Ovarian transcriptome profile from egg-laying period to incubation period of Changshun green-shell laying hens

Ovarian transcriptome profile from egg-laying period to incubation period of Changshun green-shell laying hens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ovarian transcriptome profile from egg-laying period to incubation period of Changshun green-shell laying hens Zhi Chen, Di Wen, Ren Mu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4540221/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Changshun green-shell laying hen with strong broodiness is a Chinese indigenous chicken breed. Little is known about the mechanisms responsible for ovary development of Changshun green-shell laying hens from egg-laying period (LP) to incubation period (BP). Here, RNA sequencing (RNA-seq) of ovaries from Changshun hens in LP and BP was performed to identify candidate genes and pathways associated with broodiness. Results: We identified 1650 differently expressed genes (DEGs), including 429 up-regulated and 1221 down-regulated DEGs, in chicken ovaries between LP and BP groups. GO and KEGG analysis further revealed that these DEGs were mainly involved in the pathways related to follicle development in chicken ovaries, including focal adhesion, MAPK signaling pathway, and FoxO signaling pathway, and vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathway were down-regulated in incubating ovaries. Eight candidate genes ( EGFR , VEGFRKDRL , FLT1 , KDR , PDGFRA , TEK , KIT and FGFR3 ) related to angiogenesis, folliculogenesis, steroidogenesis and oogenesis in ovaries were suggested to play important roles in the ovarian development of Changshun hens during the transition from LP to BP. Conclusions: We discovered critical genes and pathways which is closely associated with ovary development in incubating chickens, indicating the complexity of reproductive behaviour of different chicken breeds. Transcriptome analysis ovary laying period incubation period Changshun green-shell laying hen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Incubation behaviour is a common maternal behaviour of domestic chickens. Different from egg-laying chickens, the ovary and oviduct of incubating chicken are degenerate, resulting in the cessation of egg-laying. The molecular regulation mechanism of avian broodiness remains unclear. It is generally known that the onset and maintain of incubation behaviour in chicken is mainly associated with multiple neuroendocrine hormones and neurotransmitters [ 1 – 3 ]. A characteristic increase of pituitaric prolactin (PRL) in plasma is critical for the onset and maintain of chicken broodiness [ 4 – 7 ]. Meanwhile, luteinizing hormone (LH), hypothalamic gonadotropin-releasing hormone (GnRH), neuropeptide vasoactive intestinal polypeptide (VIP), neurotransmitter dopamine (DA) 5-hydroxytryptophan (5-HTP) and dynorphin are also reported to be involved in chicken broodiness [ 6 , 8 – 14 ]. In the past decades, high-throughput sequencing technology including RNA sequencing (RNA-Seq) has been widely used to explore the transcriptome profile of avian ovaries from egg-laying period (LP) to incubation period (BP). Although increasing number of candidate genes and signaling pathways in ovaries are reported to be involved in avian broodiness, there has been no specific genes and pathways elucidated which has marked effects on avian incubation behaviour. Compared to chickens, geese and ducks with high tendency to broodiness are widely used to investigate avian broodiness. In incubating Muscovy ducks ( Cairina moschata ), a total of 334 differential expression genes (DEGs) and 36 differentially abundant long noncoding RNAs (lncRNAs) transcripts were identified in ovaries, and focal adhesion, FOXO signaling pathway, Wnt signaling, oocyte meiosis and cytokine-cytokine receptor interaction pathways in ovaries might involve in the transition from egg-laying to incubation [ 15 ]. However, different expression mRNAs and the target genes of different expression lncRNAs in the ovaries of Taihe Black-Bone Silky ( Gallus gallus Domesticus Brisson ) are mainly associated with neuroactive ligand-receptor interaction, CCR6 chemokine receptor binding, G-protein coupled receptor binding, cytokine-cytokine receptor interaction, and ECM-receptor interaction [ 16 ]. iTRAQ-based quantitative proteomic analysis revealed that APOV1, GAL, SAA, GNB5, VLDLR and CDK1 might be the key molecules involved in regulation of incubation behaviour in Muscovy ducks [ 17 ]. In Zhedong white geese ( Anser cygnoides ), 572 DEGs were identified in ovaries between LP and BP geese, but FSHβ, PRL and PRLR were not observed to be expressed differentially [ 18 ]. Further analysis indicated that these DEGs are mainly associated with reproduction regulation, such as steroid hormone biosynthesis, GnRH signaling pathway, calcium signaling pathway, Wnt signaling pathway and oocyte meiosis [ 18 ]. Meanwhile, transcriptomes of different types of follicles, including small white, large white and small yellow follicles, indicated that most of DEGs are involved in hormone response, autophagy, follicular development and oxidation [ 19 , 20 ]. Additionally, FOS, HSP90AA and CDK1 in ovaries are found to consolidate and transduce signals that regulate the hypothalamic-pituitary-gonadal axis (HPGA) during broodiness in Tianfu meat geese [ 21 ]. Focal adhesion, ECM-receptor interaction and N-Glycan biosynthesis were enriched significantly in the ovaries of Xupu geese at pre-laying period, laying period and incubating period. These results further indicated the complexity of reproductive behaviour of different avian breeds. Changshun green-shell laying hen is a Chinese indigenous chicken breed with strong broodiness. Due to its strong tendency for broodiness, Changshun hen has recently become an important model to study avian incubation behaviour [ 22 ]. In the present study, we comprehensively analyzed the ovarian transcriptomes of Changshun hens at LP and BP. Candidate genes and signal pathways related to chicken broodiness were identified through KEGG and GO enrichment analysis. The results will provide new insights into the ovarian regulation of incubation behaviour in avians. Materials and methods Ethics statement Animal experimental protocols employed in this study were performed in accordance with the guidelines formulated by the Ministry of Science and Technology of the People’s Republic of China. All animal experiments of this study were approved by the Animal Ethics Committee of the College of Biological Science and Agriculture, Qiannan Normal University for Nationalities (AEC No. QNUN2021014). Sample collection A total of 6 Changshun hens, including 3 incubating and 3 egg-laying hens, were were collected from the breeding farm of Qiannan Normal University for Nationalities. Changshun hens were euthanized with exsanguination under sodium pentobarbital anesthesia (60 mg/kg), and the ovarian samples were collected swiftly. The morphologic characteristics of the ovaries were used to further evaluate the physiological stage of Changshun hens. All the ovarian samples were frozen in liquid nitrogen, and then transferred to -80°C until analysis. RNA isolation and sequencing Total RNA was extracted from ovaries using the Trizol reagent (Life technologies, California, USA). The concentration and quality of the total RNA were determined using the NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and electrophoresis. The integrity of ovarian samples were assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The resulting libraries in this study were then sequenced on an Illumina NovaSeq6000 platform using 150 bp paired-end read mode. Principal component analysis and differential expression analysis Firstly, raw sequences were transformed into clean reads through discarding low-quality sequences and adaptor sequences. Subsequently, Hisat2 (v2.2.1) was applied to construct the reference genome index and align the clean data to the reference genome (Gallus_gallus.GRCg6a_release106.genome.fa) [ 23 ]. All transcripts from Hisat2 alignment results were detected by the method of the StringTie Reference Annotation Based Transcript (v2.2.1) [ 24 ]. The gene function was annotated with the Eukaryotic Orthologous Groups of proteins, Protein family, National Center for Biotechnology Information for non-redundant proteins, KEGG, GO and Swiss-Prot databases. The similarity between LP and BP groups was assessed by principal component analysis (PCA). DEGs between LP and BP groups were identified using DESeq2 package (v1.30.1) [ 25 ]. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. Fold change (FC) ≥ 1.5 and False Discovery Rate (FDR) < 0.01 were set as the criteria for DEGs. FDR and FC refer to adjusted P value and the ratio of gene expression in LP and BP samples. KEGG pathway, GO enrichment analysis and protein-protein interaction The key pathways of DEGs were determined by performing GO and KEGG analysis. The clusterProfiler package (v3.16.1) was used to test the statistical enrichment of DEGs in GO terms and KEGG pathways [ 26 , 27 ]. Cytoscape (v3.5.1) was used to perform the protein-protein interaction (PPI) analysis [ 28 ]. Circle and rectangle node represent a protein and a KEGG pathway, respectively. The solid and dashed line represent an interaction between two proteins and the edge of KEGG pathway in PPI network, respectively. Gene expression analysis by quantitative real-time PCR Four DEGs, including ANGPT2 , TEK , EGFR and PTEN , were selected randomly to further validate the results of RNA-seq by quantitative real-time PCR (qRT-PCR). The table S1 listed the primers which were designed with Primer 5. The relative mRNA expression levels were estimated using the 2 −ΔΔCt method and normalized using Beta-actin [ 29 ]. Data was analyzed using the Student’s t-test after testing for the homogeneity of variance with Levene’s test. All data are presented as the mean ± SD, and statistical significance was shown as * P < 0.05. Results RNA sequencing quality assessment and transcriptome alignment Total RNA was extracted from ovary of Changshun green-shell laying hens at LP and BP. Six cDNA sequencing libraries (three egg-laying period samples and three incubation period samples) were sequenced on the Illumina NovaSeq6000 platform. As shown in Table 1 , more than 19.2 × 10 6 clean reads per ovarian sample were generated after filtering. The base percentage of the Q20 and Q30 was above 98.34% and 94.87%, respectively, and the GC content of six ovarian samples ranged from 47.89–51.22%. More than 93.16% of clean reads were perfectly mapped to the reference genome of Gallus gallus to generate a read count value. The percentage of uniquely and multiple mapped reads in clean reads ranged from 90.60–93.84% and 1.81–2.43%, respectively. The results indicated that the transcriptome data were suitable for subsequent analysis. Table 1 Data quality and mapping data statistics of RNA-seq libraries Sample Clean reads Clean bases Q20 (%) Q30 (%) GC (%) Total mapped Uniquely mapped Multiple mapped LP1 22,097,938 6,611,280,448 98.46 95.39 49.5 41,172,905 (93.16%) 40,260,429 (91.10%) 912,476 (2.06%) LP2 19,277,102 5,768,363,046 98.54 95.51 48.36 36,607,544 (94.95%) 35,884,081 (93.07%) 723,463 (1.88%) LP3 23,428,349 7,006,988,490 98.41 95.25 49.60 43,906,095 (93.70%) 42,884,740 (91.52%) 1,021,355 (2.18%) BP1 20,509,236 6,134,357,836 98.45 95.56 51.22 38,159,435 (93.03%) 37,161,046 (90.60%) 998,389 (2.43%) BP2 21,578,186 6,454,693,384 98.34 94.87 47.94 41,280,264 (95.65%) 40,498,880 (93.84%) 781,384 (1.81%) BP3 20,424,383 6,112,551,828 98.46 95.21 47.89 38,963,103 (95.38%) 38,206,570 (93.53%) 756,533 (1.85%) LP, ovarian samples of egg-laying group; BP, ovarian samples of incubation group; Q20, sequencing error rates lower than 1%; Q30, sequencing error rates lower than 0.1%; GC, the percentage of G and C bases in clean data. Differentially expressed analysis Firstly, the ovarian samples were analyzed by performing PCA. As shown in Fig. 1 A, six ovarian samples were divided into two parts in PCA, indicating an obvious difference between the LP and BP groups. In accordance with the results of PCA, good sample repeatability were observed in ovarian transcriptome (Fig. 1 B), indicating the reliable and reasonable analysis of DEGs in the following study. The cutoff criteria were |log2(FC)| ≥ 1.5, P value < 0.01. Further analysis using DESeq2 package revealed that a total of 1650 DEGs were identified in chicken ovaries, including 429 up-regulated and 1221 down-regulated genes (Fig. 2 and Table S2 ). The hierarchical clustering analysis of DEGs showed that ovarian samples from the same group were clustered together, and heatmap was then used to visualize the expression patterns of genes in ovaries between LP and BP groups (Figure S1 ). KEGG pathway and GO enrichment analysis Subsequently, KEGG and GO enrichment analysis were performed to determine the biological functions and key pathways of DEGs identified in ovaries. Functional classification of DEGs using KEGG pathway enrichment analysis demonstrated that the ovarian DEGs were associated with 209 pathways. The top 20 significantly enriched KEGG pathways were listed in Fig. 3 . We found that focal adhesion, vascular smooth muscle contraction, MAPK signaling pathway, adrenergic signaling in cardiomyocytes, ECM-receptor interaction, Notch signaling pathway, apelin signaling pathway, FoxO signaling pathway, GnRH signaling pathway, and Wnt signaling pathway in ovaries may play important roles in broodiness process of Changshun hens. Notably, down-regulated vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathways were observed in incubating ovaries. GO enrichment analysis demonstrated that a total of 37 GO terms were enriched, including 12 molecular function terms, 3 cell component, and 22 biological process terms (Table S3 ). The top 10 significantly enriched GO terms were biomineralization, biological adhesion, immune system process, reproduction, reproductive process, small molecule sensor activity, transcription regulator activity, detoxification, molecular carrier activity and molecular function regulator (Fig. 4 ). Taken together, our results indicated that these GO terms and KEGG pathways might play the critical roles in chicken ovaries during the transition from egg-laying to incubation. Interaction network construction of DEGs Ultimately, a PPI network of ovarian DEGs was constructed and visualized by Cytoscape to further identify hub genes associated with broodiness. As shown in Fig. 5 , the PPI network contained 455 nodes and 2392 edges, and were mainly enriched into 10 important pathways including focal adhesion, vascular smooth muscle contraction, MAPK signaling pathway, adrenergic signaling in cardiomyocytes, ECM-receptor interaction, Notch signaling pathway, apelin signaling pathway, FoxO signaling pathway, GnRH signaling pathway, and Wnt signaling pathway. The top nine hub genes with the highest interaction node degrees in PPI network were EGFR , VEGFRKDRL , FLT1 , KDR , PDGFRA , TEK , KIT, MAPK11 and FGFR3 , implying their potential roles in the transition from egg-laying to incubation in chicken ovaries. Validation of DEGs by qRT-PCR Of the nine hub genes, we found that TEK and ANGPT2 were involved in angiogenesis. EGFR and PTEN were reported to be related to ovarian development. The mRNA expression levels of ANGPT2 , TEK , EGFR and PTEN were detected by qRT-PCR. The results of qRT-PCR showed that the expression levels of four mRNA selected were decreased significantly in BP group compared with those in LP group (Fig. 6 ). It is inspiring that the expression trends validated via qRT-PCR were consistent with our RNA-Seq results, confirming that the RNA-seq results were reliable. Discussion Broodiness is known to be a maternal behavior of poultry, and it is closely associated with HPGA. During the transition from egg-laying to incubation, the transcriptome changes of avian ovaries, however, need to be further explored. In this study, we analyzed the ovarian transcriptome of Changshun hens at LP and BP, and further identified the candidate genes and signal pathways related to broodiness through KEGG and GO enrichment analysis. This study not only provides novel insights into understanding of avian broodiness, but also contributes valuable information to improve Changshun chicken breeding with low nestability in the future. Considering the fact that avian ovary contains follicles at distinct developmental stages including cortical follicles, white follicles, yellow follicles, and preovulatory follicles, it is therefore deemed to be an ideal model to study the mechanisms of follicular development [ 30 , 31 ]. Broodiness in avians is generally accompanied by the atresia of follicles and the degradation of reproductive performance. In the present study, we identified several critical signaling pathways related with follicle development in chicken ovaries, including focal adhesion, MAPK signaling pathway, FOXO signaling pathway, and Wnt signaling. The transcriptome profile of geese revealed that ovarian development-related focal adhesion and ECM-receptor interaction were the top two pathways enriched with the most DEGs in incubating ovaries, indicating the potential central roles of these two pathways in the entire ovulation cycle [ 32 ]. Similarly, focal adhesion is found to play important roles in prehierarchal follicles of laying and incubating geese [ 33 ]. In chickens, focal adhesion is observed to be closely associated with egg production and the process of follicle selection [ 34 , 35 ]. These findings indicates an important role of focal adhesion in regulating avian ovarian function and egg production. MAPK signaling pathway is one of the most important pathways in ovaries associated with avian age at first egg and egg production [ 36 – 38 ]. MAPK signaling pathway is demonstrated to be involved in cell proliferation and progesterone secretion of granulosa cells from the prehierarchical follicles in chickens [ 39 – 41 ]. Additionally, it is suggested that the granulosa layer within chicken follicles remains undifferentiated and steroidogenically inactive due to the inhibitory actions of MAPK and/or protein kinase C signaling [ 42 ]. Wnt pathway is known to be an evolutionarily conserved signaling pathway. In humans, it has been shown to play a pivotal role during human follicle formation and follicle maintenance [ 43 ]. In domastic waterfowl, Wnt signaling pathway might be implicated in the follicular development [ 17 , 33 , 44 ]. It was considered to be one of the most important signaling pathways in regulating broodiness of Muscovy ducks [ 17 ]. In accordance to these findings, our results further indicated the important role of Wnt signaling pathway in chicken ovaries during broodiness. It was previously suggested that FOXO signaling pathway in ovaries had important functions in the regulation of broodiness in ducks [ 15 ]. In the present study, we identified 20 DEGs, including PRKAB2 , IGF1 and PTEN , classified into FOXO signaling pathway. PRKAB2 gene is deemed to be associated with live-weight, carcass-weight, leg-muscle-weight and abdomen-fat-weight [ 45 , 46 ]. IGF1 and PTEN has been reported to be potential key genes that regulate ovulation of ducks [ 47 ]. In chickens, IGF1 as an autocrine/paracrine regulator of follicular growth and differentiation is involved in the regulation of follicular development [ 48 – 50 ]. Meanwhile, it stimulates the release of progesterone and affects egg production of chickens [ 51 ]. PTEN is the phosphatase of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which