Ikegami+et+al Endocrinology Vol156 p647-659

REPRODUCTION-DEVELOPMENT
Low Temperature-Induced Circulating

Triiodothyronine Accelerates Seasonal
Testicular Regression
Keisuke Ikegami,* Yusuke Atsumi,* Eriko Yorinaga, Hiroko Ono, Itaru Murayama,
Yusuke Nakane, Wataru Ota, Natsumi Arai, Akinori Tega, Masayuki Iigo,
Veerle M. Darras, Kazuyoshi Tsutsui, Yoshitaka Hayashi, Shosei Yoshida,
and Takashi Yoshimura
Laboratory of Animal Physiology (K.I., Y.A., E.Y., H.O., I.M., Y.N., W.O., T.Y.), Avian Bioscience Research
Center (Y.A., T.Y.), Graduate School of Bioagricultural Sciences, Department of Genetics (Y.H.), Division
of Stress Adaptation and Recognition, Research Institute of Environmental Medicine, and Institute of
Transformative Bio-molecules (T.Y.), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan;
Department of Applied Biochemistry (N.A., A.T., M.I.), Faculty of Agriculture, Center for Bioscience
Research and Education (M.I.), Utsunomiya University, Utsunomiya 321-8505, Japan; Utsunomiya
University Center for Optical Research and Education (M.I.), Utsunomiya, Tochigi 321-8585, Japan;
Department of Biology and Center for Medical Life Science (K.T.), Waseda University, Tokyo 162-8480,
Japan; Division of Germ Cell Biology (S.Y.), National Institute for Basic Biology, Okazaki 444-8787,
Japan; Division of Seasonal Biology (T.Y.), National Institute for Basic Biology, Okazaki 444-8585, Japan;
and Animal Physiology and Neurobiology Section (V.M.D.), Department of Biology, Laboratory of
Comparative Endocrinology, KU Leuven, B-3000 Leuven, Belgium
In temperate zones, animals restrict breeding to specific seasons to maximize the survival of their
offspring. Birds have evolved highly sophisticated mechanisms of seasonal regulation, and their
testicular mass can change 100-fold within a few weeks. Recent studies on Japanese quail revealed
that seasonal gonadal development is regulated by central thyroid hormone activation within the
hypothalamus, depending on the photoperiodic changes. By contrast, the mechanisms underlying
seasonal testicular regression remain unclear. Here we show the effects of short day and low
temperature on testicular regression in quail. Low temperature stimulus accelerated short dayinduced testicular regression by shutting down the hypothalamus-pituitary-gonadal axis and inducing meiotic arrest and germ cell apoptosis. Induction of T3 coincided with the climax of testicular
regression. Temporal gene expression analysis over the course of apoptosis revealed the suppression of LH response genes and activation of T3 response genes involved in amphibian metamorphosis within the testis. Daily ip administration of T3 mimicked the effects of low temperature
stimulus on germ cell apoptosis and testicular mass. Although type 2 deiodinase, a thyroid hormone-activating enzyme, in the brown adipose tissue generates circulating T3 under low-temperature conditions in mammals, there is no distinct brown adipose tissue in birds. In birds, type 2
deiodinase is induced by low temperature exclusively in the liver, which appears to be caused by
increased food consumption. We conclude that birds use low temperature-induced circulating T3
not only for adaptive thermoregulation but also to trigger apoptosis to accelerate seasonal testicular regression. (Endocrinology 156: 647 659, 2015)
ISSN Print 0013-7227 ISSN Online 1945-7170

Printed in U.S.A.
Copyright 2015 by the Endocrine Society
Received September 6, 2014. Accepted November 12, 2014.
First Published Online November 18, 2014
doi: 10.1210/en.2014-1741
* K.I. and Y.A. contributed equally to this work.

Abbreviations: BAT, brown adipose tissue; DIO2, type 2 deiodinase; H-P-G, hypothalamuspituitary-gonadal; LD, long day; 6L18D, 6 h light, 18 h dark; 30LD, LD conditions for 30
days; MBH, mediobasal hypothalamus; SCP3, synaptonemal complex protein 3; SD, short
day; SL, SD/low temperature; TH, thyroid hormone; TUNEL, terminal deoxynucleotidyl
transferase-mediated deoxyuridine triphosphate nick end labeling.
Endocrinology, February 2015, 156(2):647 659
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Mechanism of Seasonal Testicular Regression
ost animals living outside the tropics measure

