Comparative Transcriptomic Analysis of Hu Sheep Pituitary Gland Prolificacy at the Follicular and Luteal Phases

Abstract

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

Keywords: LHB; fecundity; follicular phase; lncRNA; luteal phase; pituitary gland.

Figure 1

LncRNA identification and classification in the pituitary gland. (A) The lncRNAs were identified by two prediction software programs, CNCI and CPC. (B) The number statistics of different types of lncRNAs.

Figure 2

Comparison analysis of lncRNAs and mRNAs in sheep pituitary gland. (A) The boxplot shows the sample expression levels (log₁₀ (FPKM)) of lncRNAs and mRNAs in the H-F and H-L groups. (B) The chromosome distribution of lncRNAs and mRNAs. (C) The length comparison of lncRNAs and mRNAs. (D) Exon content of lncRNAs and mRNAs. (E) Length of the open reading frame (ORF) of lncRNAs and mRNAs.

Figure 3

Comparisons of the number of DE lncRNAs and DE genes (DEGs) in H-F and H-L groups. (A) The hierarchical cluster of DE lncRNAs. (B) Total number of upregulated and downregulated DE lncRNAs in each comparison. (C) Total number of upregulated and downregulated DEGs in each comparison. (D) The hierarchical cluster of DEGs.

Figure 4

The verification of expression level of DE lncRNAs and DE mRNAs in different groups. (A) The relative expression level of four DE mRNAs and lncRNAs in different groups determined by RNA-Seq. (B) The relative expression level of four DE mRNAs and lncRNAs in different groups determined by RT-PCR (* p < 0.05; ** p < 0.01).

Figure 5

The top gene ontology (GO) and KEGG enrichment analysis. (A) The top 20 GO enrichment analysis of differential expressed genes (DEGs). (B) The top 20 KEGG enrichment analysis of DEGs.

Figure 6

The network of 61 screened DE genes. The network of 61 screened DE genes enriched for Hu sheep pituitary functions and reproduction-related pathways were constructed, the red and green circles represent upregulated and downregulated DE genes, respectively. Node size represents the fold change of a node.

Figure 7

43 DE lncRNAs and their 36 predictably interactive cis- and trans-targeted genes comprised this interactive network. The red and green colors represent up and downregulation, quadrilaterals represent lncRNA, the box represents targeted genes, the straight line and dotted line represent the interaction relationship of trans- and cis-regulation, respectively.

Figure 8

Identification of lncRNA XR_001039544.4. (A) lncRNA XR_001039544.4 localization in Hu sheep pituitary cells as detected by fluorescence in situ hybridization (FISH); scale bar, 20 μm. (B) Expression of lncRNA XR_001039544.4 in the Hu sheep hypothalamus, ovary and pituitary gland (HPO axis). (C,D) Expression of lncRNA XR_001039544.4 and its target gene LHB in different stages of Hu sheep pituitary gland. The data are shown as the mean ± SEM of at least three independent experiments. Statistical significance was analyzed by one-way ANOVA and Student t-test (** p < 0.01).

Figure 9

LncRNA XR_001039544.4 knockdown inhibits the secretion of gonadotropins in pituitary cells. (A,B) mRNA expression of lncRNA XR_001039544.4 and LHB in sheep pituitary cells treated with siRNAs were decreased compared to the negative control (NC), as determined by qPCR. (C) LH secretion was detected by ELISA. (E) mRNA expression of PCNA in lncRNA XR_001039544.4-knockdown pituitary cells. (D,F) Pituitary-cell proliferation was detected using EDU. Scale bars, 100 μm. The data are shown as the mean ± SEM of at least three independent experiments. Statistical significance was analyzed by one-way ANOVA and Student t-test (** p < 0.01).