The expression characteristics of the hypothalamic–pituitary–gonadal (HPG)
axis-related candidate genes, DIO2, EYA3, KISS1 and GPR54, were analyzed in year-round estrous
rams (small-tail Han sheep, STH) and seasonally estrous rams (Sunite sheep,
SNT) using qPCR. The results were as follows: DIO2 was mainly expressed in
pituitary, and KISS1 was specifically expressed in hypothalamus in the two
groups. However, EYA3 and GPR54 were widely expressed in the cerebrum, cerebellum,
hypothalamus, pituitary, testis, epididymis, vas deferens and adrenal gland
tissues in both breeds, with significant differences in the cerebellum,
hypothalamus, pituitary, testis and vas deferens tissues. We speculated that DIO2 and
KISS1 may have positive roles in different regions in ram year-round estrus.
Moreover, the expression patterns of EYA3 and GPR54 suggested that they may regulate
the estrous mode of ram via testis and vas deferens. This is the first study
to systematically analyze the expression patterns of HPG axis-related genes
in rams.
Background
Seasonal estrus is an important physiological behavior that animals have
evolved to adapt to the environment, which is regulated by photoperiods
(Dupré et al., 2010; Dardente et al., 2019). The photoperiodic mechanism
in vertebrates is known to involve seasonal regulation of thyroid hormones,
mediated in mammals via specialized cells (thyrotrophs) in the pars
tuberalis (PT). Normally, the female estrous cycle is initiated by the
melatonin from the hypothalamus, which functions in the pituitary and
regulates the expression of photoperiodically induced genes (eyes absent 3
(EYA3), thyrotrophin β subunit (TSHβ) and chromogranin (CGA)). Then it
induces a neuroendocrine cascade along the hypothalamic–pituitary–gonadal (HPG) axis in turn (Dupré et
al., 2010; Tavolaro et al., 2014).
To date, some major genes affecting seasonal estrus in sheep have already
been identified, such as period (PER), cryptochrome (CRY), BMAL1 and CLOCK, which all belong
to the circadian rhythm genes. The expression levels of these genes are
closely related to cycles in behavior and physiology (Marcheva et al., 2010;
Janich et al., 2011; Nam et al., 2015).
Research has also found that HPG axis-related genes, type 2 deiodinase
(DIO2), eyes absent 3 (EYA3), G protein-coupled receptor 54 (GPR54) and kisspeptin-1
(KISS1), are involved in regulating the mechanism of seasonal estrus in mammals
(Dupré et al., 2010; Herbison et al., 2010; Tariq et al., 2013; Liu et
al., 2017). The variations of DIO2/3 expression patterns in hypothalamic ependymal
cells are regulated by melatonin secretion, which is associated with
inactivation of THs and related to gonadal function (Lincoln, 1999; Ikegami
and Yoshimura, 2012). EYA3, as the strongest long photoperiod-induced gene in
sheep, is activated by a long photoperiod, revealing a common photoperiodic
molecular response in birds and mammals (Dupré et al., 2010). GPR54 has been
identified as the receptor of KISS1 (Muir et al., 2001; Ohtaki et al., 2001); it
is vital for puberty onset and male fertility due to its central regulatory
roles (Han et al., 2020). The loss-of-function mutations in GPR54 for humans and
mice lead to the symptoms of idiopathic hypogonadotropic hypogonadism with
the phenomenon of retarded sexual development and failure to reach puberty
(Funes et al., 2003; Seminara et al., 2003; Roux et al., 2003). KISS1 neurons in
the arcuate nucleus may regulate the negative feedback effect of gonadal
steroids on GnRH and gonadotropin secretion in both sexes (Popa et al.,
2008). Kisspeptins are potent secretagogues for GnRH, and the KISS1 gene is a
target for regulation by gonadal steroids (e.g., estradiol and
testosterone), metabolic factors (e.g., leptin), photoperiods, and seasons (Li
et al., 2008). With an understanding of the associations of the four genes
with seasonal estrus in female animals, there was a desire to gain further
knowledge of how these genes could affect male reproduction.
Sheep (Ovis aries) are a typical seasonally estrous species (Tang et al., 2018). Small-tail Han sheep (STH) and Sunite sheep (SNT) are two native Chinese sheep
breeds. In addition, STH has comparatively excellent characters, such as
crude feed tolerance, rapid growth and good meat quality. Therefore,
STH sheep provide an ideal model breed to explore the molecular genetic
mechanisms related to year-round estrus in certain breeds (Miao et al.,
2016). In contrast, Sunite sheep develop gonads and display seasonal
reproductive behavior during specific times of the year (La et al., 2020).
Therefore, the molecular mechanism of seasonal reproduction of sheep can be
better studied by using Sunite sheep as a model. Results of all these
previous studies indicated the DIO2, EYA3, KISS1 and GPR54 genes have important functions in
female estrus. However, there have been few studies on the expression
analyses in rams. Whether the four genes affect rams estrus remains to be
elucidated. Therefore, in this study, we compared the tissue expression
profiles and mRNA expression levels of the four genes in eight
reproduction-related tissues between STH and SNT rams. Our study paves the
way for an in-depth study of the seasonally estrous mode of rams.
Materials and methodsEthics statement and sample collection
All animals used in this study were approved by the Science Research
Department (in charge of animal welfare issues) of the Institute of Animal
Science, Chinese Academy of Agricultural Sciences (IAS-CAAS; Beijing, PR
China). In addition, there was ethics approval by the animal ethics
committee of IAS-CAAS (no. IAS2020-82, 28 July 2020).