changes in day length to breed at a specific time of
year, largely to ensure that offspring are born only when
the climate is moderate and abundant food is available.
Birds have evolved highly sophisticated photoperiodic
mechanisms, probably due to the adaptations necessary
for flight. Seasonal changes in testicular mass are typically
of the order of 100-fold in birds, as compared with severalfold in mammals (1). Among birds, Japanese quail
provides a particularly attractive model for studying these
phenomena; recent studies using quail have uncovered the
signal transduction pathway that triggers photoperiodic
gonadal development.
Light information is received by deep-brain photoreceptors, including Opsin 5-positive cerebrospinal fluidcontacting neurons in the paraventricular organ and then
transmitted to the pars tuberalis of the pituitary gland
(2 4). The long day (LD)-induced TSH in the pars tuberalis acts on TSH receptors in the ependymal cells within
the mediobasal hypothalamus (MBH) to induce DIO2
and to reduce DIO3 expression (5). The regulatory mechanism of pars tuberalis-derived TSH is completely different from that of the classical pars distalis-derived TSH (6).
TSH in the pars distalis is regulated by the hypothalamuspituitary-thyroid axis. However, there is no TRH receptor
and thyroid hormone (TH) receptor in the pars tuberalis,
and TSH in the pars tuberalis is independent of the hypothalamus-pituitary-thyroid axis (7). DIO2 and DIO3 encode TH-activating (type 2 iodothyronine deiodinase) and
-inactivating (type 3 iodothyronine deiodinase) enzymes,
respectively. These two genes act as switches to regulate
local TH concentration, and LD-induced T3 within the
MBH causes testicular development (8, 9). TH is involved
in the development and plasticity of the brain (10). Indeed, morphological changes in GnRH nerve terminals
and glial processes are observed in the median eminence; these changes are likely to modulate seasonal
GnRH secretion (11).
In contrast to the mechanisms that trigger seasonal testicular development, described above, the mechanisms underlying seasonal testicular regression remain unclear. In
quail, a low temperature stimulus accelerates short day
(SD)-induced testicular regression (12, 13). Therefore, in
this study, we examined the effect of SD and short day/low
temperature (SL) on quail testis. To this end, we first evaluated germ cell differentiation and apoptosis in the testis.
Next, we assessed expression profiles of key genes regulating seasonal reproduction in the brain. We also determined serum hormone levels and detected changes in both
LH and T. In addition, low temperature-induced serum T3
was observed at the climax of testicular regression. It is
well established that TH is involved in amphibian meta-
morphosis (14). Therefore, we examined temporal expression profiles of T3 response genes involved in amphibian
metamorphosis in the quail testis. We then confirmed that
T3 administration mimics the effect of low temperature. In
mammals, type 2 deiodinase (DIO2) in the brown adipose
tissue (BAT) generates T3, and BAT is considered to be a
major source of local and circulating T3 under exposure to
a low temperature (1517). However, birds have no distinct BAT or related thermogenic tissue (18). To identify
the tissue responsible for low temperature-induced serum
T3, we measured expression of DIO2 in various tissues
and detected induction of this gene exclusively in the liver.
It has been known that feeding regulates diet-induced thermogenesis, but the involvement of hepatic DIO2 in the
avian adaptive thermogenesis remains unclear. Therefore,
we also examined the effect of fasting on hepatic DIO2
expression.
Ikegami et al
Materials and Methods

Animals
Male, 4-week-old Japanese quail (Coturnix japonica) were
obtained from a local dealer and kept under SD conditions [6 h
light, 18 h dark (6L18D)] for 4 weeks in light-tight boxes in a
room held at a temperature of 23 1C. At 8 weeks of age, quail
were transferred to LD conditions (20 h light, 4 h dark). Thirty
days after the transfer to LD conditions, quail were transferred
to SD or SL conditions (6L18D, 9C). In the castration experiment, quail were castrated before they were transferred into SL
conditions. In the fasting experiment, 3 weeks after the transfer
to SD or SL conditions, the measurement of core body temperature by ip loggers (0.7C, Thermochron Type-SL; KN
Laboratories) and food intake were performed. This study
was approved by the Animal Experiment Committee of Nagoya University.
Histology
Paraffin sections (4 m) of Bouins fixed testes were used for
synaptonemal complex protein 3 (SCP3) immunohistochemistry
and analysis of apoptosis. Immunohistochemistry for SCP3 was
performed using an anti-SCP3 antibody (NB300-231, 1:1500;
Novus Biologicals) as previously described (19, 20). Apoptosis
was analyzed by in situ terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL)
labeling with an ApopTag peroxidase staining kit (Millipore),
which was followed by periodic acid-Schiff counterstaining.
Gene expression analysis

