Egg-laying and brooding stage-specific hormonal response and transcriptional regulation in pituitary of Muscovy duck (Cairina moschata)

Egg-laying and brooding stage-specific hormonal response and transcriptional regulation in pituitary of Muscovy duck (Cairina moschata)

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,∗,†,2 Kai Ge,∗,†,‡,2 Min Li,∗,† Lei Yang,∗,† Sihua Jin,∗,† Cheng Zhang,∗,† Xingyong Chen,∗,† and Zhaoyu Geng∗,†,1

College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; † Anhui province key laboratory of local livestock and poultry genetic resource conservation and bio-breeding, Anhui Agricultural University, Hefei 230036, P.R. China; and ‡ College of biological and pharmaceutical engineering, West Anhui University, Liuan 237012, China at the 2 reproductive stages. A total of 398 differentially expressed genes included 20 transcription factors were identified (fold change ≥ 1.5, P < 0.01). There were 109 upregulated and 289 downregulated genes at brooding phases (n = 6) compared with egg-laying phases (n = 6). Real-time quantitative PCR analysis was carried out to verify the transcriptome results. The present study suggested that neuroactive ligand-receptor interaction pathway, calcium signaling pathway, and response to steroid hormones biological process are critical for controlling broodiness in the ducks. Further analysis revealed that SHH, PTGS2, RLN3, and transcription factor AP-1 may act as central signal modulators of hormonal and behavioral regulation mechanism associated with broodiness.

ABSTRACT Broodiness is an interesting topic in reproductive biology for its reduced egg production. The strong brooding trait of Muscovy duck has become a major factor restricting the development of its industry. Broody phenotype and environmental factors influencing broodiness in poultry have been extensively studied, but the molecular regulation mechanism of broodiness remains unclear. In this research, the Muscovy duck reproductive endocrine hormones and pituitary transcriptome profiles during egg-laying phases (LP) and brooding phases (BP) were studied. During BP (n = 19), prolactin (PRL) levels was higher, while progesterone (P4) and estradiol (E2) were lower as compared to ducks during their LP (n = 20) (P < 0.01). We then examined the pituitary transcriptome of Muscovy duck

Key words: pituitary, hormone, transcriptome, broodiness, egg-laying 2019 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pez433

INTRODUCTION

ness make this duck species a suitable material to study egg-laying and broody behavior. Broodiness, a common habit of the most birds, results from the interaction between the hormonal system and the egg-laying environment of the birds (Sharp, 2009). The transition from the egg-laying to brooding phase is a complex process that ultimately leads to gonadal regression, changes in neuroendocrine patterns, manifestation of brooding behavior, and termination of ovulation (Romanov, 2001). In poultry, the reproductive endocrine system and reproductive activity are strictly controlled by the hypothalamic–pituitary– gonadal axis (HPG axis) (Sharp, 2009; Chaiseha and El Halawani, 2015). The neuroendocrine basis of incubation behavior has been studied extensively in some domestic species, including the domestic fowls like chicken (Gallus gallus) (Sharp et al., 1984), turkeys (Meleagris gallopavo) (Harvey et al., 1981), domesticated doves (Streptopelia risoria) (Lea et al., 1992), and geese (Huang et al., 2008). It is believed that the altered levels of reproductive endocrine hormones, including gonadotrophin (GnRH), growth hormone (GH), prolactin (PRL), luteinizing hormone (LH), progesterone

The domestic Muscovy duck (Cairina moschata) is an economically important species around the world for its unique meat taste and low-caloric content. The ancestor of Muscovy duck is different from nearly all domestic duck breeds, which are domesticated from mallard (Anas platyrhynchos) (Veeramani et al., 2016). Muscovy duck has a long incubation period and strong broodiness, which has led to a decrease in egg production and limited the development of the Muscovy duck industry (Jiang et al., 2010). Incubation behavior of the Muscovy duck is characterized by persistent nesting, tilting head backwards, clucking, and nest defence (Wu et al., 2014). The stable laying broodiness cycles and easily identifiable characteristics of broodi-

© 2019 Poultry Science Association Inc. Received November 14, 2018. Accepted July 16, 2019. 1 Corresponding author: [email protected] 2 These authors contributed equally to this work.

