The circadian rhythm is a biological rhythm that is closely related to
the rhythmic expression of a series of clock genes. Results from several
studies have indicated that clock genes are associated with the estrous cycle in
female animals. Until now, the relationship between estrus cycle transition
and clock gene expression in reproductive-axis-related tissues has remained
unknown in Small-tailed Han (STH) sheep. This study was conducted to analyze
the expression patterns of six canonical clock genes (Clock, BMAL1, Per1, Per2, Cry1, and Cry2) in the follicle
phase and luteal phase of STH sheep. We found that all six genes were
expressed in the brain, cerebellum, hypothalamus, pituitary, ovary, uterus,
and oviduct in follicle and luteal phases. The results indicated that Clock expression
was significantly higher in the cerebellum, hypothalamus, and uterus of
the luteal phase than that of the follicle phase, whereas BMAL1 expression was
significantly higher in the hypothalamus of the luteal phase than that of
the follicle phase. Per1 expression was significantly higher in the brain,
cerebellum, hypothalamus, and pituitary of the luteal phase than that of the follicle
phase, and Per2 expression was significantly higher in the hypothalamus,
pituitary, and uterus of the luteal phase than that of the follicle phase. Cry1
expression was significantly higher in the brain, cerebellum, and
hypothalamus of the luteal phase than that of the follicle phase, whereas Cry2 expression
was significantly higher in the pituitary of the luteal phase than that of the
follicle phase. The clock gene expression in all tissues was different
between follicle and luteal phases, but all clock gene mRNA levels were
found to exhibit higher expression among seven tissues in the luteal
phase. Our results suggest that estrous cycles may be associated
with clock gene expression in the STH sheep. This is the first study to
systematically analyze the expression patterns of clock genes of different
estrous cycle in ewes, which could form a basis for further studies to
develop the relationship between clock genes and the estrous cycle.
Introduction
Circadian rhythms are the nearly 24 h processes that allow an organism to
coordinate appropriate physiological responses to the environmental
light–dark changes associated with the rotation of the Earth (Goldstein and Smith, 2016). In mammals, various behaviors and physiological functions of the
body can present a typical circadian rhythm, such as the sleep–wake cycle,
food intake, body temperature fluctuation, hormone secretion, and energy
metabolism (Chacon et al., 2004; Buhr et al., 2010; Mohawk et al., 2012).
Like many other functional activities, animal reproduction is closely
related to the circadian rhythm. Previous studies have shown that many aspects
of the reproductive biology of males and females are regulated by the circadian
rhythm (Brown-Grant et al., 1977; Peterlin et al., 2019; Mills and Kuohung,
2019), including the estrus cycle, levels of luteinizing hormone (LH),
ovulation, production and maturation of sperm, fertilization, insemination,
and embryo implantation (Gray et al., 1978; Christian et al., 2005).
However, disruptions to the circadian rhythm have noticeable negative effects
on female reproductive health, such as irregular ovulatory cycles, reduced
fertility, increased miscarriage rates, and anomalous fetal development
(Gotlieb et al., 2020). Moreover, studies in rodents have shown that
ablation of the master circadian clock in the brain can block relevant clock
output signals or disrupt the genes driving the circadian clock function at the
cellular level, leading to pronounced deficits in ovulation and reproductive
success (Gotlieb et al., 2020).
The circadian rhythm is controlled by the central clock in the hypothalamic
suprachiasmatic nucleus (SCN) and the peripheral clocks in various tissues
(Zhang et al., 2016). It is accepted wisdom that circadian rhythms are
generated and maintained by an autoregulatory transcription–translation
feedback loop consisting of clock genes and their protein products
(Takahashi, 2015; Honma et al., 2018). Currently, more than 10 genes
have been identified that form the basis of cellular rhythmicity in mammals,
including two transcriptional activators, Clock and BMAL1, the
transcriptional repressors called Period (Per1, Per2), and Cryptochrome (Cry1, Cry2)
(Preitner et al., 2002; Leloup and Goldbeter, 2003), which are considered to be the
core clock genes (Sen and Hoffmann, 2020). These genes contribute to
reproductive processes in mammals (Pan et al., 2020). Multiple studies have
indicated a connection between clock gene expression and reproduction. For
example, clock gene expression is related to ovarian follicular development
(Sen and Sellix, 2016; Nagao et al., 2019) and steroidogenesis (Liu et al.,
2014; Sellix, 2015), and they can regulate LH surge to affect
ovulation (Simonneaux et al., 2017). Studies on the mouse estrous ovary
have shown that the expression of clock protein is rhythmic in four follicular
stages (Wiggins and Legge, 2016). In addition, clock gene expression abnormalities
resulted in increased rates of placental abruption later in pregnancy (Qiu
et al., 2016). Studies in humans have suggested that disruption of
the cellular clocks also perturbs reproductive cycles including ovulation
(Mahoney, 2010).
