Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing

Abstract

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms from 5592 gene loci, and 8761 isoforms from 5115 gene loci in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis- expressed genes in pigs. At the gene locus level, we found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing (AS) events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long non-coding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Long- and short-read RNA sequencing from five reproductive organs of boar

“…The Banna mini-pig inbred line (BMI) was developed through rigorous selection and continuous inbreeding using the Diannan small-ear (DSE) pigs via a full-sibling or parent-offspring mating strategy 1 , 2 . Due to its high degree of homozygosity in the genome, BMI serves as a valuable experimental resource for biomedical research and as an organ donor for xenotransplantation 1 .…”

“…Due to its high degree of homozygosity in the genome, BMI serves as a valuable experimental resource for biomedical research and as an organ donor for xenotransplantation 1 . However, after more than 20 generations of inbreeding 2 , the reproductive capacity of boars decreases, thereby limiting the propagation and utilization of BMI. Male fertility requires the coordination of various organs of the male urogenital system, such as the testis, epididymis, and male accessory glands (vesicular gland, prostate gland, and bulbourethral gland), which secrete seminal plasma responsible for regulating capacitation and acrosomal exocytosis 3 , 4 .…”

Integrated single cell transcriptome sequencing analysis reveals species‐specific genes and molecular pathways for pig spermiogenesis

“…Next, at five different developmental timepoints, testicular somatic cells were extensively examined and compared with human scRNA‐seq data sets (Zhang, Guo, et al., 2022; Zhang, Ma, et al., 2022). On the testis and epididymis of the Banna mini‐pigs, single‐molecule real‐time sequencing identified alternative splicing and long noncoding RNAs (Wang, Zhang, et al., 2023). Spermiogenesis refers to the metamorphosis and maturation of spermatids, and is vital to the spermiation process and male fertility.…”