Identification and Functional Analysis of miRNAs in the Cauda Epididymis of Yak and Cattle

Abstract

The yak represents a distinct domestic animal species that predominantly inhabits the Qinghai-Tibet Plateau and adjacent areas, possessing considerable value in both scientific and economic contexts. Compared to animals that mainly dwell on plains, such as cattle, the sperm maturation process in yak exhibits a certain degree of species specificity to adapt to their unique reproductive needs in high-altitude environments. Serving as the main storage site for functionally competent sperm, the cauda epididymis plays an integral role in mediating their post-testicular maturation. MiRNAs are vital regulatory molecules in the epididymis, influencing sperm maturation by modulating gene expression after transcription. To investigate the unique regulatory mechanisms of sperm maturation in yak, this study compared the miRNA expression profiles in the cauda epididymis of yak and cattle using high-throughput small RNA (sRNA) sequencing. The comparative analysis identified and characterized sRNA populations in the cauda epididymis of yak and cattle, revealing a similar length distribution that peaked at 22 nt and a predominance of known miRNAs. Notably, eight miRNAs were found to be highly expressed in both species. Furthermore, the first-nucleotide bias differed significantly between known and novel miRNAs within each species. A total of 31 differentially expressed (DE) miRNAs were identified, with 11 upregulated and 20 downregulated in yak compared to cattle. Among these, bta-miR-1298 exhibited the most significant upregulation, while bta-miR-2344 displayed the most pronounced downregulation. Bioinformatic analysis linked the predicted target genes of these miRNAs to numerous critical signaling pathways, including calcium signaling, the mitogen-activated protein kinase (MAPK) signaling pathway, the Ras-associated protein 1 (Rap1) signaling pathway, and the cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway. Furthermore, eight significantly DE miRNAs, including bta-miR-2443, bta-miR-503-3p, bta-miR-6517, bta-miR-2440, bta-miR-2431-3p, bta-miR-2436-3p, bta-miR-6523a, and bta-miR-6775, were predicted to target genes involved in various aspects of sperm structural and functional maturation. These aspects include flagellum formation, sperm motility, chromatin remodeling, acrosome reaction, acrosome structure, sperm capacitation, chemotaxis, and nuclear chromatin condensation. Multiple miRNAs and their corresponding predicted target genes were analyzed by quantitative real-time PCR (qPCR), demonstrating an inverse correlation between miRNA expression and target gene levels. These findings reveal a distinct, species-specific miRNA signature in the yak cauda epididymis, which suggests a potential contribution to regulating the epididymal luminal environment and the process of sperm maturation. This study provides preliminary foundational data for elucidating the differences in sperm maturation mechanisms between yak and cattle, and offers potential novel targets for improving reproductive efficiency in plateau livestock.

Keywords: cauda epididymis; high-throughput sequencing; microRNA; yak.

Figure 1

Identification of miRNAs in the cauda epididymis of yak and cattle. (A) sRNA length distribution. (B) Annotation and classification of sRNAs. (C) Top 10 most abundant miRNAs. Among these, the miRNA indicated by the green bar was highly expressed in both species, whereas those denoted by blue bars exhibited high expression exclusively in either yak or cattle.

Figure 2

Nucleotide composition of 18–25 nt miRNAs in the cauda epididymis of yak and cattle. (A,B) First nucleotide bias performance of known miRNAs in yak and cattle. (C,D) First nucleotide bias performance of novel miRNAs in yak and cattle.

Figure 3

DE miRNAs in the cauda epididymis of yak and cattle. (A) Volcano plot illustrating the comparison of all miRNAs between yak and cattle. The Y-axis represents −log₁₀(adjusted p-value) and the X-axis represents log₂ fold change. Red dots depict upregulated miRNAs, green dots depict downregulated miRNAs, and blue dots represent non-significant miRNAs. (B) Heatmap displaying miRNA expression profiles across samples. Columns represent different samples, and rows represent different miRNAs. The clustering was based on the lg (TPM + 1 × 10⁻⁶) value. Color scale ranges from blue (low expression) to red (high expression), with white indicating intermediate levels. (C) Top 10 most significantly DE miRNAs. The Y-axis represents log₂ fold change.

Figure 4

KEGG pathway enrichment analysis of target genes for DE miRNAs in the cauda epididymis of yak and cattle. (A) Bar plot displaying the Top 20 most significantly enriched pathways, with pathway names shown on the y-axis and the number of target genes annotated in each pathway indicated above the bars. (B) Bubble chart summarizing the enrichment results, where the y-axis lists pathway names and the x-axis represents the ratio of target genes mapped to a pathway relative to the total number of DE genes.

Figure 5

Expression detection of several DE miRNAs and their target genes related to spermatogenesis in the cauda epididymis of yak and cattle. (A–D) The levels of four distinct DE miRNAs—bta-miR-6517, bta-miR-2443, bta-miR-2431-3p, and bta-miR-505-3p—were measured and adjusted to the reference U6. (E–H) Expression detection of predicted target genes corresponding to the miRNAs in (A–D), including SPEF2 (target of bta-miR-6517), UCP2 (bta-miR-2443), MMP2 (bta-miR-2431-3p), and TEKTIP1 (bta-miR-503-3p), and adjusted to the reference GAPDH. Statistical significance at different thresholds was denoted as follows: * p < 0.05 and ** p < 0.01. Comparisons were analyzed using Student’s unpaired t-test.