PGAM enzymatic activity assays in vitro and in vivo.

<p>(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. ***<i>p</i><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.</p

Prevalence of PGAM4 SNPs in infertile and proven-fertile human populations.

<p>Translation start site was +1.</p

Gene expression and localization of PGAM1 and PGAM4.

<p>(A) Western blot analysis of total protein from leukocytes, testes, spermatozoa and transfected cell lysates. Mouse brain lysate was loaded as a positive control for PGAM1. (B–F) Immunohistochemical observations. Immunofluorescence in the testis was measured using the anti-PGAM1 (B) and anti-PGAM4 (D, F) antibodies. (F) High-power field of D. Immunofluorescence in the spermatozoa was measured using the anti-PGAM1 (C) and anti-PGAM4 (E) antibodies. PGAM4 protein was detected postmeiosis in spermatids and spermatozoa (white arrowhead) and was localized to the principal piece (white arrow) and acrosome (white arrowhead) in ejaculated spermatozoa. Stars indicate nonspecific fluorescence signals from the secondary antibody in interstitial Leydig cells, because negative control experiments without primary antibodies showed the signals in the interstitial area (data not shown). White bars = 20 µm; red bars = 10 µm.</p

Gene expression analysis of <i>PGAM1</i> and <i>PGAM4</i>.

<p>(A) <i>PGAM4</i> mRNA detection in human leukocytes, testes and ejaculated spermatozoa. Total RNA samples from leukocytes, testes and spermatozoa were subjected to RT–PCR analysis. RT–PCR without reverse transcriptase confirmed the absence of DNA contamination after DNase treatment. M, 100-bp ladder DNA marker. (B) Western blot analysis of transfected COS7 cell lysates to determine the specificity of the anti-PGAM1 and anti-PGAM4 antibodies for each antigen. Approximately 8 µg of protein from the transfected cell lysate was loaded. COS7 and pcDNA3.1+ indicate proteins from untransfected cells and cells that were transfected with the empty pcDNA3.1+ vector, respectively. (C) Immunofluorescence analysis of COS7 cells engineered to overexpress PGAM1 or PGAM4. pcDNA3.1+ indicates cells that were transfected with pcDNA3.1+ as a negative control. The nuclei were counterstained with DAPI (blue). PGAM1 Ab, PGAM1-specific antibody; PGAM4 Ab, PGAM4-specific antibody.</p