Study Of Sperm Plasma Membrane Translocation Of Phosphatidylserin, Mitochondrial Transmembrane Potential And Plasma Membrane Scrambling

Successful fertilization requires a sperm with normal integrity plasma membrane and fine mitochondrial transmembrane potential. The numerous functions of the membrane and mitochondrial transmembrane potential are related to cell metabolism, for maintaining sperm motility, capacitation, acrosome reaction and sperm-oocyte interaction. Therefore, the identification of plasma membrane and mitochondrial dysfunctions, sensitive and early markers of programmed cell death in somatic cells, might allow us to gain further insight into this process and its clinical significance in human ejaculated spermatozoa.One of the objectives of the present study was to examine mitochondrial loss of transmembrane potential in fertile donors and in subfertile patients. We therefore compared the effect of Rhl23/PI dual fluorescent staining and JC-1 fluorescent staining related to mitochondrial loss of transmembrane potential. To evaluate the feasibility of two methods on analysis seminal quality by flow cytometry. We found there was a significant correlation not only between mitochondrial membrane potential and sperm motility, "a" sperm and "a+b" sperm. On the other hand,we studied the relation between PS externalization and disturbances of mitochondrial transmembrane potential in human sperm, a significant correlation was found.At last, we collected eight normal sperm samples and divided the samples into two purified subpopulation of sperm with high (90%) and low (45%) motility by discontinuous Percoll gradient (90% and 45% layers) centrifugalization. The ram sperm and both fractions were further subdivided into two aliquots, and one aliquot was incubated with 0.3% HSA of BWW, the other aliquot was incubated with 3% HSA of BWW for 0, 1, 3, 6 and 12 hours respectively. The PS externalization, loss of mitochondrial transmembrane potential and plasma membrane scrambling were evaluated at different times. We found low motility sperm were characterized by a statistically significant presence of PS externalization, loss of mitochondrial transmembrane potential and plasma membrane scrambling when compared with ram samples. But the phenomenon was opposite in high motility sperm population. Futhermore, increased the percentage of necrosis sperm was observed in vitro incubation by a time-dependent manner, however, no change was observed with PS externalization, loss of mitochondrial transmembrane potential and plasma membrane scrambling.These results demonstrate that ejaculated spermatozoa lack the capacity of initiating the apoptotic pathway of cell death, and their demise occurs mainly by necrosis, at least under in 0.3% HSA of BWW and in 3% HSA of BWW conditions.