Amyloid Structures Localize in the Acrosome of Ejaculated Bull Spermatoza Carmen Gorder and Nathaly Cormier University of Wisconsin – Whitewater, Department of Biology Introduction Amyloids are aggregated proteins, with specific cross-beta-sheet structures, typically associated with neurodegenerative diseases such as Alzheimer.1 Recent studies in mice revealed the presence of amyloid structures within the acrosomal matrix (AM) of epididymal spermatozoa, along with zona pellucida binding-proteins 2, suggesting a physiological role for during the early events of mammalian fertilization. However, previous information cannot be directly transposed in other mammalian species due to biochemical and structural modifications that sperm cells undergo following ejaculation.3 In this study, we hypothesized that amyloid structures will localize in the acrosome of ejaculated bull spermatozoa. Conclusion Results obtained by fluorescence microscopy revealed that amyloid structures localized over the acrosomal region of ejaculated bull spermatozoa. Following sperm cell fractionation, the detection of amyloids structures in disrupted acrosomes using the aggresome dye and the labelling with the PNA-FITC suggests that amyloids are present in the AM core. Future Experiments Examine the biological function of amyloids during the acrosome reaction using conformation-dependent antibodies to detect both mature and immature forms of amyloid proteins with indirect immunofluorescence. Investigate the interactions between amyloids and other proteins found within the acrosomal matrix and their role in mammalian fertilization using specific antibodies with indirect immunofluorescence. Methods Semen Preparation, Mechanical Disruption of Sperm Acrosomes and Isolation of Acrosomal Matrix (AM) Bull semen was washed twice (280 x g, 10 min) in a phosphate-buffered saline + protease inhibitor cocktail (DPBS+PI). A differential centrifugation procedure was used to mechanically disrupt the acrocromes and isolate AM. 2 The supernatant solutions from centrifugations at 4ºC (12,000 x g for 10 min; 500 x g for 10 min) contained the total AM, and the resulting pellet contains spermatozoa with disrupted acrosomes. Isolation of AM Core Previous pellet was incubated in 20 mM sodium acetate (pH 3) + 1% SDS (37ºC, 15 min), then centrifuged (42,000 x g, 5 min, 25ºC). Resulting supernatant (S1) was centrifuged at high speed (250,000 x g, 30 min, 25ºC), resulting in a pellet (P2) containing the extracted total AM. Pellet (P2) was incubated in 20 mM sodium acetate (pH 3) + 5% SDS, then centrifuged at high speed as above, resulting in a pellet (P3) containing the AM core while the supernatant (S3) contains the AM shroud. Fluorescence Microscopy Sperm samples (±acrosomes) in DPBS and AM (shrouds and core) were spread on a slide, dried, and fixed in 100% methanol (10 min, RT). Sperm and AM were labelled using peanut agglutinin conjugated to fluorescein isothiocyanate PNA-FITC (0.1 mg/mL in PBS; 15 min in the dark, RT) 4, and visualized using an upright fluorescent microscope and Triple Filter set. A specific fluorescent dye (Proteostat Aggresome Detection Kit, Enzo Life Sciences, NY) was used to localize endogenous amyloids according to the manufacturer’s instructions.5 Amyloids were visualized using an upright fluorescent microscope and Triple Filter set. http://www.uww.edu/icit/services/ga-labs#tab_ServiceRequest ). You can request media printing to send the converted file for approval before printing. Allow 5 working days between contacting gaps lab and having to use the poster. If you want your poster laminated, it may take a bit longer. Acknowledgements University of Wisconsin-Whitewater Undergraduate Research Program for their financial support. Ganser Biology Research Award for additional funding. Dr. Steve Tardif (International Center for Biotechnologies, Mount Horeb) for providing the bull semen. References Fowler, D. M., Koulov, A. V., Alory-Jost, C., Marks, M. S., Balch, W. E., & Kelly, J. W. (2005). “Functional Amyloid Formation within Mammalian Tissue.” PLoS Biol, 4(1), e6. Guyonnet, B., Egge, N., & Cornwall, G. A. (2014). “Functional Amyloids in the Mouse Sperm Acrosome.” Molecular and Cellular Biology, 34(14), 2624-2634. Knobil, E., & Neill, J. D. (2006). Knobil and Neill's Physiology of Reproduction(Vol. 2). Gulf Professional Publishing. Tardif S., Sirard M. A., Sullivan R., Bailey J.L. (1999) “Identification of Capacitation-Associated Phosphoproteins in Porcine Sperm Electroporated with ATP-gamma-(32)P.” Mol. Reprod. Dev. 54: 292-302. Usmani, S. M., Zirafi, O., Müller, J. A., Sandi-Monroy, N. L., Yadav, J. K., Meier, C., ... & Nilsson, K. P. R. (2014). “Direct Visualization of HIV-Enhancing Endogenous Amyloid Fibrils in Human Semen.” Nature Communications, 5.