Relative mRNA levels were determined by in situ hybridization using 33P-labeled oligonucleotide probes (Supplemental Table 1), as previously described (21). OD was normalized by subtracting the OD at an area of the same section in which no
hybridization signal was observed. Densitometric analysis of hybridization signals was performed using Image Gauge (Fujifilm
Co Ltd).
doi: 10.1210/en.2014-1741
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Radioimmunoassay
Serum LH concentrations were determined by a RIA using the chicken LH
RIA kit (kindly supplied by Dr John A.
Proudman, US Department of Agriculture-Agricultural Research Service Beltsville Agricultural Research Center) (5).
The labeled antigen was prepared by iodinating purified chicken LH (USDAcLH-I-3) with Na125I (NEZ033A; Perkin
Elmer Japan) by the lactoperoxidase
method. USDA-cLH-K-3 and USDAAcLH-5 were used as the standard and
the antibody, respectively. For T determination, samples were extracted with
diethylether and subjected to a RIA using
[1,2,6,7-3H(N)]-T (PerkinElmer Japan)
and the rabbit antitestosterone serum
(FKA-102; CosmoBio) (22). T3 and T4
concentrations were determined by a
RIA using L-3,5,3-[125I]T3 (NEX110H;
PerkinElmer Japan) and anti-T3 (Hycor Biomedicals), and L-[5-125I]T4
(NEX111H; PerkinElmer Japan) and anti-T4 (Wien Laboratories), respectively
(8). Parallelism of inhibition curves was
proven between the standard and serial
2-fold dilution of quail samples for each
hormone (Supplemental Figure 1). Intraand interassay coefficients of variation
were 7.6% (n 3) and 11.1% (n 4) for
LH, 5.1% (n 6) and 7.8% (n 3)
for T, 6.9% (n 6) and 10.7% (n 4)
for T3, and 7.5% (n 6) and 11.0% (n
4) for T4, respectively.
T3 treatment
Quail kept under LD conditions for
30 days (30LD) were transferred to SD
conditions or kept continuously under
LD conditions. These birds were ip injected with T3 (0, 1, 10, or 100 g/100 g
body weight, once a day) (T2877; Sigma)
dissolved in vehicle (0.02 mol/L NaOH)
for 4 weeks from 30LD onward. Sera and
testes were collected 24 hours after the
final injection.
Measurement of DIO2 activity
Figure 1. Effect of changing day length and temperature on quail testicular weight and meiosis.
A, top panels, Testes of quail kept under SD and LD conditions. Scale bars, 1 cm. Bottom panels,
Changes in testicular mass in quail transferred from SD to LD and then to short day/low
temperature (SL; solid line) or SD (dashed line). *, P .05; **, P .01 vs 0 day of LD condition
(0LD) (ANOVA); , P .05 SL vs SD (t test, n 8 10). B, Representative photomicrographs for
immunohistochemistry of SCP3, a marker of meiosis (arrowhead). Scale bars, 25 m. C, Changes
in number of SCP3-positive cells. *, P .05; **, P .01 vs 30LD (corresponding to 0SD/SL)
(ANOVA); , P .05; , P .01 SL vs. SD (t test, n 4 5).
DIO2 activities were measured in

homogenates of the tissues, as previously described (23). Reactions contained 1 nM T4 as the substrate, 100
nM T3 0.1 mM propylthiouracil to
block the possible interference by other
deiodinases, and 25 mM dithiothreitol
as the cofactor. The protein concentrations used in the different tests varied
from 25 to 2000 g/mL and the incubation time from 30 240 minutes, de-
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Ikegami et al
pending on the tissue. All activities

were calculated as the amount of substrate deiodinated per milligram of
protein per minute.
Data analysis
Normally distributed data from timecourse samples were analyzed by a parametric test (one way ANOVA with Dunnetts multiple comparison post hoc
test), and nonnormally distributed data
were analyzed by a nonparametric test
(Steels multiple comparison test) to determine the significance. A Students t
test was performed to compare the results of SD and SL conditions at each time
point. All values reported are means
SEM.
Results
Figure 2. Changes in number of apoptotic germ cells and Sertoli cells. A, Representative
photomicrographs of TUNEL-labeled germ cells (arrowhead). B, The number of apoptotic
germ cells per seminiferous tubule. **, P .01 vs 0LD (ANOVA); , P .05 SL vs SD (t test,
n 4 5). C, Representative photomicrograph of a TUNEL-positive Sertoli cell and the
number of such cells per cross-section (but not per seminiferous tubule). Scale bars, 25 m
(A) and 10 m (C).
Low temperature stimulus

