Transcriptome analysis revealed the possible regulatory pathways initiating female geese broodiness within the hypothalamic-pituitary-gonadal axis

Abstract

Geese have the strongest tendency toward broodiness among all poultry. The mechanisms initiating broodiness within the goose hypothalamic-pituitary-gonadal axis (HPGA) are still unclear. Here, we reported the transcriptome differences between laying and initial nesting within the HPGA tissues of geese. We constructed a unigene database based on HPGA tissues and identified 128,148 unigenes, 100% of which have been annotated. By using Digital Gene Expression (DGE) sequencing, we screened 19, 110, 289, and 211 differentially expressed genes (DEGs) in the hypothalamus, pituitary gland, stroma ovarii, and follicles, respectively, between laying and nesting geese. Expression changes of hypocretin (HCRT) and pro-opiomelanocortin (POMC) in the hypothalamus of nesting geese may cause appetite reduction, which is possibly the first step and a prerequisite to initiate broodiness. In addition to prolactin (PRL), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), genes including oxytocin-neurophysin (OXT), chordin-like protein 1 (CHRDL1) and growth hormone (GH), expressed in the pituitary gland, are new candidate molecules that may be involved in broodiness in geese. Heme oxygenase 1 (HMOX1) in the pituitary gland, the proto-oncogene c-Fos (FOS), heat shock protein 90-alpha (HSP90AA), and cyclin-dependent kinase 1 (CDK1) in the ovary that may consolidate and transduce signals regulating the HPGA during broodiness in geese.

Fig 1. The entire experimental design and inclusion criteria for the groups of geese.

A, The entire experimental design and sequencing procedure. L_Hy, L_Pitu, L_Ov and L_ThB, respectively, mean hypothalamus, pituitary gland, stroma ovarii and walls of follicles with a diameter range of 8–10 mm from laying geese. N_Hy, N_Pitu, N_Ov and N_ThB refer to the corresponding tissues from geese at the beginning of nesting. B, The standards for dividing geese into initial laying and nesting basing on the status of hierarchical follicles status (before the anatomical observation, the geese were distinguished first by their nesting behaviors, such as nesting, foraging times and fluffy feathers).

Fig 2. Volcano map of differential gene expression.

The horizontal coordinates represent the fold changes of genes between the laying and initial nesting groups. The longitudinal coordinates represent the statistical significance of the changes in gene expression. The smaller the p value, the greater the value of -log₁₀p. Each dot in the image represents one gene; the blue ones indicate no significant difference in the gene, while the red ones indicate a significant difference. All the DEGs enriched in the hypothalamus, pituitary gland, stroma ovarii and walls of follicles with diameters of 8–10 mm between laying geese and early nesting geese are provided in S3–S6 Tables.

Fig 3. Validation of the sequencing data by qRT-PCR.

A-D, Validation of results for the hypothalamus, the pituitary gland, the stroma ovarii and the walls of follicles (diameter range 8–10 mm), respectively. E, The correlation coefficient between qRT-PCR and DEG data was analyzed based on all validated genes. The genes were selected randomly from S3–S6 Tables, and the primers designed for qRT-PCR are listed in S1 Table. For both the qRT-PCR and DEG results, the fold changes were calculated based on the expression levels in laying and nesting group and were then converted to log₂(Fold change).

Fig 4. KEGG enrichment scatter plot.

A-D, The enriched pathways based on DEGs distributed in the hypothalamus, the pituitary gland, the stroma ovarii and the walls of follicles (diameter range 8–10 mm); the top 20 enriched pathways, sorted by enrichment factor, in each comparison group. The enrichment factor was the ratio of the number of DEGs enriched in the pathway to the total number of all annotated genes enriched in this pathway.

Fig 5. Protein-protein interaction relationships based on the DEGs.

A, Table 4 lists the DEGs that were analyzed. B, Networks are based on the DEGs that code for secreted proteins in the hypothalamus (Table 4) and all DEGs from the pituitary gland (S4 Table). C, Networks based on the DEGs that code for secreted proteins in the hypothalamus and pituitary gland (Table 4) and all DEGs in the stroma ovarii and follicle wall (S4 and S6 Tables). The information on the interactions between the proteins was downloaded from the STRING database (http://string-db.org/).

Fig 6. Hierarchical clusters of DEGs.

A, Clustering of the DEGs listed in Table 4. B, Clustering of groups based on gene expression patterns among groups.

Fig 7. Regulatory processes within the HPGA that may take part in initiating broodiness in geese.