regulates cell proliferation cycles and inhibits cell migration. PTEN is observed to involve in the ovarian function remodeling of laying hens [ 52 ]. We found that both IGF1 and PTEN were down-regulated in incubating ovaries in the present study, indicating that they might have critical roles to play in the regulation of ovarian function in incubating chickens. Vascular smooth muscle contraction, ECM-receptor interaction and GnRH signaling pathways are classical signaling pathways associated with follicle development. Notably, vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathways in the present study were all down-regulated in the ovaries of incubating chickens. Development of ovarian follicles generally requires the coordinated interactions between theca cells, oocytes and granulosa cells. Transcriptome analysis of circRNA and mRNA in theca cells from different types of follicles reveals the important roles of vascular smooth muscle contraction in follicular development in chickens [ 53 ]. In Taihe black-bone silky fowls, vascular smooth muscle contraction is deemed as a critical signaling pathway that would affect ovarian development at different egg-laying stages, and ECM-receptor interaction in ovaries might be essential for the transition from laying to broodiness [ 16 , 54 ]. In pigeons, ECM-receptor interaction and vascular smooth muscle contraction were reported to be closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles [ 55 ]. In geese, vascular smooth muscle contraction was involved in follicular development from F4 to F1 stage, and ECM-receptor interaction was significantly enriched in ovaries from the pre-laying period to the broody period [ 32 , 56 ]. GnRH signaling pathway is known to involve in ovarian function of chickens [ 57 ]. In this study, a total of 14 down-regulated DEGs in this study, including CACNA1C , GNAQ , ADCY5 , and MMP2 , were mapped to GnRH signaling pathway. GNAQ and CACNA1C has been found to be associated with the development of ovarian follicles and onset of the reproductive maturation in animals [ 58 , 59 ]. In chickens, the expression of GNAQ and CACNA1C were demonstrated to be regulated by MSTRG.19756.2, a novel lncRNA, in trans, and then they acted upstream factors of GnRH signaling pathway to affect the downstream genes involved in ovarian development [ 60 ]. ADCY5 as a member of the adenylatecyclases family is reported to be responsible for egg production in Muscovy ducks [ 37 ]. Furthermore, ADCY5 has been identified to be closely associated with ovarian morphological related traits of animals [ 61 ]. MMP2 is reported to be associated with ovary development of chickens [ 62 ]. It was reported that there was lower expression of MMP2 in the atrophy of chicken ovaries [ 62 ]. It is now generally recognized that the control of avian ovarian development involves pituitary gonadotropins and various local paracrine and autocrine growth factors such as epidermal growth factor (EGF). In the present study, EGFR, the receptor of EGF, was observed to decrease significantly in ovaries from incubating Changshun hens. Similarly, previous study revealed the dynamic expressions of EGFR in chicken ovarian follicles [ 63 ]. Transcriptomic analysis reveals that EGFR is abundantly but differentially expressed in granulosa cells proximal and distal to the germinal disc of chicken preovulatory follicles [ 64 ]. EGFR has been shown to be expressed highly in granulosa cells from the prehierarchical follicles, and thereafter its expression is found to decrease markedly to the stage of the largest preovulatory follicles [ 63 , 65 ]. EGF functions as a ligand of EGFR to stimulate the proliferation of chicken granulosa cells [ 31 , 63 , 66 ]. However, the in vitro experiments have shown that EGF or FSH-induced proliferation of granulosa cell can be reversed by EGFR inhibitor AG1478 [ 63 ]. Similarly, AG1478 can also inhibit significantly EGF or FSH-reduced apoptosis of granulosa cells [ 63 ]. These results indicate the important roles of EGFR in chicken reproduction and broodiness. Growth and maturation of ovarian follicles in chickens require a complex network of blood vessels. Compared to prehierarchical follicles which have limited number and size of blood vessels, the large yellow follicles and preovulatory follicles are highly vascularized [ 49 ]. The vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin and their receptors are involved in angiogenesis. VEGF is a key regulator of physiological angiogenesis as it can facilitate blood vessel growth and remodeling processes. The network of ovarian blood vessels is closely associated with VEGF. VEGF exerts biological effects by binding to its tyrosine kinase family receptor FLT1, KDR, FLT4 and KDRL (also known as VEGFRKDRL) [ 67 , 68 ]. Interestingly, VEGF seemly binds to its receptor FLT1 and KDR with high affinity [ 69 ]. KDR is mainly expressed in the vascular endothelium of the theca layer in chicken follicles. KDR might be the most important receptor which is involved in VEGF-induced angiogenesis [ 70 – 72 ]. FLT1 can regulate the VEGF activity via interacting with VEGF and making it less available to KDR [ 73 ]. Decreased expression of VEGF, FLT1 and KDR might be related to follicle atresia in chickens [ 49 ]. Consistently, results from the present study showed that there was a significant decrease in expression of VEGF, including VEGFA and VEGFD, and its receptors, FLT1, KDR and VEGFRKDRL, in ovaries from incubating chickens, indicating the important roles of VEGF and its receptors in chicken broodiness. Simultaneously, our results showed that there was a significant decrease of PDGF and its receptor (PDGFR) in ovaries of incubating chickens compared with egg-laying hens. There are four PDGF, including PDGFA, PDGFB, PDGFC and PDGFD, and two receptors, including PDGFRA and PDGFRB, identified in vertebrates [ 74 , 75 ]. The importance of PDGFRA has been confirmed by studies that showed the presence of PDGFRA in follicular cells in the ovaries of different species. In humans, PDGFRA is widely expressed in oocytes, theca cells and ovarian stroma cells [ 76 ]. In rats, PDGFRA is also identified in oocytes and granulosa cells [ 77 ]. The expression pattern of PDGFRA indicated that it might be involved in the ovarian folliculogenesis, selection and growth initiation of follicles and the formation of thecal layer [ 78 , 79 ]. Meanwhile, PDGFRA is reported to be a requirement in steroid-producing cells in mice ovaries, and is involved in the steroidogenesis through regulating the downstream target gene Sgpl1 , Plekha1 , Tiparp , Schip1 , and BC058969 [ 80 ]. Additionally, signaling downstream of PDGFRA has been reported to induce both apoptotic and antiapoptotic responses [ 81 , 82 ]. The identification of PDGFRA but not PDGFRB in ovaries from incubating chickens in the present study further suggests that PDGFRA may be important for ovarian development during chicken broodiness. TEK is a tyrosine kinase receptor and can bind with angiopoietins. The angiopoietin 1 (ANGPT1), angiopoietin 2 (ANGPT2), and TEK receptor tyrosine kinase are mainly expressed in ovaries. In this study, we found TEK and angiopoietins were down-regulated in ovaries from incubating Changshun chickens. The angiopoietin-TEK system is observed to play a crucial role in blood vessel formation and stability, follicular development and atresia [ 83 , 84 ]. The ANGPT1 and ANGPT2 can bind to TEK, inducing opposite effects. ANGPT1 elicits an activation of TEK by increased tyrosine phosphorylation of TEK when they bind. ANGPT2 as a natural antagonist acts to inhibit the activation of TEK and disrupts ANGPT1-dependent TEK-mediated angiogenesis [ 84 ]. ANGPT2 is reported to destabilize existing vessels, loosening the supporting cell matrix to allow angiogenic factors such as VEGF to stimulate cell proliferation and migration during early angiogenesis [ 85 ]. Additionally, TEK is hypothesized to play an important role in folliculogenesis [ 86 ]. Functionality of the mammalian Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor (KIT), has been shown that they have multiple roles during oogenesis, folliculogenesis, and melanogenesis [ 87 , 88 ]. The ovarian expression pattern revealed that mammalian KIT is mainly expressed in theca cells, oocytes and follicular fluid [ 89 – 91 ]. Similarly, the chicken KIT is observed to express in very small follicles (< 1 mm), theca cell layer and the ovarian stroma, indicating that Kit system might promote the transition from quiescence to slow growing follicles in chickens [ 92 ]. Furthermore, KIT is reported to be detected in many other chicken organs including the testis, brain, bursa, spleen, thymus, heart and kidney, indicating that Kit system might be implicated in a variety of non-ovarian functions [ 93 ]. Fibroblast growth factor receptor 3 (FGFR3) is known to be a receptor for fibroblast growth factors (FGF). In mammals, FGFR3 is identified in oocytes, granulosa cells, theca cells and stromal cells [ 94 , 95 ]. Additionally, FGFR3 is expressed by human primordial germ cells during the first and second trimester, and is then repressed after meiotic initiation to form primordial oocytes [ 96 ]. Study from buffalos revealed that FGFR3 is expressed widely in ovarian follicles during different stages of development [ 97 ]. FGFs and their receptors are reported to be involved in multiple biologic processes in angiogenesis, hematopoiesis, wound healing and even embryonic development [ 98 ]. Studies based on transgenic mice models showed that mutant FGFR3 leads to dwarfism and infertility [ 99 ]. In chickens, FGFR3 is identified to be a candidate gonadal sex differentiation gene in embryo [ 100 ]. Decreased FGFR3 in incubating hens in the present study further supports the assumption that FGFR3 might play critical roles