accelerates meiotic arrest
LD (20L4D) stimulus induced testicular development (Figure 1A).
Thereafter, the SD (6L18D) stimulus
induced partial testicular regression,
whereas the SL (6L18D, 9C) stimulus induced a full and rapid testicular regression (Figure 1A). Between
initial SD conditions (LD d 0) to LD
conditions (LD d 30), testicular mass
increased by 126-fold. To evaluate
the occurrence of germ cell differentiation during testicular development and regression, we used immunohistochemistry to examine the
expression of a meiotic marker,
SCP3 (also known as Sycp3) (19). Although no SCP3-immunopositive
cells were observed at the time of the
initiation of the LD condition, the
number of SCP3-positive cells increased over the course of testicular
development (Figure 1, B and C).
SD exposure gradually reduced the
number of SCP3-positive cells, but
these cells did not disappear entirely
under the SD condition. In contrast,
when a low temperature stimulus
was administered in combination
with an SD stimulus, only a few
SCP3-positive cells were observed at
the climax of testicular regression
doi: 10.1210/en.2014-1741
(ie, 20 or 25 d after transfer to SL), suggesting that SL

stimuli significantly arrest meiosis (Figure 1, B and C).
Low temperature stimulus-induced germ cell
apoptosis causes rapid testicular regression
We next assessed apoptosis by in situ TUNEL labeling.
A relatively low level of apoptotic cells was observed under
the LD conditions (Figure 2, A and B). A substantial number of germ cells underwent apoptosis 15 and 20 days after
the transfer to SL conditions (Figure 2, A and B). Although
some apoptotic germ cells were observed under SD conditions, their number did not increase significantly (Figure
2, A and B). We observed a few apoptotic Sertoli cells in
the fully matured testes but considerably less than germ
cells (Figure 2C).
Photoperiodic response of key genes that regulate
seasonal reproduction
When we examined the expression profiles of key genes
involved in the regulation of seasonal reproduction, we
observed an LD induction of TSHB in the pars tuberalis of
the pituitary gland (Figure 3A). In addition, LD exposure
induced DIO2 and suppressed DIO3 in the ependymal
cells within the MBH (Figure 3, B and C). Although the
responses of TSHB and DIO3 tended to occur slightly
earlier under the SL conditions than under the SD conditions, there was no significant difference between the two
conditions.
Changes in serum hormone concentrations
We next assessed serum hormone concentrations by
RIA. LH levels changed rapidly, increasing significantly by
5 days after the transfer to LD conditions and decreasing
significantly 5 days after the transfer to SL conditions (Figure 4A). In contrast, serum T increased more gradually. A
significant increase was first observed 15 days after the
651
transfer to LD conditions, and a further increase was observed after the transfer to SL conditions. However, T
rapidly decreased between 10 and 15 days after the transfer to SL conditions (Figure 4B). Although a decrease in
serum LH and T was also observed under SD conditions,
these reductions were partial and more gradual than under
SL conditions (Figure 4, A and B). Because circulating TH
is increased by a low temperature stimulus for the purpose
of adaptive thermogenesis, we also determined TH concentrations and observed a significant increase in T3 during the climax of testicular regression under SL conditions
(ie, 20 and 25 d after transfer to SL conditions) but no such
increase under SD conditions (Figure 4C). This result was
consistent with the notion that an increase in the capacity
for classical adaptive nonshivering thermogenesis takes
weeks to develop (24). T4 was slightly higher during the
transition from initial SD to LD conditions (Figure 4D).
Induction of T3 response genes and reduction of
LH response genes during the climax of testicular
regression
We next performed an analysis of temporal gene expression in the quail testis. Genes encoding the LH receptor (LHR) and steroidogenic acute regulatory protein
(StAR), a rate-limiting factor of steroidogenesis, are both
expressed in Leydig cells in an LH-dependent manner (25,
26). Expression of these genes was rapidly induced by an
LD stimulus and sustained until 10 days after the transfer
to SL conditions, followed by a rapid decrease (Figure 5,
A and B). When we examined the expression of T3 response genes involved in amphibian metamorphosis, we
observed a high expression of TH receptors (THRA,
THRB) and its dimeric partner (RXRA) during the transition from the initial SD to LD conditions as well as during the climax of testicular regression under SL conditions
Figure 3. Temporal expression profiles of key genes involved in regulating seasonal reproduction in the quail brain. AC, Representative
autoradiograms and densitometric quantitation are shown: TSHB in the pars tuberalis (A), DIO2 (B), and DIO3 (C) in the ependymal cells within the
MBH. *, P .05; **, P .01 vs 0LD (n 4 5, ANOVA). Solid line, SL; dashed line, SD. Note that in Figures 15, all samples were collected at 16
hours after dawn to examine the expressions of TSHB and DIO2 in the brain in the same animals. These genes are rhythmically expressed and
show high expression levels at around this time of the day.
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Ikegami et al
compared with the annual molt,

