Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

Abstract

Background: As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals.

Methods: Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray.

Results: Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data.

Conclusions: In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

Figure 1

Effect of ACH treatment on sperm motility (left) and progressive motility (right). Data are represented as mean ± S.EM. * P < 0.05 versus the control group, n = 8.

Figure 2

Electron microscopic analysis of the effect of ACH treatment on sperm morphology. A: In the control, there are no mid-piece sperm that are surrounded by cytoplasmic droplets; B: After ACH treatment, there are some mid-piece sperm surrounded by cytoplasmic droplets (black arrows); C: Comparison of the percentage of sperm droplet retention between the control and ACH groups. *P < 0.05 versus the control group.

Figure 3

Effect of ACH treatment on serum T and DHT levels. Data are represented as mean ± S.EM.

Figure 4

Scatter plot of epididymal-expressed genes between the two groups. The yellow points represent genes that are not expressed in either group. The blue points indicate the genes that are expressed in either of the two groups. The red points denote genes that are expressed in both groups. The four upper-left diagonal lines represent the fold of up-regulated gene expression, which is 2, 3, 10 and 30 times, respectively; meanwhile, the four lower-right diagonal lines represent the fold of down-regulated gene expression, which is 1/2, 1/3, 1/10 and 1/30, respectively.

Figure 5

The functional classification of the differentially expressed genes between the two groups.

Figure 6

RT-PCR analysis of selected genes demonstrates the alteration in mRNA expression.