Table 1.
rs12676 and rs1025689 SNP distribution frequencies in the screened population.
Figure 1.
On Day 1, all subjects gave informed consent, completed a health questionnaire form and blood samples were collected. Plasma was separated from other blood components and reserved for measures of choline metabolites. Genomic DNA was isolated from purified lymphocytes and used to genotype subjects for rs12676 and rs1025689 SNPs. Individuals with the genotypes of interest were invited to complete Day 2 of the study which entailed leaving a semen sample for measures of semen characteristics, sperm motility, ATP concentration and mitochondrial morphology.
Table 2.
Characteristics of the study population.
Table 3.
Semen parameters by rs12676 and rs1025689 genotype.
Figure 2.
Motility characteristics of sperm change with rs12676 genotype.
Mean velocity (MVUS, A), curvilinear velocity (VCL, B), straight line velocity (VSL, C) and mean tortuosity (MT, D) were determined for sperm. Sperm were prepared and motility characteristics measured as described in the Methods section. N = 2756 (GG), 2553 (GT), 827 (TT) sperm. Statistical analyses were performed as described in the Methods section; refer to Table 4 for p-values. X-axis values indicate the upper limit of each quartile; the line at 25% represents values for sperm from GG subjects.
Table 4.
Significance values for sperm motility analyses.
Figure 3.
Motility characteristics change with rs1025689 genotype.
Mean velocity (MVUS, A), curvilinear velocity (VCL, B), straight-line velocity (VSL, C) and mean tortuosity (MT, D) were determined for sperm. Sperm were prepared and motility characteristics measured as described in the Methods section. N = 1708 (GG), 2709 (GC), 1719 (CC) sperm. Statistical analyses were performed as described in the Methods section; refer to Table 4 for p-values. X-axis values indicate the upper limit of each quartile except for last label, which indicates the lower limit of the fourth quartile; the line at 25% represents values for sperm from GG subjects.
Figure 4.
rs12676 TT genotype is associated with dysmorphic mitochondrial structure in sperm.
Sperm were fixed and processed for transmission electron microscopy as described in the Methods section. Longitudinal and cross-sectional sections of sperm were examined for mitochondria structure anomalies. Representative images for rs12676 genotypes (GG, panel A and D; GT, panel B and E; TT, panel C and F) are shown. N = 5 per genotype. Micrographs shown are at 80,000× magnification and arrows indicate mitochondria.
Figure 5.
The rs12676 minor T allele is associated with decreased ATP concentrations in sperm.
ATP concentrations were determined in sperm using a commercially available bioluminescent assay kit and ATP concentration was normalized to number of sperm assayed. A, Men who were heterozygous or homozygous for the rs12676 variant T allele have reduced ATP concentrations in their sperm. N = 17 (GG), 18 (GT) and 5 (TT). * indicates difference from GG by ANOVA and Tukey-Kramer HSD, p-value <0.05. B, ATP concentrations are not different with rs1025689 genotype. N = 10 (GG), 21 (GC) and 9 (CC). Data presented are mean ± SEM.
Figure 6.
Sperm mitochondrial morphology and ATP concentrations are not changed in samples from men who are homozygous for rs1025689, but not rs12676.
A. Representative longitudinal section of sperm midpiece. B. Representative cross-sectional section of sperm midpiece. Arrows indicate mitochondria. C. ATP concentrations as measured previously; “CC" group only contains data from men who are CC for rs1025689 and not TT for rs12676. Data presented are mean ± SEM. N = 9 (GG), 22 (GC) and 5 (CC).
Figure 7.
Expression of CHDH protein is decreased in sperm and primary hepatocytes from humans who are heterozygous or homozygous for the rs12676 SNP.
The abundance of CHDH protein in sperm (A) and primary hepatocyte (B) lysates was measured by western blot. Blots were probed for ∝-TUBULIN (sperm) or β-ACTIN (hepatocytes) and data presented are the mean ± SEM of the ratio of CHDH: ∝-TUBULIN or CHDH:β-ACTIN protein. Statistical differences were tested by ANOVA and Student's t test. * indicates different from GG, p-value <0.05. ** indicates different from GG, p-value <0.01. N = 3 per genotype.
Table 5.
Choline metabolite concentrations in plasma and sperm.