Ultrastick ultrafrost adhesion slides

Testis pieces were fixed in Bouin’s fluid for 16 h at room temperature, dehydrated and embedded in paraplast (Sigma-Aldrich). The testis biopsies from all treatment groups were oriented in the molds in the same manner to obtain sagital sections of the same testicular area. Sections of 7 μm in thickness were further attached to UltraStick/UltraFrost Adhesion slides (Electron Microscopy Sciences) and stained with hematoxylin and eosin as previously described [18 (link)].
Somatic (Sertoli and Leydig cells) and germ cells were identified following the criteria by García-López et al. [14 (link)]. The area of the seminiferous tubules and the number of Leydig cells and each type of germ cell in the cortical and medullar regions of the testis were assessed using the NIS-element AR 4.30.02 software (Nikon). The number of spermatogonia (type A and B), spermatocytes, spermatids and spermatozoa was scored in 10 tubules of 5–10 different areas of the cortex and medulla for each fish and normalized by the area of the whole tubule. Similarly, the number of Leydig cells was counted in six foci in 5–10 different areas of the cortex and medulla per fish. To estimate the number of spermatozoa in the lumen of the sperm duct, the spermatozoa were counted in nine different areas of 0.22 mm² of the lumen of the duct per fish.

Immotile ejaculated spermatozoa were attached to UltraStick/UltraFrost Adhesion slides (Electron Microscopy Sciences, Hatfield, PA, USA), activated in SW or NAM for 2 min and directly fixed on the slide in 4% paraformaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 100 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) for 15 min. Different antigen retrieval protocols were then applied depending on the primary antibody employed (Supplementary Table S1). After blocking for 1 h in PBST (PBS with 0.1% Tween-20) containing 5% normal goat serum and 0.1% BSA, sections were incubated with primary antibodies (Supplementary Table S1) overnight at 4 °C in a humidified chamber. Anti-mouse or anti-rabbit secondary antibodies were applied for 1 h at room temperature at 1:800, subsequently cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:3000; Merck, G8294) for 3 min in PBS to stain the nuclei. The sections were mounted with fluoromount aqueous anti-fading medium (Merck, F4680), and images were acquired at 100× magnification with a Zeiss Axio Imager Z1/ApoTome fluorescence microscope (Carl Zeiss Corp., Oberkochen, Germany).

Sorted germ cells and SPZ_EJ were processed as described previously^{7 (link),38 (link)} and attached to UltraStick/UltraFrost Adhesion slides (Electron Microscopy Sciences). Samples were fixed in 4% PFA in PBS for 15 min before antigen retrieval in three consecutive 5-min incubations with boiling citrate (10 mM at pH 6), followed by triton X-100 (0.2% in PBS) for 15 min. After blocking for one hour in PBST with 5% normal goat serum (Merck G9023) and 0.1% BSA, antibodies were applied overnight at 4 °C in a humidified chamber. The primary antibodies were α-tubulin (Merck T9026; 1:1000), H3K9ac (Abcam ab4441; 1:1000), and Spo11 (Santa Cruz Biotechnology sc-33146; 1:1000). Anti-mouse or anti-rabbit IgG coupled with Alexa-555 (Invitrogen A-21422 and Merck AP510C, respectively) were applied for 1 h at room temperature and cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Merck G8294; 1:3000) before mounting with Fluoromount™.