Figure 1.

Sperm Head Is Round and the Acrosome Is Absent or Atrophied and Misplaced in Globozoocephalic Spermatozoa

Panels (A–C) show control spermatozoa, (D–F) show globozoocephalic spermatozoa with no acrosome, and (G–I) show globozoocephalic spermatozoa with atrophied and misplaced acrosome.

(A, D, G) Confocal microscopy. Sperm were double stained with TopRo3 (blue), for evidencing the nucleus, and with lectins from Pisum sativum conjugated to fluorescein isothiocyanate (PSA-FITC, Sigma Aldrich, France) (green), for evidencing the acrosome. In control sperm, the acrosome is displayed as a bell surrounding the tip of the sperm head (A). In globozoocephalic sperm, the acrosome is absent (D) or atrophied and misplaced (G). Lectin from PSA-FITC was used to label the acrosomal matrix, and TopRo3 was used to label the nucleus. Slide cells were washed in PBS and fixed in 4% PFA for 30 min on ice. Fixed spermatozoa were washed in PBS for 3–5 min and incubated with PSA-FITC (10 μg/ml in PBS). Slides were analyzed on a Leica TCS-SP2 (Mannheim, France) confocal laser scanning microscope.

(B, E, H) Electronic microscopy. The different organites, acrosome (A), and nucleus (N) of control sperm are clearly identified (B). In globozoocephalic sperm, the acrosome is absent and the plasma membrane (PM) surrounds the nucleus (E). In some cells, redundant nuclear membrane (N Mb) is clearly evidenced (H). Sperm cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer of pH 7.4 for 2 hr at room temperature. Cells were washed with buffer and postfixed with 1% osmium tetroxyde in the same buffer for 1 hr at 4°C. Ultrathin sections of the cell pellet were cut with an ultramicrotome (Leica, Nanterre, France) after dehydration and epon inclusion. Sections were poststained with 4% uranile acetate and 1% lead citrate before being observed in a 80 kV electron microscope (JEOL 1200EX).

(C, F, I) Scanning microscopy. The surface of control sperm appears smooth (C), whereas that of globozoocephalic spermatozoa appears wrinkled (F) and with a probable remnant of atrophic acrosome (I). Transmission electron microscopy was performed as detailed previously.¹²