Increasing reproductive efficiency with regard to litter size (also known as
fecundity) is one of the economic objectives of the sheep industry. Most
sheep breeds produce one lamb per gestation, and only a few produce twins,
which substantially affects the overall reproductive efficiency. Compared
with other sheep, the Small-tailed Han (STH) sheep is an excellent local breed in China, which is
well known for high fecundity, especially year-round estrus, and an average
lambing rate of 250 %. Accordingly, they are considered to be a good
breeding source (Di et al., 2012; Wang et al., 2015). It is generally known
that seasonal reproduction is regulated by the
hypothalamus–pituitary–gonadal (HPG) axis system. However, the clock genes
play important roles in regulating the HPG axis, especially the secretion of
GnRH and LH (Chappell et al., 2003), hinting that the clock genes may have
an effect on the change in the estrus pattern in animals. However, there are few
reports about the functions of clock genes in sheep reproduction.
In the present study, to ascertain the potential role of Clock, BMAL1, Per1, Per2, Cry1, and Cry2 in STH
sheep, we compare and analyze the mRNA expression levels of these genes in
HPG-related tissues between the follicular phase and luteal phase of STH.
Our study paves the way for an in-depth study of the estrus mode transition of
STH sheep.
Materials and methodsSelection of experimental sheep and sample collection
The six Small-tailed Han adult ewes (3 years old) used for study were selected
from the Sheep & Goat Breeding Farm of Tianjin Institute of Animal Sciences
(Tianjin, China). All sheep were kept in a sheltered outdoor paddock and
were provided with alfalfa hay and concentrate, with clean water available
ad libitum. All sheep were subjected to estrus synchronization
administration of progesterone (CIDR device, InterAg Co., Ltd., New Zealand)
for 12 d. Then three estrus-synchronized sheep were euthanized
(intravenous pentobarbital at 100 mg per kilogram) within 45–48 h of CIDR
removal (follicular phase); the remaining three estrus-synchronized sheep
were euthanized (intravenous pentobarbital at 100 mg per kilogram) 9 d
after CIDR removal (luteal phase). All animals were euthanized (intravenous
pentobarbital at 100 mg per kilogram), and seven tissues (brain, cerebellum,
hypothalamus, pituitary, ovary, uterus, oviduct) were collected from each
animal. All tissues were snap-frozen in liquid nitrogen and then stored at
-80∘C to be used for RNA extraction.
All animals used in the present 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, P.R.
China). Ethical approval was given by the animal ethics committee of
IAS-CAAS (no. IAS2020-82, 28 July 2020).
Total RNA extraction and cDNA synthesis
Total RNA in the different tissues (each tissue smashed, mixed, and 50–100 mg used for RNA extraction) was extracted using the Trizol reagent according
to the manufacturer's instructions (Invitrogen Inc., Carlsbad, CA, USA). The
concentration and integrity of the RNA samples were detected by ultraviolet
spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan) and 1.5 % agarose gel
electrophoresis (U = 160 V; 10 min). Then, total RNA (500 ng) was
reverse-transcribed into cDNA using a PrimeScript™ RT reagent kit
(TaKaRa Bio Inc., Dalian, China) following the method provided by the
manufacturer. Briefly, each 10 µL reaction mix contained 5×
PrimeScript Buffer (for real time) 2.0 µL, PrimeScript RT Enzyme 0.5 µL, oligo dT Primer 0.5 µL, random 6-mers 0.5 µL, and total
RNA 500 ng, with ddH2O accounting for the rest of the volume. The PCR
thermocycler program was as follows: 37 ∘C for 15 min, followed
by 85 ∘C for 5 s. After reaction, the cDNA was stored at -20∘C until use.
Primer design
The corresponding quantitative real-time polymerase chain reaction (qRT-PCR)
primers were designed using Primer 5.0 (Palo Alto, CA, USA) software based
on the GenBank sequence of target genes (Clock, BMAL1, Per1, Per2, Cry1, Cry2, and RPL19). All primers were synthesized by
Beijing Tianyi Biotechnology Co., Ltd. (Beijing, China). The
corresponding qRT-PCR primers are shown in Table 1.
