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 (NA 1.4, Olympus) and a sCMOS camera (Hamamatsu ORCA-Flash4.0 V3 Digital CMOS camera). The exposure time was 10 msec. The acquisition frequency was 100 images every 30 sec for 20 min. For excitation a 561 nm laser was used, the emission was collected in channel 1 (665-705 nm). Cells were stained with FM4-64 0.5 μM. In Figure 2G transgenic EGFP-DsRed2 sperm were used. Images were taken with a Zeiss LSM880 microscope equipped with an 63X oil-immersion objective (NA 1.4) and a EMCCD Andor iXon 897 camera. The laser power was: for DsRed 5%, for EGFP 1.2% and for Alexa Fluor 647 24%. Immunofluorescence was performed using ɑ-GLUT3 1:100 and Alexa Fluor 647 1:200. Figure 4 dataset.zip In Figure 4A and 4C hybrid F1 (C57BL/6 × Balb/C) 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 500-200 msec for both Fluo4 and FM4-64. The acquisition frequency was 1 image every 10 sec, for 9 min. The filters used were: for Fluo4, the 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 and Fluo4 1 µM. In Figure 4C CD1 sperm were used. Images were taken with a NanoImager S microscope (Oxford Nanoimaging Ltd), equipped with an 100X oil-immersion objective (NA 1.4, Olympus) and a sCMOS camera (Hamamatsu ORCA-Flash4.0 V3 Digital CMOS camera). The exposure time was 10 msec. The acquisition frequency was 100 images every 30 sec for 20 min. The filters used were: for excitation of Fluo4 a 473 nm laser was used, the emission was collected in channel 0 (band 1: 498-551 nm); for excitation of FM4-64 a 561 nm laser was used, the emission was collected in channel 1 (665-705 nm). Cells were stained with FM4-64 0.5 µM and Fluo4 1 µM. Figure 5 dataset.zip In Figure 5A and 5B CD1 sperm were used. Images were taken with a NanoImager S microscope (Oxford Nanoimaging Ltd), equipped with an 100X oil-immersion objective (NA 1.4, Olympus) and a sCMOS camera (Hamamatsu ORCA-Flash4.0 V3 Digital CMOS camera). The exposure time was 10 msec. The acquisition frequency was 100 images every 30 sec for 20 min. The filters used were: for excitation of FM4-64 a 561 nm laser was used, the emission was collected in channel 1 (665-705 nm); for excitation of SiR-actin a 640 nm laser was used, the emission was collected in channel 1 (665-705 nm). Cells were stained with FM4-64 0.5 µM and SiR-actin 0.1 µM. Figure 6 dataset.zip In Figure 6 transgenic EGFP-DsRed2 sperm were used. Images were taken with a Fluorescence spinning disk confocal microscope (Olympus IX83-DSU) and a 20X (UPLSAPO, NA 0.75) or 40X magnification objective (UPLSAPO, NA 0.95) with a sCMOS camera (Andor, Zyla). The exposure time was 10 msec for DIC, 50 msec for Hoechst, 120 ms for EGFP and DsRed2* and 500 msec for FM4-64. The acquisition frequency was 1 image every 5 µm with a total of 10-15 planes. The filters used were: for Hoechst 33342, LF405-B Ex 368-415 nm/Em 426-477 nm; for EGFP, U-FBWA Ex 460-495 nm/Em 510-550 nm; for DsRed2*, U-FGWA Ex 530-550 nm /Em 575-625 nm and for FM4-64 ETCY5 Ex 590-650 nm/Em 665-735 nm. Cells were stained with FM4-64 10 µM and Hoechst 33342 10 µg/ml. Figure 7 dataset.zip In Figure 7 transgenic EGFP-DsRed2 sperm were used. Images were taken with a Fluorescence spinning disk confocal microscope (Olympus IX83-DSU) and a 10X (UPLSAPO, NA 0.4) or 20X magnification objective (UPLSAPO, NA 0.75) with a sCMOS camera (Andor, Zyla). The exposure time was 10 msec for DIC, 50 msec for Hoechst, 120 msec for EGFP and DsRed2-FM4-64 and 500 msec for FM4-64. The acquisition frequency was 1 image every 1 min for 60-120 min and 1 image every 7 µm with a total of 10-15 planes. The filters used were: for Hoechst 33342, LF405-B Ex 368-415 nm/Em 426-477 nm; for EGFP, U-FBWA Ex 460-495 nm/Em 510-550 nm; for DsRed2*, U-FGWA Ex 530-550 nm /Em 575-625 nm and for FM4-64 ETCY5 Ex 590-650 nm/Em 665-735 nm. Sperm and eggs were stained with FM4-64 10 µM and eggs with Hoechst 33342 1 µg/ml. Figure 8 dataset.zip In Figure 8 hybrid F1 (C57BL/6 × Balb/C) sperm were used. Images were taken with a fluorescence spinning disk confocal microscope (Olympus IX83-DSU) and a 20X magnification objective (UPLSAPO, NA 0.75) with a sCMOS camera (Andor, Zyla). The exposure time was 10 msec for DIC, 40 msec for Hoechst, 300 msec for Fluo4 and 100 msec for FM4-64. The acquisition frequency was 1 image every 1 min for 60-120 min and 1 image every 7 µm with a total of 10-15 planes. The filters used were: for Hoechst 33342, LF405-B Ex 368-415 nm /Em 426-477 nm; for Fluo4, U-FBWA Ex 460-495 nm /Em 510-550 nm and for FM4-64, U-FGWA Ex 530-550 nm /Em 575-625 nm. Sperm were stained with Fluo4 1 µM, eggs were stained with Hoechst 33342 1 µg/ml and both sperm and eggs were stained with FM4-64 10 µM.