Microinjection of antisense oligonucleotides into living mouse testis enables lncRNA function study

Area of Science:

• Molecular Biology
• Genetics
• Reproductive Biology

Background:

• Long non-coding RNAs (lncRNAs) are involved in various cellular processes.
• lncRNAs are abundant in mammalian testes, but their functions are largely unknown.

Purpose of the Study:

• To establish an efficient method for lncRNA knockdown in mouse testes.
• To investigate the function of the lncRNA Tsx in spermatogenesis.

Main Methods:

• Development of an antisense oligonucleotides (ASOs)-based targeting approach for lncRNA knockdown.
• Microinjection of ASOs into mouse seminiferous tubules to deplete Tsx.
• Comparison of ASOs with small interfering RNAs (siRNAs) for knockdown efficiency and duration.

Main Results:

• ASOs efficiently reduced Tsx levels in both nuclear and cytoplasmic compartments of testicular cells.
• The knockdown effect of ASOs persisted for at least 10 days.
• ASOs demonstrated superior depletion of nuclear Tsx and longer-lasting effects compared to siRNAs.
• Tsx depletion led to increased germ cell apoptosis and a specific loss of meiotic spermatocytes.
• No overall impact on meiosis or male fertility was observed.

Conclusions:

• ASOs are an effective tool for functional interrogation of lncRNAs in spermatogenesis.
• The study characterized the lncRNA Tsx and its role in male germ cell development.