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Pengfei Ye

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MATERIALS AND METHODS Ethics Statement All experimental procedures and sample collection were performed according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004) and approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology, Anhui Agricultural University, Hefei, China (permit No. AHAU20101025).

Animals and Sample Preparation The experimental female Muscovy ducks obtained from Anqing Yongqiang Agricultural Science and Technology Stock Co., Ltd., China, were raised according to the standard procedure. There is great variation of reproductive performance and broodiness between individual ducks. At 42 wk of age, there are ducks in the stages of laying and brooding. At 42 wk of age, 25 ducks from egg-laying (LP) status and 25 ducks in broodiness (BP) were first selected according to their behavior (Figure 1A). Blood, ovary, and pituitary gland samples were taken after euthanized by exsanguination at nearby slaughterhouse. The morphologic characteristics of the ovaries were used to further evaluate individual physiological stage of Muscovy duck. The pituitary gland samples were wrapped in freezing tube, frozen in liquid nitrogen, and then stored at –80°C until needed. The blood samples were kept for 1 h at room temperature, then centrifuged at 3,000 × g, 4°C, for 10 min. Serum samples were collected and stored at −20°C until analysis.

Assay of Hormones A total of 20 egg-laying ducks and 19 broody ducks were selected according to the ovary morphology for hormone determination. Hormone concentrations were assayed using commercial radioimmunoassay kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) (Xiao, 2015) for E2 (sensitivity of 1 pg/mL), P (sensitivity of 0.1 ng/mL), and PRL (sensitivity of 10 μIU/mL). The accuracy (correlation coefficient of R) of the assays was at least 0.9900. The intra-assay CVs for PRL, P, and E2 levels were 11.4, 7.5, and 6.2%, respectively. All the analyses were carried out according to the kit protocols.

Library Construction and Sequencing Based on the broodiness development and hormone data, 6 individuals with similar physiological conditions were selected from each group for pituitary transcriptome studies. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments

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(P), and estradiol (E2), are the major factors inducing the occurrence of broodiness (Lea et al., 1992; Romanov, 2001; Sharp, 2009; Chaiseha and El Halawani, 2015). In addition, various neuropeptide involved in the reproductive process were widely studied, such as serotonin (5-HT), vasoactive intestinal polypeptide (VIP), and dopamine (DA) (Zhou et al., 2008; Xu et al., 2010). Neuroendocrine components of the reproductive system include GnRH/FSH/LH and VIP/PRL systems both influenced by dopaminergic and serotonergic neurotransmission. On the other hand, the combined action of estrogen, progesterone, and nesting activity further stimulates the VIP/PRL system. These systems govern the period of egg-laying, initiate and maintain maternal behaviors, and may influence the onset of gonadal regression (Kagyaagye et al., 2012; Chaiseha and El Halawani, 2015; Ye et al., 2019). As next-generation sequencing, RNA-Seq has provided a powerful, highly reproducible and cost-efficient tool for transcriptomic research without the genome information of the species (Morozova and Marra, 2008). RNA-Seq has been applied in the hypothalamus, pituitary, and ovaries to investigate gene expression profiles during the laying period and broody period. Hehe Liu analyzed the transcriptome in the hypothalamus and concluded that the expression changes of hypocretin and pro-opiomelanocortin in the hypothalamus of nesting geese may cause appetite reduction, which is associated with broody behavior in geese (Liu et al., 2018). Next-generation sequencing was used to identify differentially expressed miRNAs in the hypothalamus of goose, and 52 known miRNAs and 208 novel miRNAs were found between the broody and egg-laying groups (Chen et al., 2014). Hehe Liu and Xu Shen analyzed the pituitary transcriptome between laying and brooding using geese and chicken, respectively. They concluded that steroid biosynthesis and hormonal interactions are responsible for brooding behavior (Shen et al., 2016; Liu et al., 2018). Fallahshahroudi et al. (2018) compared the genes expression of pituitary in White Leghorn chicken and the ancestral Red Junglefowl, and found that the expression of TSHB, DIO2, PRLR, NR3C1, and CRHR2 is the basis of multiple domesticated traits. Moreover, previous transcriptome analysis of follicles revealed the important mechanisms of hormones, autophagy, and oxidation in regulating broodiness and egg-laying in geese (Xu et al., 2013; Yu et al., 2016). These studies have confirmed the importance of the HPG axis for broodiness in poultry. However, within the pituitary, the behavioral-endocrine mechanisms of broodiness in Muscovy duck remain unclear. In the present study, we systematically examined the dynamics of the Muscovy duck plasma hormones and pituitary transcriptome at egg-laying and brooding stages and focused on the gene expression differences between those 2 periods. The purpose of this study was to understand the genetic basis of the transition between the laying and brooding phases at the transcriptome level and to provide new insights into poultry reproductive behavior.

