Comparative transcriptome analyses of organ or tissues at different developmental stages can provide valuable insights into the question of how regulatory gene interaction networks control specific biological processes and how diseases can arise . Recently, increasing evidence has confirmed that lncRNAs are important regulatory factors of gene expression, regulating target genes by cis-acting (neighboring genes) or trans-acting (distant genes) mechanisms . Furthermore, RNA-seq has been performed to provide an extensive lncRNA and gene expression profile in different tissues of livestock and poultry (e.g., cell differentiation and development , cancer , and skeletal muscle development ). Previous studies of the hen uterus transcriptome and gene expression profiling during formation of the eggshell demonstrate a large number of DEGs that participate in ion transport for eggshell mineralization and the secretion of matrix proteins [19–23]. Most of the previous studies report the roles of mRNAs in the avian eggshell gland, but systematic identification of the functions of lncRNAs remained unclear in the development of the chicken shell gland. Therefore, in this study, we performed transcriptome sequencing of the shell gland of laying hens in the peak and late laying periods and analyzed the DE-lncRNAs and DEGs to reveal their roles in eggshell quality. To the best of our knowledge, this study represents the first systematic genome-wide analysis of lncRNAs and mRNAs in the chicken shell gland, providing a valuable catalog of functional lncRNAs and mRNAs associated with eggshell quality.
In the present study, we developed a highly stringent filtering pipeline to minimize the selection of false positive lncRNAs, which aimed to remove transcripts with evidence of protein-coding potential, and performed co-location mRNA prediction and co-expression mRNA prediction for the lncRNAs obtained from the chicken eggshell gland. Ultimately, we identified 176 DE-lncRNAs and 383 DE-mRNAs. To gain insight into how interactions between DE-lncRNAs and their corresponding target genes regulate shell gland development, we constructed co-expression interaction networks between DE-lncRNAs and their predicted cis- and trans-target genes. Then, four DE-lncRNAs and their target genes related to eggshell quality were selected for qPCR validation, and the results were consistent with the RNA-seq data, which demonstrated that lncRNA TCONS_01464392 can target the GPX8 gene, and they are all down-regulated. LncRNAs TCONS_00181492, TCONS_03234147, and TCONS_03123639 target FGF14, COL25A1, and GRXCR1, respectively, and these six genes are up-regulated. Together, these results confirmed that the identified lncRNAs and mRNAs were of high quality.
The oviduct of hens is composed of the infundibulum, magnum, isthmus, shell gland, and vagina. Especially, the shell gland is the place where the eggshell is deposited . The formation of the eggshell is a complex process involving the precipitation of calcium carbonate . Mature follicles reach the shell gland and calcify layer by layer. After the mature follicles reach the shell gland, they need to go through the calcification process, eventually form the eggshell, and the whole process takes about 15–16 h. Approximately 94% of minerals in the eggshell are calcium carbonate, with other inorganic minerals being calcium phosphate, magnesium phosphate, and magnesium carbonate . Previous studies suggest that eggshell calcification requires the interaction of numerous processes, including transcellular and paracellular transport of minerals and the secretion of different matrix proteins [26–28]. Particularly, ion transportation plays a crucial role in the process of eggshell formation. The ion channels contribute to the transportation of Ca2+ from the plasma to the uterine lumen, which includes Na+, Ca2+, and K+ channels . Moreover, the characteristics of egg shell calcification in poultry are that the body rapidly and massively transports Ca2+ from blood to the lumen of the eggshell gland, and a calcium ATPase (calcium pump) is a key enzyme involved in Ca2+ transport in the uterus during eggshell formation . Apart from Ca2+, inorganic phosphate (Pi) is also essential in the formation of eggshells. Pi is involved in many biological processes, including nucleic acid synthesis, skeletal development, signaling cascades, and tooth mineralization [31–33]. More meaningfully, phosphorus participates in the transport mechanism of the calcium pump (calcium ATPase).
In the present study, we conducted GO and KEGG pathway enrichment analyses on DE-mRNAs and DE-lncRNAs and found that the most of identified DEGs were involved in eggshell calcification and cuticularization pathways, such as “inorganic anion transport”, “inorganic anion transmembrane transporter activity”, “phosphate-containing compound metabolic process”, “phosphorus metabolic process”, “protein metabolic process”, “mitochondrial proton-transporting ATP synthase complex”, “proton-transporting ATP synthase complex”, and “calcium ion binding”. Notably, SPP1 was significantly enriched in the “Toll-like receptor signaling pathway”, and the authors of a previous study suggest that SPP1 is differentially expressed in the uterus between a low eggshell strength group and normal eggshell strength group during eggshell formation . In addition, another study indicates that the PHGDH gene is highly over-expressed in the white isthmus during deposition of the eggshell membranes . The PHGDH gene was also differentially expressed between two groups and enriched in the “Glycine, serine, and threonine metabolism pathway” in this study. Hence, all of these results indicate that the formulation of the eggshell is significantly affected by the shell gland of laying hens with different ages.
