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

## TL;DR

This study compares microRNA profiles in the cauda epididymis of yaks and cattle to understand how these molecules regulate sperm maturation in high-altitude environments.

## Contribution

The study identifies species-specific miRNA signatures in yaks that may regulate sperm maturation in extreme environments.

## Key findings

- 31 differentially expressed miRNAs were identified in yak compared to cattle cauda epididymis.
- Eight miRNAs were predicted to target genes involved in sperm motility, flagellum formation, and chromatin remodeling.
- qPCR confirmed inverse correlations between miRNA expression and target gene levels.

## Abstract

Yak is an essential livestock species found on the Tibetan Plateau. Compared to animals that primarily inhabit plains, yak have a unique sperm maturation process tailored to their reproductive needs in high-altitude environments. The cauda epididymis is crucial for sperm development and is susceptible to post-transcriptional regulation by microRNAs (miRNAs). To investigate the regulatory dynamics of yak sperm maturation, miRNAs from this tissue in both yak and cattle were sequenced. A total of 497 miRNAs were identified across the two species. Comparative analysis revealed 31 miRNAs that were differentially expressed between them. Among these, 16 predicted target genes associated with eight miRNAs were found to be potentially linked to the structural or functional maturation of sperm. The expression of some miRNAs was validated, revealing an opposite trend compared to their corresponding predicted target genes. This study provides foundational data and new research perspectives for further understanding the gene regulatory network underlying yak sperm maturation in extreme environments.

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.