Three STH rams were supplied by the Yuncheng Breeding Sheep Farm (Shandong
province, China), and three SNT rams were from Urad Middle Banner, Inner
Mongolia of China. All rams were healthy, approximately 2.5 years old and
were kept in a sheltered outdoor paddock. All animals were euthanized
(intravenous pentobarbital at 100 mg per kilogram), and eight tissues (cerebrum,
cerebellum, hypothalamus, pituitary, epididymis, testis, vas deferens and
adrenal gland) were collected from each animal. All tissues were frozen in
liquid nitrogen immediately and then stored at -80 ∘C to be used
for RNA extraction.
Total RNA extraction and cDNA synthesis
Total RNA was extracted from the eight collected tissues using a total RNA
extraction kit for animal tissue (Tiangen, Beijing, China). Trizol
(Invitrogen Inc., Carlsbad, CA, USA) was used to dissolve the tissues. The
quantity and quality of total RNA were monitored using 1.5 % agarose gel
electrophoresis (U = 150 V; 10 min) and ultraviolet spectrophotometry
(UV-1201, Shimadzu, Kyoto, Japan), respectively. Then, the RNAs were stored
at -80 ∘C until use.
The first strand of cDNA was prepared using a PrimeScript™ RT Reagent Kit
according to the manufacturer's instructions (TaKaRa Bio Inc., Dalian,
China). The PCR thermocycler program was as follows: 37 ∘C for 15 min, followed by 85 ∘C for 5 s. The reaction mixture contained
1.0 µL of PrimeScript RT Enzyme, 1.0 µL of random 6-mers, 4.0 µL of 5 × PrimeScript Buffer (for real time), 1.0 µL
of total RNA and 13 µL of RNase-free ddH2O (total volume, 20 µL). Prior to storage at -80 ∘C, the standard working
concentration of cDNA was 200 ng/µL. The quality of cDNA was
evaluated by housekeeping gene (GAPDH) amplification, and cDNA was stored at -20 ∘C until use.
Primer design
Primers were designed with Premier 3.0 (version 4.1.0)
(https://bioinfo.ut.ee/primer3-0.4.0/, last access: 15 October 2019). A total of five pairs of primers were designed to
amplify different fragments of the ovine DIO2 (GenBank: XM_027972090.1), EYA3 (GenBank: NM_001161733.1), GPR54 (GenBank:
NM_001318077.1), KISS1 (GenBank: NM_001306104.1) and
GAPDH (GenBank: NM_001190390.1) genes, based on their assembled
sequences in GenBank. All primers were synthesized by Beijing Tianyi
Biotechnology Co., Ltd. (Beijing, China). The housekeeping gene (GAPDH) was used
as an internal control to normalize the threshold cycle (Ct) values. Primer
details are given in Table 1.
The expression of DIO2 in eight tissues of small-tail Han sheep (STH)
and Sunite sheep (SNT): (a) the comparison of the expression between SNT and
STH, (b) the expression in SNT and (c) the expression in STH. Means with
different superscripts are significantly different (P<0.05). The
significant results with a p value lower than 0.05 are given one asterisk (∗)
and lower than 0.01 are given two asterisks (∗∗), respectively.
qPCR
Real-time polymerase chain reaction (qPCR) amplification was performed in 20 µL of reaction mixture that contained 10 µL of SYBR Premix EX
Taq II (TaKaRa Bio Inc., Dalian, China), 0.4 µL of each forward and
reverse primer, 6.4 µL of RNase-Free ddH2O, and 2 µL of
cDNA. PCR amplification was performed in triplicate wells using the
following conditions: initial denaturation at 95 ∘C for 5 min,
followed by 40 cycles of 95 ∘C for 10 s and 60 ∘C for
30 s. The dissociation curve was analyzed after amplification. A melting
temperature (Tm) peak at 85 ∘C ± 0.8 on the dissociation
curve was used to determine the specificity of PCR amplification.
The expression of EYA3 in eight tissues of small-tail Han sheep (STH)
and Sunite sheep (SNT): (a) the comparison of the expression between SNT and
STH, (b) the expression in SNT and (c) the expression in STH. Means with
different superscripts are significantly different (P<0.05). The
significant results with a p value lower than 0.05 are given one asterisk (∗)
and lower than 0.01 are given two asterisks (∗∗), respectively.
Statistical analysis
The relative gene expression levels were calculated by the 2-ΔΔCt method (Livak and Schmittgen, 2001; Livak, 2008).
Statistical analyses were carried out using SPSS 19.0 software (IBM, Armonk,
NY, USA). The levels of gene expression were analyzed for significant
differences by one-way analysis of variance (ANOVA) followed by Fisher's
least significant difference test as a multiple comparison test (Meier,
2006). All experimental data are presented as mean ± standard error of
the mean (SEM).
ResultsExpression level of DIO2
As shown in Fig. 1, the expression level of DIO2 was similar in SNT and STH,
with the highest level being in pituitary, followed by hypothalamus and
cerebrum, with no significant difference between the two sheep breeds (Fig. 1a). However, DIO2 expression in pituitary was significantly higher than in
the other seven tissues both in SNT and STH (P<0.05), and the expression
level in hypothalamus was significantly higher than that in cerebellum,
epididymis, testis, vas deferens and adrenal gland in STH (P<0.05)
(Fig. 1b, c).
