The mammalian biological clock system has a multi-level structure, including the main clock located in the suprachiasmatic nucleus and the sub-clocks of peripheral organs and tissues (Schibler, et al. 2003). The ovary is a pivotal reproductive organ in female animal, and the expression of the biological clock gene has been found in the ovaries of many organisms (Sellix 2015). In this study, we analyzed the expression profile and alternative splicing of the biological clock gene in Xiang pig ovaries in estrous cycle from the RNA-seq data. The study found out a total of 90 rhythms genes that were expressed in ovaries. The expression abundance of these genes was ranging from 0.1 to 532.6 CPM. A lot of the genes were expressed in ovaries at medium or high levels (48.9% and 27.4%), including the core clock components, arntl, clock, per1/2/3, and cry1/2. Furthermore, 33 genes were detected to undergo differentially expression and 34 genes were detected to undergo differential alternative splicing between estrous and diestrous ovaries. The DEGs and DSGs related with rhythms might have a connection with the regulation of estrus process in Xiang pig.
Transcription and translation of core clock components genes (clock or npas2, arntl/bmal1, or arntl2/bmal2, per1/2/3, and cry1/2) play a critical role in rhythm generation process. CLOCK and BMAL1 heterodimerize to activate transcription of circadian target genes including per1/2/3 and cry1/2. PER and CRY interact and conversely inhibit transcription of bmal1 and clock genes. These genes and their protein products are organized into interlocking positive and negative transcriptional and translational feedback loops, which regulates circadian rhythm generation in the brain suprachiasmatic nucleus (SCN) and peripheral organs (Rosenwasser and Turek 2015). In this study, we found that three core clock genes were differentially expressed between estrus and diestrus ovaries. Clock and per3 were down-regulated and per1 was up-regulated in the estrous ovaries. CLOCK plays a key role in maintaining the circadian rhythm and activating downstream elements. Inhibition of CLOCK can inhibit cell growth and increase the rate of apoptosis (Li, et al. 2015). In vivo experiments show that female mice is injected with CLOCK-shRNA have fewer oocytes, fewer litters, and a higher rate of apoptosis. The results indicate that clock plays an important role in fertility, and down-regulation of clock leads to cell apoptosis and decrease reproductive capacity (Li, et al. 2015). It was proposed that the down-regulation of clock in the ovary at estrus stage might be related to the low litter size of the Xiang pig. The core clock gene period 1 (per1) may be a prolific gene in Drosophila (Cushman, et al. 2007). Female mice with the per1 mutation showed a normal number of implantation sites but reduced litter size (Pilorz and Steinlechner 2008). Per1 mRNA locates in the secondary oocytes and follicles of ruminants (sheep, cattle) and found that there was no relationship between its transcription level and prolificacy, and this gene did not map to the known QTL region of ovulation rate in cattle (Cushman, et al. 2007). Treatment with Progesterone for 1 hour could induce the expression of per1 mRNA in MCF-7 cells (Nakamura, et al. 2010). In fact, the up-regulation of per1 was a common feature of many tissues in response to certain types of hormonal stress(For xample: luteinising hormone) (Mansuy, et al. 2009; Mendoza, et al. 2011; Smarr, et al. 2013). This expression pattern indicated that PER1 may be the most sensitive effector in the biological clock system (Zhang, et al. 2019). Some studies have found that estrus leads to changes in the expression time and amplitude of the biological clock genes (López-Rodríguez, et al. 2019). Rising estrogen cause female animal to go into estrous stage (Ravinder, et al. 2016).The estrus in Xiang pig might be speculated to reset the biological clock by up-regulating the per1 gene to response the stimulus of steroid hormone.In this study, per3 was down-regulated in the ovaries of estrus pigs. There is little information on the function of per3 gene in reproduction except for brain development (Noda, et al. 2019). In contrast, the expression patterns of clock genes in other pig breeds are much different from that in Xiang pig. In the ovarian follicles of Large White pigs and Mi pigs, the core circadian clock genes were all down-regulated and not differentially expressed between estrus and diestrus periods (Chu, et al. 2017). Compared with Large White pigs and Mi pigs, the characteristics of Xiang pig are specific, such as low birth rate and insignificant estrous behavior. It would be interesting to prove whether the expression profiles of core biological clock genes in ovary is a reason for the special reproductive traits such as estrus performance and litter size in different pig breeds.
