Ecm0101l

Spreads were prepared as previously described [42 (link), 61 (link), 86 (link)]. Briefly, testes were taken off from juvenile euthanized mice and the tunica albuginea was removed. Next, the seminiferous tubules were placed in DMEM high glucose (Euroclone, ECM0101L) and disrupted and mixed using a razor blade. The supernatant was centrifuged at 7200 rpm for 1 min and the pellet was resuspended in 0.5 M sucrose (VWR, 27480.294). Cell suspension was fixed on slides (Thermo Sientific, Menzel-Glaser Superfrost Plus, J1800AMNZ) using 1% paraformaldehyde (PFA) (ChemCruz, SC-281692)/0.015% Triton X-100 (Sigma-Aldrich, 9002-93-1)/dH2O pH 9.2) and incubated for 2 h in a humidified chamber at room temperature (RT). When the slides were completely fixed, they were washed twice with Washing Buffer 2 [WB2, 0.4% Photo-Flo (Kodak Professional 200 Solution, 1464510)/dH2O] and left air-drying at RT. Slides were either stained soon after or stored at − 80 °C for up to 6 months.

IPEC-J2 cells (porcine jejunal epithelial cells, IZSLER Cell Bank code BS CL 205) were cultured in a complete medium consisting of a mixture (1:1) of Dulbecco’s modified Eagle high glucose medium (DMEM, cod. ECM0101L, Euroclone, Milan, Italy) and nutrient mixture F-12 (F12, cod. ECB7502L, Euroclone, Milan, Italy) enriched with 10% fetal bovine serum (FBS, GIBCO™, cod. A38401, Thermofisher scientific, Milan, Italy), 1% L-glutamine 2 mM solution (cod. ECB3000D, Euroclone, Milan, Italy) and 1% penicillin/streptomycin solution (cod. ECB3001D, Euroclone, Milan, Italy). Cells were seeded into 12 well plates (1 mL per well, 3 × 10⁵ cells/mL; Euroclone, Milan, Italy) and then incubated at 37 °C, 5% CO₂, until confluence.

Wild type (A3 strain) and Zfp57 -/- (28 ,29 (link)), hybrid JB1 and BJ1 generated from F1 hybrids derived from JF1 x C57Bl/6 (henceforth B6) and B6 x JF1 crosses, respectively (30 (link)) and TC-1 (31 (link)) ESC lines were cultured under standard feeder-free conditions on gelatinized tissue culture dishes with media containing DMEM (EuroClone ECM0101L) supplemented with 2-mercaptoethanol, non-essential amino acids, sodium pyruvate, 10% fetal calf serum (A3 and Zfp57 -/-) or 15% fetal calf serum (JB1, BJ1 and TC-1), and leukemia inhibitory factor (LIF) at 37°C under an atmosphere of 5% CO₂. The culture medium of TC-1 cells was supplied with 25 μg/ml hygromycin B as described by Feng et al. (31 (link)).