The expression level of EYA3
The results of the EYA3 expression analysis are shown in Fig. 2. EYA3 was widely
expressed in the eight tissues in the two breeds. And EYA3 was expressed
significantly higher in STH than SNT except for cerebrum and adrenal gland
(P<0.05, P<0.01) (Fig. 2a). Additionally, EYA3 expression was
significant higher in adrenal gland than that in other tissues both in SNT
and STH (P<0.05) (Fig. 2b, c). Its expression in testis was
significantly higher than other six tissues, with pituitary significantly
higher than those of cerebrum, cerebellum, hypothalamus and epididymis in
STH (P<0.05) (Fig. 2c). The expression of EYA3 in cerebellum,
hypothalamus and epididymis had no significant difference in STH (Fig. 2c).
The expression of GPR54 in eight tissues of small-tail Han sheep (STH)
and Sunite sheep (SNT): (a) the comparison of the expression between SNT and
STH, (b) the expression in SNT and (c) the expression in STH. Means with
different superscripts are significantly different (P<0.05). The
significant results with a p value lower than 0.05 are given one asterisk (∗)
and lower than 0.01 are given two asterisks (∗∗), respectively.
The expression level of GPR54
Figure 3 clearly shows that GPR54 was widely expressed in all tissues in the two
breeds, with a higher level in STH than SNT. The expression levels of GPR54 were
reached at significant difference in hypothalamus and adrenal gland
(P<0.05), with extremely significant difference in cerebrum,
cerebellum, pituitary, testis and vas deferens between the two breeds
(P<0.01) (Fig. 3a). There was no significant difference in SNT (Fig. 3b). And it was the highest in testis and the lowest in epididymis in STH
(P<0.05) (Fig. 3c).
The expression level of KISS1
The results are shown in Fig. 4a. KISS1 was specifically expressed in
hypothalamus, and the expression of KISS1 in hypothalamus is significantly higher
in STH than SNT (P<0.01). The expression level of KISS1 was significantly
higher in hypothalamus than that in pituitary, vas deferens and adrenal
gland (P<0.05) in SNT (Fig. 4b); it was significantly higher in
hypothalamus than other tissues in STH (P<0.05) (Fig. 4c).
Discussion DIO2 expression in seasonally estrous and year-round estrous sheep
DIO2 is known to be crucial for the seasonal estrous mode of female animals. It
plays an important role in thyroid hormone metabolism and regulation (Park
et al., 2018). DIO2 converts inactive (thyroxine, T4) to “active” thyroid
hormone (3,5,3′-triiodothyronine, T3) and plays a significant role
as a determinant of the final concentration of T3 (Dunn et al., 2017; Lomet
et al., 2018; Dardente et al., 2019). Previous reports had found that DIO2 was
upregulated in breeding compared to nonbreeding testes in male lizards,
while DIO3 was upregulated in breeding ovaries in female lizards (Kang et al.,
2020). Trivedi et al. (2019) found that DIO2 and EYA3 were highly expressed, and
DIO3 and GnIH were lowly expressed in long photoperiods, concomitant with testis
recrudescence in male migratory red-headed buntings. Therefore, the DIO2 gene has
a certain impact on male reproduction. To our knowledge, no research on the
expression of DIO2 in rams has ever been reported. In the present study, the
highest expression of DIO2 was detected mainly in pituitary in STH and SNT rams.
It further confirmed that DIO2 is associated with the pituitary function.
The expression of KISS1 in eight tissues of small-tail Han sheep (STH)
and Sunite sheep (SNT): (a) the comparison of the expression between SNT and
STH, (b) the expression in SNT and (c) the expression in STH. Means with
different superscripts are significantly different (P<0.05). The
significant results with a p value lower than 0.05 are given one asterisk (∗)
and lower than 0.01 are given two asterisks (∗∗), respectively.
Studies had found that DIO2 is widely expressed in the cerebrum, cerebellum,
thyroid and testis in Brandt's vole and found that the core function of
the DIO2 gene should be restricted in response to the photoperiod rather than
factors directly regulating gonadal development (Liu et al., 2017). In
addition, some researchers had found that DIO2 was expressed in the testes in
rat and found DIO2 expression in adult rat testis was consistent with the
participation of thyroid hormone in testicular function (Romano et al.,
2017). In rodents, DIO2 was highly expressed in the hypothalamus (Tavolaro et
al., 2014). In this research, DIO2 was shown to be highly expressed in the
pituitary and widely expressed in the eight tissues. The result is similar
to the study on Jining Grey goat and Liaoning cashmere goat, in which it was
found that DIO2 was expressed in multiple tissues such as the cerebrum,
cerebellum, hypothalamus, pituitary, ovary and uterus, and it was highly
expressed in the pituitary and uterus (Huang et al., 2016). These indicated
that its function is diverse and that it plays a critical role in the
pituitary for ram seasonal estrus.
The expression of DIO2 in the hypothalamus, pituitary and testis is higher in
STH than in SNT. This observation was different from previous studies
comparing seasonally estrous and year-round estrous sheep (An et al., 2019a, b), in which ewes with seasonal estrus were reported to have a higher
expression of DIO2 in the gonad tissues, which implies that rams may have a
different regulation mechanism in estrus compared to ewes. Considering the
function of DIO2 in conversion of thyroid hormone activity, it seems plausible
that DIO2 may have a certain positive effect on ram year-round estrus. Of
course, further studies should be performed to deeply investigate the
relationship between DIO2 and ram estrus.
EYA3 expression in seasonally estrous and year-round estrous sheep
EYA genes had been identified as homologues of Drosophila eyes absent (EYA) genes.
