Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization

Figure 1 dataset.zip In Figure 1A transgenic EGFP-DsRed2 sperm were used. Images were taken with a Fluorescence spinning disk confocal microscope (Olympus IX83-DSU) and a 10X objective (UPLSAPO, NA 0.4) with a sCMOS camera (Andor, Zyla). The exposure time was 500 msec for both EGFP and DsRed2. The acquisition frequency was 1 image every 1 min, for 15 min. The filters used were: for EGFP, U-FBWA Ex 460-495 nm /Em 510-550 nm and for DsRed2, U-FGWA Ex 530-550 nm/Em 575-625 nm. In Figure 1B transgenic EGFP-DsRed2 sperm were used. Images were taken with a Eclipse TE 300 Nikon microscope with a 40X oil immersion objective (PlanApo N, NA 1.42) with EMCCD camera (Andor Technology, Belfast, UK). The exposure time was 500 msec. The acquisition frequency was 2 images every 1 sec for 5 min. The filter used were: Led cyan (3.15 A, Luminus Devices, Woburn, MA) Bandpass, excitation filter (HQ 480 nm /40X), dichroic mirror (Q505lp), and emission filter (HQ 535 nm /50M, Chroma Technology, Bellows Falls, VT). In Figure 1D transgenic EGFP-DsRed2 sperm were used. Images were taken with a Fluorescence spinning disk confocal microscope (Olympus IX83-DSU) and a 10X objective (UPLSAPO, NA 0.4) with a sCMOS camera (Andor, Zyla). The exposure time was 500 msec for both EGFP and FM4-64. The acquisition frequency was 1 image every 1 min, for 15 min. The filters used were: for EGFP, U-FBWA Ex 460-495 nm /Em 510-550 nm and for FM4-64, ETCY5 Ex 590-650 nm/Em 665-735 nm. Cells were stained with FM4-64 10 µM. In Figure 1E, 1F and 1G left panel transgenic EGFP-DsRed2 sperm were used. Images were taken with a Nikon (Melville, NY,USA)-inverted microscope (Eclipse Ti-U) and a 60X oil immersion objective (plan Apo TIRF DIC, NA 1.45, Nikon) with an Andor Ixon 3 EMCCD camera model DU-8970-C00#B (Andor Technology, Belfast UK) or Hammamatsu Orca-100 CCD model C4742-95 (Hammamatsu Photonics, Bridgewater, NJ USA). The exposure time was 100 msec for FM4-64, 350 msec for EGFP. The acquisition frequency was 1 image every 2 sec, for 5 min. The filters used were: for EGFP, filter set GFP 96343, dichroic mirror (D): 495 nm, Ex 470 nm/40, barrier 525 nm/50 (Nikon); for FM4-64, the filter set Wide Green 11007v2, D: 565 nm dcxt, Ex 535 nm/50, Em 590 nm lpv2 (Chroma Technology Corporation) Cells were stained with FM4-64 10 µM. Figure 2 dataset.zip In Figure 2A and 2B CD1 sperm were used. Images were taken with a NanoImager S microscope (Oxford Nanoimaging Ltd), equipped with a 100X oil-immersion objective (N