Integrating the transcriptome and proteome to identify important functional genes for laying hens with hard- or weak-shelled eggs

Background Eggshell strength and thickness are critical factors for reducing the egg breaking rate and preventing economic losses. The calcite biomineralization process is very important for eggshell quality. Therefore, we employed transcriptional sequencing and proteomics to investigate the differences between the uteruses of laying hens with high- or low-breaking-strength shells. We identied 1028 differentially expressed genes (DEGs) and 270 differentially expressed proteins (DEPs). The analysis results of GO terms and KEGG pathways showed most of the DEGs and DEPs were enriched in vital pathways related to such processes as calcium metabolism, hormone and amino acid biosynthesis, and cell proliferation and apoptosis. Some DEGs and DEPs that were coexpressed at mRNA and protein levels were veried. adjusted pH to 10.0 using ammonium hydroxide) and B (98% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) were employed to develop a gradient elution. The eluates were monitored at UV 214 nm, collected into a tube each minute and merged into 10 fractions. All fractions were dried under vacuum and reconstituted in 0.1% (v/v) formic acid (FA) for subsequent analyses.


Protein identi cations and DEP analyses by TMT proteome
To elucidate the mechanism underlying differences in eggshell strength, the differentially expressed proteins of the HE and LE groups were detected by TMT proteomics. In this experiment, 6786 proteins were identi ed. There were 270 differentially expressed proteins between the LE and HE groups that were signi cantly different (Supplement Table S4), including 161 upregulated proteins and 109 downregulated proteins (LE vs HE).
Analysis of these DEPs by GO analysis showed that the DEPs were primarily enriched in biological processes, molecular functions and cellular components including calcium ion binding, molecular function regulators, and extracellular regions (Fig. 2a). The top 20 results of KEGG enrichment analysis further indicated that these DEPs were most enriched in some vital pathways related to such processes as calcium metabolism, hormone and amino acid biosynthesis, and cell proliferation and apoptosis; these proteins were involved in the calcium signaling pathway, cell adhesion molecules (CAMs), ECM-receptor interactions, the p53 signaling pathway, biosynthesis of amino acids, and steroid hormone biosynthesis (Fig. 2b).
To further demonstrate the function of DEPs, we constructed a protein-protein interaction network of the DEPs (Fig. 2c). This study provides a new method and perspective for future research studying protein-protein interactions.

Correlation between differentially expressed transcriptomes and proteomes
To elucidate the relationship between the transcriptome and proteome in the HE and LE groups, two different omics datasets were integrated and analyzed. The results indicated that the number of DEGs and DEPs varied considerably in different eggshellstrength groups (Fig. 3a, 3b, 3c), and some genes (or proteins) were differentially expressed at the gene level but not at the protein level, possibly because they were late-response genes. There were 20 genes and proteins detected at both the gene and protein levels ( Table 1). The GO enrichment analysis results showed that the correlated-expression DEGs/DEPs were primarily enriched in such processes as transporter activity, molecular function regulation, single-organism processes, membrane, and structural molecule activity (Fig. 3d); meanwhile, KEGG enrichment analysis results showed that the correlated-expression DEGs/DEPs were primarily enriched in such processes as metabolic pathways, the p53 signaling pathway, focal adhesion, the calcium signaling pathway, and SNARE interactions in vesicular transport (Fig. 3e).

The validation of differentially expressed genes and proteins
In this study, the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) between LE and HE hens were identi ed. Seven upregulated and seven downregulated genes were randomly selected for veri cation via qPCR; eight proteins that were coexpressed at the mRNA and protein levels were randomly selected for veri cation by Western blotting. The results are shown in Fig. 4a and Fig. 4b. The log2 fold changes (LE/HE) were calculated based on the RNA-seq and qPCR results for DEGs, and the fold changes (LE/HE) were calculated based on the TMT and Western blotting results for DEPs. The expression trends indicated that the two methods produced consistent results. Additionally, we investigated the relative expression levels of KRT14, ANXA2 and DKK3 in different chicken tissues (i.e., heart, liver, spleen, lung, kidney, muscle and uterus) (Fig. 4c, 4d, 4e). In particular, the KRT14, ANXA2 and DKK3 genes exhibited high expression levels in the uterus compared with other tissues, and these genes were expressed at the lowest levels in the spleens and livers of hens. Thus, the tissue expression pro le of the KRT14, ANXA2 and DKK3 genes demonstrated tissue speci city.

