Thyroid hormone action on
the development of neuroendocrine anestrus is known to be
limited to the specific seasonal window. This long-day sup-
pression of Dio2 may provide a mechanism for the lack of
responsiveness to thyroxine during mid to late anestrus.
Finally, in the future, we plan to determine the mechanism
of thyroid hormone action on the reproductive neuroendo-
crine axis.
Acknowledgments
We thank Nagoya University Radioisotope Center for use of facilities.
Received April 28, 2005. Accepted September 19, 2005.
Address all correspondence and requests for reprints to: Takashi
Yoshimura, Ph.D., Division of Biomodeling, Graduate School of Bioag-
ricultural Sciences, & Institute for Advanced Research, Nagoya Univer-
sity, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: takashiy@
agr.nagoya-u.ac.jp.
T.Yo. was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN) and a Grant-in-Aid
for Encouragement of Young Scientists from the Ministry of Education,
Science, Sports and Culture.
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