LncRNA THOR regulates spermatogenesis by interacting with and stabilizing Sycp3 protein in zebrafish†

Testis-associated highly conserved oncogenic long non-coding RNA (THOR) is a highly conserved and testis-enriched long non-coding RNA (lncRNA) across vertebrates that plays diverse roles in various cancers. However, its physiological function and regulatory mechanism in testes remain largely unknown. Here, we investigated the genomic location and expression pattern of THOR in the model organism zebrafish, and generated a homozygous THOR knockout model using clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 technology. Loss of THOR in zebrafish impaired spermatogenesis, leading to oligospermia (38.7% reduction in sperm count), reduced sperm motility, sperm ultrastructural defects, and decreased fertilization rates. RNA sequencing (RNA-seq) analysis of wild type (WT) and THOR knockout testes revealed dysregulation of cell cycle-related genes, including cyclin-dependent kinase inhibitor 1D (cdkn1d), forkhead box O1 a (foxo1a), TSC complex subunit 1a (tsc1a), TSC complex subunit 2 (tsc2), ATRX chromatin remodeler (atrx), and RAD21 cohesin complex component b (rad21b). RNA pulldown assays in zebrafish testes identified 486 potential THOR-interacting proteins primarily involved in ribosome biogenesis, RNA splicing, chromatin architecture, and meiotic progression. Notably, the core synaptonemal complex components synaptonemal complex protein 1 (Sycp1), synaptonemal complex protein 2 (Sycp2), and synaptonemal complex protein 3 (Sycp3) were all captured as THOR-binding partners. We further demonstrated that THOR directly interacts with Sycp3 and positively regulates its protein levels. Immunostaining assays on chromosome spreads revealed a significantly higher frequency of discontinuous Sycp3 signals in THOR-/- testes, suggesting the presence of meiosis defects caused by Sycp3 downregulation. Our findings expand the understanding of lncRNA-mediated control of spermatogenesis and male infertility by providing the first evidence that lncRNA THOR interacts with the synaptonemal complex to regulate meiosis progression.