Effect of folate deficiency on promoter methylation and gene expression of Esr1, Cav1, and Elavl1, and its influence on spermatogenesis

Abstract

This study aims to investigate the effect of folate deficiency on the male reproductive function and the underlying mechanism. A total of 269 screened participants from 421 recruitments were enrolled in this study. An animal model of folate deficiency was constructed. Folate concentration was measured in the ejaculate, and its association with semen parameters was then determined. The expression and promoter methylation status of ESR1, CAV1, and ELAVL1 were also evaluated. Results showed that seminal plasma folate level was significantly lower among subjects with azoospermia than those with normozoospermia. Low folate level was significantly correlated with low sperm concentration in men with normozoospermia. Folate deficiency significantly reduced the expression of ESR1, CAV1, and ELAVL1, which are critical to spermatogenesis. However, low folate levels did not increase the methylation levels of the promoter regions of ESR1, CAV1, and ELAVL1 in human sperm DNA. Thus, folate deficiency impairs spermatogenesis may partly due to inhibiting the expression of these genes. Thus future research should determine the significance of sufficient folate status in male fertilization and subsequent pregnancy outcomes.

Keywords: Pathology Section; folate deficiency; gene expression; male infertility; methylation; spermatogenesis.

Figure 1. Distribution of folate level in seminal plasma in two groups

Box plots of seminal plasma folate concentrations in the 269 study subjects. In each plot, the solid horizontal lines mark the 10th, 25th, 50th, 75th and 90th percentile points of the data. The box encompasses the 25th through the 75th percentiles.

Figure 2. Effect of folate deficiency on sperm counts in mice

Means ± SD of determinations (each group, n = 6) are shown. ^*P < 0.05 by Student's t-test.

Figure 3. Haematoxilin/eosin staining

A. control group, B. folate deficiency group, Scale bars, 10 μm.

Figure 4. Effect of low folate level in human seminal fluid on gene expression of Esr1, Cav1 and Elavl1, ^**P < 0.01

Figure 5. Effect of low dietary folate on gene expression of Esr1, Cav1 and Elavl1 of mice, FD: folate deficiency, **P < 0.01, *P < 0.05

Figure 6. Effect of low dietary folate on Expression levels of Esr1, Cav1 and Elavl1 proteins in each mice group, FD: folate deficiency, **P < 0.01

Figure 7. The methylation status of the Esr1, Cav1 and Elavl1 promoter region in human sperm sample

Schematic diagram A., B. and C. indicates the classic 25 (Esr1), 19(Cav1) and 18 (Elavl1) CpG sites in the 5′-flanking region, respectively. Red columns represent CpG sites. Graphical representation of data from subjects with low folate and high folate level in seminal plasma, respectively, showing distribution of methylated CpGs in two groups.

Figure 8. Flowchart of included men in this study