in chicken reproduction and broodiness. In conclusion, we characterized and evaluated the ovarian transcriptome in LP and BP Changshun hens. The results suggest that focal adhesion, MAPK signaling pathway, FoxO signaling pathway, vascular smooth muscle contraction, ECM-receptor interaction and GnRH signaling pathway are critical for incubation behaviour in Changshun hens. Eight candidate genes ( EGFR , VEGFRKDRL , FLT1 , KDR , PDGFRA , TEK , KIT and FGFR3 ) in ovaries might play crucial roles in chicken broodiness. Our results provide a theoretical basis for further exploration of the molecular mechanism of broodiness in chickens. Abbreviations Egg-laying period: LP; Incubation period: BP; RNA sequencing: RNA-seq; Differently expressed genes: DEGs; Prolactin: PRL; Gonadotropin-releasing hormone: GnRH; Vasoactive intestinal polypeptide: VIP; Dopamine: DA; 5-hydroxytryptophan: 5-HTP; Hypothalamic-pituitary-gonadal axis: HPGA; Principal component analysis: PCA; False discovery rate: FDR; Protein-protein interaction: PPI; Quantitative real-time PCR: qRT-PCR. Declarations Acknowledgments We thank Biomarker Technologies Co., Ltd for assisting in sequencing and bioinformatics analysis. Authors’contributions ZC designed this study, ZC, DW and RM performed the transcriptome analysis. ZC wrote the manuscript. All authors approved this manuscript. Funding This study was supported by Guizhou Provincial Basic Research Program (Natural Science) (ZK[2021]167, ZK[2023]455); Natural Science Research Project of the Department of Education of Guizhou Province of China (KY[2020]071, Innovation team No.[2023]089). Availability of data and materials The raw sequence data in the present study have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA016819) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa. 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Supplementary Files FigureS1.jpg TableS1.PrimersusedforqRTPCR..docx TableS2.ListofalltheDEGsdetectedinovaries.xlsx TableS3.ListofGOenrichmentanalysisofDEGs..docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4540221","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317072863,"identity":"3e97e838-0c76-4185-8a84-c525c708ee4e","order_by":0,"name":"Zhi Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACefbmgw8kKv7LsbE3EKnFsOdYsoHFGWZjfp4DxFpzI8dMorKFOXHmjAQidTA2pCVI3GxgMza4+XjjDYYam2iCWtgZDh8wnLmDR87gdlqxBcOxtNwGgrY0tiUkS56RMDa4DXQhY8NhwloYDvMYHP7bZpC44eYZYrUc4zFskGxLAHqfh0gthj1syQwSZw4AAxnolwRi/CIv//j4D4mKA8CoPLzxxocaGyIchgQMJBJIUQ7RQqqOUTAKRsEoGBkAAIRxQyDQA1fMAAAAAElFTkSuQmCC","orcid":"","institution":"Qiannan Normal University for Nationalities","correspondingAuthor":true,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Chen","suffix":""},{"id":317072864,"identity":"99f01697-3ea6-470c-81e1-e97311875a98","order_by":1,"name":"Di Wen","email":"","orcid":"","institution":"Qiannan Normal University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Wen","suffix":""},{"id":317072865,"identity":"cc9d2d24-d661-4744-a8a3-0f3a2aae3a9f","order_by":2,"name":"Ren Mu","email":"","orcid":"","institution":"Qiannan Normal University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Mu","suffix":""}],"badges":[],"createdAt":"2024-06-06 12:06:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4540221/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4540221/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58803700,"identity":"482e6dc5-4495-4e47-8626-bae93f26bac4","added_by":"auto","created_at":"2024-06-21 10:19:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165375,"visible":true,"origin":"","legend":"\u003cp\u003eFeatures of sequencing data. (A) PCA score plot of ovarian transcriptomes. Red and green nodes represent individuals from BP and LP, respectively. (B) Pearson correlation analysis of LP and BP groups. LP1, LP2, LP3 and LP4 are ovariansamples from egg-laying hens, and BP1, BP2, BP3 and BP4 are ovarian samples from incubation hens.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/e7e96afd4dbce51124811a8d.png"},{"id":58803701,"identity":"479285ae-20b0-45ff-8dcf-20dfce51e231","added_by":"auto","created_at":"2024-06-21 10:19:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212437,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano map of all expressed genes. The horizontal and longitudinal coordinates represent the fold changes of genes and the statistical significance of the changes in gene expression, respectively. Blue plots represent significantly down-regulated genes (|log2(FC)| ≥ 1.5, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.01), and black plots are genes without significant difference.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/fd0188a3a9896511d873f4ae.png"},{"id":58803328,"identity":"98b6b3a2-bc11-47f1-92f2-8f511bd47598","added_by":"auto","created_at":"2024-06-21 10:11:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":294683,"visible":true,"origin":"","legend":"\u003cp\u003eTop 20 KEGG pathways enriched by the ovarian DEGs. The longitudinal and horizontal coordinates represent the name of pathway and the pathway corresponding rich factor, respectively. Each bubble represents a KEGG pathway. Bubble size indicates the number of DEGs in each pathway, and the colour corresponds to the q value of each pathway.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/5ed315d3fedc54d87a99cdfe.png"},{"id":58804173,"identity":"0eab59ba-4852-4cac-989d-4968d74e92a5","added_by":"auto","created_at":"2024-06-21 10:27:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500405,"visible":true,"origin":"","legend":"\u003cp\u003eGO classification of ovarian DEGs. The longitudinal and horizontal coordinates represent the GO term and the number of DEGs annotated to the term, respectively. Green, orange and purple indicate the molecular function, cellular component and biological process, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/84b97671381b2a4e11965cfb.png"},{"id":58803333,"identity":"3f7b6a28-c319-498e-8ab8-e322a5a78911","added_by":"auto","created_at":"2024-06-21 10:11:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":515528,"visible":true,"origin":"","legend":"\u003cp\u003ePPI networks of ovarian DEGs. Red and green circle nodes indicate down-regulated and up-regulated DEGs, respectively. The size of node represents the expression of DEGs. Blue rectangles indicate the KEGG pathway. Interactions were shown as solid lines between proteins, and edges of KEGG pathway in dashed lines.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/92cd939c74d579209b985781.png"},{"id":58803703,"identity":"ceb1df6f-c333-4e1f-b125-59b8d1e1798b","added_by":"auto","created_at":"2024-06-21 10:19:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100216,"visible":true,"origin":"","legend":"\u003cp\u003eqRT-PCR validation of ovarian DEGs. The results were expressed as mean ± SD. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. LP, egg-laying group; BP, incubation group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/888741af99a8155d5dd503aa.png"},{"id":58946975,"identity":"1421c58e-b57c-4322-9e8c-b7469dfe6acd","added_by":"auto","created_at":"2024-06-24 12:59:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2398817,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/12d058da-6d7c-47a1-8ae1-9b826a45f9e7.pdf"},{"id":58803330,"identity":"c07646b4-9458-4a80-8cea-ad4fbfdc4342","added_by":"auto","created_at":"2024-06-21 10:11:24","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":214050,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/c21e5eddea9e47652ad0c9e9.jpg"},{"id":58803325,"identity":"d97623e0-ce90-46fb-b604-db33c4f4766c","added_by":"auto","created_at":"2024-06-21 10:11:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12087,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.PrimersusedforqRTPCR..docx","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/d01b2685ce3f0a5c50a237ce.docx"},{"id":58803705,"identity":"09fa5d65-ca72-4bc7-9ac1-9ec9790dfc5a","added_by":"auto","created_at":"2024-06-21 10:19:24","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":402408,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.ListofalltheDEGsdetectedinovaries.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/bcbc55d6222235645bbe7e41.xlsx"},{"id":58804174,"identity":"f7089ad7-0af3-4881-946b-64c40b1d6e34","added_by":"auto","created_at":"2024-06-21 10:27:24","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15363,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.ListofGOenrichmentanalysisofDEGs..docx","url":"https://assets-eu.researchsquare.com/files/rs-4540221/v1/b726fb8a978aeb13ff824be7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ovarian transcriptome profile from egg-laying period to incubation period of Changshun green-shell laying hens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIncubation behaviour is a common maternal behaviour of domestic chickens. Different from egg-laying chickens, the ovary and oviduct of incubating chicken are degenerate, resulting in the cessation of egg-laying. The molecular regulation mechanism of avian broodiness remains unclear. It is generally known that the onset and maintain of incubation behaviour in chicken is mainly associated with multiple neuroendocrine hormones and neurotransmitters [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A characteristic increase of pituitaric prolactin (PRL) in plasma is critical for the onset and maintain of chicken broodiness [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Meanwhile, luteinizing hormone (LH), hypothalamic gonadotropin-releasing hormone (GnRH), neuropeptide vasoactive intestinal polypeptide (VIP), neurotransmitter dopamine (DA) 5-hydroxytryptophan (5-HTP) and dynorphin are also reported to be involved in chicken broodiness [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the