The eyes absent family has a protein phosphatase function, and its
enzymatic activity is required for regulating genes encoding growth control
and signaling molecules, modulating precursor cell proliferation (Li et al.,
2003). The highly conserved vertebrate homologues Six1–6 (Oliver et al.,
1996), Eya1-4 (Xu et al., 1997) and Dach1-2/Ski/Sno (Hammond et al., 1998) are co-expressed in multiple organs, including eye, inner ear, pituitary gland, muscle and kidney.
EYA3 can be expressed in thyroid-stimulating cells in the PT region, and it
activates or inhibits the hypothalamic–pituitary–gonadal axis through the
TSHβ-DIO2-TH pathway to achieve the regulation of seasonal estrus in
sheep (Yoshimura, 2013; Dardente et al., 2014). Previous research had
revealed that EYA3 plays an important role in regulating TSHβ in male CBA/N
mice. EYA3 and its partner SIX1 synergistically activate TSHβ expression, and
this activation is further enhanced by TEF and HLF (Masumoto et al., 2010).
Likewise, long photoperiods caused a significant increase in EYA3, CGA, TSHβ
and DIO2, followed by changes in testis size and plasma level of testosterone
in male red-headed buntings (Park et al., 2018). Thus, EYA3 is an important
candidate gene in male seasonal estrus.
Previous studies had shown that EYA3 as a photoperiod-induced gene is highly
expressed in pituitary in female sheep, mice and birds (Dupré et al.,
2010; Mishra et al., 2017; Masumoto et al., 2010). In this study, EYA3 was
expressed in all eight tissues in rams, which implies that it plays a role in
promoting the differentiation of many tissues. This is in agreement with
EYA3 being widely expressed in the pituitary, pineal gland, cerebrum,
cerebellum, hypothalamus, fallopian tube, ovary, adrenal gland and kidney in
seasonal estrous and year-round estrous ewes (Xia et al., 2018). The highest
expression of EYA3 in testis in STH ram indicated that EYA3 is associated with the
testis function.
In addition, we compared the expression level of EYA3 in the selected eight tissues
between two sheep breeds. We found the expression levels of EYA3 were
significantly higher in cerebellum, hypothalamus, pituitary, testis and vas
deferens of STH rams compared with SNT rams. Given the expression of EYA3 gene in
the pituitary in ewes (Xu et al., 1997; Dupréet al., 2010), we
speculated that its regulatory mechanism was similar in rams. EYA3 may have a
certain positive effect on ram estrus.
KISS1 and GPR54 expression in seasonally estrous and year-round estrous sheep
Besides DIO2 and EYA3, the molecular mechanism of seasonal estrus involves the
downstream signaling factor, including KISS1 and GPR54. They can regulate the release
of hypothalamic gonadotropin-releasing hormone (GnRH), which in turn affects
the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone
(LH), and are closely related to the onset of puberty and may be key genes
for mammalian sexual maturity (Novaira et al., 2009; Papaoiconomou et al.,
2011). KISS1 and GPR54 expression in mouse, rat, rhesus monkey and human testes
indicates that this system has an autocrine or paracrine effect in the
testis (Ohtaki et al., 2001; Terao et al., 2004; Tariq et al., 2013). In
addition, they are contributing to stimulate testosterone secretion, testis
maturation, gametogenesis and spermatozoid maturation in epididymis
through FSH and LH (Scarlet et al., 2017; Feng et al., 2019). Smith et al. (2005a, b) found that KISS1 mediates testosterone and estrogen feedbacks on
GnRH neurons and differentially responds to sex steroids. Exogenous
administration of kisspeptins to sexually inactive male Syrian hamsters
reverses the inhibitory effect of nonbreeding season by re-activating the
HPG axis (Revel et al., 2006). Therefore, KISS1 and GPR54 have a certain impact on
male seasonal estrus. But no evidence of the effect of KISS1 and GPR54 on seasonal
estrus in rams has been reported so far. Therefore, KISS1 and GPR54 were selected for
investigation of the seasonal estrus of rams in this study.
KISS1 was expressed in cerebrum and placenta in humans (Muir et al., 2001).
Further studies revealed that KISS1 was expressed with high level in the
hypothalamus in pigs (Tomikawa et al., 2010). GPR54 gene was widely expressed in
many tissues in pigs, mice and Siberian hamsters (Li et al., 2008; Shahed et
al., 2009; Herbison et al., 2010). KISS1 and GPR54 have also been implicated in regulating
the estrus cycle of seasonal breeders and in the control of lactational
amenorrhea (Colledge, 2008). Our results demonstrated that KISS1 was specifically
expressed in hypothalamus both in SNT and STH. This is consistent with
previous studies about seasonal estrous and year-round estrous goats (Huang
et al., 2015) and ewes (An et al., 2019a). In addition, GPR54 was widely
expressed in all selected tissues and highly expressed in the testis,
cerebellum, pituitary and vas deferens, which implied that GPR54 may be
positively correlated with the seasonal estrus of rams.
Numerous studies revealed that KISS1 and GPR54 had effects on testis in goats, mice,
monkeys and humans (Terao et al., 2004; Tariq et al., 2013; Samir et al.,
2015; Mei et al., 2013). In the present study, we compared the expression
level of KISS1 and GPR54 in the testis, epididymis, and vas deferens between two sheep
breeds. We found the expression levels of GPR54 in the testis and vas deferens of
STH are significantly higher than in SNT rams. The KISS1 expression had no
significant difference in testis between STH and SNT, while it was
significantly higher in STH than in SNT in hypothalamus. Considering the
core function of the hypothalamus in seasonal estrus, we speculated that
KISS1 may play a key role in year-round estrus in hypothalamus of rams, similar
to their reported functions in other mammals. The results of GPR54 was in
agreement with Han et al. (2020), who found GPR54 was expressed in sertoli cells,
leydig cells and spermatids, suggesting the local expression of GPR54 in goats'
testes and its autocrine role in leydig cells. Therefore, we concluded that
the year-round estrus of rams might be due to high expression level of the
GPR54 gene. Of course, further studies are needed to investigate the relationship
between KISS1 and GPR54 and ram estrus in more depth.
