Comparative Transcriptomics Reveals the Key lncRNA and mRNA of Sunite Sheep Adrenal Gland Affecting Seasonal Reproduction

Abstract

The hypothalamic-pituitary-adrenal (HPA) axis plays an important role in the growth and development of mammals. Recently, lncRNA transcripts have emerged as an area of importance in sheep photoperiod and seasonal estrus studies. This research aims to identify lncRNA and mRNA that are differentially expressed in the sheep adrenal gland in long (LP) or short (SP) photoperiods using transcriptome sequencing and bioinformatics analysis based on the OVX + E₂ (Bilateral ovariectomy and estradiol-implanted) model. We found significant differences in the expression of lncRNAs in LP42 (where LP is for 42 days) vs. SP-LP42 (where SP is for 42 days followed by LP for 42 days) (n = 304), SP42 (where SP is for 42 days) vs. SP-LP42 (n = 1,110) and SP42 vs. LP42 (n = 928). Cluster analysis and enrichment analysis identified SP42 vs. LP42 as a comparable group of interest and found the following candidate genes related to reproductive phenotype: FGF16, PLGF, CDKN1A, SEMA7A, EDG1, CACNA1C and ADCY5. FGF16 (Up-regulated lncRNA MSTRG.242136 and MSTRG.236582) is the only up-regulated gene that is closely related to oocyte maturation. However, EDG1 (Down-regulated lncRNA MSTRG.43609) and CACNA1C may be related to precocious puberty in sheep. PLGF (Down-regulated lncRNA MSTRG.146618 and MSTRG.247208) and CDKN1A (Up-regulated lncRNA MSTRG.203610 and MSTRG.129663) are involved in the growth and differentiation of placental and retinal vessels, and SEMA7A (Up-regulated lncRNA MSTRG.250579) is essential for the development of gonadotropin-releasing hormone (GnRH) neurons. These results identify novel candidate genes that may regulate sheep seasonality and may lead to new methods for the management of sheep reproduction. This study provides a basis for further explanation of the basic molecular mechanism of the adrenal gland, but also provides a new idea for a comprehensive understanding of seasonal estrus characteristics in Sunite sheep.

Keywords: HPA axis; candidate gene; photoperiod; seasonality; sheep.

Figure 1

Identification of lncRNAs and mRNA in sunite sheep adrenal gland. (A) The regions of identified long non-coding RNAs (lncRNAs) in short photoperiod for 42 days (SP42), long photoperiod for 42 days (LP42), and short photoperiod for 42 days and turn to long photoperiod for 42 days (SP-LP42). (B) The exon number of lncRNA and mRNA. (C) The results of novel lncRNA predictions by using CNCI, CPC, PFAM, and CPAT software tools. (D) The length of lncRNA and mRNA.

Figure 2

The histogram of DELs and DEMs in different comparable groups. (A) DELs (B) DEMs. Where red and green represent up- or down regulation, respectively.

Figure 3

The differentially expressed lncRNAs and mRNAs in each sample using Heat maps. According to a base logarithm of 2 of expression about DEMs and DELs in each sample and the Euclidean distance was calculated, and then the overall clustering results of the samples were obtained by systematic clustering method (Hierarchical Cluster), (A) lncRNAs, (B) mRNAs.

Figure 4

Validation of RNA-Sequencing (RNA-Seq) data using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Different types of rectangles represent different light period processing. The figure ** and *** represents the p value ≤ 0.05 and 0.01, respectively. (A) RT-qPCR, (B) RNA-seq.

Figure 5

Histogram of GO enrichment of target gene of DELs. Heat maps showing the GO items enriched in the three comparison groups of LP42 vs. SPLP42, SP42 vs. LP42 and SP42 vs. SPLP42.

Figure 6

KEGG pathway enrichment analysis of target gene of DELs. The significant enriched KEGG pathways in the three comparison groups of LP42 vs. SPLP42, SP42 vs. LP42 and SP42 vs. SPLP42.

Figure 7

lncRNA-mRNA interaction networks. The interaction networks of lncRNAs and their corresponding target genes in SP42 vs. LP42, where the solid and dashed lines represent trans- and cis-regulation functions, respectively; red and green represent up- and downregulation, respectively; and round rectangle, V and triangle represent mRNAs, novel lncRNAs and known lncRNA, respectively.