BACKGROUND: The testis is a key reservoir of long non-coding RNAs, yet their physiological roles in male reproduction remain debated. Notably, long non-coding RNAs 1700101O22Rik (22Rik) and 1700027A15Rik (15Rik) are uniquely expressed in the mouse testis. Previous research indicates that both 22Rik and 15Rik play roles in male reproductive processes
however, it is still unclear whether their effects on fertility are cumulative or compensatory. OBJECTIVES: To investigate the influence of simultaneous deletion of 22Rik and 15Rik on male reproduction and whether there are additive effects. MATERIALS AND METHODS: 22Rik and 15Rik knockout mice were generated using CRISPR-Cas9, and double knockout mice were obtained through co-caging. To investigate reproductive phenotypes, we utilized computer-aided sperm analysis, acrosome reaction assessments, in vitro fertilization techniques, and sperm morphology analysis. Additionally, RNA sequencing and RNA binding protein immunoprecipitation were employed to explore the regulatory mechanisms of 22Rik and 15Rik. RESULTS: The simultaneous deletion of 22Rik and 15Rik led to abnormal sperm morphology, impaired acrosome reaction, and reduced in vitro fertilization. Sperm count and fertility were also decreased in double knockout male mice. Compared to the knockout of long non-coding RNA 22Rik, reproductive abnormalities were somewhat exacerbated but largely similar to those observed with 15Rik knockout alone because of shared targeted genes, particularly Y chromosome-linked genes. Additionally, these abnormal phenotypes may be linked to reduced expression of transition protein 1 and dysfunction of the HSF2‒Rik22‒Rik15 complex in double knockout mice. DISCUSSION AND CONCLUSION: Our study demonstrates for the first time that simultaneous knockout of these two long non-coding RNAs adversely affects sperm morphology and function by disrupting the HSF2‒Rik22‒Rik15 complex. Moreover, many overlapping regulated genes suggest that 22Rik and 15Rik may share similar regulatory mechanisms at the molecular level. This research sheds light on the causes and mechanisms behind sperm malformation and impaired male fertility.