QRT-PCR analysis for mRNA expression
QRT-PCR was performed to examine the expression levels of Clock, BMAL1, Per1, Per2, Cry1, and
Cry2 in seven tissues from the follicular phase and luteal phase of STH sheep.
We performed qRT-PCR on a LightCycler480 system (Roche, Basel, Sweden). Each
10 µL qRT-PCR reaction mix contained 5 µL of TB Green Premix
Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio Inc., Dalian, China), 2 µL of
cDNA, 0.4 µL of forward primer, and 0.4 µL of reverse primer,
with ddH2O accounting for the rest of the volume. The PCR program
consisted of initial denaturation at 95 ∘C for 5 min, followed by
40 cycles of amplification at 95 ∘C for 10 s, annealing at
60 ∘C for 30 s, extension at 72 ∘C for 60 s, a
melting curve step (65–95 ∘C, starting fluorescence acquisition at
65 ∘C with measurements every 10 s to 95 ∘C), and a
final cooling step to 4 ∘C. Three replicates were performed for
each reaction, and mRNA levels were normalized to the expression level of
the housekeeping gene RPL19 in each sample.
Primers used for real-time reverse transcription polymerase chain
reaction.
All the experiments were repeated at least three times, and the results are
shown as mean ± standard error of the mean (SEM). The relative gene
expression levels were calculated based on the 2-ΔΔCt
method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008). Statistical analysis
was performed using SPSS 22.0 software (IBM Armonk, NY, USA) and GraphPad
Prism 7.0 software (GraphPad Prism Software Inc., San Diego, CA, USA). The
Student's t test was used to compare the levels of gene expression between two groups. To compare the
means of more than two groups, one-way analysis of variance (ANOVA) was used
followed by the Tukey' honest significant difference (HSD) test.
Differences were considered statistically significant at P<0.05.
ResultsExpression levels of Clock
As shown in Fig. 1a, Clock was expressed in seven tissues of the follicular and
luteal phases in STH sheep, with the highest level being in the brain. In
the follicular phase, the expression level of Clock in the brain was
significantly higher than that in the cerebellum, hypothalamus, pituitary,
ovary, uterus, and oviduct (P<0.05), and the expression level in the
cerebellum was significantly higher than that in the pituitary, ovary,
uterus, and oviduct (P<0.05). Furthermore, the expression levels of
Clock in the ovary and uterus were lower than other tissues (P<0.05). In
the luteal phase, there was no significant difference in the pituitary,
ovary, uterus, and oviduct (P>0.05), but the expression levels of
Clock in the ovary and oviduct were significantly lower than that in the brain,
cerebellum, and hypothalamus (P<0.05), and Clock expression in the pituitary and
uterus was significantly lower than that in the brain and cerebellum
(P<0.05). In addition, the expression level of Clock in the cerebellum
was not different from that in the brain and hypothalamus (P>0.05), but there was a significant difference between the brain and
hypothalamus (P<0.05).
As depicted in Fig. 1b, there was no significant difference in the
expression level of Clock in the brain, pituitary, ovary, and oviduct between two
phases, but its expression in the cerebellum, hypothalamus, and uterus of the
luteal phase was higher than that of the follicular phase (P<0.01, P<0.01, P<0.05).
Comparison of expression of Clock among two phases (a) and among
tissues (b). Different letters mean significant difference (P< 0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Expression levels of BMAL1
The results of the BMAL1 analysis are shown in Fig. 2. BMAL1 was expressed in seven
tissues of the follicular and luteal phases in STH sheep, with the highest
level being in the brain, followed by the hypothalamus, with a significant
difference between the two tissues (P<0.05). And the expression
levels of BMAL1 in them were higher than that in the pituitary, ovary, uterus, and
oviduct (P<0.05). Additionally, there was no significant difference
among the pituitary, ovary, uterus, and oviduct (P>0.05) (Fig. 2a).
In addition, the tissue expression profiles of the two phases in STH sheep
were analyzed, and the expression level of BMAL1 in the hypothalamus of the luteal
phase was significantly higher than that of the follicular phase
(P<0.01); however, there was no significant difference in other
tissues (P>0.05) (Fig. 2b).
