Transcriptome Analysis During Follicle Development in Turkey Hens With Low and High Egg Production

Abstract

Low and high egg producing hens exhibit gene expression differences related to ovarian steroidogenesis. High egg producing hens display increased expression of genes involved in progesterone and estradiol production, in the granulosa layer of the largest follicle (F1G) and small white follicles (SWF), respectively, whereas low egg producing hens display increased expression of genes related to progesterone and androgen production in the granulosa (F5G) and theca interna layer (F5I) of the fifth largest follicle, respectively. Transcriptome analysis was performed on F1G, F5G, F5I, and SWF samples from low and high egg producing hens to identify novel regulators of ovarian steroidogenesis. In total, 12,221 differentially expressed genes (DEGs) were identified between low and high egg producing hens across the four cell types examined. Pathway analysis implied differential regulation of the hypothalamo-pituitary-thyroid (HPT) axis, particularly thyroid hormone transporters and thyroid hormone receptors, and of estradiol signaling in low and high egg producing hens. The HPT axis showed up-regulation in high egg producing hens in less mature follicles but up-regulation in low egg producing hens in more mature follicles. Estradiol signaling exclusively exhibited up-regulation in high egg producing hens. Treatment of SWF cells from low and high egg producing hens with thyroid hormone in vitro decreased estradiol production in cells from high egg producing hens to the levels seen in cells from low egg producing hens, whereas thyroid hormone treatment did not impact estradiol production in cells from low egg producing hens. Transcriptome analysis of the major cell types involved in steroidogenesis inferred the involvement of the HPT axis and estradiol signaling in the regulation of differential steroid hormone production seen among hens with different egg production levels.

Keywords: HPG axis; HPT axis; RNA-seq; egg production; estradiol; steroidogenesis; thyroid hormone; turkey.

FIGURE 1

Number of differentially expressed genes. Numbers of total, up-regulated in high egg producing hens (HEPH), and up-regulated in low egg producing hens (LEPH) differentially expressed genes (DEGs) between LEPH and HEPH in (A) the F1 granulosa, (B) the F5 granulosa, (C) the F5 theca interna, and (D) the small white follicles (RPKM > 0.2, P < 0.05). The portion of genes that are unannotated in the turkey genome are represented in dark gray and the portion of gene that are annotated in the turkey genome are represented in light gray.

FIGURE 2

Overview of differentially expressed genes. (A) Venn diagram showing the number of differentially expressed genes (DEGs) between low egg producing hens (LEPH) and high egg producing hens (HEPH) that are unique to the F1 granulosa, F5 theca interna, and small white follicles as well as the number of DEGs common to one or more follicle cell types (RPKM > 0.2, P < 0.05). (B) Heat map showing the expression profiles of the DEGs common to the F1 granulosa, F5 theca interna, and small white follicles displaying high expression in each of the follicle cell types examined (RPKM > 10, P < 0.05). Blue represents genes up-regulated in LEPH, whereas orange represents genes up-regulated in HEPH. (C) Venn diagram showing the number of DEGs in unique to the F1 granulosa and F5 granulosa as well as the number of DEGs common both the F1 granulosa and F5 granulosa (RPKM > 0.2, P < 0.05). (D) DEGs common to both the F1 granulosa and F5 granulosa displaying high expression in both follicle cell types broken down by expression pattern in LEPH and HEPH (RPKM > 10, P < 0.05).

