Journal: Reproductive biology and endocrinology : RB&E

Article Title: Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

doi: 10.1186/s12958-021-00880-4

Figure Lengend Snippet: Fig. 3 Expression analysis of SLO3 mRNA and protein and localization of the SLO3 protein in sperm flagella. A Quantitative real-time PCR analysis of the expression of SLO3 mRNA in the spermatozoa from normal control and the SLO3-mutated individual. Compared with control, the expression of SLO3 mRNA is significantly reduced in the sperm from the SLO3-mutated individual. β-actin was used as an internal control. Data are presented as the mean ± SEM. ***P < 0.001; Student’s t-test. B Immunoblotting of sperm lysates from a normal control and the SLO3-mutated individual using an anti-SLO3 antibody. Na+/K+-ATPase α1 was used as loading control. C Representative images of spermatozoa from controls and from the SLO3-mutated individual stained with anti-SLO3 antibody, anti-Ac-tubulin antibody, and Hoechst. SLO3 staining is concentrated at the mid-piece of sperm flagella and faintly along the flagella in the fertile control but is significantly reduced in the sperm flagella of the individual harbouring the SLO3 mutation. Scale bar, 20 μm

Article Snippet: Membranes were then immunoblotted using the following primary antibodies: rabbit polyclonal anti-LRRC52 (1:1000; PA5–107159, Invitrogen, Carlsbad, CA 92008 USA), rabbit polyclonal anti-CatSper1 (1:1000; DF9349, Affinity Biosciences, Beijing, China), rabbit polyclonal anti-Na+/K+-ATPaseα1 (1:2000; ABP51894, Abbkine, China), anti-HSP60 antibody (1:1000; ab13532, Abcam, Cambridge, UK), and HRP-conjugated β-actin (1:2000; HRP-60008, Proteintech, Rosemont, IL, USA), at 4 °C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining, Mutagenesis

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

doi: 10.1186/s12958-021-00880-4

Figure Lengend Snippet: Fig. 5 Effects of SLO3 deficiency on mitochondrial function. A Influence of the SLO3 mutation on mitochondrial membrane potential (MMP). Assessment of mitochondrial membrane potential by flow cytometry acquisition for JC-1-stained (a marker of MMP) cells was performed through FL1 for green and FL2 for red fluorescence. MMP is significantly reduced in mutant sperm compared with control. B Immunoblotting of sperm lysates from controls and the SLO3-mutated individual using the anti-HSP60 antibody. β-actin was used as loading control. C The expression of HSP60 is almost diminished in SLO3 mutant spermatozoa. HSP60 staining is present in the flagellar midpiece of spermatozoa from normal controls, but significantly reduced in spermatozoa from A-132. Scale bars, 20 μm

Article Snippet: Membranes were then immunoblotted using the following primary antibodies: rabbit polyclonal anti-LRRC52 (1:1000; PA5–107159, Invitrogen, Carlsbad, CA 92008 USA), rabbit polyclonal anti-CatSper1 (1:1000; DF9349, Affinity Biosciences, Beijing, China), rabbit polyclonal anti-Na+/K+-ATPaseα1 (1:2000; ABP51894, Abbkine, China), anti-HSP60 antibody (1:1000; ab13532, Abcam, Cambridge, UK), and HRP-conjugated β-actin (1:2000; HRP-60008, Proteintech, Rosemont, IL, USA), at 4 °C overnight.

Techniques: Mutagenesis, Membrane, Flow Cytometry, Staining, Marker, Fluorescence, Control, Western Blot, Expressing