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using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP, and buffer. Then, the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced on the Illumina HiSeq 4000 sequencing platform by Gene Denovo Biotechnology Co. (Guangzhou, China) (Reuter et al., 2015). The average depth of sequencing was 20X, and the paired reads were 150 BP lengths each sample.

Transcriptome Analysis All the sequences filtered adaptor sequences and lowquality sequences were mapped to the reference transcriptome using short reads alignment tool Bowtie2 (Li et al., 2009) by default parameters. The Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession number GGZN00000000, which used for reference transcriptome in this study. The unigene expression was calculated and normalized to RPKM (Reads Per kb per Million reads) (Mortazavi et al., 2008). The formula is as follows: RPKM = (1,000,000*C)/(N*L/1000). To identify differentially expressed genes (DEGs) across

samples or groups, the edgeR package (http://www. r-project.org/) was used. We identified genes with a fold change ≥1.5 and P < 0.01 in a comparison as significant DEGs (Butte et al., 2001).

Quantitative RT-PCR Verification Gene expression profiles in the 12 pituitary gland samples were validated by qRT-PCR on a CFX96 qRT-PCR detection system (Bio-Rad, Hercules, CA). cDNA reverse-transcribed from total RNA was used as the template along with SuperReal PreMix Plus with SYBR Green (Tiangen Biotech, Beijing, China) and gene-specific primers designed with Primer Premier 6 software (Premier Biosoft, Palo Alto, CA). Relative abundance of the transcripts was calculated by the comparative cycle threshold method with beta-actin used as an endogenous control. Reactions were prepared in triplicate for each sample. Relative expression levels were calculated by the 2−ΔΔCT method.

Statistical Analysis Hormone and qRT-PCR data were compared by SPSS 22.0 for Windows (IBM, Chicago, IL) using 1-way analysis of variance followed by independent sample t-test at 5% significance level. DEGs were mapped to GO terms in the Gene Ontology database

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Figure 1. Broodiness, egg-laying, and endocrine study of Muscovy duck. (A)1. Broody Muscovy duck and its ovary morphology. 2. Egg-laying Muscovy duck and its ovary morphology. (B) Concentration of prolactin (PRL), progesterone (P), and estradiol (E2) in serum at egg-laying phases (LP, n = 20) and brooding phases (BP, n = 19). See also Supplementary Table S1. BP are represented in white and LP are represented in gray. Box plots show the interquartile range (IQR) of each group analyzed with whiskers extending to 1.5 × the IQR. Horizontal lines inside the box represent medians, and blue crosses “+” represent means. Scattered points within each boxplot represent the individual values used to generate each plot.

