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Sperm mobility is defined as the net movement of a sperm population against resistance. Previous research demonstrated that the cell-permeant Ca2+ indicator rhod-2 AM gave a fluorescent signal predominantly in the head region of fowl sperm that differed between sperm mobility phenotypes. This thesis served to explore the role of intracellular calcium in high and low mobility fowl sperm using fluorimetric techniques. Experiments were performed with the cell-permeant Ca2+ indicator fluo-3 AM. Confocal microscopy revealed two distinct staining patterns of fluo-3 AM in fowl sperm. Eighty-nine percent of high mobility sperm cells were stained solely in the midpiece and sixty percent of low mobility sperm cells were stained throughout the entire cell. Both confocal microscopy and flow cytometry showed that there was no difference in fluo-3 AM signal between mobility phenotypes (p = 0.18, p = 0.11). A posteriori analysis revealed that there was a difference in midpiece volume between cells with different fluo-3 AM distributions (p <0.0001). Cells stained solely in the midpiece, had larger midpieces than those stained throughout the whole cell. This research demonstrates that fluo-3 AM targets Ca2+ in fowl sperm differently than rhod-2 AM and that fluo-3 AM can be used as an indicator of mitochondrial Ca2+ content in fowl sperm. These findings suggest that mitochondrial Ca2+ content is equivalent between sperm cells of differing mobility while Ca2+ in the perinuclear space acts as a supply for mitochondrial Ca2+ cycling. Future research must address the questions of why fluo-3 AM shows a different staining pattern than rhod-2 AM, how intracellular Ca2+ moves between compartments, and whether or not there is a direct relationship between fluo-3 AM distribution and sperm cell function. |
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