Polyvinylpyrrolidone pvp

Sperm preparation was essentially as previously described21 (link)22 (link). Briefly, cauda epididymidal sperm from 8~12-week-old males were triturated for 45 sec in nuclear isolation medium (NIM; 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 3.0 mM EDTA; pH 7.0) containing 1.0% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at room temperature (25°C). Sperm were washed twice in NIM and pelleted (1,890 g) at ambient temperature; head-tail detachment was enhanced by trituration during pellet resuspension. Sperm were resuspended in ice-cold NIM (~0.5 ml per epididymis) and stored at 4°C for up to 3 h until injection. Typically, ~50 μl of each suspension was mixed with 20 μl of polyvinylpyrrolidone (PVP, average M_r ≈ 360,000; Sigma, UK) solution (15% [w/v]) and sperm injected (ICSI) using a piezo-actuated pipette (Prime Tech, Japan) into oocytes in a droplet of M2 within ~60 min, essentially as described34 (link). Assuming an inner pipette tip diameter of 6 μm, we estimate that 2~2.5 pl were introduced per injection. After a brief (5~15 min) recovery period, injected oocytes were transferred to KSOM under mineral oil equilibrated in humidified 5% CO₂ (v/v air) at 37°C.

Following micro-CT analysis, femurs were decalcified in 14% EDTA (Sigma-Aldrich, Sydney, NSW, Australia) at 37 ℃ for 7 days, and then embedded into paraffin for sectioning. Hematoxylin and eosin (H&E) staining and TRAcP activity staining were performed. Section images were acquired using Aperio Scanscope (Mt Waverley, VIC, Australia), and bone histomorphometric analyses were performed using BIOQUANT OSTEO software (Bioquant Image Analysis Corporation, Nashville, TN, USA).
For in vivo ROS fluorescence detection, cryosections of bone tissues were prepared as previously described 39 (link). Fresh bone tissues were dissected, cleaned and immediately fixed in 4% paraformaldehyde (Sigma-Aldrich, Sydney, NSW, Australia) solution at 4 ℃ for 4 h. Next, decalcification was carried out with 0.5 M EDTA overnight at 4 ℃ under constant rotation, after which the EDTA was exchanged for cryoprotective solution composed of 20% sucrose and 2% polyvinylpyrrolidone (PVP) (Sigma-Aldrich, Sydney, NSW, Australia) for 24 h. Finally, the resultant tissues were embedded and frozen in 8% gelatin in the presence of 20% sucrose and 2% PVP. 5 μm sections were generated and air-dried at room temperature prior to permeabilization for 10 min in 0.3% Triton X-100 (Sigma-Aldrich, Sydney, NSW, Australia). Nuclei were stained with DAPI for 30 min. After washing with PBS, sections were mounted, and coverslips were sealed with nail polish. A NIKON A1Si confocal microscope (Nikon Corporation, Minato, Tokyo, Japan) was used for imaging. The fluorescence intensity of 6 random areas of each group were quantified and analyzed using NIS-Elements software (Nikon Corporation, Minato, Tokyo, Japan).