mRNA-Seq of testis and liver tissues reveals a testis-specific gene and alternative splicing associated with hybrid male sterility in dzo

Abstract

Alternative splicing (AS) plays an important role in the co-transcription and post-transcriptional regulation of gene expression during mammalian spermatogenesis. The dzo is the male F1 offspring of an interspecific hybrid between a domestic bull (Bos taurus ♂) and a yak (Bos grunniens ♀) which exhibits male sterility. This study aimed to identify the testis-specific genes and AS associated with hybrid male sterility in dzo. The iDEP90 program and rMATS software were used to identify the differentially expressed genes (DEG) and differential alternative splicing genes (DSG) based on RNA-seq data from the liver (n = 9) and testis (n = 6) tissues of domestic cattle, yak, and dzo. Splicing factors (SF) were obtained from the AmiGO2 and the NCBI databases, and Pearson correlation analysis was performed on the differentially expressed SFs and DSGs. We focused on the testis-specific DEGs and DSGs between dzo and cattle and yak. Among the top 3,000 genes with the most significant variations between these 15 samples, a large number of genes showed testis-specific expression involved with spermatogenesis. Cluster analysis showed that the expression levels of these testis-specific genes were dysregulated during mitosis with a burst downregulation during the pachynema spermatocyte stage. The occurrence of AS events in the testis was about 2.5 fold greater than in the liver, with exon skipping being the major AS event (81.89% to 82.73%). A total of 74 DSGs were specifically expressed in the testis and were significantly enriched during meiosis I, synapsis, and in the piRNA biosynthesis pathways. Notably, STAG3 and DDX4 were of the exon skipping type, and DMC1 was a mutually exclusive exon. A total of 36 SFs were significantly different in dzo testis, compared with cattle and yak. DDX4, SUGP1, and EFTUD2 were potential SFs leading to abnormal AS of testis-specific genes in dzo. These results show that AS of testis-specific genes can affect synapsis and the piRNA biosynthetic processes in dzo, which may be important factors associated with hybrid male sterility in dzo.

Keywords: alternative splicing; dzo; male sterility; splicing factor.

Figure 1.

Disorder of testis-specific gene expression in dzo. (A) Principal component analysis of gene expression differences between the liver and testis tissues of domestic cattle, yak, and dzo. (B) Standard deviations of 21,043 genes. (C) The elbow rule determines the optimal K value of the top 3,000 genes using K-means with the largest difference. (D) Heat map of the aggregation of differentially expressed genes between the tissues of cattle, yak, and dzo. (E) Functional enrichment of A–F cluster genes. (F) The scatterplot compares the differences in gene expression levels between dzo and its parental species (cattle, x-axis; yak, and y-axis). The green box contains the scatter plot of testicular tissue gene expression levels.

Figure 2.

Analysis of alternative splicing events and differential alternative splicing genes. (A) Five types of alternative splicing (AS) events. (B) Five types of AS differentially splicing genes (DSG) in the liver tissue samples. (C) Five types of AS DSGs in testis tissue samples. (D) Venn plots of DSGs showing gene clusters C, D, E, and F in dzo testis tissues. The numbers represent the number of genes in each category. (E) Enrichment analysis of the GO biological process clusters of C and D overlapping differential genes (ODG). (F) Enrichment analysis of the GO biological processes of ODGs in clusters E and F. (G) The expression of ODGs (STAG3, DMC1, KASH5, GPAT2, DDX4, and TDRD9) involved in synapsis and piRNA biosynthetic process in testicular tissue of cattle, yak and dzo. (H) The KEGG pathways of DSGs occurring in the testis of dzo, compared with cattle and yak. (I) The mRNA expression of DSGs in the liver and testis tissues of dzo, compared with cattle and yak. The x-axis represents dzo and cattle; the y-axis represents dzo and yak.

Figure 3.

Splicing factor difference analysis and correlation analysis. (A) Volcano plot of the differences in splicing factors (SF). Red indicates upregulation and blue indicates down-regulation in dzo testis. Green circles show overlapping differential genes (ODG) for SFs. (B) Basic characteristics of SFs: DSG_SF refers to AS level differences in SFs; DEG_SF refers to gene level differences in SFs (left); the expression of 7 SFs with different mRNA levels and AS levels (right). (C) Expression patterns of genes in purified mouse spermatogenic cell populations: 2C, most contained somatic cells; LZ, leptotene and zygotene spermatocytes; PS, pachytene spermatocytes; RS, round spermatids. Data from da Cruz et al. (2016). (D) ODGs correlation network diagram for SFs and clusters E–F in dzo testis tissue. The colors represent log₂fold change: red indicates upregulation of gene expression; blue indicates downregulation. The inner circle shows ODGs and the outer circle shows differential SFs. The green area in the outer circle shows the ODGs of SFs. (E) Sequence alignment of DDX4 and MYBL1 full-length transcripts and splicing variants (left); agarose gel electrophoresis of genes and the β-actin internal reference (right). (F) mRNA expression levels of DDX4 and MYBL1 full-length transcripts and splicing variants based on reverse transcription-quantitative PCR. The full-length transcripts and splice variants of DDX4 and MYBL1 are represented by DDX4-Full, DDX4-ES, MYBL1-Full, and MYBL1-ES, respectively.