Molecular characteristics and regulatory role of insulin-like growth factor 1 gene in testicular Leydig cells of Tibetan sheep

Abstract

This study aimed to analyze the molecular characteristics of insulin-like growth factor 1 (IGF1) gene in the testes of Tibetan sheep and its role in the testosterone synthesis and cell development. First, we cloned IGF1 gene for bioinformatics analysis, and the primary Leydig cells (LCs) of Tibetan sheep were isolated to explore its effect on the proliferation, apoptosis and function of LCs. Finally, the specific regulatory mechanism of IGF1 on LCs was analyzed by transcriptome sequencing. Results showed that overexpression of IGF1 increased the proliferation rate and decreased apoptosis of LCs. In addition, overexpression of IGF1 altered expression of genes related to testosterone synthesis and transformation and significantly increased amount of the final product testosterone. Mechanistically, IGF1 stimulated the expression of the proliferating cell nuclear antigen and IGF1R and promoted the proliferation of LCs via the PI3K/Akt signaling pathway. Collectively, what should be clear from the results reported here is that IGF1 might play roles in the proliferation or differentiation and testosterone synthesis of LCs. These findings add to our understanding on the regulation of testosterone synthesis in sheep and other mammals.

Keywords: Insulin-like growth factor 1; Leydig cell; Testosterone; Tibetan sheep.

Figure 1

Cloning results of Tibetan sheep IGF1 gene. (A) PCR amplification products of the Tibetan sheep IGF1 coding sequence (CDS). M, DL2000 marker; 1–2, PCR product. (B) Alignment of NCBI nucleotide sequence and cloned nucleotide sequence of Tibetan sheep IGF1. (C) Amino acid composition. (D) Multiple alignment of IGF1 amino acid sequences from 6 different mammalian species. (E) The amino acid composition. (F) Secondary molecular structure. Different lines with different colors denote different secondary structures: blue, alpha helix; red, extended strand; green, beta turn; purple, random coil. (G) Tertiary molecular. (H) Neighbor-joining phylogenetic tree based on nucleotide sequences of IGF1 gene among different mammals. Original blots are presented in Supplementary Fig. S2.

Figure 2

Identification of LCs, evaluation of recombinant plasmid and detection of transfection efficiency. (A) The primary Leydig cells of Tibetan sheep. (B) PCR results of HSD3B1 and GAPDH gene. (C) Immunofluorescence staining of primary LCs of Tibetan sheep (200 μm). (D) Evaluation of recombination plasmids. (E-G) Detection of transfection efficiency. Original blots are presented in Supplementary Fig. S2.

Figure 3

Effect of IGF1 gene silencing and overexpression on the proliferation, apoptosis, and cycle of LCs. (A) CCK-8 detects the cell proliferation rate after overexpression and silencing of IGF1. (B) Flow cytometry was used to detect the apoptosis of LCs after overexpression and silencing of IGF1. (C) The effect of overexpression and silencing of IGF1 gene on the expression of proliferation, cycle, and apoptosis related genes. (D, E) The expression of IGF1R mRNA and protein. Original blots are presented in Supplementary Fig. S3.

Figure 4

The effect of IGF1 on testosterone content of Leydig cells. (A) Testosterone content in culture medium and intracellular. Graphic area represents testosterone content. (B, D) Changes in expression of HSD11B1, CYP11A1, HSD3B1, SRD5A2, HSD17B3, and STAR after overexpression or silencing of IGF1 gene. (C) Western blot analysis of SRD5A2, HSD17B3, CYP11A1 and β-actin expression in the four groups, original blots are presented in Supplementary Fig. S3.

Figure 5

Differential Genes expression profiles. (A) Volcano plot showing DEGs expression in two groups. (B) Cluster analysis of DEGs in two groups. (C) Correlation heat map in two groups.

Figure 6

The GO and GSEA annotation of DEGs in the two groups. (A) GO enrichment terms. (B) KEGG enrichment terms. (C-F) GSEA enrichment result maps in two groups, respectively. BP: biological process; CC: cellular component; MF: molecular function.

Figure 7

Identification of testosterone synthesis related genes and validation of RNA-Seq by RT-qPCR analysis. (A) The PPI network of gene sets enriched on testosterone synthesis-related pathways. (B) The gene-pathway network according to cholesterol synthesis and androgen synthesis-related genes and their enriched pathways. (C) OE and NC represent pcDNA3.1(+) -IGF1 and LCs, respectively.