Figure 4. Apoptotic sperm cells are efficiently phagocytosed in the presence of fibrils.
(A) Incubation of spermatozoa for 24 hr at 25°C increases the proportion of apoptotic sperm cells. Spermatozoa from fresh ejaculates were purified by the swim-up technique and then assessed immediately for cell surface expression of the apoptotic marker Annexin V by flow cytometry, or incubated for 24 hr at 25°C prior to staining and analysis. Results are representative of 3 independent donors. Presented are flow cytometric plots showing the percentages of Annexin-negative (non-apoptotic) and Annexin-positive (apoptotic) spermatozoa as indicated. (B) Results from experimental triplicates of each condition described in panel A. *p<0.05 (two-tailed Student’s t test). Results are representative of 3 independent donors. (C) Phagocytosis of fresh spermatozoa or spermatozoa incubated for 24 hr at 25°C. Motile spermatozoa purified by the swim-up method were labeled with eFluor 670 and then fed immediately to macrophages or incubated for 24 hr at 25°C to induce apoptosis prior to incubation with macrophages. Assays were conducted in the presence or absence of 100 µg/ml SEM fibrils, and in all cases phagocytosis was allowed to proceed for 0.5 hr prior to flow cytometric analysis. Macrophages were identified by gating on CD14+CD33+ cells, and phagocytosis was assessed by determining the percentages of macrophages that were eFluor 670+. Results are representative of data from three different donors.