Conclusions
This study describes the expression pattern of four HPG axis-related genes
in year-round estrous (STH) and seasonal estrous (SNT) rams. We found that
DIO2 was highly expressed in pituitary, and KISS1 was specifically highly expressed in
hypothalamus. The expression levels of EYA3 and GPR54 were high in hypothalamus,
pituitary and testis, and the expression levels of the four genes are all
higher in STH than SNT. The results suggested that the four genes may
regulate the estrous mode of ram via HPG axis, and they may play positive
functions in the ram year-round estrus. However, the specific mechanism
remains to be further explored. This is the first systematic analysis of
tissue expression patterns of the HPG axis-related genes in rams.
Data availability
The data sets are available upon request from the corresponding author.
Author contributions
These studies were designed by QX and MXC, who performed the
experimental analyses and prepared the figures and tables. QX analyzed
the data and drafted the manuscript. MXC contributed to revisions
of the manuscript. RD, XYH and CHW assisted in
interpreting the results and revised the final version of the manuscript.
All authors read and approved the final manuscript for publication.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Mingxing Chu, Ran Di and all the facilities involved including Chinese Academy of Agricultural Sciences, as well as the local abattoir for their support during this study.
Financial support
This research has been supported by the National Natural Science Foundation of China (grant nos. 31472078 and 31772580), the Earmarked Fund for China Agriculture Research System (grant no. CARS-38), the Agricultural Science and Technology Innovation Program of China (grant no. ASTIP-IAS13), the China Agricultural Scientific Research Outstanding Talents and Their Innovative Teams Program, China High-level Talents Special Support Plan Scientific and Technological Innovation Leading Talents Program (grant no. W02020274), the Tianjin Agricultural Science and Technology Achievements Transformation and Popularization Program (grant no. 201704020), the Youth Innovative Research and Experimental Project of Tianjin Academy of Agricultural Sciences (grant no. 201915), and the article processing charges were funded by the National Natural Science Foundation of China (grant no. 31472078).
Review statement
This paper was edited by Steffen Maak and reviewed by two anonymous referees.
ReferencesAn, X. J., Pan, Z. Y., Zhao, S. G., Li, C. Y, Tian, Z. L., Di, R., Liu, Q.
Y, Hu, W. P., Wang, X. Y., Zhang, X. S., Zhang, J. L., Cai, Y., and Chu, M.
X.: Expression of KiSS-1 and RFRP-3 in Small Tail Han sheep at luteal and
follicular periods, Journal of China Agricultural University, 24, 88–96,
10.11841/j.issn.1007-4333.2019.02.09, 2019a.An, X. J., Zhao, S. G., Pan, Z. Y., Tian, Z. L., Zhang, X. S., Zhang, J. L.,
Cai, Y., and Chu, M. X.: Tissue expression of DIO2 and DIO3 in Small Tail
Han sheep at luteal and follicular periods, Chinese Journal of Animal
Science, 55, 73–77, 10.19556/j.0258-7033.2019-01-068, 2019b.Colledge, W. H.: GPR54 and kisspeptins: Results, Probl. Cell. Differ.,
46, 117–143, 10.1007/400_2007_050,
2008.Dardente, H., Hazlerigg, D. G., and Ebling, F. J. P.: Thyroid hormone and
seasonal rhythmicity. Front. Endocrinol (Lausanne), 5, 19, 10.3389/fendo.2014.00019, 2014.Dardente, H., Lomet, D., Chesneau, D., Pellicer-Rubio, M. T., and Hazlerigg,
D.: Discontinuity in the molecular neuroendocrine response to increasing
daylengths in Ile-de-France ewes: is transient Dio2 induction a key feature
of circannual timing? J. Neuroendocrinol., 31, e12775, 10.1111/jne.12775, 2019.Dunn, I. C., Wilson, P. W., Shi, Y., Burt, D. W., Loudon, A. S. I., and
Sharp, P. J.: Diurnal and photoperiodic changes in thyrotrophin-stimulating
hormone β expression and associated regulation of deiodinase enzymes
(DIO2, DIO3) in the female juvenile chicken hypothalamus, J.
Neuroendocrinol., 29, e12554, 10.1111/jne.12554, 2017.Dupré, S. M., Miedzinska, K., Duval, C. V., Yu, L., Goodman, R. L.,
Lincoln, G. A., Davis, J. R. E., Mcneilly, A. S., Burt, D. D., and Loudon,
A. S. I.: Identification of Eya3 and TAC1 as long-day signals in the sheep
pituitary, Curr. Biol., 20, 829–835, 10.1016/j.cub.2010.02.066,
2010.Feng, T., Bai, J. H., Xu, X. L., and Liu, Y.: Kisspeptin and its effect on
mammalian spermatogensis, Curr. Drug. Metab., 20, 9–14, 10.2174/1389200219666180129112406, 2019.Funes, S., Hedrick, J. A., Vassileva, G., Markowitz, L., Abbondanzo, S.,
Golovko, A., Yang, S., Monsma, F. J., and Gustafson, E. L.: The KiSS-1
receptor GPR54 is essential for the development of the murine reproductive
system, Biochem. Biophys. Res. Commun., 312, 1357–1363, 10.1016/j.bbrc.2003.11.066, 2003.Hammond, K. L., Hanson, I. M., Brown, A. G., Lettice, L. A., and Hill, R.
