Identification and functional characterization of developmental-stage-dependent piRNAs in Tibetan sheep testes

The core function of the testes is to produce sperms, which is the prerequisite for maintaining male fertility. PIWI-interacting RNAs (piRNAs) are a class of non-coding small RNAs that are mainly enriched in the reproductive organ and play a key role in germ cell development and spermatogenesis. However, the expression and function of piRNAs in the testes of Tibetan sheep, a domestic animal endemic to the Tibetan Plateau, remain unknown. In this study, we evaluated the sequence structure, expression profile, and potential function of piRNAs in testicular tissues from Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years of age, respectively) by small RNA sequencing. Of the identified piRNAs, the sequence lengths of 24-26 nt and 29 nt dominate. Most piRNA sequences begin with uracil and have a distinct ping-pong structure which mainly distributes in exons, repeat regions, introns, and other unannotated regions of the genome. The piRNAs in the repeat region are primarily derived from the retrotransposons: long terminal repeats, long interspersed nuclear elements, and short interspersed elements. These piRNAs constitute 2,568 piRNA clusters, which mainly distribute on chromosomes 1, 2, 3, 5, 11, 13, 14, and 24, and of these clusters, a total of 529 piRNA clusters were differentially expressed in at least two age groups. Most of the piRNAs were expressed in a low abundance in the testes of developing Tibetan sheep. A total of 41,552 and 2,529 differential piRNAs were identified in testes from 3 months vs. 1 year, and 1 year vs. 3 years, respectively, presenting significantly increased abundance for most piRNAs in 1 year and 3 years compared with 3 months. The functional evaluation of the target genes showed that the differential piRNAs are mainly involved in regulating gene expression, transcription, protein modification, and cell development during spermatogenesis and testicular development. In conclusion, this study focused on the sequence structure and expression characteristics of piRNAs in the testis of Tibetan sheep and provided new insights into the functional mechanism of piRNAs in testicular development and spermatogenesis of sheep. This study unraveled the unique sets of piRNAs and their expression profiles for the first time, as well as potential functions in developmental Tibetan sheep. These findings broaden our knowledge on the complexity of transcriptional regulation during Tibetan sheep spermatogenesis, also providing novel and valuable information for further investigating the mechanism of spermatogenesis in sheep. Lay Summary The testis in mammals plays an indispensable role in male fertility, in which the development and function are strictly regulated by a variety of non-coding small RNAs. PIWI-interacting RNAs (piRNAs) are the most abundant non-coding small RNAs in mammalian testes, playing an irreplaceable role in testicular development and spermatogenesis. To characterize the piRNA expression and investigate their potential biological function in developmental Tibetan sheep testes, we carried out RNA sequencing. Our results revealed that the length distribution of the identified piRNAs is mostly 24-26 nt and 29 nt, exhibiting a preference for uracil at their 5' end and significant ping-pong structure. Most piRNAs were differentially expressed in Tibetan sheep testes in a development-age-dependent manner, and the vast majority of them were upregulated in postpubertal and adult testes. Based on the functional annotation of piRNA target genes, they were mainly implicated in the regulation of gene expression, transcription, protein modification and development during testicular development and spermatogenesis.

Automated summary

This study investigated the characteristics and function of piRNAs in the testes of Tibetan sheep. The results showed that piRNAs in Tibetan sheep testes have specific sequence structures and expression profiles. They play a crucial role in testicular development and spermatogenesis by regulating gene expression, transcription, protein modification, and cell development.