Mitotracker mitochondria red cmxros probe

Mouse caudal epididymides from sexually mature males (12–16 weeks old) were excised, several slits were cut using iridectomy scissors, and the tissues were transferred to wells of a cell culture plate containing 2 ml of Biggers, Whitten and Whittingham (BWW) medium pre-warmed at 37°C.12 (link) After incubation for 5 min, the caudal epididymal sperm were collected and adjusted to 5 × 10⁶ – 10 × 10⁶ sperm ml⁻¹ for the following assays.
To determine the mitochondrial membrane potential of collected sperm, the samples were incubated with a pre-warmed staining solution of 250 nmol l⁻¹ MitoTracker mitochondria Red CMXRos probe (MITO; Molecular Probes) at 37°C for 10 min.5 (link) After washed twice with pre-warmed fresh PBS, sperm were then attached to poly-L-lysine-coated slides and dried at room temperature (RT) for the immunofluorescence double staining.
To block AKT activity in collected mouse sperm, the samples were treated for 3 h in a 5% CO₂ incubator at 37°C with different concentrations of PI3K inhibitor wortmannin (Cell Signaling Technology) at 0, 1, 5, 10, and 20 μmol l⁻¹, respectively. The viability of treated sperm was then evaluated using MitoTracker mitochondria Red CMXRos probe (MITO) as described above.5 (link) The level of sperm motility and phosphorylation levels of AKT and PHB were examined as described below.

All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Phosphate-buffered saline (PBS) solution was purchased from Ambion Inc. (Austin, TX, USA). MitoTracker mitochondria Red CMXRos probe (M7512) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Normal goat serum (NGS), mouse immunoglobulin G (IgG), rabbit IgG, and VECTASHIELD Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI; H1200) were from Vector Laboratories, Inc. (Burlingame, CA, USA). The PI3K inhibitor (wortmannin; #9951) was from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protein A/G PLUS-Agarose (sc-2003) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). 3-([3-Cholamidopropyl] dimethylammonio)-1-propane sulfonate (CHAPS), urea, thiourea, dithiothreitol (DTT), and acrylamide were purchased from Amersham Biosciences (Uppsala, Sweden). All buffers were prepared using Milli-Q water (Millipore, Bedford, MA, USA).