Discussion
It is well-known that the formation of eggshells consists of several stages [23]. The formation of the mammillary knob layer follows the formation of the outer surface of the outer shell membrane and is the rst calci ed layer to be deposited, and its tips are embedded in the outer shell membrane [24]. The palisade layer formed with the mammillary knobs fuse, and the vertical crystalline layer is the last calci ed layer to be deposited, consisting of short crystals running perpendicular to the shell membrane [25,26]. Previous studies have shown that shell quality is dependent on the mammillary layer [27], and another study suggested that the organization of the columns of the palisade layer is one of the major determinants of the rigidity of the shell and the strength and shell resistance of the eggs [28]. When eggshells are calci ed in the uterus, large amounts of calcium ions and matrix proteins are required. Calcium metabolism and uterine proteins exhibit changes during the calci cation periods (initiation, growth, and termination). Eggshell quality is determined by its ultrastructure [29], especially the palisade layer [30]. Therefore, many studies have focused on the growth period when the palisade layer is formed [31,32].
Previous studies have shown that calcium could disrupt the endocrine system of females due to its wide spectrum of toxic effects on the uterus and mammary gland of rats and the developing human reproductive tract [33,34]. Zhang et al. (2019) [35] reported that the initiation period of calci cation determines eggshell strength. However, differences in the uterine transcriptome between hens laying eggs with high eggshell breaking strength and those producing eggs with low eggshell breaking strength during synchronous calci cation periods have not been reported. In this study, the differences in gene expression and protein expression were identi ed in the uteri of LE and HE hens. The results showed that the numbers of DEPs were signi cantly lower than those of DEGs. Fewer proteins were detected than genes in the Venn diagram analysis, probably due to the short length of the sample treatment period, modi cation and activation at the protein level, or the limitations of protein detection technologies [36]. Previous studies have shown that most genes are divided into two categories according to their different activation pathways in response to stimuli. The early response genes were induced without de novo protein synthesis [37], and the late response genes were induced more slowly with de novo protein synthesis because this procedure requires signaling molecules or cytokines [38].
The DEGs and DEPs were classi ed according to KEGG analysis, and most DEGs and DEPs were determined to be enriched in vital pathways related to calcium metabolism, hormone or amino acid biosynthesis or cell proliferation or apoptosis. Thus, we hypothesized that the quality of eggshells is closely related to the deposition of calcium ions. GO analysis revealed that many DEGs and DEPs were enriched in ion transport functions or cytoskeletal functions associated with eggshell calci cation. These results suggested that during normal calci cation, there were differences in ion transport between the uteri of hens producing high-or low-breaking-strength eggshells, which helped to elucidate the eggshell calci cation process. Aside from the common ion transport genes identi ed in previous reports, some novel genes were also described in this study (e.g., ANAX2, DKK3, and KRT14).
Annexin A2 (ANXA2) is a multifunctional calcium (Ca) and phospholipid-binding protein that is expressed in a wide spectrum of cells, including those participating in the in ammatory response [39]. Dickkopf 3 (Dkk3) is a secreted protein that belongs to the Dkk family and is encoded by the orthologous gene REIC. It was reported that Dkk3 is a physiological ER stressor in the mouse adrenal gland [40]. Keratin-14 (KRT14) is a key regulator of spheroid integrity, mesothelial attachment, and invasion into the submesothelial matrix. Keratin-14 (KRT14)-positive leader cells mediate mesothelial clearance and invasion by ovarian cancer cells [41]. In this study, ANXA2, KRT14 and DKK3 were determined to be signi cantly upregulated in LE hens compared to HE hens. In addition, the mRNA expression levels of the ANXA2, KRT14 and DKK3 genes were observed signi cantly elevated in the uterus. In summary, our results indicate that ANXA2, KRT14 and DKK3 may be related to the deposition of calcium in eggshells.

Conclusions
Based on the sequencing results and transcriptome and proteome analyses, our results were in keeping with previous ndings indicating that the initiation period of calci cation determines eggshell strength in the uterus. During normal calci cation, differences in ion transport were observed between the uteri of hens producing high-breaking-strength eggshells and those producing low-breaking-strength eggshells, which may help to elucidate the eggshell calci cation process.

