Journal: Acta biochimica et biophysica Sinica

Article Title: Low levels of PRSS37 protein in sperm are associated with many cases of unexplained male infertility.

doi: 10.1093/abbs/gmw096

Figure Lengend Snippet: Figure 4. Sperm with low PRSS37 contents exhibit abnormal activation of the proacrosin/acrosin system and premature proteolysis of ADAM2 ACROSIN immunoblotting detected proacrosin bands (55 kDa) in sperm from men with PF with high PRSS37 content (N1–N3) but not in sperm from patients with UMI with low PRSS37 content (L1–L4). ADAM2 immunoblotting detected only the precursor form (98 kDa or 72 kDa) in sperm from men with PF with high PRSS37 content (N1–N3), whereas only the mature form (44 kDa) existed in sperm from patients with UMI with low PRSS37 content (L1–L4). The sizes of specific protein bands are marked on the right.

Article Snippet: Antibodies used were rabbit polyclonal antibody raised against PRSS37 (1:1000 dilution, code HPA020541; Sigma), ADAM2 (1:1000 dilution, code HPA024621; Sigma), ACROSIN (1:1000 dilution, code SC-67151; Santa Cruz Biotechnology, Santa Cruz, USA), and mouse monoclonal antibody against GAPDH (1:10,000 dilution, code KC-5G4; Kangchen, Shanghai, China).

Techniques: Activation Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: SPINK 2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes

doi: 10.15252/emmm.201607461

Figure Lengend Snippet: Representative kinetics of HEK293 cell proliferation measured with Real‐Time Cell Analysis (RTCA) technology in different conditions as indicated. Each point corresponds to the mean of four technical replicates measured simultaneously. Black arrows indicate the time of cell plating ( t = 0 h) and introduction of the different plasmids in the cell chambers ( t = 16 h). Scatter plots showing the mean and SD of the cell index measured at 40 h after plating (corresponding to cell proliferation and detachment) in different transfection conditions and measured for three independent biological replicates. Statistical differences were assessed using t ‐test, P ‐values as indicated. Western blot using an anti‐SPINK2 antibody showing the expression of SPINK2 in cell extracts of HEK293 cells transfected with different plasmids containing SPINK2 (SP) or acrosin and SPINK2. Representative Western blot using an anti‐acrosin antibody. In extracts of HEK293 cells transfected with proacrosin only (lane “acrosin”), two bands were observed, one at around 34 kDa and corresponding to the active form of acrosin (red arrowhead) and one at 46 kDa and corresponding to the zymogen form, whereas in extracts of HEK293 cells transfected with proacrosin and SPINK2 (lane “Acr + SP”), only the zymogen form was observed. Equal protein loading was verified by stain‐free gel technology (Taylor & Posch, ) and Western blots against tubulin ( Appendix Fig S6 ). Note that the zymogen form in lane “acrosin” has a slightly lower mass and that the band is less intense than that in lane “Acr + SP”. Representative Western blot using an anti‐DDK antibody showing the expression of the proacrosin zymogen form in HEK293 cells transfected with different plasmids as indicated. Note that once more, the zymogen form in the lane “acrosin” has a slightly lower mass and that the band is less intense than that in lane “Acr + SP”. Similarly, equal protein loading was verified by stain‐free gel technology ( Appendix Fig S6 ).

Article Snippet: Mycoplasma‐free HEK293 cells were a gift from A. Andrieux from Grenoble Neuroscience Institute and grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS (Invitrogen, France) and 50 μg/ml gentamicin (Sigma) in a 37°C, 5% CO 2 cell culture incubator and transiently transfected with Cter‐DDK‐tagged human acrosin (RC214256; OriGene, Rockville, MD, USA) and/or human SPINK2 (RC205388; OriGene) and/or human c.1A>T mutated SPINK2 ‐containing pCMV6 plasmids, using JetPRIME Transfection Reagent (Polyplus, France) according to the manufacturer's instructions.

Techniques: Cell Analysis, Transfection, Western Blot, Expressing, Staining

Journal: International journal of molecular sciences

Article Title: Effect of Granulocyte Colony-Stimulating Factor on the Development of Spermatogenesis in the Adulthood of Juvenile AML Mice Model Treated with Cytarabine.

doi: 10.3390/ijms241512229

Figure Lengend Snippet: Figure 7. Effect of GCSF on the presence and expression levels of the meiotic/post-meiotic marker (ACROSIN). Mice were treated as described in Figure 1. Testes were removed after 2, 4, or 5.5 weeks and fixed or used for RNA extraction. Fixed testicular tissues were immune stained with specific anti-ACROSIN antibody to identify meiotic cells (A). Tubules with more than 15 ACROSIN- positive cells (B–D) are presented as % of tubules. The RNA expression levels of Acrosin (D–F) in the testes isolated from each treatment group were examined by qPCR analysis using specific primers, and housekeeping gene (Gapdh) was used as internal control. The results are presented as fold of increase compared to Gapdh (B–D). Arrows indicate the location of ACROSIN-positive stained cells. N (number of repeats of all treatments) = 3; n = (number of mice examined from all the experiments for each treatment (immunostaining) = 6–9/group; number of tubules/group = 40–120; n = 5–7/group (RNA expression). * (p < 0.05), ** (p < 0.01), *** (p < 0.001) significant relative to control group. ### (p < 0.001) significant in relative to the same group without GCSF. Scale bar: 100 µm. Data are presented as mean ± SEM.

Article Snippet: For IF staining of ACROSIN, primary antibodies (Polyclonal rabbit anti-mouse, 1:1000; Novus Biologicals, LLC, Centennial, CO, USA, Cat. No. A113694) were used.

Techniques: Expressing, Marker, RNA Extraction, Staining, RNA Expression, Isolation, Control, Immunostaining