past decades, high-throughput sequencing technology including RNA sequencing (RNA-Seq) has been widely used to explore the transcriptome profile of avian ovaries from egg-laying period (LP) to incubation period (BP). Although increasing number of candidate genes and signaling pathways in ovaries are reported to be involved in avian broodiness, there has been no specific genes and pathways elucidated which has marked effects on avian incubation behaviour. Compared to chickens, geese and ducks with high tendency to broodiness are widely used to investigate avian broodiness. In incubating Muscovy ducks (\u003cem\u003eCairina moschata\u003c/em\u003e), a total of 334 differential expression genes (DEGs) and 36 differentially abundant long noncoding RNAs (lncRNAs) transcripts were identified in ovaries, and focal adhesion, FOXO signaling pathway, Wnt signaling, oocyte meiosis and cytokine-cytokine receptor interaction pathways in ovaries might involve in the transition from egg-laying to incubation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, different expression mRNAs and the target genes of different expression lncRNAs in the ovaries of Taihe Black-Bone Silky (\u003cem\u003eGallus gallus Domesticus Brisson\u003c/em\u003e) are mainly associated with neuroactive ligand-receptor interaction, CCR6 chemokine receptor binding, G-protein coupled receptor binding, cytokine-cytokine receptor interaction, and ECM-receptor interaction [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. iTRAQ-based quantitative proteomic analysis revealed that APOV1, GAL, SAA, GNB5, VLDLR and CDK1 might be the key molecules involved in regulation of incubation behaviour in Muscovy ducks [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In Zhedong white geese (\u003cem\u003eAnser cygnoides\u003c/em\u003e), 572 DEGs were identified in ovaries between LP and BP geese, but FSHβ, PRL and PRLR were not observed to be expressed differentially [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Further analysis indicated that these DEGs are mainly associated with reproduction regulation, such as steroid hormone biosynthesis, GnRH signaling pathway, calcium signaling pathway, Wnt signaling pathway and oocyte meiosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Meanwhile, transcriptomes of different types of follicles, including small white, large white and small yellow follicles, indicated that most of DEGs are involved in hormone response, autophagy, follicular development and oxidation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, FOS, HSP90AA and CDK1 in ovaries are found to consolidate and transduce signals that regulate the hypothalamic-pituitary-gonadal axis (HPGA) during broodiness in Tianfu meat geese [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Focal adhesion, ECM-receptor interaction and N-Glycan biosynthesis were enriched significantly in the ovaries of Xupu geese at pre-laying period, laying period and incubating period. These results further indicated the complexity of reproductive behaviour of different avian breeds.\u003c/p\u003e \u003cp\u003eChangshun green-shell laying hen is a Chinese indigenous chicken breed with strong broodiness. Due to its strong tendency for broodiness, Changshun hen has recently become an important model to study avian incubation behaviour [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, we comprehensively analyzed the ovarian transcriptomes of Changshun hens at LP and BP. Candidate genes and signal pathways related to chicken broodiness were identified through KEGG and GO enrichment analysis. The results will provide new insights into the ovarian regulation of incubation behaviour in avians.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e Animal experimental protocols employed in this study were performed in accordance with the guidelines formulated by the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China. All animal experiments of this study were approved by the Animal Ethics Committee of the College of Biological Science and Agriculture, Qiannan Normal University for Nationalities (AEC No. QNUN2021014).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eA total of 6 Changshun hens, including 3 incubating and 3 egg-laying hens, were were collected from the breeding farm of Qiannan Normal University for Nationalities. Changshun hens were euthanized with exsanguination under sodium pentobarbital anesthesia (60 mg/kg), and the ovarian samples were collected swiftly. The morphologic characteristics of the ovaries were used to further evaluate the physiological stage of Changshun hens. All the ovarian samples were frozen in liquid nitrogen, and then transferred to -80\u0026deg;C until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from ovaries using the Trizol reagent (Life technologies, California, USA). The concentration and quality of the total RNA were determined using the NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and electrophoresis. The integrity of ovarian samples were assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The resulting libraries in this study were then sequenced on an Illumina NovaSeq6000 platform using 150 bp paired-end read mode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis and differential expression analysis\u003c/h2\u003e \u003cp\u003eFirstly, raw sequences were transformed into clean reads through discarding low-quality sequences and adaptor sequences. Subsequently, Hisat2 (v2.2.1) was applied to construct the reference genome index and align the clean data to the reference genome (Gallus_gallus.GRCg6a_release106.genome.fa) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. All transcripts from Hisat2 alignment results were detected by the method of the StringTie Reference Annotation Based Transcript (v2.2.1) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The gene function was annotated with the Eukaryotic Orthologous Groups of proteins, Protein family, National Center for Biotechnology Information for non-redundant proteins, KEGG, GO and Swiss-Prot databases. The similarity between LP and BP groups was assessed by principal component analysis (PCA). DEGs between LP and BP groups were identified using DESeq2 package (v1.30.1) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. Fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;1.5 and False Discovery Rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were set as the criteria for DEGs. FDR and FC refer to adjusted \u003cem\u003eP\u003c/em\u003e value and the ratio of gene expression in LP and BP samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eKEGG pathway, GO enrichment analysis and protein-protein interaction\u003c/h2\u003e \u003cp\u003eThe key pathways of DEGs were determined by performing GO and KEGG analysis. The clusterProfiler package (v3.16.1) was used to test the statistical enrichment of DEGs in GO terms and KEGG pathways [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Cytoscape (v3.5.1) was used to perform the protein-protein interaction (PPI) analysis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Circle and rectangle node represent a protein and a KEGG pathway, respectively. The solid and dashed line represent an interaction between two proteins and the edge of KEGG pathway in PPI network, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis by quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eFour DEGs, including \u003cem\u003eANGPT2\u003c/em\u003e, \u003cem\u003eTEK\u003c/em\u003e, \u003cem\u003eEGFR\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e, were selected randomly to further validate the results of RNA-seq by quantitative real-time PCR (qRT-PCR). The table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e listed the primers which were designed with Primer 5. The relative mRNA expression levels were estimated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method and normalized using \u003cem\u003eBeta-actin\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Data was analyzed using the Student\u0026rsquo;s t-test after testing for the homogeneity of variance with Levene\u0026rsquo;s test. All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and statistical significance was shown as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing quality assessment and transcriptome alignment\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from ovary of Changshun green-shell laying hens at LP and BP. Six cDNA sequencing libraries (three egg-laying period samples and three incubation period samples) were sequenced on the Illumina NovaSeq6000 platform. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, more than 19.2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e clean reads per ovarian sample were generated after filtering. The base percentage of the Q20 and Q30 was above 98.34% and 94.87%, respectively, and the GC content of six ovarian samples ranged from 47.89\u0026ndash;51.22%. More than 93.16% of clean reads were perfectly mapped to the reference genome of \u003cem\u003eGallus gallus\u003c/em\u003e to generate a read count value. The percentage of uniquely and multiple mapped reads in clean reads ranged from 90.60\u0026ndash;93.84% and 1.81\u0026ndash;2.43%, respectively. The results indicated that the transcriptome data were suitable for subsequent analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eData quality and mapping data statistics of RNA-seq libraries\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClean reads\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClean bases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ20 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQ30 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal mapped\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUniquely mapped\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMultiple mapped\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22,097,938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,611,280,448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e41,172,905 (93.16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40,260,429 (91.10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e912,476 (2.06%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19,277,102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5,768,363,046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36,607,544 (94.95%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e35,884,081 (93.07%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e723,463 (1.88%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23,428,349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7,006,988,490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e43,906,095 (93.70%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e42,884,740 (91.52%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1,021,355 (2.18%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20,509,236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,134,357,836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38,159,435 (93.03%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e37,161,046 (90.60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e998,389 (2.43%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21,578,186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,454,693,384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e41,280,264 (95.65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40,498,880 (93.84%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e781,384 (1.81%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20,424,383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6,112,551,828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38,963,103 (95.38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e38,206,570 (93.53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e756,533 (1.85%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLP, ovarian samples of egg-laying group; BP, ovarian samples of incubation group; Q20, sequencing error rates lower than 1%; Q30, sequencing error rates lower than 0.1%; GC, the percentage of G and C bases in clean data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferentially expressed analysis\u003c/h2\u003e \u003cp\u003eFirstly, the ovarian samples were analyzed by performing PCA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, six ovarian samples were divided into two parts in PCA, indicating an obvious difference between the LP and BP groups. In accordance with the results of PCA, good sample repeatability were observed in ovarian transcriptome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating the reliable and reasonable analysis of DEGs in the following study. The cutoff criteria were |log2(FC)| \u0026ge; 1.5, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Further analysis using DESeq2 package revealed that a total of 1650 DEGs were identified in chicken ovaries, including 429 up-regulated and 1221 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The hierarchical clustering analysis of DEGs showed that ovarian samples from the same group were clustered together, and heatmap was then used to visualize the expression patterns of genes in ovaries between LP and BP groups (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eKEGG pathway and GO enrichment analysis\u003c/h2\u003e \u003cp\u003eSubsequently, KEGG and GO enrichment analysis were performed to determine the biological functions and key pathways of DEGs identified in ovaries. Functional classification of DEGs using KEGG pathway enrichment analysis demonstrated that the ovarian DEGs were associated with 209 pathways. The top 20 significantly enriched KEGG pathways were listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. We found that focal adhesion, vascular smooth muscle contraction, MAPK signaling pathway, adrenergic signaling in cardiomyocytes, ECM-receptor interaction, Notch signaling pathway, apelin signaling pathway, FoxO signaling pathway, GnRH signaling pathway, and Wnt signaling pathway in ovaries may play important roles in broodiness process of Changshun hens. Notably, down-regulated vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathways were observed in incubating ovaries. GO enrichment analysis demonstrated that a total of 37 GO terms were enriched, including 12 molecular function terms, 3 cell component, and 22 biological process terms (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The top 10 significantly enriched GO terms were biomineralization, biological adhesion, immune system process, reproduction, reproductive process, small molecule sensor activity, transcription regulator activity, detoxification, molecular carrier activity and molecular function regulator (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Taken together, our results indicated that these GO terms and KEGG pathways might play the critical roles in chicken ovaries during the transition from egg-laying to incubation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInteraction network construction of DEGs\u003c/h2\u003e \u003cp\u003eUltimately, a PPI network of ovarian DEGs was constructed and visualized by Cytoscape to further identify hub genes associated with broodiness. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the PPI network contained 455 nodes and 2392 edges, and were mainly enriched into 10 important pathways including focal adhesion, vascular smooth muscle contraction, MAPK signaling pathway, adrenergic signaling in cardiomyocytes, ECM-receptor interaction, Notch signaling pathway, apelin signaling pathway, FoxO signaling pathway, GnRH signaling pathway, and Wnt signaling pathway. The top nine hub genes with the highest interaction node degrees in PPI network were \u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eVEGFRKDRL\u003c/em\u003e, \u003cem\u003eFLT1\u003c/em\u003e, \u003cem\u003eKDR\u003c/em\u003e, \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eTEK\u003c/em\u003e, \u003cem\u003eKIT, MAPK11\u003c/em\u003e and \u003cem\u003eFGFR3\u003c/em\u003e, implying their potential roles in the transition from egg-laying to incubation in chicken ovaries.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eValidation of DEGs by qRT-PCR\u003c/h2\u003e \u003cp\u003eOf the nine hub genes, we found that \u003cem\u003eTEK\u003c/em\u003e and \u003cem\u003eANGPT2\u003c/em\u003e were involved in angiogenesis. \u003cem\u003eEGFR\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e were reported to be related to ovarian development. The mRNA expression levels of \u003cem\u003eANGPT2\u003c/em\u003e, \u003cem\u003eTEK\u003c/em\u003e, \u003cem\u003eEGFR\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e were detected by qRT-PCR. The results of qRT-PCR showed that the expression levels of four mRNA selected were decreased significantly in BP group compared with those in LP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It is inspiring that the expression trends validated via qRT-PCR were consistent with our RNA-Seq results, confirming that the RNA-seq results were reliable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBroodiness is known to be a maternal behavior of poultry, and it is closely associated with HPGA. During the transition from egg-laying to incubation, the transcriptome changes of avian ovaries, however, need to be further explored. In this study, we analyzed the ovarian transcriptome of Changshun hens at LP and BP, and further identified the candidate genes and signal pathways related to broodiness through KEGG and GO enrichment analysis. This study not only provides novel insights into understanding of avian broodiness, but also contributes valuable information to improve Changshun chicken breeding with low nestability in the future.\u003c/p\u003e \u003cp\u003eConsidering the fact that avian ovary contains follicles at distinct developmental stages including cortical follicles, white follicles, yellow follicles, and preovulatory follicles, it is therefore deemed to be an ideal model to study the mechanisms of follicular development [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Broodiness in avians is generally accompanied by the atresia of follicles and the degradation of reproductive performance. In the present study, we identified several critical signaling pathways related with follicle development in chicken ovaries, including focal adhesion, MAPK signaling pathway, FOXO signaling pathway, and Wnt signaling. The transcriptome profile of geese revealed that ovarian development-related focal adhesion and ECM-receptor interaction were the top two pathways enriched with the most DEGs in incubating ovaries, indicating the potential central roles of these two pathways in the entire ovulation cycle [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, focal adhesion is found to play important roles in prehierarchal follicles of laying and incubating geese [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In chickens, focal adhesion is observed to be closely associated with egg production and the process of follicle selection [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These findings indicates an important role of focal adhesion in regulating avian ovarian function and egg production. MAPK signaling pathway is one of the most important pathways in ovaries associated with avian age at first egg and egg production [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. MAPK signaling pathway is demonstrated to be involved in cell proliferation and progesterone secretion of granulosa cells from the prehierarchical follicles in chickens [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, it is suggested that the granulosa layer within chicken follicles remains undifferentiated and steroidogenically inactive due to the inhibitory actions of MAPK and/or protein kinase C signaling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Wnt pathway is known to be an evolutionarily conserved signaling pathway. In humans, it has been shown to play a pivotal role during human follicle formation and follicle maintenance [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In domastic waterfowl, Wnt signaling pathway might be implicated in the follicular development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It was considered to be one of the most important signaling pathways in regulating broodiness of Muscovy ducks [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In accordance to these findings, our results further indicated the important role of Wnt signaling pathway in chicken ovaries during broodiness. It was previously suggested that FOXO signaling pathway in ovaries had important functions in the regulation of broodiness in ducks [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the present study, we identified 20 DEGs, including \u003cem\u003ePRKAB2\u003c/em\u003e, \u003cem\u003eIGF1\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e, classified into FOXO signaling pathway. \u003cem\u003ePRKAB2\u003c/em\u003e gene is deemed to be associated with live-weight, carcass-weight, leg-muscle-weight and abdomen-fat-weight [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. \u003cem\u003eIGF1\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e has been reported to be potential key genes that regulate ovulation of ducks [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In chickens, \u003cem\u003eIGF1\u003c/em\u003e as an autocrine/paracrine regulator of follicular growth and differentiation is involved in the regulation of follicular development [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Meanwhile, it stimulates the release of progesterone and affects egg production of chickens [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. PTEN is the phosphatase of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which regulates cell proliferation cycles and inhibits cell migration. PTEN is observed to involve in the ovarian function remodeling of laying hens [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We found that both \u003cem\u003eIGF1\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e were down-regulated in incubating ovaries in the present study, indicating that they might have critical roles to play in the regulation of ovarian function in incubating chickens.\u003c/p\u003e \u003cp\u003eVascular smooth muscle contraction, ECM-receptor interaction and GnRH signaling pathways are classical signaling pathways associated with follicle development. Notably, vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathways in the present study were all down-regulated in the ovaries of incubating chickens. Development of ovarian follicles generally requires the coordinated interactions between theca cells, oocytes and granulosa cells. Transcriptome analysis of circRNA and mRNA in theca cells from different types of follicles reveals the important roles of vascular smooth muscle contraction in follicular development in chickens [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In Taihe black-bone silky fowls, vascular smooth muscle contraction is deemed as a critical signaling pathway that would affect ovarian development at different egg-laying stages, and ECM-receptor interaction in ovaries might be essential for the transition from laying to broodiness [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In pigeons, ECM-receptor interaction and vascular smooth muscle contraction were reported to be closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In geese, vascular smooth muscle contraction was involved in follicular development from F4 to F1 stage, and ECM-receptor interaction was significantly enriched in ovaries from the pre-laying period to the broody period [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. GnRH signaling pathway is known to involve in ovarian function of chickens [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this study, a total of 14 down-regulated DEGs in this study, including \u003cem\u003eCACNA1C\u003c/em\u003e, \u003cem\u003eGNAQ\u003c/em\u003e, \u003cem\u003eADCY5\u003c/em\u003e, and \u003cem\u003eMMP2\u003c/em\u003e, were mapped to GnRH signaling pathway. GNAQ and CACNA1C has been found to be associated with the development of ovarian follicles and onset of the reproductive maturation in animals [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In chickens, the expression of GNAQ and CACNA1C were demonstrated to be regulated by MSTRG.19756.2, a novel lncRNA, in trans, and then they acted upstream factors of GnRH signaling pathway to affect the downstream genes involved in ovarian development [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. ADCY5 as a member of the adenylatecyclases family is reported to be responsible for egg production in Muscovy ducks [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, ADCY5 has been identified to be closely associated with ovarian morphological related traits of animals [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. MMP2 is reported to be associated with ovary development of chickens [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. It was reported that there was lower expression of MMP2 in the atrophy of chicken ovaries [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is now generally recognized that the control of avian ovarian development involves pituitary gonadotropins and various local paracrine and autocrine growth factors such as epidermal growth factor (EGF). In the present study, EGFR, the receptor of EGF, was observed to decrease significantly in ovaries from incubating Changshun hens. Similarly, previous study revealed the dynamic expressions of EGFR in chicken ovarian follicles [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Transcriptomic analysis reveals that EGFR is abundantly but differentially expressed in granulosa cells proximal and distal to the germinal disc of chicken preovulatory follicles [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. EGFR has been shown to be expressed highly in granulosa cells from the prehierarchical follicles, and thereafter its expression is found to decrease markedly to the stage of the largest preovulatory follicles [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. EGF functions as a ligand of EGFR to stimulate the proliferation of chicken granulosa cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, the \u003cem\u003ein vitro\u003c/em\u003e experiments have shown that EGF or FSH-induced proliferation of granulosa cell can be reversed by EGFR inhibitor AG1478 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Similarly, AG1478 can also inhibit significantly EGF or FSH-reduced apoptosis of granulosa cells [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. These results indicate the important roles of EGFR in chicken reproduction and broodiness.\u003c/p\u003e \u003cp\u003eGrowth and maturation of ovarian follicles in chickens require a complex network of blood vessels. Compared to prehierarchical follicles which have limited number and size of blood vessels, the large yellow follicles and preovulatory follicles are highly vascularized [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin and their receptors are involved in angiogenesis. VEGF is a key regulator of physiological angiogenesis as it can facilitate blood vessel growth and remodeling processes. The network of ovarian blood vessels is closely associated with VEGF. VEGF exerts biological effects by binding to its tyrosine kinase family receptor FLT1, KDR, FLT4 and KDRL (also known as VEGFRKDRL) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Interestingly, VEGF seemly binds to its receptor FLT1 and KDR with high affinity [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. KDR is mainly expressed in the vascular endothelium of the theca layer in chicken follicles. KDR might be the most important receptor which is involved in VEGF-induced angiogenesis [\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. FLT1 can regulate the VEGF activity via interacting with VEGF and making it less available to KDR [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Decreased expression of VEGF, FLT1 and KDR might be related to follicle atresia in chickens [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consistently, results from the present study showed that there was a significant decrease in expression of VEGF, including VEGFA and VEGFD, and its receptors, FLT1, KDR and VEGFRKDRL, in ovaries from incubating chickens, indicating the important roles of VEGF and its receptors in chicken broodiness.