Syntaxin and b-actin interact with acrosome-expressing protein VAD1.2/AEP2 in spermatogenesis

Abstract

Spermatogenesis involves meiosis of the diploid spermatogonia to form the haploid spermatids and spermiogenesis to transform the round spermatids into mature spermatozoa. Normal fertilization requires the sperm to come into contact with the egg and release its enzymes from the head (acrosome). Acrosomal defects are known to cause infertility in human. To understand the molecular regulation of spermatogenesis in vivo, we used differential display RT-PCR to identify testis-specific genes in a retinol-supplemented vitamin A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressing protein 2, AEP2) gene, which was expressed strongly in the rat testis from postnatal day 32 to adult stage. However, the functional roles of this protein on acrosome formation during spermatogenesis remain elusive. The aims of this study were to (1) study the spatiotemporal expression of VAD1.2 during spermatogenesis and (2) study the interaction of this protein with their binding protein(s) in the mouse, rat and human testis. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 colocalized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with defective spermatogenesis. Co-immunoprecipitation using polyclonal VAD1.2 antibody followed by Western blotting and mass spectrometry (MS-MS) identified syntaxin-1, b-actin and myosin heavy chain (MHC) but not VAD1.3/AEP1, TRS4, SEC23, synaptotagmin and cyclophilin B as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin) and structural (b-actin and MHC) proteins suggest that VAD1.2 is involved in acrosome formation during spermiogenesis. Research supported by CERG HKU7537/05M and NSFC/RGC N_HKU712/06 to KFL.