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Table 1. Characteristics of the sequence data from 12 libraries in Muscovy duck. Before filter

After filter

Reads num

BP1 BP2 BP3 BP4 BP5 BP6 LP1 LP2 LP3 LP4 LP5 LP6

51,633,044 58,415,318 54,427,968 61,225,274 69,593,496 72,405,518 74,061,022 46,104,298 51,229,562 69,730,862 56,623,468 73,248,270

7.21 8.16 7.6 8.55 9.72 10.11 10.35 6.44 7.16 9.74 7.91 10.23

Reads num (%) 50,247,974 57,074,010 52,833,952 59,456,942 67,399,026 70,042,916 71,836,744 44,492,300 49,605,352 67,337,420 54,852,818 71,013,534

(97.32%) (97.7%) (97.07%) (97.11%) (96.85%) (96.74%) (97%) (96.5%) (96.83%) (96.57%) (96.87%) (96.95%)

After filter data (G)

Q201 (%)

Q302 (%)

GC content3 (%)

6.93 7.88 7.29 8.21 9.31 9.69 9.92 6.13 6.85 9.29 7.57 9.78

97.79 97.9 97.7 97.73 97.63 97.62 97.58 97.31 97.55 97.42 97.67 97.32

94.31 94.56 94.08 94.1 93.98 93.99 93.93 93.41 93.87 93.66 94.11 93.6

0.4767 0.4814 0.4742 0.486 0.4772 0.4726 0.4788 0.4824 0.4776 0.4826 0.4774 0.4632

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Q20: the proportion of read bases whose error rate is less than 1%. Q30: the proportion of read bases whose error rate is less than 0.1%. 3 GC content: guanine-cytosine content. 2

(http://www.geneontology.org/). Pathway enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Significantly enriched GO or pathway terms in DEGs comparing to the genome background were defined by the hypergeometric test, taking P ≤ 0.05 as a threshold. The protein–protein interaction (PPI) analysis was performed using Cytoscape_v3.2.1. The PPI information was gained with default confidence cutoff of 400 from STRING (https://string-db.org/).

44,492,300 to 71,836,744 reads, with the valid ratios being more than 96% for 12 libraries. Clean reads from each sample were mapped to the constructed reference genes and the mapped reads were counted to RPKM for quantification. The mapping results showed that 84.16 to 88.19% of the reads from each library were perfectly matched to the reference genome (Supplementary Table S2). All raw data are available at the NCBI SRA database using the project ID: SRP115365.

Analysis of DEGs RESULTS Analysis of Endogenous Hormones Level A total of 20 egg-laying and 19 broody Muscovy ducks were selected according to their behavior observation and ovary morphologic examination (Figure 1A). We measured serum PRL, P, and E2 concentrations of the BP and LP and shown in box and whisker plots of Figure 1B. The concentrations of plasma PRL during the broody period were significantly higher than those during the laying period. However, the level of P and E2 was markedly lower (P < 0.05) in broody stage than in laying period (P < 0.01) (Figure 1B, Supplementary Table S1). The outliers are shown as dots that lie outside the range of Box and whisker plot (Dawson, 2011). After removing individuals with outliers of hormone concentrations, 6 pituitary gland samples in each group were randomly selected for transcriptome study.

RNA Sequencing of Muscovy Duck Pituitary A total of 46,104,298 to 74,061,022 raw reads were generated in each library. The data quality was good, with Q20 (base sequencing error probability < 1%) > 97% and Q30 (base sequencing error probability < 0.1%) > 93% in each library (Table 1). After the raw data has been filtered by removing adaptor sequences and low-quality sequences, the valid data were