\u003c/p\u003e \u003cp\u003eSimultaneously, our results showed that there was a significant decrease of PDGF and its receptor (PDGFR) in ovaries of incubating chickens compared with egg-laying hens. There are four PDGF, including PDGFA, PDGFB, PDGFC and PDGFD, and two receptors, including PDGFRA and PDGFRB, identified in vertebrates [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The importance of PDGFRA has been confirmed by studies that showed the presence of PDGFRA in follicular cells in the ovaries of different species. In humans, PDGFRA is widely expressed in oocytes, theca cells and ovarian stroma cells [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. In rats, PDGFRA is also identified in oocytes and granulosa cells [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The expression pattern of PDGFRA indicated that it might be involved in the ovarian folliculogenesis, selection and growth initiation of follicles and the formation of thecal layer [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Meanwhile, PDGFRA is reported to be a requirement in steroid-producing cells in mice ovaries, and is involved in the steroidogenesis through regulating the downstream target gene \u003cem\u003eSgpl1\u003c/em\u003e, \u003cem\u003ePlekha1\u003c/em\u003e, \u003cem\u003eTiparp\u003c/em\u003e, \u003cem\u003eSchip1\u003c/em\u003e, and \u003cem\u003eBC058969\u003c/em\u003e [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Additionally, signaling downstream of PDGFRA has been reported to induce both apoptotic and antiapoptotic responses [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The identification of PDGFRA but not PDGFRB in ovaries from incubating chickens in the present study further suggests that PDGFRA may be important for ovarian development during chicken broodiness.\u003c/p\u003e \u003cp\u003eTEK is a tyrosine kinase receptor and can bind with angiopoietins. The angiopoietin 1 (ANGPT1), angiopoietin 2 (ANGPT2), and TEK receptor tyrosine kinase are mainly expressed in ovaries. In this study, we found TEK and angiopoietins were down-regulated in ovaries from incubating Changshun chickens. The angiopoietin-TEK system is observed to play a crucial role in blood vessel formation and stability, follicular development and atresia [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The ANGPT1 and ANGPT2 can bind to TEK, inducing opposite effects. ANGPT1 elicits an activation of TEK by increased tyrosine phosphorylation of TEK when they bind. ANGPT2 as a natural antagonist acts to inhibit the activation of TEK and disrupts ANGPT1-dependent TEK-mediated angiogenesis [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. ANGPT2 is reported to destabilize existing vessels, loosening the supporting cell matrix to allow angiogenic factors such as VEGF to stimulate cell proliferation and migration during early angiogenesis [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Additionally, TEK is hypothesized to play an important role in folliculogenesis [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFunctionality of the mammalian Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor (KIT), has been shown that they have multiple roles during oogenesis, folliculogenesis, and melanogenesis [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. The ovarian expression pattern revealed that mammalian KIT is mainly expressed in theca cells, oocytes and follicular fluid [\u003cspan additionalcitationids=\"CR90\" citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Similarly, the chicken KIT is observed to express in very small follicles (\u0026lt;\u0026thinsp;1 mm), theca cell layer and the ovarian stroma, indicating that Kit system might promote the transition from quiescence to slow growing follicles in chickens [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Furthermore, KIT is reported to be detected in many other chicken organs including the testis, brain, bursa, spleen, thymus, heart and kidney, indicating that Kit system might be implicated in a variety of non-ovarian functions [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibroblast growth factor receptor 3 (FGFR3) is known to be a receptor for fibroblast growth factors (FGF). In mammals, FGFR3 is identified in oocytes, granulosa cells, theca cells and stromal cells [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Additionally, FGFR3 is expressed by human primordial germ cells during the first and second trimester, and is then repressed after meiotic initiation to form primordial oocytes [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. Study from buffalos revealed that FGFR3 is expressed widely in ovarian follicles during different stages of development [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. FGFs and their receptors are reported to be involved in multiple biologic processes in angiogenesis, hematopoiesis, wound healing and even embryonic development [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Studies based on transgenic mice models showed that mutant FGFR3 leads to dwarfism and infertility [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. In chickens, FGFR3 is identified to be a candidate gonadal sex differentiation gene in embryo [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. Decreased FGFR3 in incubating hens in the present study further supports the assumption that FGFR3 might play critical roles in chicken reproduction and broodiness.\u003c/p\u003e \u003cp\u003eIn conclusion, we characterized and evaluated the ovarian transcriptome in LP and BP Changshun hens. The results suggest that focal adhesion, MAPK signaling pathway, FoxO signaling pathway, vascular smooth muscle contraction, ECM-receptor interaction and GnRH signaling pathway are critical for incubation behaviour in Changshun hens. Eight candidate genes (\u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eVEGFRKDRL\u003c/em\u003e, \u003cem\u003eFLT1\u003c/em\u003e, \u003cem\u003eKDR\u003c/em\u003e, \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eTEK\u003c/em\u003e, \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFGFR3\u003c/em\u003e) in ovaries might play crucial roles in chicken broodiness. Our results provide a theoretical basis for further exploration of the molecular mechanism of broodiness in chickens.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEgg-laying period: LP; Incubation period: BP; RNA sequencing: RNA-seq; Differently expressed genes: DEGs; Prolactin: PRL; Gonadotropin-releasing hormone: GnRH; Vasoactive intestinal polypeptide: VIP; Dopamine: DA; 5-hydroxytryptophan: 5-HTP; Hypothalamic-pituitary-gonadal axis: HPGA; Principal component analysis: PCA;\u0026nbsp;False discovery rate: FDR; Protein-protein interaction: PPI; Quantitative real-time PCR: qRT-PCR.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Biomarker Technologies Co., Ltd for assisting in sequencing and bioinformatics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZC designed this study, ZC, DW and RM performed the transcriptome analysis. ZC wrote the manuscript. All authors approved this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Guizhou Provincial Basic Research Program (Natural Science) (ZK[2021]167, ZK[2023]455); Natural Science Research Project of the Department of Education of Guizhou Province of China (KY[2020]071, Innovation team No.[2023]089).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data in the present study have been deposited in the Genome Sequence Archive (Genomics, Proteomics \u0026amp; Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA016819) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal protocol was approved by the Animal Ethics Committee of the Qiannan Normal University for Nationalities (AEC No. QNUN2021014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e Riddle O, Baxter RW, Lahr EL. 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BMC Genomics. 2015; 16(1): 704.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Transcriptome analysis, ovary, laying period, incubation period, Changshun green-shell laying hen","lastPublishedDoi":"10.21203/rs.3.rs-4540221/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4540221/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eChangshun green-shell laying hen with strong broodiness is a Chinese indigenous chicken breed. Little is known about the mechanisms responsible for ovary development of Changshun green-shell laying hens from egg-laying period (LP) to incubation period (BP). Here, RNA sequencing (RNA-seq) of ovaries from Changshun hens in LP and BP was performed to identify candidate genes and pathways associated with broodiness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe identified 1650 differently expressed genes (DEGs), including 429 up-regulated and 1221 down-regulated DEGs, in chicken ovaries between LP and BP groups. GO and KEGG analysis further revealed that these DEGs were mainly involved in the pathways related to follicle development in chicken ovaries, including focal adhesion, MAPK signaling pathway, and FoxO signaling pathway, and vascular smooth muscle contraction, ECM-receptor interaction, and GnRH signaling pathway were down-regulated in incubating ovaries. Eight candidate genes (\u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eVEGFRKDRL\u003c/em\u003e, \u003cem\u003eFLT1\u003c/em\u003e, \u003cem\u003eKDR\u003c/em\u003e, \u003cem\u003ePDGFRA\u003c/em\u003e, \u003cem\u003eTEK\u003c/em\u003e, \u003cem\u003eKIT \u003c/em\u003eand \u003cem\u003eFGFR3\u003c/em\u003e) related to angiogenesis, folliculogenesis, steroidogenesis and oogenesis in ovaries were suggested to play important roles in the ovarian development of Changshun hens during the transition from LP to BP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe discovered critical genes and pathways which is closely associated with ovary development in incubating chickens, indicating the complexity of reproductive behaviour of different chicken breeds.\u003c/p\u003e","manuscriptTitle":"Ovarian transcriptome profile from egg-laying period to incubation period of Changshun green-shell laying hens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-21 10:11:19","doi":"10.21203/rs.3.rs-4540221/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c66fad2f-59b8-4825-b485-4d4389aabc3a","owner":[],"postedDate":"June 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T12:51:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-21 10:11:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4540221","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4540221","identity":"rs-4540221","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}