The biological clock system consists of an input pathway, a core oscillator and an output pathway. The post-translational modification of clock proteins is essential to maintain the accuracy and robustness of the evolutionarily conserved circadian clock (Gossan, et al. 2014). Post-translational modification and degradation of the clock proteins are key steps in determining the length of the circadian clock cycle (Kennaway, et al. 2015). Our research found that the products of most differentially expressed genes were related to the post-translational modification of the core clock protein. For example, PPP1CB and PPP1CA can reduce the phosphorylation of PER2, and affect the nuclear localization of the protein PER2, which may at least partially change the cycle and phase shift characteristics of the biological clock (Schmutz, et al. 2011). Also, highlighted DYRK1A is the enzyme responsible for the phosphorylation of CRY2 at Ser557 and plays a key role in regulating the protein level of CRY2 (Kurabayashi, et al. 2010). The casein kinases CSNK1D and CSNK1E phosphorylate the PER protein and provide a marker for subsequent degradation (Kennaway, et al. 2015). The UBE3A binds and degrades BMAL1 in a ubiquitin ligase-dependent manner (Gossan, et al. 2014). Furthermore, MAGEL2 regulates the ubiquitination and stability of CRY1, and changes its nuclear and cytoplasmic distribution (Devos J 2011). Lastly, SIRT1 deacetylates PER2 and BMAL1, thereby participates in biological rhythm regulation (Wang, et al. 2016). The previous reports illustrate that the estrus cycle affects the localization and degradation of the core clock protein by changing their post-translational modification. And transcription and post-transcriptional regulation are the basis of clock system component activities, and post-transcriptional mechanisms accounts for more than half of the regulatory network (Koike, et al. 2012). The results of this study further strengthened the view that the post-translational mechanism participated in the circadian gene regulatory network. In addition, we detected some differentially expressed genes from Xiang pig ovaries (Table S2) that might affect the expression of core clock genes at the transcriptional level. For example, TOP2A binds to the unique GC-rich open chromatin structure of the bmal1 promoter region, indicating that TOP2 on the bmal1 promoter affects transcription of bmal1 (Ogawa, et al. 2014). Also, CBP /P300 and tissue-specific cofactors regulate CLOCK /BMAL1 transcription positively or negatively (Hosoda, et al. 2009). Accordingly, ID (DNA binding inhibitor) is an important transcription repressor. Each ID protein contains a helix-loop-helix domain through which it can interact with bHLH protein. However, ID lacks the basic domain that allows binding to the E-box element, which leads to the ID being able to modify the transactivation of clock genes and CCG by interfering with the CLOCK-BMAL1 heterodimer, bHLH orange factor and other bHLH factors (Hou, et al. 2009). These data indicate that the estrus cycle promoted the time change of the biological clock in reproductive tissues at the level of transcription and post-translational modifications (Fig. 4).
In addition, we detected several genes controlled by the circadian rhythm, which were differentially expressed in the ovaries of the two periods including klf9, star, ptgs2. Other studies have shown that klf9 is a clock output gene, and CLOCK and BMAL1 complexes can bind and activate transcription at the 5' flanking region of klf9, which is blocked by the co-expression of PER1. Also, KLF9 may play a role in regulating the effects of CLOCK/BMAL1 and in the expression of DBP, other clock and clock output genes, thereby changing the timing and amplitude of the circadian oscillations of gene transcription (Knoedler, et al. 2020). The previous studies have found that steroidogenic enzymes (STAR), prostaglandin synthase (PTGS2), which are related to ovarian progesterone synthesis, are clock-controlled genes (CCGs). The E-box enhancer exists in the 5'-flanking region of star, which binds to the CLOCK-BMAL1 heterodimer and activates the transcription of gene star (Nakao, et al. 2007). Co-expression of the negative regulators PER and CRY attenuates this activation (Wu, et al. 2011). The promoter of ptgs2 has an E-box element and a REV-ERBα/RORα response element (RORE). The secretion of PGF2α can be balanced by the inhibition or stimulation of transcriptional regulation on REV-ERBα and BMAL1/CLOCK, respectively (Isayama, et al. 2014). It is thus inferred that the peripheral circadian oscillator, such as ovary, could play an essential role in synchronizing local physiology through regulation of the expression of clock-controlled genes (Mohawk, et al. 2012). In this study, star and ptgs2 in the ovaries of the estrus pigs were up-regulated by 6.42 and 5.55 times, respectively. It might be possible that the core clock genes might regulate the production of steroid hormones by controlling the ovarian-specific CCG, thereby affecting the estrus cycle of Xiang pigs.
Pre-mRNA splicing is a basic biological process through which introns of nascent RNA are removed and exons are merged to form mature RNA, which is then translated into protein (Lee and Rio 2015). Through alternative splicing (AS), different proteins are produced, resulting in a wider variety of cellular functions (Genov, et al. 2019). Except for transcription or post-translational controls, alternative splicing also plays a key role in circadian rhythms in a cell-type-specific manner (Shakhmantsir, et al. 2018). Recent research reports several cases, in which AS is involved in the regulation of biological clocks in plants, mice, and fruit flies (Sanchez, et al. 2010; Petrillo, et al. 2011; Hughes, et al. 2012; McGlincy, et al. 2012; Preußner, et al. 2014). In this study, Arntl only underwent differential alternative splicing, while clock and per1 experienced both of differential alternative splicing and differential expression. The differential alternative splicing event occurred in per1 was the retention of introns 14–15. This event would result in the deletion of amino acid residues from positions 545 to 1278 in the encoded protein, and the deletion site was located in the core domain to activate the protein. This intron retention event occurred more frequently in the ovaries of Xiang pigs in the diestrus period, and the active PER1 protein decreased. During the estrus period, the occurrence of the intron retention event decreased, and the active PER1 protein level increased, which further increased the expression of PER1. It indicated that the core clock component PER1 may respond to the changes of sterol hormones in the ovary through transcriptional regulation and post-transcriptional regulation. Clock underwent the differential exon 17 skipping event. When this event occurred, the encoded protein would be deleted amino acid residues from positions 484 to 513. The deleted amino acids were not located in the core domain and the influence on the protein activity was still unclear. These findings suggested that AS might directly change the structure of core clock components to regulate the ovarian biological clock, and the AS regulation of biological clock genes might be independent in the gene expression. However, more evidence needs to be accumulated whether the AS events in these biological clock genes would turn on and turn off the circadian rhythm in pig.