We focused on discovering genes differentially expressed in pituitaries between egg-laying and brooding ducks. A cutoff value of 1.0 RPKM was established for either of the pituitary gland samples to evaluate the relative abundance levels of transcripts that were considered for further analysis. Between the 6 LP and 6 BP libraries, 398 significantly DEGs (P < 0.01 and fold change≥1.5) composed of 109 upregulated and 289 downregulated genes were identified (Figure 2A, Supplementary Table S3). The expression levels of 18 randomly selected genes in the 12 pituitary gland samples were confirmed by RT-qPCR which is consistent with the sequencing results (Figure 2B). Primer sequences for qPCR experiments are shown in Supplementary Table S4. To determine the function of DEGs, we mapped them according to terms of the GO database. A total of 398 genes were categorized into the 3 main categories of GO classification, including biological processes, cellular components, and molecular functions (Supplementary Table S5). Among them, the most important biological processes included “developmental process,” “reproductive process,” and “hormone-related process” (Figure 3A). The important cellular components included the “extracellular region,” “membrane,” “organelle,” and “macromolecular complex.” For molecular function, genes were involved in “nucleic acid binding transcription factor activity,” “signal transducer

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Sample

Before filter data (G)

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Figure 3. GO and KEGG enrichment analysis for the different expressed genes. (A) Biological process of GO analysis. (B) Top 20 significantly enriched KEGG pathway.

activity,” and “molecular transducer activity and binding.” In this study, we identified 63 genes in hormonerelated process differently regulated in pituitaries of broody ducks, compared to these of egg-laying ducks (Supplementary Table S6). The hormone-related genes were divided into endocrine system-contained, neuroactive ligand-receptor interaction, reproductive processrelated, and response to steroid hormone groups. For KEGG pathway analysis in LP and BP libraries, 398 genes were mapped onto 99 pathways (Figure 3B; Supplementary Table S7). The genes were found significantly enriched in the main pathways including “neu-

roactive ligand-receptor interaction (13.04%),” “calcium signaling pathway (8.7%),” “protein processing in endoplasmic reticulum (7.83%),” and “GnRH signaling pathway (5.22%).”

Differentially Expressed Transcription Factors Considering the functional importance of transcription factors, we identified a total of 634 transcription factors expressed in pituitaries. In the LP and

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Figure 2. The heatmap of DEGs and validation of the gene expression profile by qPCR. (A) The heatmap showing the different gene expression patterns of the egg-laying phases (LP) and brooding phases (BP). (B) The relative expression levels of 18 selected genes were calculated according to the 2−ΔΔCt method using beta-actin as an internal reference gene. Error bars represent the standard error. The x-axis indicates different genes in the 2 tissues. The P values were calculated from t-tests, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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BP libraries, 20 genes were significantly differentially expressed, including 3 upregulated and 17 downregulated (Supplementary Table S8). Of the 17 upregulated transcription factors, 9 were involved in the biological process of response to hormone, including FOSB, HEY1, ISL1, JUN, JUND, LOC107199665, NR0B1, NR2F1, and NR2F2 among which LOC107199665, JUND, JUN, and FOSB belonged to TF_bZIP family. More importantly, 6 reproductive process-related transcription factors (TFs) included FOSB, GATA2, HEY1, HEY2, NR2F2, and SALL1. Of the 3 downregulated transcription factors, CREB3L1 was TF_bZIP gene involved in endocrine system, NEUROG2 was bHLH gene related to nervous system development and LOC101791228 belonged to THR-like family associated with response to hormone.

PPI Analysis To further extract relevant information from the identified transcriptome data, a more comprehensive bioinformatics analysis of PPI networks of DEGs was performed (Figure 4). Protein–protein interaction network was used to select the hub genes that were associated with egg-laying and broodiness. The following network model is generated with cytoscape based on information gained up to 4 levels of functional analysis: fold change of genes, PPI, KEGG pathway, and biological process enrichment. The PPI network of DEGs consisted of 180 nodes and 423 edges and 8 significantly important pathways/biological processes. The hub genes with the highest degrees in the PPI network included SHH, NCOR2, CALB2, PR, PTGS2, IGF1R, PRL, and RLN3 and transcription factors GATA2, ISL1, JUN, JUND, FOSB, NR2F2, and HEY1.

