Fig. 1: SPATA33 and ATG16L1 are colocalized in germline cells in mice.

a Immunofluorescence of SPATA33 and ATG16L1 proteins in mouse testis. Anti-SPATA33, anti-ATG16L1 (Alexa Fluor^®488), and TRITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect SPATA33 (red) and ATG16L1 (green). The nuclei were stained by Hoechst (blue). Images were taken by confocal fluorescence microscopy (SP8, Leica, Wetzlar, Germany). Positive signals were detected in Leydig cells (LC), Sertoli cells (Sn), spermatogonia (Sg), spermatocytes (Sc), and spermatids (Sp). Scale bar, 25 μm; scale bar in enlarged panels: 20 μm. b Cell sorting of spermatogonia (Sg), spermatocytes (Sc), and the round spermatids (Sp) from testis by cell flow sorter. Serial charts of cells were indicated. c Expression of Spata33 was detected in spermatogonia (Sg), spermatocyte (Sc), the round spermatids (Sp), and spermatozoa (sperm). The sperm cells were isolated from epididymis of adult mouse. RT-PCR was performed from mRNAs isolated from these cells. Actin was used as an internal control. d SPATA33 was colocalized with ATG16L1, LC3B, and VDAC2 in the mid-piece region (mitochondria region) of the sperm cells. The spermatozoa were extracted from epididymis of adult mice. The cells were stained with MitoTracker (mitochondria matrix marker, red). Immunofluorescence analysis were performed with anti-SPATA33, anti-ATG16L1 (Alexa Fluor^®488), anti-VDAC2, anti-LC3B (Alexa Fluor^®488), TRITC-conjugated goat anti-rabbit IgG (H + L), and FITC-conjugated rabbit anti-goat IgG (H + L) antibodies. The enlarged images were originated from the squares in the merged panels. The nuclei were stained by Hoechst (blue). Images were taken by confocal fluorescence microscopy (SP8, Leica). The graph on the right indicates sperm ultrastructure, highlighting cross-section of the mid-piece region, including axoneme, outer dense fiber, and mitochondrial sheath (red). Scale bar: 25 μm; scale bar in enlarged panels: 6 μm.