Western Blotting To study the expression profile of scyreprocin, total proteins extracted from the tissues of adult male and female crabs (= 3) were submitted to the Tricine-SDS-PAGE assay and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham, Sunnyvale, CA, USA). and sperm AR. To our knowledge, this is the first report around the direct involvement of AMPs in sperm AR, which would expand the current understanding of the roles of AMPs in reproduction. in our previous study . It is male specific and dominantly expressed in the ejaculatory duct (ED). During mating, SCY2 showed cross-gender transmission and was thought to exert reproductive immune functions . Interestingly, the expression level of SCY2 is found significantly induced by PG, but not by lipopolysaccharides (LPS) . Those previous results led us to presume whether SCY2 may not only exert antimicrobial activity but also have multiple functions in reproductive processes, especially in hormone-modulated post-mating events. Meanwhile, a novel SCY2-interacting protein, scyreprocin (“type”:”entrez-nucleotide”,”attrs”:”text”:”MH488960″,”term_id”:”1682038632″,”term_text”:”MH488960″MH488960), was revealed Rabbit Polyclonal to RBM26 and proved to exert potent, broad-spectrum antibacterial, antifungal, and antibiofilm activities in vitro by multiple action modes . Unexpectedly, recombinant products of SCY2 and scyreprocin showed no synergistic antimicrobial activity in vitro . Considering both AMPs were dominantly expressed in the male reproductive system, whether their synergistic functions were reflected in other aspects of the reproduction Prosapogenin CP6 processes as reported in other animals drawn us to explore their potential physiological roles in the present study. In this work, the in vivo expression profiles of scyreprocin and SCY2 were investigated to confirm their roles in the reproductive immunity of mud crab. Interestingly, we found that both scyreprocin and SCY2 were also expressed in sperm, and their localizations were dynamically changed during the AR. To better understand sperm AR of mud crab, we used flow cytometry and Ca2+ fluorescent probes to investigate whether PG was one of the ARIS of crab Prosapogenin CP6 sperm and to assess the change in Ca2+i during AR. The potential functions of scyreprocin and SCY2 in AR were further explored by antibody blockade assays. In Prosapogenin CP6 addition, the binding properties of scyreprocin and SCY2 to PG and Ca2+ were evaluated via functional experiments to further verify their roles in PG-induced AR. 2. Results 2.1. Expression Pattern of Scyreprocin and SCY2 In Vivo Under natural conditions, the scyreprocin transcript was predominantly expressed in male gonads, with the highest expression level in testes, followed by anterior vas deferens, while relatively low expression was observed in female crabs, with the highest expression in ovaries (Physique 1A). High levels of scyreprocin protein expression were detected in gonads of adult male crabs, while no scyreprocin expression was detected in adult female crabs. (Physique 1B). In juvenile male crabs, scyreprocin was mainly expressed in spermatophores isolated from testes and seminal plasma isolated from ED (Physique 1C). In adult males, scyreprocin was detected in the spermatophore, and seminal plasma was collected from anterior vas deferens, seminal vesicle, ED, and posterior ejaculatory duct (Physique 1C). Detection of in situ expression indicated that scyreprocin was mainly expressed in spermatophores and epithelial cells of the testis, while SCY2 was expressed in interspaces between spermatophores in the testes of Prosapogenin CP6 adult mud crabs (Physique 1D). Only weak signals of both proteins were detected in testicular sections of juvenile males (Physique 1D). Scyreprocin and SCY2 were not detected in spermathecae of pre-mating females. In post-mating females, scyreprocin and SCY2 signals were detected in the contents of spermathecae, moreover, strong SCY2 fluorescent signals were observed in epithelial cells (Physique 1E and Physique S1). Open in a separate window Physique 1 Scyreprocin and SCY2 expressed in reproductive system of adult male mud crabs and transferred to female spermathecae via mating. (A) Scyreprocin transcriptional expression level in adult male (= 3) and female (= 3) under natural conditions. Data are presented as the mean standard deviation (SD). * 0.05, one-way analysis.