Semen samples were allowed to liquefy for 20 min at room temperature. Seminal plasma, containing exosomes, was separated from the cell fraction by centrifugation at 1000 ×g for 10 min at 20°C. Cell debris was removed by subsequent centrifugation at 2400×g for 30 min at 20°C followed by 0.45 and 0.22 μm syringe filtration (Millex HA, Darmstadt, Germany). The cell pellet was cryopreserved (both in PWID and control semen samples) in 1 ml of freezing medium containing 90% heat-inactivated fetal bovine serum (Nucleus Biologics, San Diego, CA, USA) and 10% dimethyl sulfoxide. Cryovials were placed in a –80°C freezer in a Mr. Frosty™ freezing device containing isopropanol; the approximate freezing rate was –1°C/min. For long-term storage, cryovials were transferred to vapor-phase nitrogen.
Mature spermatozoa were purified using an adapted Percoll gradient protocol (Claassens et al., 1998 (link)). Isotonic Percoll solution was prepared by mixing 9 ml of Percoll (Sigma-Aldrich, St. Louis, MO, USA) with 1 ml of 10× concentrated Ham’s F10 solution (MP Biomedicals, Solon, OH, USA). The bottom fraction of the Percoll gradient (90%) contained 1 ml of 90% isotonic Percoll mixed with 10% X-VIVO 15 (Lonza, Basel, Switzerland) or human tubal fluid ‘HTF’ culture media (in-house). The upper fraction (45%) contained 1 ml of 45% isotonic Percoll mixed with 55% X-VIVO 15 (Lonza, Basel, Switzerland) or HTF culture media (in-house). The gradient was prepared in 15 ml Falcon conical tubes. Semen samples from both groups (PWID and controls) were thawed quickly in a 37°C water bath and 1 ml of X-VIVO 15 was added dropwise to each cryovial. Thawed semen samples were washed in 25 ml of X-VIVO 15, centrifuged at 400×g for 10 min at 20°C, resuspended in ∼500 µl of X-VIVO15 and layered on top of the gradient and centrifuged again at 400×g for 20 min (brake off). After centrifugation, ∼1.5 ml of the supernatant was aspirated off and discarded. The pellet and remaining ∼1 ml of Percoll were washed with 14 ml of X-VIVO 15 or PBS and spun down (400×g for 10 min at 20°C). The final pellet was resuspended in 500 µl of X-VIVO 15 and used to prepare smears on coverslips for Wright and hematoxylin and eosin (H&E) staining and/or aliquoted and frozen at –80°C for later RNA purification. Both stainings were performed at the Histology and Imaging Core (HIC) of the University of Washington, South Lake Union campus (Seattle, WA, USA).
Mature spermatozoa were purified using an adapted Percoll gradient protocol (Claassens et al., 1998 (link)). Isotonic Percoll solution was prepared by mixing 9 ml of Percoll (Sigma-Aldrich, St. Louis, MO, USA) with 1 ml of 10× concentrated Ham’s F10 solution (MP Biomedicals, Solon, OH, USA). The bottom fraction of the Percoll gradient (90%) contained 1 ml of 90% isotonic Percoll mixed with 10% X-VIVO 15 (Lonza, Basel, Switzerland) or human tubal fluid ‘HTF’ culture media (in-house). The upper fraction (45%) contained 1 ml of 45% isotonic Percoll mixed with 55% X-VIVO 15 (Lonza, Basel, Switzerland) or HTF culture media (in-house). The gradient was prepared in 15 ml Falcon conical tubes. Semen samples from both groups (PWID and controls) were thawed quickly in a 37°C water bath and 1 ml of X-VIVO 15 was added dropwise to each cryovial. Thawed semen samples were washed in 25 ml of X-VIVO 15, centrifuged at 400×g for 10 min at 20°C, resuspended in ∼500 µl of X-VIVO15 and layered on top of the gradient and centrifuged again at 400×g for 20 min (brake off). After centrifugation, ∼1.5 ml of the supernatant was aspirated off and discarded. The pellet and remaining ∼1 ml of Percoll were washed with 14 ml of X-VIVO 15 or PBS and spun down (400×g for 10 min at 20°C). The final pellet was resuspended in 500 µl of X-VIVO 15 and used to prepare smears on coverslips for Wright and hematoxylin and eosin (H&E) staining and/or aliquoted and frozen at –80°C for later RNA purification. Both stainings were performed at the Histology and Imaging Core (HIC) of the University of Washington, South Lake Union campus (Seattle, WA, USA).
