Fitc pna

After the male mice were anesthetized, their epididymal tails were removed, cut into pieces with ophthalmic scissors, and placed in PBS. The liquid was placed in a 37°C incubator for 10 min for the spermatozoa to swim up. Using a large pipette, the liquid containing the spermatozoa was aspirated and placed in a centrifuge tube. After centrifugation at 1000 × g for 1 minute, the supernatant was aspirated, an appropriate amount of PBS was added to mix and wash the spermatozoa, and the samples were centrifuged again at the same speed and time as before. Washed spermatozoa were diluted with PBS to adjust the final sperm concentration to 1×10⁴ spermatozoa/ml. Add 20 µl of the diluted liquid to each smear so that the number of sperm on each smear is 200. Then, the cells were fixed with 4% PFA and stained with 0.5 mM PNA-FITC (L7381, Sigma‐Aldrich) and DAPI (D9542, Sigma‐Aldrich) as previously described. The stained spermatozoa were then covered with a cover slip (24 × 50 mm) (20 (link)).

For each data point, a minimum of 500 sperm per straw were assessed using previously described methods (Lieberman et al., 2016 (link)). Briefly, frozen semen straws were thawed in a water bath set at 35 °C for 30 s. Semen was then transferred to a 1.7-mL microcentrifuge tube and incubated at 35 °C for 1 h. Twenty microliters of semen solution was spread on a microscope slides and air-dried for 15 min. The slides were fixed by immersion in absolute methanol for 15 min. Slides were rinsed in PBS baths twice for 5 min each, transferred to a bath containing 25 μg/mL PNA-FITC (Sigma, St. Louise, MO) for 30 min, and rinsed in three phosphate buffered saline baths for 5 min each. Slides were gently dried using compressed air. One drop of Fluoroshield with DAPI Histology Mounting Medium (Sigma, St. Louise, MO) was then applied to each slide and acrosome integrity was measured using fluorescence microscopy.

3-Morpholinosydnonimine (SIN-1) and dihydrorhodamine 123 (DHR 123) probe were from ENZO Life Science Inc. (Farmingdate, NY, USA); Propidium iodide (PI), SYBR-14, and M540 and YoPro-1 probes were purchased from Molecular Probes (Leiden, The Netherlands); PNA-FITC and calcium ionophore A23187 were from Sigma-Aldrich (St Louis, MO, USA); C11-BODIPY 581/591 (4,4-difluoro-5- (4-phenyl-1,3-butadienyl)-4- bora-3a, 4a-diaza-s-indacene-3-undecanoic acid)and JC-1 probes from Life Technologies Ltd (Grand Island, NY, USA); coulter isotone II diluent from Beckman Coulter Inc. (Brea, CA, USA); DC ™ Protein Assays and 2× Laemmli Sample Buffer from Bio-Rad (Hercules, CA, USA). ECL detection kit and LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit (L10120) were from Thermo Scientific (Rockford, USA). Furthermore, the anti-phospho (Ser/Thr) PKA Substrate (#9624) and anti-phospho (Ser21/9) GSK3α/β (#9331) polyclonal antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA); the anti-α-tubuline antibody (TU-02, #SC-8035) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All reagents used to prepare incubation media were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Spermatozoa were collected from the caput, corpus, and cauda epididymis of adult mice. Immunofluorescence was enhanced as previously described [36 (link)]. Briefly, sperm cells on slides were fixed with 4% PFA, incubated with 0.5% Triton X-100, and blocked with 10% goat serum (AR1009, Boster Biological Technology Co., Ltd., Wuhan, China). The slides were then incubated with antibody against ATP1B3 (1:100; ab231671, Abcam, Cambridge, MA, USA) at 4 °C overnight, and anti-rabbit Alexa Fluor 568 (1:1000; ab175471, Abcam, Cambridge, MA, USA) was used as the secondary antibody for incubation at 37 °C for 1 h. Peanut agglutinin (PNA) is a lectin that binds specifically to the outer acrosome membrane of sperm [37 (link)]. Here, FITC-labeled PNA (PNA-FITC; 1:2000, L7381, Sigma-Aldrich, St. Louis, MO, USA) was used to stain the sperm acrosome and co-incubated with secondary antibodies. Finally, sperm nuclei were revealed using 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, D9542, Sigma-Aldrich, St. Louis, MO, USA) stain at 37 °C for 10 min. Images were acquired with confocal laser scanning microscopy (Leica, Heidelberg, Germany).