FIGURE 3

Confirmation of RNAseq gene expression results. Confirmation of RNA sequencing results in the F1 granulosa (F1G), F5 granulosa (F5G), F5 theca interna (F5I), and small white follicles (SWF). A total of 6 genes common to all four tissues that displayed high expression and different expression patterns among the four tissues were selected for confirmation in each tissue through RT-qPCR. The expression differences (P < 0.05) between low egg producing hens (LEPH) and high egg producing hens (HEPH) for each gene seen in RNA sequencing results are listed above each graph. Normalized data are presented relative to LEPH expression for each follicle type for each gene. Significant expression differences between LEPH and HEPH for a given follicle type are denoted with an asterisk (*). (A) Relative mRNA levels for ceramide synthase 2 (CERS2), which was up-regulated in HEPH in all four tissues in RNA sequencing results. (B) Relative mRNA levels for insulin-like growth factor 1 receptor (IGFR1), which showed increased expression in HEPH in the F1G and SWF and increased expression in LEPH in the F5G in RNA sequencing results. (C) Relative mRNA levels for extended synaptotagmin 3 (ESYT3), which was up-regulated in HEPH in the F5I in RNA sequencing results (D) Relative mRNA levels for biogenesis of lysosomal organelles complex 1 subunit 4 (BLOC1S4), which showed increased expression in HEPH in the F1G, F5G, and F5I in RNA sequencing results. (E) Relative mRNA levels for beta-actin (ACTB), which showed up-regulation in HEPH in the F5I in RNA sequencing results. (F) Relative mRNA levels for phosphoglycerate kinase 1 (PGK1), which showed up-regulation in HEPH in the F5G and F5I in RNA sequencing results.

FIGURE 4

(A,B) F1 granulosa pathway analysis. Network analysis in the F1 granulosa comparing low egg producing hen (LEPH) and high egg producing hens (HEPH) gene expression (RPKM > 0.2, P < 0.05, | fold change| > 1.5) generated using Ingenuity^® Pathway Analysis (Qiagen, Valencia, CA). Blue represents genes up-regulated in LEPH, whereas orange represents genes up-regulated in HEPH. Solid lines represent direct relationships between molecules, whereas dotted lines represent indirect relationships between molecules.

FIGURE 5

(A,B) F5 granulosa pathway analysis. Network analysis in the F5 granulosa comparing low egg producing hens (LEPH) and high egg producing hens (HEPH) gene expression (RPKM > 0.2, P < 0.05, | fold change| > 1.5) generated using Ingenuity^® Pathway Analysis (Qiagen, Valencia, CA). Blue represents genes up-regulated in LEPH, whereas orange represents genes up-regulated in HEPH. Solid lines represent direct relationships between molecules, whereas dotted lines represent indirect relationships between molecules.

FIGURE 6

(A,B) F5 theca interna pathway analysis. Network analysis in the F5 theca interna comparing low egg producing hens (LEPH) and high egg producing hens (HEPH) gene expression (RPKM > 0.2, P < 0.05, | fold change| > 1.5) generated using Ingenuity^® Pathway Analysis (Qiagen, Valencia, CA). Blue represents genes up-regulated in LEPH, whereas orange represents genes up-regulated in HEPH. Solid lines represent direct relationships between molecules, whereas dotted lines represent indirect relationships between molecules.

FIGURE 7

(A,B) Small white follicle pathway analysis. Network analysis in the small white follicles comparing low egg producing hens (LEPH) and high egg producing hens (HEPH) gene expression (RPKM > 0.2, P < 0.05, | fold change| > 1.5) generated using Ingenuity^® Pathway Analysis (Qiagen, Valencia, CA). Blue represents genes up-regulated in LEPH, whereas orange represents genes up-regulated in HEPH. Solid lines represent direct relationships between molecules, whereas dotted lines represent indirect relationships between molecules.

FIGURE 8

Impact of thyroid hormone on estradiol production. Estradiol production in small white follicle cells from low egg producing hens (LEPH) and high egg producing hens (HEPH) after pretreatment with no pretreatment (NPT) or thyroid hormone (T₃) followed by treatment with follicle stimulating hormone (FSH). Significant expression differences between LEPH and HEPH for a given condition are denoted with an asterisk (*) (P < 0.05). Significant differences between FSH treatments for a given egg production group are denoted with a dagger (†) (P < 0.05). Significant differences between pretreatments for a given egg production group are denoted with a double dagger (‡) (P < 0.05).