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Figure 4. Protein–protein interaction (PPI) networks of DEGs. Circle nodes for genes/proteins, pentagon for transcription factors, rectangle nodes for KEGG pathway or biological process. In case of fold change analysis, genes/proteins were colored in red (upregulation) and green (downregulation). Interactions were shown as solid lines between genes/proteins, and edges of KEGG pathway/biological process in dashed lines.

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DISCUSSION

ratio between EB and P (Halawani et al., 1983). Glucocorticoids have been shown to be involved in induction of growth hormone expression during pituitary development (Ellestad et al., 2015) and induction AP-1 transcription factor mediating GNRHR expression in pituitary (Mayanunez and Conn, 2003). Inspired by glucocorticoid can be used to treat hyperprolactinemia in human (Gutenberg et al., 2006), we hypothesized that glucocorticoid addition may terminate the duck’s broodiness behavior through inhibiting prolactin secretion and promoting GH expression. Further research is needed to determine therapeutic potential of steroid hormones against broodiness in early brooding ducks. Results of both GO and KEGG analysis suggested that some genes were highly enriched in calcium ionrelated biological process or calcium signaling pathway (Supplementary Tables S5 and S7). It is well known that intracellular Ca2+ concentration is a key signaling molecule controlling exocytosis to regulate neurotransmitters and endocrine hormones release (Martin, 2003), and pituitary hormone secretion is a Ca2+ dependent process (Bates and Conn, 1984). Feeding a calcium-deficient diet to laying hens significantly reduced plasma Ca2+ concentration, reduced or completely stopped egg production, and the ovaries degenerated within 6 to 9 D. The basal concentrations of LH and GH in birds fed a low calcium diet were significantly lower than those on a calcium-rich diet (Luck and Scanes, 1979; Bonga and Pang, 1991; Xia et al., 2015). We speculated that calcium ion signaling pathway and related genes were potential hormonal regulation mechanisms induced in the different hormone level. We also identified new pathways associated with reproductive cycle transitions, the neuroactive ligandreceptor interaction pathway significantly changed during the transition from laying to brooding. In this functional pathway, 12 genes ADRB2, GLP1R, CGA, F2RL1, HRH3, GH1, GHRHR, GNRHR, LHB, PRL, SSTR3, and TACR3 were involved. As we know, genes of CGA, GH1, GHRHR, GNRHR, and SSTR3 are directly related to hormones synthesis. The β 2-adrenergic receptor (ADRB2) is a key regulator of a wide range of metabolic processes that include steroid metabolism in the body (Braadland et al., 2016). Previous reports had shown that activation of expressed ADRB2 led to increases in expression of PTGS2 (Nagaraja et al., 2014). Our result showed that the expressions of both ADRB2 and PTGS2 were upregulated in the brooding. Multiple evidences had suggested that activation of GLP1R expressed on central nervous system or peripheral vagal afferents decreases food intake and improves glucose tolerance while inhibition increases food intake and worsens glucose tolerance. GLP1R was increased in broody ducks; this may be the reason for poor appetite of ducks during brooding (Sisley et al., 2014). Histamine receptor H3 (HRH3) is a receptor for histamine also increased in the broody pituitaries, which interact with N-type voltage-gated calcium channels, to reduce action potential-mediated influx of calcium and hence involved