Comparison of expression of BMAL1 among two phases (a) and among
tissues (b). Different letters mean significant difference (P<0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Expression levels of Per1
As shown in Fig. 3a, Per1 was widely expressed in all selected tissues. There
was no difference in the expression of Per1 in the brain, cerebellum, and
pituitary, but the expression of Per1 in them was significantly higher than that
in the ovary and oviduct (P<0.05).
In the comparison between the follicular phase and luteal phase (Fig. 3b), the
expression levels of Per1 in the brain, cerebellum, hypothalamus, and pituitary
were much higher in the luteal phase than in the follicular phase
(P<0.05), but there was no significant difference in other tissues
(P>0.05).
Comparison of expression of Per1 among two phases (a) and among
tissues (b). Different letters mean significant difference (P< 0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Expression levels of Per2
Subsequently, we evaluated the expression levels of Per2 in seven tissues of the
follicle and luteal phases. As depicted in Fig. 4a, Per2 was expressed in all
selected tissues, with the highest level being in the cerebellum. In the
follicular phase, the expression levels of Per2 in the brain, cerebellum, and
oviduct had no significant difference (P>0.05), but its expression in the
cerebellum was significantly higher than that in the hypothalamus,
pituitary, ovary, and uterus (P<0.05). Furthermore, in the luteal phase, the
expression level of Per2 in the cerebellum was significantly higher than those of
other tissues (P<0.05).
As shown in Fig. 4b, compared with the follicular phase, Per2 expression
levels of the luteal phase were significantly higher in the hypothalamus,
pituitary, and uterus (P<0.05), but there was no difference in other tissues
(P>0.05).
Comparison of expression of Per2 among two phases (a) and among
tissues (b). Different letters mean significant difference (P<0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Expression levels of Cry1
As depicted in Fig. 5a, the mRNA expression of Cry1 was detected in all tissues,
with the highest level being in the cerebellum, and its expression was
significantly higher than those of other tissues (P<0.05). In
addition, the expression levels of Cry1 in the brain and hypothalamus were
significantly higher than that in the pituitary, ovary, uterus, and oviduct
(P<0.05). As depicted in Fig. 5b, the expression levels
of Cry1 were markedly higher in the brain, cerebellum, and hypothalamus of the luteal
phase than that of the follicular phase (P<0.01, P<0.05, P<0.05), but
there was no difference in other tissues (P>0.05).
Comparison of expression of Cry1 among two phases (a) and among
tissues (b). Different letters mean significant difference (P<0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Expression levels of Cry2
Figure 6a clearly shows that the expression pattern of Cry2 in the seven tissues
was similar in the follicular phase and luteal phase. Cry2 is expressed among
the seven tissues, with the highest level being in the brain. In addition,
the expression levels of Cry2 in the brain, cerebellum, and hypothalamus were
significantly higher than that in the pituitary, ovary, uterus, and oviduct
(P<0.05). As shown in Fig. 6b, the Cry2 expression level of the luteal
phase was significantly higher in the pituitary than that of the follicular
phase (P<0.01), but there was no difference in other tissues
(P>0.05).
Comparison of expression of Cry2 among two phases (a) and among
tissues (b). Different letters mean significant difference (P< 0.05).
One asterisk denotes differences at P<0.05, and two asterisks denote
differences at P<0.01. Data are presented as the mean ± SEM.
Discussion
The Clock was the first mammalian circadian clock gene to be discovered (King et
al., 1997). Accumulating evidence indicates that Clock plays an important role in female reproduction.
For example, Clock mutant mice showed irregular estrous cycles, with no normal
LH surge on the day of proestrus, as well as failing to show circadian rhythms
of clock gene expression in the uterus (Dolatshad et al., 2006). In
addition, prior studies have indicated that Clock–Clock mutant mice have differences in pregnancies, with a higher
rate of fetal absorption, serious dystocia, morphological abnormalities, and
lower serum progesterone and estradiol levels (Miller and Takahashi, 2014; Pilorz
et al., 2018). Thus, Clock is an important gene in female reproduction. In the
present study, we found that the Clock gene was expressed in all the selected
tissues. This result is consistent with that of previous studies, which
shows that Clock mRNA expression is not limited to the SCN but lies in diverse
tissues (King et al., 1997). However, the highest expression of Clock in the brain
of STH sheep differs from reports on mice; this discrepancy may be
attributed to the differences between species and sampling time points.
Furthermore, the expression of Clock in the uterus in the luteal phase was found
to be significantly higher than in the follicular phase, which may be
beneficial to pregnancy.