E.: Mammalian and Drosophila dachshund genes are related to the Ski
proto-oncogene and are expressed in eye and limb, Mech. Dev., 74, 121–131,
10.1016/s0925-4773(98)00071-9, 1998.Han, Y. G., Zhao, Y. H. T., Si, W. J., Jiang, X. P., Wu, J. Y., Na, R. S.,
Han, Y. Q., Li, K., Yang, L. G., E,G. X., Zeng, Y., Zhao, Y. J., and Huang,
Y. F.: Temporal expression of the KISS1/GPR54 system in goats' testes and
epididymides and its spatial expression in pubertal goats, Theriogenology,
152, 114–121, 10.1016/j.theriogenology.2020.04.022, 2020.Herbison, A. E., Xavier, d. A. d. T., Joanne, D., and Colledge, W. H.:
Distribution and postnatal development of Gpr54 gene expression in mouse
brain and gonadotropin-releasing hormone neurons, Endocrinology, 151,
312–321, 10.1210/en.2009-0552, 2010.Huang, D. W., Chu, M. X., Di, R., Liu, Q. Y., Hu, W. P., Wang, X. Y., Pan,
Z. Y., and Guo, X. F.: Study on expression of KISS1 and RFRP genes related
to reproductive seasonality in goats, Acta Veterinaria et Zootechnica
Sinica, 46, 924–931, 10.11843/j.issn.0366-6964.2015.06.007,
2015.Huang, D. W., Di, R., Hu, W. P., Wang, X. Y., Pan, Z. Y., Guo, X. F., Cao,
X. H., Liu, Q. Y., and Chu, M. X.: Expression analysis of DIO2 and DIO3
genes related to reproductive seasonality in goats (Capra hircus), Journal of
Agricultural Biotechnology, 24, 1536–1543, 10.3969/j.issn.1674-7968.2016.10.010, 2016.Ikegami, K. and Yoshimura, T.: Circadian clocks and the measurement of
daylength in seasonal reproduction, Mol. Cell. Endocrinol., 349, 76–81,
10.1016/j.mce.2011.06.040, 2012.Janich, P., Pascual, G., Merlos-Suarez, A., Batlle, E., Ripperger, J.,
Albrecht, U., Cheng, H. Y. M., Obrietan, K., Croce, L. D., and Benitah, S.
A.: The circadian molecular clock creates epidermal stem cell heterogeneity,
Nature, 480, 209–214, 10.1038/nature10649, 2011.Kang, H., Kenealy, T. M., and Cohen, R. E.: The
hypothalamic-pituitary-gonadal axis and thyroid hormone regulation interact
to influence seasonal breeding in green anole lizards (Anolis carolinensis), Gen. Comp.
Endocrinol., 292, 113446, 10.1016/j.ygcen.2020.113446, 2020.La Y. F., He, X. Y., Zhang, L. P., Di, R., Wang, X. Y., Gan, S. Q., Zhang, X. S., Zhang, J. L., Hu, W. P., and
Chu, M. X.: Comprehensive analysis of differentially expressed
profiles of mRNA, lncRNA, and circRNA in the uterus of seasonal reproduction
sheep, Genes (Basel), 11, 301, 10.3390/genes11030301, 2020.Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C.
K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., and Rosenfeld,
M. G.: Eya protein phosphatase activity regulates Six1-Dach-Eya
transcriptional effects in mammalian organogenesis, Nature, 426, 247–254,
10.1038/nature02083, 2003.Li, S., Ren, J., Yang, G., Guo, Y., and Huang, L.: Characterization of the
porcine Kisspeptins receptor gene and evaluation as candidate for timing of
puberty in sows, J. Animal. Breed. Genet., 125, 219–227, 10.1111/j.1439-0388.2008.00732.x, 2008.Lincoln, G.: Melatonin modulation of prolactin and gonadotrophin
secretion, Adv. Exp. Med. Biol., 460, 137–153, 10.1007/0-306-46814-x_16, 1999.Liu, L., Chen, Y., Wang, D., Li, N., Guo, C., and Liu, X.: Cloning and
expression characterization in hypothalamic Dio2/3 under a natural
photoperiod in the domesticated Brandt's vole (Lasiopodomys brandtii), Gen. Comp. Endocrinol.,
259, 45–53, 10.1016/j.ygcen.2017.11.002, 2018.Livak, K. J.: Analyzing real-time PCR data by the comparative c(t) method,
Nat. Protoc., 3, 1101–1108, 10.1038/nprot.2008.73, 2008.Livak, K. J. and Schmittgen, T. D.: Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-delta delta C(T)) method,
Methods, 25, 402–408, 10.1006/meth.2001.1262, 2001.Lomet, D., Cognie, J., Chesneau, D., Dubois, E., Hazlerigg, D., and
Dardente, H.: The impact of thyroid hormone in seasonal breeding has a
restricted transcriptional signature, Cell. Mol. Life. Sci., 75, 905–919,
10.1007/s00018-017-2667-x, 2018.Marcheva, B., Ramsey, K. M., Buhr, E. D., Kobayashi, Y., Su, H., Ko, C. H.,
Ivanova, G., Omura, C., Mo, S., and Vitaterna, M. H.: Disruption of the
clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes,
Nature, 466, 627–631, 10.1038/nature09253, 2010.Masumoto, K. H., Ukai-Tadenuma, M.,Kasukawa, T., Nagano, M., Uno, K.