when birds exhibit anorexia and lose
20% 40% of their body weight
(32). Accordingly, we believe that
the daily T3 injections mimicked
physiological conditions.
Identification of the tissue
responsible for low
temperature-induced circulating
T3 in birds
Although DIO2 in the BAT is responsible for low temperature-induced circulating T3 in mammals,
there is no distinct BAT or related
thermogenic tissue in birds. To identify the tissue responsible for low
temperature-induced serum T3, we
collected serum and various tissue
samples every 7 days after the transfer to SD/SL conditions. Consistent
with Figure 4C, a significant increase
Figure 4. Changes in serum hormone concentrations. A, LH. B, T. C, T3. D, T4. *, P .05;
**, P .01 vs trough value (ANOVA); , P .05; , P .01 SL vs SD (t test, n 510). Solid
in serum T3 level was observed under
line, SL; dashed line, SD.
SL conditions but not under SD conditions (Figure 7, A and B). When we
examined the expression of DIO2 in
(Figure 5, CE). Low temperature-induced expression of
various
tissues
(eg,
thyroid
gland, pectoral muscle, gasother T3 response genes, including deiodinases (DIO2,
DIO3) and apoptosis-related genes (CASP6, MMP13, trocnemius, kidney, and liver), we observed low-temperTGFB2, TNFAIP2) (14, 2729), were also observed (Fig- ature induction of DIO2 only in the liver (Figure 7D), and
ure 5, FK). The magnitude of the reduction in LH re- this induction did not occur under SD conditions (Figure
sponse genes and the induction in T3 response genes 7C). Although an increased thyroid gland weight in rewere smaller under the SD than under the SL conditions sponse to low temperature stimulus was reported several
decades ago (33, 34), we did not observe changes in thy(Figure 5).
roid gland weight (Figure 7E). We also observed low temperature-induced expression of DIO2 in the testis (Figure
Daily ip T3 administration mimics the effect of low
5F), but the magnitude of this induction was much smaller
temperature
To determine whether T3 mimics the effects of cold than in the liver. Furthermore, DIO2 enzyme activity was
stimulus, we examined its effects of T3 administration on significantly increased by low temperature in the liver but
germ cell apoptosis, testicular weight, and the size of the not in the testis (Figure 7F), and castration did not attencloacal gland area, a T-dependent structure located at the uate the serum level of T3 induced by low temperature
caudal end of the cloaca (30). In a dose-dependent manner, (Figure 7G). These results suggest that the liver, but not the
T3 administration increased germ cell apoptosis (Figure 6, testis, appears to be the source of low temperature-inA and B) and reduced testicular weight and the size of the duced circulating T3. Although we also examined the efcloacal gland area (Figure 6, C and D) under both SD and fect of SL on DIO3 expression in the liver, we observed no
LD conditions. Effects of T3 administration were more changes in the expressions of these genes (Supplemental
prominent under SD than under LD conditions (Figure 6, Figure 2). Therefore, we focused on the regulation of liver
AD). However, T3 treatment did not completely mimic DIO2 expression.
the effect of low temperature under SD (also see Figure
1A). This is probably due to the short half-life of T3 (4 Low temperature-induced hepatic DIO2 requires
h) during circulation (31). Although changes in body increased food intake
In mammals and birds, increased food intake has been
weight were also observed, these differences were less than
10% of body weight (Figure 6E), a relatively small change reported under low-temperature conditions (3537). To
doi: 10.1210/en.2014-1741
653
day-night variation: high during the day and low during

the night (Figure 8B, left panel). However, when animals
were fasted, their body temperature decreased profoundly
during the night phase, regardless of ambient temperature
(Figure 8, B right panel, and C). This observation of hypothermia during the inactive phase is consistent with a
previous report in quail (38). When we examined hepatic
DIO2 expression in these animals, the suppression of the
low-temperature DIO2 induction was observed under
fasting conditions (Figure 8D). Although a similar tendency was observed under SD conditions, no statistically
significant difference was observed. It is interesting to note
that the serum T3 levels were suppressed by fasting under
both the SL and SD conditions (Figure 8E). These results
suggest that increased food intake is required for the lowtemperature induction of DIO2 in the liver.
Discussion
Figure 5. Temporal change in gene expression in the quail testis. A

and B, LH-driven genes (LHR, StAR). CE, TH receptors (THRA, THRB)
and their dimeric partner (RXRA). F and G, Deiodinase genes (DIO2,
DIO3). HK, Apoptosis-related genes (CASP6, MMP13, TGFB2,
TNFAIP2). *, P .05; **, P .01 vs trough value; , P .05; , P
.01 SL vs SD (t test, n 4 5). Solid line, SL; dashed line, SD.
test whether food intake is related to cold-induced hepatic