Previous research has also demonstrated that BMAL1 is necessary to maintain normal
female reproduction (Brown et al., 2012; Wang et al., 2017). BMAL1 null mice
showed irregular estrous cycles, late onset of puberty, absence of proestrus
LH surges, implantation failure, and progesterone-dependent implantation
failure (Boden et al., 2010, 2013). Additionally, studies on
mice and porcine luteinized granulosa cells in vitro have suggested that BMAL1 knockdown
reduces transcriptional levels of steroidogenesis-associated genes and
deteriorates P4 and E2 production, with promoting granulosa cell apoptosis
(Wang et al., 2017, 2020). Therefore, the BMAL1 gene has a certain
impact on follicle development and female reproduction. In the current
study, our results demonstrate that BMAL1 is expressed among seven tissues,
implicating BMAL1 as involved in STH sheep reproduction. Recently,
as physiologically verified by immunohistochemistry, BMAL1 expression significantly
increased during mouse corpus luteum formation (Wiggins et al., 2016).
Likewise, Kobayashi et al. (2018) found that BMAL1 expression reached a maximum 16 h
after hCG (human chorionic gonadotropin) administration when follicle
luteinization occurred. These results suggest
a role of BMAL1 in ovarian luteinization. Consistent with these previous
reports, our present data demonstrate that the expression levels of BMAL1 were
higher in the brain, cerebellum, hypothalamus, pituitary, ovary, and uterus of
the luteal phase compared with the follicular phase. However, there is only a
significant difference in the hypothalamus. One potential explanation is
that the differences in the genetic models or sampling position led to these
results.
Period genes are a class of circadian clock genes that act as transcriptional
repressors, forming a core component of the circadian clock (Dibner and Schibler,
2018). Evidence from previous studies has suggested that mice deficient in
Per1 or Per2 had a short circadian period. Additionally, double mutations of
Per1–Per2 genes in mice disrupted circadian rhythms in locomotor activity and the
expression of key clock genes as well as clock-regulated genes (Zheng et
al., 1999, 2001). However, Per3 mutation mice did not show any
effect on the circadian rhythm, implying that Per3 is considered to be unnecessary
for maintaining the circadian rhythm (Shiromani et al., 2004). Therefore, Per1 and Per2
were selected for the investigation of the STH sheep in this study. Previous
studies reported that Per1 and Per2 are widely expressed throughout the body (Nakamura et
al., 2005; Lamont et al., 2007). Further studies revealed that the
expression of Per1 and Per2 was detected in many tissues of Sunite sheep (Xiang et al.,
2019a, b). Our data also show that Per1 and Per2 are expressed in the
brain, cerebellum, and hypothalamic–pituitary–gonadal axis. And we found
that Per1 and Per2 are more highly expressed in the hypothalamus and pituitary of the luteal phase
than that of the follicle phase, whereas the hypothalamus and pituitary play
central roles in the production and release of reproductive hormones.
Additionally, a previous study reported that Per1 and Per2 mRNA may participate in the
coordination of GnRH (gonadotropin-releasing hormone) and LH (luteinizing
hormone) surge (Zheng et al., 2019). This research implies that Per1 and Per2 may have a
certain effect on STH sheep reproduction. Of course, further research is
needed in this regard.
In mammals, the Cryptochrome gene family has two members, Cry1 and Cry2, which are negative
feedback regulators of the circadian clock (Duong et al., 2011). Cry1 knockout
mice present a short-period circadian rhythm at behavioral and tissue as well as cell
levels, whereas Cry2 knockout mice exhibit a completely opposite phenotype (van der Horst et al., 1999). The reason behind these opposing phenotypes
is still unclear. In addition, double mutations of Cry1 and Cry2 genes in mice resulted in
complete loss of the circadian rhythm (van der Horst et al., 1999). This
research indicated that Cry1 and Cry2 are key for producing and maintaining the
circadian rhythm for the body. Previous studies have shown that Cry1 is widely
expressed in the human and mouse heart, ovary, and testis (Kobayashi et al.,
1998). Subsequently, studies on sheep demonstrated that the Cry1 gene was
expressed in pituitary tissues at different ages (Zhan et al.,
2012). Next, Gao et al. found that Cry1 was expressed in the
hypothalamus–pituitary–ovary axis, suggesting that it may initiate estrus
and seasonal reproduction (Gao et al., 2013). Likewise, the expression of
Cry1 mRNA and protein was also detected in the male yak reproductive axis (Chen et
al., 2019). Consistent with previous studies, our results show that Cry1 is
expressed in all selected tissues of STH sheep, implying that Cry1 is closely
related to sheep reproduction. To date, the Cry2 gene has only been shown to be
involved in the reproduction of diapausing animals through the seasons (Pan
et al., 2020). In this study, we also found that Cry2 was expressed in all
selected tissues of STH sheep. The results suggest that Cry2 may play a role in
sheep reproduction. However, what part Cry2 plays in reproduction remains
largely unclear, and more work will have to be done to identify this exact
role in the future.