D., Tsujino, K., Horikawa, K., Shigeyoshi, Y., and Ueda, H. R.: Acute
induction of Eya3 by late-night light stimulation triggers TSHβ
expression in photoperiodism, Curr. Biol., 20, 2199–2206, 10.1016/j.cub.2010.11.038, 2010.Mei, H., Doran, J., Kyle, V., Yeo, S. H., and Colledge, W. H.: Does
kisspeptin signaling have a role in the testes?, Front. Endocrinol
(Lausanne), 4, 198, 10.3389/fendo.2013.00198, 2013.Meier, U.: A note on the power of sher's least significant difference
procedure, Pharm. Stat., 5, 253–263, 10.1002/pst.210, 2006.Miao, X. Y., Luo, Q. M., Zhao, H. J., and Qin, X. Y.: Co-expression analysis
and identification of fecundity-related long non-coding RNAs in sheep
ovaries, Sci. Rep., 6, 39398, 10.1038/srep39398, 2016.Mishra, I., Bhardwaj, S. K., Malik, S., and Kumar, V.: Concurrent
hypothalamic gene expression under acute and chronic long days: Implications
for initiation and maintenance of photoperiodic response in migratory
songbirds, Mol. Cell. Endocrinol., 439, 81–94, 10.1016/j.mce.2016.10.023, 2017.Muir, A. I., Chamberlain, L., Elshourbagy, N. A., Michalovich, D., Moore, D.
J., Calamari, A., Szekeres, P. G., Sarau, H. M., Chambers, J. K., Murdock,
P., Steplewski, K., Shabon, U. Miller, J. E., Middleton, S. E., Darker, J.
G., Larminie, C. G., Wilson, S., Bergsma, D. J., Emson, P., Faull,
R., Philpott, K. L., and Harrison, D. C.: AXOR12, a novel human G
protein-coupled receptor, activated by the peptide KiSS-1, J. Biol. Chem.,
276, 28969–28975, 10.1074/jbc.M102743200, 2001.Nam, D., Yechoor, V. K., and Ma, K.: Molecular clock integration of brown
adipose tissue formation and function, Adipocyte, 5, 243–250, 10.1080/21623945.2015.1082015, 2015.Novaira, H. J., Ng, Y., Wolfe, A., and Radovick, S.: Kisspeptin increases
GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines,
Mol. Cell. Endocrinol., 311, 126–134, 10.1016/j.mce.2009.06.011, 2009.Ohtaki, T., Shintani, Y., Honda, S., Matsumoto, H., Hori, A., Kanehashi, K.,
Kumano, S., Takatsu, Y., Masuda, Y., Ishibashi, Y., Watanabe, T., Asada, M.,
Yamada, T., Suenaga, M., Kitada, C., Usuki, S., Kurokawa, T., Onda, H.,
Nishimura, O., and Fujino, M.: Metastasis suppressor gene KiSS-1 encodes
peptide ligand of a G-protein-coupled receptor, Nature, 411, 613–617,
10.1038/35079135, 2001.Oliver, G., Mailhos, A., Wehr, R., Copeland, N. G., and Jenkins, N. A.:
Six3, a murine homologue of the sine oculis gene, demarcates the most
anterior border of the developing neural plate and is expressed during eye
development, Development, 121, 4045–4055,
10.1111/j.1365-2303.1995.tb00491.x, 1996.Papaoiconomou, E., Msaouel, P., Makri, A., Diamanti-Kandarakis, E., and
Koutsilieris, M.: The role of kisspeptin/GPR54 in the reproductive system,
Vivo., 25, 343–354, 10.1038/gt.2010.177, 2011.Park, E., Jung, J., Araki, O., Tsunekawa, K., Park, S. Y., Kim, J.,
Murakami, M., Jeong, S. Y., and Lee, S.: Concurrent TSHR mutations and DIO2
T92A polymorphism result in abnormal thyroid hormone metabolism, Sci. Rep.,
8, 10090, 10.1038/s41598-018-28480-0, 2018.Popa, S. M., Clifton, D. K., and Steiner, R. A.: The role of kisspeptins and
GPR54 in the neuroendocrine regulation of reproduction, Annu. Rev. Physiol.,
70, 213–238, 10.1146/annurev.physiol.70.113006.100540, 2008.Revel, F. G., Saboureau, M., Masson-Pevet, M., Pevet, P., Mikkelsen, J. D.,
and Simonneaux, V.: Kisspeptin mediates the photoperiodic control of
reproduction in hamsters, Curr. Biol., 16, 1730–1735, 10.1016/j.cub.2006.07.025, 2006.Romano, R. M., Gomes, S. N., Cardoso, N. C. S., Schiessl, L., Romano, M. A.,
and Oliveira, C. A. New insights for male infertility revealed by
alterations in spermatic function and differential testicular expression of
thyroid-related genes, Endocrine., 55, 607–617, 10.1007/s12020-016-0952-3, 2017.Roux, N. D., Genin, E., Carel, J. C., Matsuda, F., Chaussain, J.