DIO2 expression, we first examined the effect of a lowtemperature stimulus on food intake. As expected, increased food intake was observed under SL conditions
(Figure 8A). When we measured body temperature
rhythms, the internal body temperature exhibited clear
In this study, we demonstrated dynamic seasonal changes

in the quail testis. Although quail raised under LD conditions reach puberty when they are 8 weeks old (30), the
testes of 8-week-old quail raised under SD conditions remained small and immature (Figure 1A). However, once
quail were transferred to LD conditions, meiosis and rapid
testicular development occurred. In quail, a single LD
stimulus triggers cascades of gene induction, which results
in LH secretion approximately 22 hours after dawn of the
first long day; testicular development is accomplished
within a few weeks (5, 39 41). However, meiosis continued for a time after the transfer to SD/SL conditions (Figure 1, B and C). The slow rate of initiation of the SD/SLinduced testicular regression observed in this study was in
contrast to the rapid rate of photoinduction. It appears
that repeated exposure to SD/SL stimuli is required to initiate testicular regression. This mechanism may enable
birds to avoid misinterpreting several consecutive rainy,
cloudy, or cold days as the start of the autumn stimulus.
Testicular mass gradually decreased under SD conditions (Figure 1A). Because the number of SCP3-positive
cells decreased under SD conditions and no significant
increase in the number of apoptotic germ cells was observed, the arrest of meiosis appeared to be the primary
cause of partial testicular regression under SD conditions
(Figures 1 and 2). In marked contrast to SD conditions, a
substantial number of apoptotic germ cells was observed
15 and 20 days after transfer into SL conditions. In SL
quail, the number of apoptotic cells per seminiferous tubule observed was approximately 10-fold larger than in
seasonally breeding mammals (42) but was equivalent to
that of European starlings, which exhibit photorefracto-
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Ikegami et al
Figure 6. Effect of daily T3 administration on apoptosis, testicular mass, cloacal gland area, and body weight. A, Representative
photomicrographs of TUNEL-labeled germ cells (arrowheads). B, The number of apoptotic cells. CE, Effect of daily ip T3 administration on testis
mass (C), cloacal gland area (D), and body weight (B.W.; E). *, P .05; **, P .01 vs vehicle (Veh); , P .05; , P .01 SL vs SD (t test, n
512). Red line, LD; blue line, SD.
riness (42). Photorefractoriness is the insensitivity of gonadal development to the stimulatory effects of LD in
birds. The extensive germ cell death observed in these birds
seemed to account for the rapid and drastic testicular regression observed in avian species. In addition to germ
cells, apoptotic Sertoli cells have been reported in starlings
(43). Although we found a few apoptotic Sertoli cells in
fully matured quail testes, we did not detect them during
the regression process (Figure 2). We therefore conclude
that seasonal testicular regression caused by an SL stimulus in quail is mediated by the arrest of germ cell differentiation and apoptosis.
Based on these results, we can propose mechanisms for
seasonal testicular development and regression (Supplemental Figure 3). Expression profiles of key genes regulating seasonal reproduction, TSHB, DIO2, and DIO3,
simply reflect environmental photoperiodic information
(Figure 3). Therefore, when quail raised under SD conditions are transferred to LD conditions, the photoperiodic
signaling pathway is immediately switched on and the hypothalamus-pituitary-gonadal (H-P-G) axis is activated.
LD-induced LH up-regulates the expression of the LH
response genes LHR and StAR in the testis (Figures 4 and
5 and Supplemental Figure 3). Thus, the activation of the
LH-dependent steroidogenesis pathway results in increased T production. T is a survival factor for germ cells:
withdrawal of T increases germ cell apoptosis, whereas

reintroduction decreases it (44). Consequently, activation
of the LH-dependent steroidogenesis pathway activates
germ cell differentiation and inhibits germ cell apoptosis
under LD conditions (Supplemental Figure 3).
When quail were transferred from LD to SD conditions,
the photoperiodic signaling pathway (ie, TSHB and
DIO2) was switched off, and serum LH gradually decreased (Figures 3 and 4 and Supplemental Figure 3).
However, because serum LH and T did not return to basal
levels under SD conditions, the arrest of meiosis was partial and the number of apoptotic cells did not increase
significantly (Figures 1 and 2). On the other hand, when
quail were transferred to SL conditions, we observed a
rapid decrease in serum LH (Figure 4). Expression profiles
of key genes regulating seasonal reproduction (TSHB,
DIO2, and DIO3) did not significantly differ between SD
and SL conditions (Figure 3). The photoperiodic response
of quail, unlike that of other avian species, has been proposed to be regulated primarily at the level of GnRH secretion rather than GnRH synthesis (1, 11, 45, 46). Therefore, low-temperature stimulus appeared to attenuate
GnRH secretion to enhance the effect of SD stimulus,
thereby completely shutting down the H-P-G axis (Supplemental Figure 3). LHR-null mice have smaller seminiferous tubules due to apoptosis in spermatocytes (47).
doi: 10.1210/en.2014-1741
655
Figure 7. Identification of the tissue responsible for low temperature-induced serum T3. A and B, Changes in serum T3 (A) and T4 (B) levels under
SD (dashed line) and SL (solid line) conditions. *, P .05; **, P .01 vs day 0. C and D, Changes in DIO2 mRNA in various tissues under SD (C)
and SL (D) conditions. Representative autoradiograms (top panel) and densitometric quantification (bottom panel) are shown. **, P .01 vs day 0.
Scale bars, 1 cm. E, Size of thyroid gland did not change under SD and SL conditions. F, DIO2 activity in liver and testis under SD and SL conditions.
Samples were collected 28 days after transferred to each condition. *, P .05 (t test, n 3 6). G, Castration did not affect low-temperatureinduced serum T3 level. *, P .05; **, P .01 day 0 vs day 20 (t test, n 6 8). All samples were collected at 3 hours after dawn under SD/SL
condition (ie, midday).
Therefore, once serum LH and T decrease to the basal

