|AZEVEDO, HYMERSON - Embrapa|
|GRAHAM, JAMES - Colorado State University|
|MARQUEZ, MARIA - Colorado State University|
Submitted to: Animal Reproduction Science
Publication Type: Abstract Only
Publication Acceptance Date: 4/2/2018
Publication Date: 7/1/2018
Citation: Purdy, P.H., Azevedo, H.C., Graham, J.K., Marquez, M.A. 2018. Use of anti-phosphotyrosine and PDK1 monoclonal antibodies enables observation of the effects of media on bull sperm second messenger signaling during capacitation.. Animal Reproduction Science. 10.1016.
Technical Abstract: Quantitative evaluation of sperm capacitation and acrosome reaction can lead to a more robust methodology for sperm sample evaluation. Therefore, in an exploratory experiment, we incubated (39 °C) frozen-thawed sperm (n = 3 bulls) using in vitro fertilization medium (CFDM) or 10 µM calcium TALP. Flow cytometry assessed the physiologic changes during capacitation attributed to these media. Samples were evaluated every 60 minutes for 3 h to estimate the proportion of plasma membrane intact cells with: high intracellular calcium, acrosomal integrity, highly fluid plasma membranes, and cells that demonstrate protein tyrosine (PTYR) and PDK1 (PDK1) phosphorylation which are second messengers indicative of capacitation and acrosome reaction, respectively. Statistical analysis was performed with GLM-ANOVA to determine the effects of incubation media, time, their interaction, and bull on the physiologic characteristics. A significant model was derived for each of the sperm characteristics but bull was only a significant source of variation for PTYR (P = 0.05). Media and incubation time were significant sources of variation for all response variables (P < 0.04 and P < 0.05, respectively) except for PTYR. The media by time interaction was a significant source of variation for intracellular calcium and PDK1 (P < 0.0003). These media, selected because of their physiologic (CFDM) and non-physiologic (TALP) ability to induce capacitation and the acrosome reaction, induced capacitation, evidenced by their ability to increase intracellular calcium, plasma membrane fluidity, and acrosome react (39 and 48%, respectively, P = 0.003). Although both media allowed capacitation to occur, greater proportions of sperm exhibiting PTYR (19.9%; P = 0.009) and PDK1 (49.5%; P <0.0001) intracellular signaling were observed in TALP compared with CFDM (4.3 and 2.5%, respectively) suggesting these assays can provide valuable information about sperm quality and function. Furthermore, evaluation of second messenger signaling in this manner should enable assessment of sample fertilizing potential.