L., and Milgrom, E.: Hypogonadotropic hypogonadism due to loss of function
of the KiSS1-derived peptide receptor GPR54, P. Natl. Acad. Sci. USA,
100, 10972–10976, 10.1073/pnas.1834399100, 2003.Samir, H., Nagaoka, K., Karen, A., Ahmed, E., El Sayed, M., and Watanabe,
G.: Investigation the mRNA expression of KISS1 and localization of
kisspeptin in the testes of Shiba goats and its relationship with the
puberty and steriodogenic enzymes, Small. Ruminant. Res., 133, 1–6,
10.1016/j.smallrumres.2015.10.024, 2015.Scarlet D, A. C., Ille, N., Walter, I., Weber, C., Pieler, D., Peinhopf, W.,
Wohlsein, P., and Aurich, J.: Anti-Muellerian hormone, inhibin A,
gonadotropins, and gonadotropin receptors in bull calves after partial
scrotal resection, orchidectomy, and Burdizzo castration, Theriogenology,
87, 242–249, 10.1016/j.theriogenology.2016.08.030, 2017.Seminara, S. B., Messager, S., Chatzidaki, E. E. Thresher, R. R., Acierno
Jr, J. S., Shagoury, J. K., Bo-Abbas, Y., Kuohung, W., Schwinof, K. M.,
Hendrick, A. G., Zahn, D., Dixon, J., Kaiser, U. B., Slaugenhaupt, S. A.,
Gusella, J. F., Rahilly, S. O., Carlton, M. B. L., Crowley Jr, W. F.,
Aparicio, S. A. J. R., and Colledge, W. H.: The GPR54 gene as a regulator of
puberty, N. Engl. J. Med., 349, 1589–1592, 10.1056/NEJMoa035322, 2003.Shahed, A. and Young, K. A.: Differential ovarian expression of KiSS-1 and
GPR-54 during the estrous cycle and photoperiod induced recrudescence in
Siberian hamsters (Phodopus sungorus), Mol. Reprod. Dev., 76, 444–452, 10.1002/mrd.20972, 2009.Smith, J. T., Cunningham, M. J., Rissman, E. F., Clifton, D. K., and
Steiner, R. A.: Regulation of Kiss1 gene expression in the brain of the
female mouse, Endocrinology., 146, 3686–3692, 10.1210/en.2005-0488, 2005a.Smith, J. T., Dungan, H. M., Stoll, E. A., Gottsch, M. L., Braun, R. E,
Eacker, S. M., Clifton, D. K., and Steiner, R. A.: Differential regulation
of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse,
Endocrinology, 146, 2976–2984, 10.1210/en.2005-0323, 2005b.Tang, J. S., Hu, W. P., Di, R., Liu, Q. Y., Wang, X. Y., Zhang, X. S.,
Zhang, J. L., and Chu, M. X.: Expression analysis of the prolific candidate
genes, BMPR1B, BMP15, and GDF9 in small tail han ewes with three fecundity
(FecB gene) genotypes, Animals., 8, 166, 10.3390/ani8100166,
2018.
Tariq, A. R., Shahab, M., Clarke, I. J., Pereira, A., Smith, J. T., Khan, S.
H., Sultan, J., Javed, S., and Anwar, T.: Kiss1 and Kiss1 receptor
expression in the rhesus monkey testis: a possible local regulator of
testicular function, Cent. Eur. J. Biol., 8, 968–974, 10.2478/s11535-013-0219-4, 2013.Tavolaro, F. M., Thomson, L. M., Ross, A. W., Morgan, P. J., and Helfer, G.:
Photoperiodic effects on seasonal physiology, reproductive status and
hypothalamic gene expression in young male F344 rats, J. Neuroendocrinol.,
27, 79–87, 10.1111/jne.12241, 2014.Terao, Y., Kumano, S., Takatsu, Y., Hattori, M., Nishimura, A., Ohtaki, T.,
and Shinrani, Y.: Expression of KiSS-1, a metastasis suppressor gene, in
trophoblast giant cells of the rat placenta, Biochim. Biophys. Acta., 1678,
102–110, 10.1016/j.bbaexp.2004.02.005, 2004.Tomikawa, J., Homma, T., Tajima, S., Shibata, T., Inamoto, Y., Takase, K.,
Inoue, N., Ohkura, S., Uenoyama, Y., Maeda, K. I., and Tsukamura, H.:
Molecular characterization and estrogen regulation of hypothalamic KISS1
gene in the pig, Biol. Reprod., 82, 313–319, 10.1095/biolreprod.109.079863, 2010.Trivedi, A. K., Sur, S., Sharma, A., Taufique, S. T., and Kumar, V.:
Temperature alters the hypothalamic transcription of photoperiod responsive
genes in induction of seasonal response in migratory redheaded buntings,
Mol. Cell. Endocrinol., 493, 110454, 10.1016/j.mce.2019.110454,
2019.Xia, Q., Zhang, X. S., Liu, Q. Y., Wang, X. Y., He, X. Y., Guo, X. F., Hu,
W. P., Zhang, J. L., Chu, M. X., and Di, R.: Expression patterns of EYA3 and
TSHβ in seasonal estrous and year-round estrous sheep, Acta
Veterinaria et Zootechnica Sinica, 49, 263–269, 10.11843/j.issn.0366-6964.2018.02.005, 2018.Xu, P. X., Woo, I., Her, H., Beier, D. R., and Maas, R. L.: Mouse Eya
homologues of the Drosophila eyes absent gene require Pax 6 for expression
in lens and nasal placode, Development, 124, 219–231, 10.1146/annurev.cellbio.13.1.779, 1997.Yoshimura, T.: Thyroid hormone and seasonal regulation of reproduction,
Front. Neuroendocrinol., 34, 157–166, 10.1016/j.yfrne.2013.04.002, 2013.