Based on the lncRNA-mRNA co-expression interaction networks, the predicted target gene of lncRNA TCONS_00181492 is FGF14. Prior to this analysis, little was known concerning the association between FGF14 and lncRNA. FGF14 is a well-known growth factor belonging to the FGF family. FGF family members possess broad mitogenic and cell survival activities and are involved in a variety of biological processes, including cell growth, embryonic development, tissue repair, morphogenesis, tumor growth, and invasion [34, 35]. Previous work demonstrates that FGF14 is a functionally relevant component of the neuronal voltage-gated Na+ (Nav) channel complex , and FGF14 can also regulate members of the presynaptic Cav2 Ca2+ channel family . Simultaneously, there is evidence that the transfer and concentration of Na+ can directly affect the transportation of Ca2+ and HCO3− in the chicken uterus .
In the present study, we found that the expression of FGF14 is up-regulated in the shell gland of chickens in the old group as compared to the young group. The aforementioned studies indicate that the FGF14 gene plays an important role in chicken growth . The predicted regulatory lncRNA, TCONS_00181492, was significantly more highly expressed in the shell gland in the old group than in the young group and controlled the expression of FGF14 via cis-acting mechanisms. Furthermore, TCONS_00181492 and FGF14 were positively correlated. Therefore, we had reason to speculate that TCONS_00181492 may regulate shell gland development in the chicken via the cis-acting target gene FGF14. Additionally, we found that the old hens had a higher incidence of disease than the young hens in the long-term cultivation of layers. Previous studies indicate that inherited mutations in FGF14 are linked to disease [40–42], and a study hints that the pathogenic effects of mutant FGF14 are likely mediated by dysregulation of both Ca2+ and Na+ channels . All of these results indicate the possible role of FGF14 in aging laying hens with deteriorated eggshell quality.
COL25A1 was a predicted cis-target of TCONS_03234147 that is related to the focal adhesion pathway. Collagen XXV alpha 1 (COL25A1), the extracellular matrix gene, is a collagenous type II transmembrane protein, which was first purified from senile plaques of Alzheimer’s disease (AD) brains . In recent years, work on collagen genes has attracted the attention of many researchers. Previous studies of the hen oviduct transcriptome during eggshell membrane formation identify a large number of differentially expressed collagen genes, such as collagen X (COL10A1), collagen I (COL1A1), collagen II (COL2A1), and collagen III (COL3A1) . Moreover, COL11A1 was also differentially expressed between the normal eggshell strength group and low eggshell strength group in the study integrating transcriptome and genome re-sequencing in the chicken uterus . TCONS_03234147 and its target gene COL25A1 were differentially expressed between the two groups in the present study, and their expression was higher in aging hens compared to young hens.
The GRXCR1 gene is the putative cis-target of TCONS_03123639 in the lncRNAs-genes network. The GRXCR1 gene encodes an evolutionarily conserved cysteine-rich protein with sequence similarity to the glutaredoxin family of proteins . Recently, research on the function of the GRXCR1 gene has mostly been focused on diseases [45, 46]. However, the biological function of the GRXCR1 gene is still rarely reported in livestock and poultry research. Herein, we found that GRXCR1 was enriched in the ion transport pathway, implying that GRXCR1 may play an important role in the formation of eggshells. Remarkably, the members (SLC1A3, SLC6A4, SLC20A1, SLC22A13, SLC26A3, SLC30A8, SLC39A2, SLC43A3, and SLC45A2) of the sodium-dependent phosphate transporter (SLC) family were also enriched in ion transport pathways (Table 10). Previous studies show that zinc ion transporters include two major families, SLC30 (Solute-Linked Carrier30, also named ZnT) and SLC39 (Solute-Linked Carrier 39, also named ZIP). ZnT contains 10 transporters of SLC30A1-SLC30A10, and ZIP contains 14 transporters of SLC39A1-SLC39A14. In our study, the differentially expressed SLC30A8 and SLC39A2 genes belong to ZnT family and ZIP family, respectively. Carbonic anhydrase located the eggshell gland epithelial cells is an important enzyme in the process of eggshell formation, which can reversibly catalyze the hydrolysis of H2CO3, regulate the concentration of HCO− in the eggshell gland, and then affect the Ca2+ transport process and the calcium deposition in the eggshell, changing the quality of the eggshell . Zinc ions are necessary for the activity center of carbonic anhydrase, so zinc can affect the activity of carbonic anhydrase . Moreover, zinc is also a component of alkaline phosphatase, which may regulate some phosphorylated proteins related to the mechanism of eggshell formation and affect the synthesis of calcium carbonate crystals . This provides us a vision for adding appropriate zinc to the diet of aging laying hens, which may reduce the deterioration of eggshell quality.
Through integration analysis of bioinformatics, we found that the differentially expressed TCONS_01464392 could target the GPX8 gene, whose expression was extremely significant, and their expression levels were negatively correlated. Glutathione peroxidases (GPXs) are enzymes that are present in almost all organisms, with the primary function of limiting peroxide accumulation. In mammals, GPXs consist of eight isoforms, but only two members (GPX7 and GPX8) reside in the endoplasmic reticulum [49, 50]. A previous study demonstrates that GPX8 is enriched in mitochondria-associated membranes and can regulate Ca2+ storage and fluxes . This indicates that the decline in eggshell quality of aging laying hens may be closely related to down-regulated GPX8 expression levels.