Author
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Submitted to: American Society of Andrology Meeting
Publication Type: Abstract Only Publication Acceptance Date: 12/10/2007 Publication Date: 10/20/2008 Citation: Purdy, P.H. 2008. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology. American Society of Andrology Meeting, Albuquerque, New Mexico, April 2008. Pp. 49. Interpretive Summary: Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples could be analyzed for PMP and plasma membrane integrity using flow cytometry. Fresh boar sperm samples (Fresh), diluted in BF5 cooling extender and cooled to 5ºC (Cooled), the same samples then diluted with the freezing extender diluent (FE), and after cryopreservation (Post-thaw) were evaluated. The fluorescence of the plasma membrane intact population was recorded for each sample. Increases in the fluroescence indicate greater fluorescence/greater amounts of detected protein. Analysis of the boar sperm PMP during the cryopreservation process demonstrated that detectable protein changed significantly due to temperature, diluent, cryoprotectant, and the freezing process. Analyses were performed to investigate the relationship between the detected protein in the previous analyses and sperm motilities/motion characteristics. PMP detected in the Fresh samples was correlated with post-thaw curvilinear velocity, lateral head amplitude, beat cross frequency, straightness and linearity. Similarly, PMP detected in the Cooled samples was correlated with post-thaw path velocity, curvilinear velocity, lateral head amplitude, beat cross frequency, straightness and linearity. In addition, the relationship of PMP with sperm physiology was also investigated. The frozen-thawed boar sperm samples were induced to capacitate and aliquots were removed at 60 min increments from 0 to 180 min. The samples were evaluated for PMP, PMI, and acrosomal integrity in separate flow cytometric analyses. Analyses revealed that greater amounts of detected PMP following cryopreservation results in significantly greater percentages of sperm that are plasma and acrosomal membrane intact when induced to capacitate. This research demonstrates the dynamic nature of the sperm plasma membrane during cryopreservation and through capacitation and the acrosome reaction. With additional research, this assay may potentially be adapted as a tool for indicating cryopreservation success and fertilizing potential. Technical Abstract: Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Yo-Pro 1 negative) using flow cytometry. Fresh boar sperm samples (Fresh), diluted in BF5 cooling extender and cooled to 5ºC (Cooled), the same samples then diluted with the freezing extender diluent (FE), and after cryopreservation (Post-thaw) were evaluated. The mean fluorescence of the plasma membrane intact population was recorded for each sample. Increases in the mean indicate greater fluorescence/greater amounts of detected protein. Analysis of the boar sperm PMP during the cryopreservation process demonstrated that detectable protein changed significantly due to temperature, diluent, cryoprotectant, and the freezing process. Analyses were performed to investigate the relationship between the detected protein in the previous analyses and sperm motilities/motion characteristics. PMP detected in the Fresh samples was correlated with post-thaw curvilinear velocity, lateral head amplitude, beat cross frequency, straightness and linearity. Similarly, PMP detected in the Cooled samples was correlated with post-thaw path velocity, curvilinear velocity, lateral head amplitude, beat cross frequency, straightness and linearity. In addition, the relationship of PMP with capacitation and the acrosome reaction was also investigated. The frozen-thawed boar sperm samples were induced to capacitate and aliquots were removed at 60 min increments from 0 to 180 min. The samples were evaluated for PMP (R640), PMI (Yo-Pro 1), and acrosomal integrity (FITC-PNA and propidium iodide) in separate flow cytometric analyses. Multiple regression analyses revealed that greater amounts of detected PMP following cryopreservation results in significantly greater percentages of sperm that are plasma (y = 25.24 + (3.86*R640 mean) + (-.06*Time) and acrosomal membrane intact (live and non acrosome reacted; y = 23.57 + (3.26*R640 mean) + (-.13*Time)) when induced to capacitate. This research demonstrates the dynamic nature of the sperm plasma membrane during cryopreservation and through capacitation and the acrosome reaction. With additional research, this assay may potentially be adapted as a tool for indicating cryopreservation success and fertilizing potential. |
