Flowzytometrische Untersuchung der Apoptose humaner Spermatozoen mit Hilfe des Annexin V-Tests

Aim of the study: The transfer of the phospholipid phosphatidylserine (PS) onto the outer membrane layer occurs during the early phase of apoptosis in human cells. The changes can be detected by the annexin V test and can be analyzed by flow cytometry. It was the aim of this study to analyze the influence of incubation of spermatozoa with several factors, on the outcome of the annexin V test and therefore apoptosis detection. The effects of incubation with fas-antibodies, with antisperm-autoantibodies (ASA) and with the calcium ionophore A23187 in order to induce acrosome reaction were investigated. Presence of fas-receptors, of high importance for apoptosis in somatic cells, on the membrane of spermatozoa was analyzed with conjugated fas-antibody and flow cytometry. Experimental Design: 1. After washing by centrifugation the spermatozoa were incubated for 4 hours with the fas-antibody clone EOS9.1 (19 semen samples) and CD95-PE 3.22 (17 semen samples), followed by another washing step and the annexin V test. The flow cytometric analyses focussed on direct binding of CD95-PE to fas receptors and on the rates of annexin V-binding after incubation with the fas clones. 2. 12 semen samples were prepared with the swim-up technique and incubated for 2 hours. Calcium ionophore A23187 was added for 15 minutes and after another washing step the annexin V test was performed. Flow cytometric analysis investigated rates of annexin V-binding, the number of acrosome-reacted spermatozoa after ionophore challenge (detected by conjugated CD46-FITC antibody binding to the inner acrosome membrane), and particular binding of annexin V to acrosome-reacted cells. 3. 2 semen samples were washed by centrifugation and 2 semen samples prepared with swim-up technique. These samples were incubated for 1 hour with ASA-containing seminal plasma or ASA-containig blood serum. Binding of the ASA to the spermatozoa was analyzed with the MAR-test and the samples were subsequently, after a further washing step and the annexin V test, analyzed for annexin V-binding and the results compared to the outcome of the MAR-test. All tests underwent flow cytometric analysis and all specimens were stained with the vital dye 7-Amino-Actinomycin D after incubation. Avital cells were excluded from analysis. Results: 1. A significant (p<0.001) but small amount of vital cells bearing fas receptors could be detected with a mean rate of fas bearing vital cells of all vital cells of 1.46%. No influence on the outcome of the annexin V test could be observed after incubation with the fas antibodies. 2. In the samples incubated with A23187 a mean rate of annexin V-positive vital cells of 42.17% was found after subtraction of the annexin V-positive rates of the control samples. Specimens that were simultaneously stained with CD46-FITC and phycoerythrin-conjugated annexin V after ionophore challenge could not be analyzed for annexin V-binding to acrosome reacted cells due to an unexpected shift of the main population into the avital area in the DotPlot-analysis. Vital spermatozoa that showed spontaneous acrosome reaction without ionophor challenge could be analyzed. A medium rate of 32.12% annexin V-binding was detected for these cells. 3. After incubation with ASA-containing seminal plasma or blood serum no influence whatsoever on the rate of annexin V-binding could be detected. Conclusions: 1. Only a very small amount of fas bearing spermatozoa could be detected. Fas antibodies, that are known to induce the apoptotic cascade in somatic cells, were not capable of having an influence on the rate of PS-expression indicative for early apoptosis. The fas pathway of apoptosis is obviously not functional in mature haploid spermatozoa. These findings support the theory that the main part of fas bearing germ cells is eliminated via apoptosis during spermatogenesis and residual fas receptors on mature spermatozoa are not functional. 2. Calcium ionophore treatment significantly increases the rate of vital spermatozoa detected by the annexin V test. This effect may be explained by an increased number of early apoptotic spermatozoa after ionophore challenge, implying that the ionophore bears a high potential of apoptosis induction in mature spermatozoa. Alternatively these findings may indicate a high amount of PS expression due to the typical membrane changes of the acrosome reaction. The latter explanation is supported by the fact that spermatozoa with spontaneous acrosome reaction showed increased binding to annexin V. Thus the annexin V test might be unsuitable for apoptosis detection in the haploid mature spermatozoa, as non-apoptotic changes in these cells can lead to PS-expression. 3. ASA do not seem to have any influence on the membrane changes of spermatozoa leading to increased PS-expression.