levels under SL conditions, germ cells arrest meiosis (Figure 1) and apoptosis takes place (Figure 2). However,
changes in seminiferous tubule diameter are much more
moderate in LHR-null mice (diameter in LHR null mice is

60% of that in wild type mice: ie, 40% reduction) (47)
than in quail (diameter in SD quail is 20% of that in LD
quail: ie, 80% reduction). Hence, the SL-induced shut-
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the growth and maturation of the

testis (48 50). Therefore, we speculate that highly expressed DIO2 in
the SD testis locally converts serum-derived T4 into T3 to promote
growth and maturation of the testis
during the transition from initial SD
to LD conditions as in the case of
mammalian fetal and perinatal period. On the other hand, we observed
a significant increase in serum T3
level (Figure 4) and induction of TH
receptors, deiodinases, and apoptosis-related genes during the climax of
testicular regression (Figure 5). Furthermore, daily T3 administration
mimicked the effects of low temperature on germ-cell apoptosis and
testicular regression (Figure 6). ThereFigure 8. Increased food intake is required for low temperature-induced DIO2 expression in the
fore, we conclude that low temperaliver. A, Food intake per day measured at 21 days after transfer to SL and SD conditions. **, P
.01 (t test). B and C, Effect of fasting on body temperature (Tb) at 21 days after transfer to SD
ture-induced circulating T3 acts on the
(light blue) and SL (dark blue) conditions. Light blue and gray shading indicate the SEM. D, Effect
testis to activate gene cascades similar
of fasting on hepatic DIO2 expression. Samples were collected 12 hours after dawn when the
to the ones involved in amphibian
lowest body temperature was observed. Representative autoradiograms (top panel) and
metamorphosis and causes germ cell
densitometric quantification (bottom panel) are shown. E, Effect of fasting on serum T3 levels
(mean SEM, n 4 6). **, P .01 vs SD control (ANOVA, Dunnetts post hoc test).
apoptosis in the quail testis (Supplemental Figure 3). Metamorphosis is
down of the LH-dependent steroidogenesis pathway (ie, an irreversible biological process that involves robust
the H-P-G axis) appears to be a component of the mech- changes in animal body structure, so it is somewhat suranisms regulating seasonal testicular regression (Supple- prising that genes involved in the metamorphosis of ammental Figure 3).
phibians are also activated during an annual cyclic event
TH has multiple functions in development and physi- of quail, ie, the regulation of seasonal reproductive
ology. Among them, the most visually striking function is activity.
amphibian metamorphosis. In frogs, resorption of the tail
Homeothermic animals maintain body temperature by
and formation of the fore- and hindlimbs in tadpoles are increasing heat production in response to a low-temperboth induced by TH (14). Two central questions in meta- ature stimulus. In mammals, DIO2 in the BAT generates
morphosis research are as follows: 1) how does TH induce T3, and BAT is a major source of circulating T3 during
opposite morphological responses in different tissues, exposure to low temperature (15). Because birds have no
ranging from outgrowth of limbs to the resorption of the distinct BAT or related thermogenic tissue (18), we
tail? and 2) how is its precise timing achieved (14)? In searched for the tissue responsible for low temperatureregard to the former question, it has been well established induced circulating T3. Although increases in thyroid acthat low levels of circulating TH induce limb outgrowth, tivity and thyroid weight during cold acclimation were
whereas the tail resorbs only when TH is the highest. Dur- reported several decades ago (33, 34), we did not observe
ing the climax of metamorphosis, a surge of TH activates changes in DIO2 expression or thyroid gland weight in
expression of its receptors (autoinduction), deiodinases this study (Figure 7). In addition, skeletal muscle has been
and apoptosis-related genes (14, 2729). In this study, suggested to be a source of heat generation in birds (51
serum T4 was slightly elevated during the transition 53). However, we did not observe low-temperature infrom initial SD to LD conditions (Figure 4). Expression duction of DIO2 in muscles (Figure 7), suggesting that
of DIO2 in the testis was also high during this period skeletal muscle is not the source of circulating T3. In con(Figure 5).
trast, we observed induction of DIO2 in the liver and the
In the mammalian testis, expression of TH receptors testis. The magnitude of DIO2 induction by low temperand TH response genes, including DIO2, is the highest ature was much smaller in the testis than in the liver. Induring fetal and perinatal life; TH plays a pivotal role in deed, low temperature increased the enzyme activity of
doi: 10.1210/en.2014-1741
DIO2 in the liver but not in the testis (Figure 7), and castration did not affect the low temperature-induced rise in
serum T3 (Figure 7). Because DIO2 is reportedly absent in
human and rat liver (54, 55), its physiological significance
has not been recognized. However, it should be noted that
the presence of DIO2 in the liver has been reported in mice,
chicken, and some species of fish (56 59).
In this study, food deprivation caused nocturnal hypothermia under both SL and SD conditions (Figure 8). This
fasting-induced hypothermia during the inactive phase
was consistent with a previous report in birds (38) and
suggests that food intake is important for body temperature maintenance during the inactive phase, regardless of
the ambient temperature. We also observed that fasting
suppressed the low-temperature induction of hepatic
DIO2 and serum T3 levels (Figure 8). Although serum T3
levels did not completely reflect the hepatic DIO2 expression profiles, these results clearly suggested that increased
food intake is important for the low-temperature induction of hepatic DIO2 expression. The mechanism by
which food intake induces hepatic DIO2 expression remains to be clarified in future studies. However, these
results collectively suggest that the liver is the potential
source of low-temperature-induced circulating T3 in birds.
TH is the key controller of heat production in birds as well
as in mammals (60 62). Therefore, it seems quite reasonable that birds would use T3 not only for adaptive thermoregulation but also for seasonal testicular regression in
autumn.
In conclusion, we have demonstrated that a low-temperature stimulus accelerates seasonal testicular regression by arresting meiosis and inducing drastic germ cell
apoptosis. Prolonged exposure to SL stimuli is required for
the initiation of testicular regression. However, once birds
interpret consecutive SL stimuli as the onset of autumn,
these mechanisms enable them to achieve full gonadal regression within a short period of time. The reported effects
of thyroidectomy or TH treatment on seasonal breeding
have often appeared to be contradictory (1). However, our
study reveals that TH plays dual roles in the regulation of
seasonal reproduction: central action induces seasonal testicular development, whereas peripheral action mediates
seasonal testicular regression.
Acknowledgments
We thank the Nagoya University Radioisotope Center for the use
of their facilities. We also thank Dr J. A. Proudman for providing
the chicken LH RIA kit.
657
Address all correspondence and requests for reprints to:

