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. 2012 May 9;7(5):e35195. doi: 10.1371/journal.pone.0035195

Figure 3. PGAM enzymatic activity assays in vitro and in vivo.

Figure 3

(A) Lysates of cells overexpressing PGAM1 or PGAM4 were compared using untransfected COS7 cells and cells transfected with empty pcDNA3.1+ as controls. (B) Lysates from cells overexpressing PGAM4 or PGAM4 Trp25Cys (W25C) were compared with control lysates. Equal amounts of overexpressed PGAM4 and PGAM4 with the SNP were confirmed by immunoblotting using anti-PGAM4 antibodies. (C) The supernatant and precipitated proteins that were separated from the fresh ejaculated spermatozoa of a fertile man were examined to determine the hydrophilicity of the enzyme. (D) Frozen spermatozoa from infertile men with the Trp25Cys SNP and those without the SNP. The seminal sperm densities for patients 1 to 3 were 39, 18 and 70 million/mL, respectively, and the motility levels were 35%, 30% and 2%, respectively. The mean seminal sperm density of patients without the Trp25Cys SNP was 41 million/mL and the mean motility level was 60%. **p<0.01. ***p<0.001. The experiments for PGAM1 and 4 in vitro enzymatic activity assays were performed independently 8 times. The PGAM enzymatic activities of spermatozoa were measured 3 times repeatedly by each sample and the averaged value were defined as individual enzymatic activity. Bars represent averages and standard deviations.