Application of auxin-induced protein degradation technique in<italic>Caenorhabditis</italic><italic> elegans</italic> spermatogenesis

Abstract

<p indent="0mm">SAC-1, a widely expressed PI4P phosphatase, is an essential PI signal regulator for <italic>Caenorhabditis</italic><italic> elegans</italic> different developmental stages. In order to study its specific functions for <italic>C</italic>.<italic> elegans</italic> spermatogenesis,<italic> </italic>the auxin-induced protein degradation (AID) method was employed to cause the temporal-spatial degradation of SAC-1 in developing sperm. Degron and GFP coded sequences were inserted into the endogenous <italic>sac-1</italic> locus to generate Degron::GFP::SAC-1-expressing <italic>C</italic>.<italic> elegans</italic> strain using the CRISPR/Cas9 gene-editing technique. Then, <italic>TIR1</italic>, encoding the plant-specific F-box protein, driven by the germline-specific promoter<italic> sun-1</italic>, was introduced to construct the AID strain. The sperm motility was measured by sperm activation imaging<italic> in vitro</italic> and fluorescently labeled male sperm localization in hermaphrodite after mating. In addition, we analyzed the fusion of membranous organelles (MOs) with the plasma membrane during sperm activation and examined worm fertility. The results showed that the degradation of SAC-1 in gonads affected sperm motility. The auxin treatment of AID-SAC-1 worms caused sperm to extend shorter pseudopod with less fused MOs. Furthermore, the fertility of AID-SAC-1 worms treated with auxin was significantly decreased. In conclusion, auxin-induced SAC-1 tissue-specific degradation was successfully achieved, and the loss of SAC-1 in gonads attenuated sperm activation and sperm motility. This study provides a new method and insight for studying the functions of more widely expressed developmental regulatory proteins in specific tissues such as germline.