Takashi Yoshimura, PhD, Professor of Animal Physiology,
Institute of Transformative Bio-molecules (WPI-ITbM), Laboratory of Animal Physiology, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-8601, Japan. E-mail: takashiy@agr.
nagoya-u.ac.jp.
This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas, Regulatory Mechanism of Gamete Stem Cells (Grant 21116504) and Funding Program for
Next Generation World Leading Researchers (NEXT Program)
initiated by the Council for Science and Technology Policy
(Grant LS055), and Japan Society for the Promotion of Science
KAKENHI Grant 26000013.
Current address for K.I.: Department of Anatomy and Neurobiology, Kinki University Faculty of Medicine, 377-2 OhnoHigashi, Osaka-Sayama, Osaka 589-8511, Japan.
Disclosure Summary: The authors have nothing to disclose.
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REPRODUCTION-DEVELOPMENT

Low Temperature-Induced Circulating

ISSN Print 0013-7227 ISSN Online 1945-7170

* K.I. and Y.A. contributed equally to this work.

Endocrinology, February 2015, 156(2):647 659

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

ost animals living outside the tropics measure

Materials and Methods

Gene expression analysis

Measurement of DIO2 activity

DIO2 activities were measured in

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

pending on the tissue. All activities

Low temperature stimulus

(ie, 20 or 25 d after transfer to SL), suggesting that SL

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

compared with the annual molt,

day-night variation: high during the day and low during

Figure 5. Temporal change in gene expression in the quail testis. A

test whether food intake is related to cold-induced hepatic

In this study, we demonstrated dynamic seasonal changes

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

withdrawal of T increases germ cell apoptosis, whereas

Therefore, once serum LH and T decrease to the basal

moderate in LHR-null mice (diameter in LHR null mice is

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

the growth and maturation of the

Address all correspondence and requests for reprints to:

Mechanism of Seasonal Testicular Regression

Endocrinology, February 2015, 156(2):647 659

hormone: Xenopus laevis metamorphosis. Trends Endocrionol

Increased in vitro fatty acid supply and cellular transport capacities

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