Expression of serine protease AR2 in mouse sperm and assessment of its participation in fertilization

MANJON, ANIA ANTONELLA; VERÓN, GUSTAVO LUIS; DUEK, PAULA; LANE, LYDIE; VAZQUEZ-LEVIN, MÓNICA HEBE

Congreso; Reunión Anual de Sociedades de Biociencias 2022; 2022

Proteases play important roles in biological processes, among them in mammalian fertilization. Human AR2 is an inactive testis-specific serine protease identified using an algorithm developed by our group and members of Swiss Institute of Bioinformatics. We have previously reported AR2 expression in human sperm and its role in sperm-oocyte interaction. This study aimed to begin AR2 characterization in the mouse model. Mouse AR2 is localized in chromosome 8; the gene contains 6 exons and 5 introns, and encodes 5 transcripts and 3 protein isoforms (>95% identity) that lack the catalytic triad residues and depict an overall high homology with the human isoforms (83%). To characterize its expression and biological role in mouse sperm, two commercial polyclonal anti-human AR2 antibodies were used (Ab1 towards N-term, Ab2 towards C-term). Protein alignment confirmed >85% homology of antibodies antigen sequences and the mouse isoforms. AR2 was immunolocalized in fixed acrosome-intact non-capacitated (NC) and capacitated (C) sperm, and in A23187 (10 µM) acrosome-reacted (AR) sperm. Specific immunolocalization patterns were: Ab1=Acrosomal region + Flagellum (A+F) and Flagellum (F) (NC=100% A+F; C= 16% A+F and 84% F; AR=100% F). Ab2= Head (H), head depicting a highly intense signal in the Acrosomal region (H+A), patchy head (P) and no signal (N) (NC and C=21% H, 79% H+A; AR= 21% H, 50% P, 29% N). AR2 was also immunodetected in NC live sperm: Ab1= H and F; Ab2= H. In vitro fertilization (IVF) of cumulus-oocyte complex was impaired when sperm were pre-incubated with Ab1 (46% inhibition; p