Circadian rhythms in physiology and behavior are known to be influenced by
the estrous cycle in female rodents (Nakamura et al., 2010), so we
investigated the expression of six clock genes in different tissues of
follicular and luteal phases in STH sheep. The present study suggests that
six clock genes are expressed in all selected tissues of STH sheep with
different expression levels, which may play an important role in maintaining
various physiological and behavioral rhythms of sheep, especially the
reproductive function, but their specific role needs to be further studied.
And all six clock gene mRNA levels were found to exhibit higher expression
among seven tissues in the luteal phase, which is consistent with the
findings in female rats by Nakamura et al. (2010).
Likewise, a study performed using the female cynomolgus model demonstrated
that significant differences were found in the expression of Cry1 or Per2 mRNA
between the late follicular phase and mid-luteal phase (Xu et al., 2015).
Additionally, studies on rodents and monkeys have suggested that progesterone
levels are associated with changes in clock gene expression during the
estrus cycle. Further studies found that P4, but not E2, acutely induces
clock gene expression in MCF-7 human cancer cells (Nakamura et al., 2010).
It is well known that the progesterone level in the luteal phase is much higher
than that in the follicular phase in sheep, implying that the different
expression levels of clock genes in the follicular phase and luteal phase of STH
sheep is probably caused by progesterone levels. Moreover, clock gene
knockout mice have all been shown to have deficiencies in embryonic
implantation and pregnancy maintenance (Pilorz and Steinlechner, 2008). These data
indicate that high expression of clock genes during the luteal phase may be the
natural demand of embryonic implantation and early development during the luteal
phase. Previous studies have suggested that the total amount of plasma melatonin
secretion in the luteal phase could be significantly increased compared to
the follicular phase in humans (Webley and Leidenberger, 1986), so melatonin may affect
the change in clock genes between the follicular phase and luteal phase in STH
sheep. Although the expression of clock genes in the luteal phase is higher than
that in the follicular phase, there is no significant difference in some
tissues, perhaps due to the relatively small sample size.
Conclusion
In conclusion, this study describes the expression pattern of six canonical
clock genes in the follicle and luteal phases of STH sheep. All six genes were
expressed in both reproductive and non-reproductive tissues of different
phases, with high expression levels shown in the luteal phase. Our results
suggest that the estrous cycle has an impact on clock gene expression.
However, further studies are needed to elucidate the changes in clock gene
expression in the estrous cycle and their biological role during this process.
This is the first study performed on the tissue-specific expression patterns
of the six canonical clock genes in the follicle and luteal phases of STH
sheep, providing a foundation for elucidating the molecular mechanism
underlying the effect of clock genes on the ewe estrous mode.
Data availability
Data are available upon request from the corresponding author.
Author contributions
These studies were designed by QH and MC, who conducted the
experimental analyses and prepared the figures and tables. QH analyzed
the data and drafted the paper. MC contributed to revisions
of the paper. RD and XH assisted in explaining the results
and revised the final version of the paper. All authors read and
approved the final paper for publication.
Competing interests
The authors declare that they have no conflict of interest.
Disclaimer
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We thank all the facilities involved, including the Chinese Academy of Agricultural Sciences, as well as the local abattoir for their support during this study.
Financial support
This research was funded by the following bodies: National Natural Science
Foundation of China (grant nos. 31472078 and 31772580); China Agriculture Research
System of MOF and MARA (CARS-38); the Agricultural Science and Technology
Innovation Program of China (grant nos. CAAS-ZDRW202106 and 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). The APC was funded by the
National Natural Science Foundation of China (grant no. 31472078). The funding bodies
had no role in the design of the study or the collection, analysis, and
interpretation of data in writing the paper.
Review statement
This paper was edited by Steffen Maak and reviewed by two anonymous referees.
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