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This study provided new insight into Muscovy duck reproductive endocrine and pituitary transcriptome profile of egg-laying and broody stages. We selected the experimental individuals for transcriptome study according to behavior observation, anatomical feature of ovary, and endocrine physiology. It was reported that poultry PRL secretion increased steadily during the brooding phase, while plasma levels of the somatotropin, gonadotropin (FSH, LH) and steroid hormones (P4, E2) decreased sharply compared to the laying period (Romanov, 2001; Sharp, 2009). Consistent with previous studies, our data revealed that plasma concentrations of PRL hormone increased rapidly, and PRL gene exhibited the highest mRNA level in the brooding phase. Expression of genes for GNRHR, LHβ , and CGA (glycoprotein hormones alpha chain) in the pituitary is sharply decreased, while FSHβ mRNA was not significantly altered. We conclude that the decrease in plasma FSH is mainly due to the lack of its alpha polypeptide. In the present study, the expression level of GHRHR and GH sharply declined in the pituitary glands of brooding ducks. Moreover, our finding was consistent with previous reports that the expression of progesterone receptor (PR) dropped significantly at the broody phase (Shen et al., 2016). Progesterone receptor is the nuclear receptor of the hormone progesterone, which affects every aspect in female development and reproduction. Inhibiting expression of PR results in decreased sensitivity of the pituitary to P4 and GnRH (Laudet and Gronemeyer, 2002). Many studies have widely reported the contributions of somatotropin, gonadotropin, and steroid hormones to classical regulatory pathways within the HPGA. Therefore, we speculate that pituitary GNRHR, GHRHR, and PR may be the key molecules in initiating gonadal degeneration and hormonal changes during the brooding period. Steroid hormones such as progesterone, estradiol, and glucocorticoids play a role in regulating the synthesis of avian pituitary hormones (Handa and Weiser, 2014), which are associated with fertility and reproductive behavior (Durant et al., 2013; Chaiseha and El Halawani, 2015). We found that many hormonerelated DEGs were significantly enriched into biological processes of response to progesterone (PR, ADRB2, FOSB, SOCS3), response to estradiol (ADRB2, GH1, GHRHR, KCNJ11, NEFH, NR2F2, NRIP1, PTGS2, SOCS3, SSTR3, TACR3), and response to glucocorticoid (ADRB2, ALPL, FOSB, GHRHR, HEY1, ISL1, PTGS2, SDC1, SOCS3, SSTR3, STC1). This suggested that steroid hormones interacted extensively with genes in the pituitary gland during the reproductive cycle. Previous study has indicated that daily injections of estradiol benzoate (EB, synthetic estrogen) and/or progesterone in ovariectomized turkeys have a variable effect on serum PRL and LH levels depending upon the dose, the duration of treatment, or the

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that is, SHH, PTGS2, and RLN3, were found to play important roles in the occurrence of broodiness. The SHH gene encodes a protein called Sonic Hedgehog, SHH signaling activation increased ACTH, GH, and prolactin secretion from normal and tumor anterior pituitary cells (Vila et al., 2005). PTGS2 (COX-2) is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins (PGs), which are important for inflammation and reproduction (Thorn et al., 2011). RLN3 expression level was downregulated in pituitary of brooding Muscovy ducks compared to laying ducks; Xu Shen detected the same result in chickens (Shen et al., 2016). Relaxin-3 activated a wide range of signaling pathways and stimulated AP-1 activation. Relaxin has many well-defined reproductive roles in many species, maternal circulating concentrations of the hormone relaxin have been positively correlated with duration of gestation (Bathgate et al., 2013; Thorell et al., 2015). Therefore, the 3 hub genes, SHH, PTGS2 and RLN3, had great potential to serve as new molecular biomarkers for regulating broodiness.

CONCLUSIONS Taken together, these results suggest that the functional regulation of broodiness behavior in the pituitary gland of Muscovy duck might be mainly associated with the mechanisms of hormone modification and secretion through transcription-dependent manners. At the transcriptional level, characteristic changes included neuroactive ligand-receptor interaction pathway, calcium signaling pathway, and response to steroid hormones biological process during the transition between the laying and brooding phases. DEGs of SHH, RLN3, PTGS2, and AP-1 may be the candidate genes in critical molecular regulation mechanism of hormonal and behavioral changes associated with broodiness. It would be intriguing to carry out further integrative studies of these findings, and hypotheses are expected to shed light on the molecular mechanisms of the complex and individual reproductive behavior.

SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Supplementary Table S1. Comparison of hormone levels between egg-laying and broody Muscovy ducks. Supplementary Table S2. Mapped statistical results of 12 libraries. Supplementary Table S3. All of the differently expressed genes detected in pituitary. Supplementary Table S4. Primer sequences used for quantitative PCR validation. Supplementary Table S5. Lists of GO enrichment analysis of specifically expressed genes. Supplementary Table S6. Differentially expressed genes associated with hormones.

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in neurotransmitter release and pituitary hormone secretion (Nuutinen and Panula, 2010). Coagulation factor II receptor-like 1(F2RL1) also known as proteaseactivated receptor 2 (PAR2) is a G-protein-coupled receptor that has multiple roles in inflammation, proliferation, and regulation of neurotransmitter release (Fan et al., 2014; Trevor et al., 2016). Tachykinin receptor 3 (TACR3) is critical in reproduction, inactivating mutations in human genes encoding NKB (TAC3) and NK3R (TACR3) that result in hypogonadotropic hypogonadism (Bianco and Kaiser, 2009; Grachev et al., 2014). NKB and NKB-related peptide were found to have effect on PRL and somatolactin α secretion and gene expression via differential activation of NK2R and NK3R expressed in the carp pituitary (Hu et al., 2014). IGF-I/-II and epidermal growth factor (EGF) could significantly induce NK3R expression in pituitary of fish model (Hu et al., 2016; Qin et al., 2018). It is reasonable for us to speculate that NK3R expression at the pituitary level might be a mechanism for amplifying NKBinduced pituitary hormone gene expression. Thus, we concluded the neuroactive ligand-receptor interaction pathway was important for hormonal regulation in reproductive cycle transitions of duck. In this study, we found that transcription factors play a crucial role in biological process of hormonal response and reproduction during the transition from laying to brooding (Supplementary Table S8). Transcription factor GATA2 is critical for maintenance of hormone production in both pituitary cells and thyrotropes (Charles et al., 2006). ISL1 has been shown to interact with estrogen receptor (ER); the ISL1ER interaction could differentially regulate the expression of ER and ISL1 target genes (Gay et al., 2000). Proteins FOSB, C-JUN, and JUND respectively encoded by genes FOSB, JUN, and JUND are functional subunits of transcription factor complex AP-1. AP-1 transcription factor has been shown to be involved in a wide range of cellular processes, and it is important for mediating GnRHR induction by glucocorticoids in pituitary (Mayanunez and Conn, 2003), which is consistent with the result of FOSB, JUN, JUND, and GnRHR downexpressed in BP compared to LP. COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) including COUP-TFI (NR2F1) and COUP-TFII (NR2F2) are orphan members of the nuclear receptor superfamily of transcription factors, which play important roles in the regulation of organogenesis, neurogenesis, and embryonic development (Yang et al., 2017). Previous studies have demonstrated the expression and function of COUP-TFs in pituitary gonadotropins, which regulate gonadotropin genes including LHβ (Zheng et al., 2010). The results of PPI networks showed interaction between DEGs (Figure 4), significant regulated pathways and GO terms included related genes, and TFs were already found to play important roles in broodiness or egg-laying, which are discussed above. Moreover, 3 hub genes with the highest degrees in the PPI network,

HORMONES AND TRANSCRIPTOME OF DUCK BROODINESS

Supplementary Table S7. Lists of KEGG pathway analysis of specifically expressed genes. Supplementary Table S8. Differently expressed transcription factors affecting broody behavior in Muscovy duck.

This study was financed by Anhui Provincial major special science and technology project (16030701067), the Project of Anhui Province Scientific Technology Plan (1604a0702009) and the Natural Science Foundation for Young Scholars of Anhui Agricultural University (yj2017-03).

AUTHORS’ CONTRIBUTIONS PFY, KG, and ZYG designed the study and drafted the manuscript. PFY, KG, ML, and LY carried out all the experimental analysis and prepared all figures and tables. SHJ, CZ, and XYC assisted in explaining the results and revised the final version of the manuscript. All authors have read and approved the final manuscript.

CONFLICTS OF INTEREST The authors declare that they have no competing interests.

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