Methods
Ethics approval and consent to participate All animal experiments were conducted according to the guidelines established by the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, 2004). The tissues were collected from 43week-old hens. The hens were raised under free food intake and were humanely sacri ced in the laboratory.

Samples collected
Three eggs from 1820 hens (Jianghan chicken, which is 43-week-old and is a local chicken in China) were collected. Eggshell breaking strength, eggshell thickness and egg weight were assessed. The eggshell thicknesses of eggs were tested using an electronic digital micrometer (Deli, Deli Group Co., Ltd, China). Eggshell breaking strength was assessed using an eggshell strength meter (NFN388, FHK Fujipin Co., Ltd, Japan). Eggs were weighed using an electronic balance (ES-E, Tianjin de ante Sensing Technology Co., Ltd, Tianjin, China).
The uteri of hens producing eggs with extreme eggshell thickness and eggshell breaking strength were collected immediately after oviposition from six hens after surgery. There were three uterus samples from the hard-eggshell group (HE group, exhibiting strong and thick eggshells) and three uterus samples from the week-eggshell group (LE group, exhibiting weak and thin eggshell). Then, the uteri were frozen in liquid nitrogen and used for RNA-seq analysis and experimental validation. Desalted peptides were labeled with TMT6/10-plex reagents (TMT6/10plex™ Isobaric Label Reagent Set, Thermo Fisher) following the manufacturer's instructions. Differently labeled peptides were mixed equally and subsequently desalted by peptide desalting spin columns (Thermo Fisher, 89852). TMT-labeled peptide mix was fractionated using a C18 column (Waters BEH C18 4.6 × 250 mm, 5 µm) on a Rigol L3000 HPLC operating at 1 mL/min, and the column oven was set at 50 °C. Mobile phases A (2% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) and B (98% acetonitrile, adjusted pH to 10.0 using ammonium hydroxide) were employed to develop a gradient elution. The eluates were monitored at UV 214 nm, collected into a tube each minute and merged into 10 fractions. All fractions were dried under vacuum and reconstituted in 0.1% (v/v) formic acid (FA) for subsequent analyses.
Shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operated in the data-dependent acquisition (DDA) mode.
Proteins with fold change in a comparison ≥ 1.2 or ≤ 0.83 and unadjusted signi cance level p < 0.05 were considered to be differentially expressed. Gene Ontology (GO) and InterPro (IPR) analyses were conducted using the interproscan-5 program against the nonredundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSitePro les, and PANTHER) [43], and the KEGG database (Kyoto Encyclopedia of Genes and Genomes) was employed to analyze the protein family and pathway. The probable interacting partners were predicted using the STRING-db server (http://string-db.org/) based on the related species. STRING is a database of both known and predicted protein-protein interactions [44]. The enrichment pipeline [45] was utilized to perform the enrichment analysis of GO and KEGG.

Correlation analysis between proteomic and transcriptomic results
The differentially expressed genes (DEGs) and the differentially expressed proteins (DEPs) were identi ed separately, and Venn diagrams were plotted according to the counted results. Correlation analysis was performed by R (version 3.5.1), and the maps were drawn based on changes in the transcriptome and proteome analyses. and calculated using the 2 −ΔΔCt method [46]. All primers were designed using Primer 5 (listed in Supplementary Table S1).
Western blot analysis RIPA lysis buffer (Beyotime, Beijing, China) was employed to generate uterine protein lysates. The uterine protein was extracted and separated using SDS-PAGE and transferred to PVDF membranes, which were blocked with skim milk. Then, antibodies speci c for differential proteins ( were used for immunoblotting. We quanti ed the protein expression levels compared to β-actin expression using ImageJ 1.42q (Wayne Rasband, National Institutes of Health, USA).

Statistical analysis
All data analyses were performed by GraphPad Prism 5 (San Diego, CA   Differentially expressed proteins (DEGs) identi ed for uterus of HE and LE hens. a. GO analysis of DEPs between HE and LE hens; b. The top 20 results of KEGG analysis of DEPs between HE and LE hens; c. The protein-protein interaction network of the DEPs.