Use of a mitochondrial-specific live sperm stain to confirm penetration of equine oocytes after in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is an important research and clinical tool. However, work with standard in vitro fertilization (IVF) is needed to explore sperm capacitation and the early events of fertilization. Repeatable methods for equine IVF have not yet been reported. Definitive evidence of fertilization, such as presence of the sperm tail within the cytoplasm, is critical when assessing potential methods for IVF. We have previously stained live sperm with the mitochondrial-specific stain, MitoTracker, and successfully visualized the sperm tail in the oocyte cytoplasm after ICSI. In the present study, we evaluated whether staining sperm with MitoTracker would A) be compatible with maintenance of sperm motility in culture and B) allow visualization of sperm tails within the COC or oocyte after co-culture with sperm. Semen was collected from one fertile stallion, and 200 µl of extended semen was layered under 1 mL of MOPS-buffered medium containing 10 or 100 nM Mitotracker Red CMXRos, in a 5-mL tube. The tube was incubated at a 45ᵒ angle at 38 °C for 20 min. The uppermost 750 µL of the medium was collected, washed, centrifuged, and the pellet resuspended in modified TALP. Sperm were added to 50-µL droplets of modified TALP to a concentration of 1 million sperm/mL and these were incubated in 5% CO₂ in air. Sperm motility was assessed visually after 1, 6 and 22 h in culture. At 22 h, sperm were removed, counterstained with DAPI, and evaluated under fluorescence microscopy. In some replicates, in vitro-maturedequine cumulus-oocyte complexes (COCs) were added to the sperm droplets and co-incubated for 1 to 9 h. To assess sperm progress toward oocyte penetration, oocytes were denuded, fixed, and counterstained with DAPI. Some COCs were stained intact for confocal assessment of chromatin, sperm mitochondria, and acrosome status. These were fixed, permeabilized with 0.1% Triton-X 100, labeled with fluorescein-conjugated peanut agglutinin, then stained with Hoechst 33342. A minimum of three replicates were performed with each MitoTracker concentration. MitoTracker-stained sperm showed progressive motility after 1, 6 and 22 h incubation (∼80%, 50%, and 15% respectively) but at 22 h appeared to have a higher proportion of immotile sperm, and less vigorous motility, than did unstained sperm (∼25% progressive at 22 h). Staining with 10 nM MitoTracker supported more vigorous motility than did 100 nM. Both concentrations enabled clear visualization of the sperm tail on fluorescence microscopy. In penetrated oocytes, the sperm tail was clearly visualized in the oocyte cytoplasm, associated with sperm chromatin. On confocal microscopy, acrosomal ghosts were visualized at the periphery of the zona pellucida, indicating that sperm underwent the acrosome reaction in response to the zona. We conclude that staining of live sperm with 10 nM MitoTracker during swim-up provides a simple method to allow confirmation of sperm penetration of oocytes after sperm-oocyte coincubation.