Demethylation and microRNA differential expression regulate plasma-induced improvement of chicken sperm quality

Abstract

The sperm quality is a vital economical requisite of poultry production. Our previous study found non-thermal dielectric barrier discharge plasma exposure on fertilized eggs could increase the chicken growth and the male reproduction. However, it is unclear how plasma treatment regulates the reproductive capacity in male chickens. In this study, we used the optimal plasma treatment condition (2.81 W for 2 min) which has been applied on 3.5-day-incubated fertilized eggs in the previous work and investigated the reproductive performance in male chickens aged at 20 and 40 weeks. The results showed that plasma exposure increased sperm count, motility, fertility rate, and fertilization period of male chickens. The sperm quality-promoting effect of plasma treatment was regulated by the significant improvements of adenosine triphosphate production and testosterone level, and by the modulation of reactive oxygen species balance and adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathway in the spermatozoa. Additionally, the plasma effect suggested that DNA demethylation and microRNA differential expression (a total number of 39 microRNAs were up-regulated whereas 53 microRNAs down-regulated in the testis) regulated the increases of adenosine triphosphate synthesis and testosterone level for promoting the chicken sperm quality. This finding might be beneficial to elevate the fertilization rate and embryo quality for the next generation in poultry breeding.

Figure 1

Sperm quality, testosterone level, sperm structure, and mitochondrial respiratory enzyme level in the spermatozoa. (a) Sperm count, viability, motility, integrities of acrosome and DNA, and fertility rate in 40-week-old male chickens. Sperm quality of 20-week-old male chickens are shown in Supplementary Fig. S1. (b) Testosterone levels in the serum of male chickens on days 30, 60, and 90, and weeks 20 and 40. For the sperm quality and testosterone level, data are presented as the mean ± standard deviation (SD) (n = 10) of three replicates; n represents an individual chicken. (c) Relative mRNA levels of testosterone biosynthesis genes [steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), and hydroxysteroid 17-beta dehydrogenase 3 (HSD17B3)] and androgen receptor (AR) gene in the testis of 40-week-old male chickens. (d) Representative sperm optical microstructure and average sperm size in 40-week-old male chickens. Scale bar: 5.0 μm. (e) Representative ultrastructure of transverse section of spermatozoa and mitochondria number. Mitochondria located around the outer dense fibers of the sperm midpiece are photographed. The red arrow shows the mitochondrion. Scale bar: 2.0 μm. (f) Nicotinamide adenine dinucleotide hydrogen (NADH) levels and activities of (g) cytochrome c oxidase and (h) ATPase synthase in the mitochondria of spermatozoa. For the mRNA level, mitochondrial number, and mitochondrial respiratory enzyme level, data are presented as the mean ± SD (n = 3) of three replicates; n represents an individual chicken. *p < 0.05 versus control; **p < 0.01 versus control, according to the one-way ANOVA with a least significant difference (LSD) test.

Figure 2

Adenosine triphosphate (ATP) level and adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway. (a) ATP concentrations in the serum and spermatozoa of 40-week-old male chickens. For the ATP concentration, data are presented as the mean ± SD (n = 10) of three replicates; n represents an individual chicken. Relative mRNA levels of (b) ATP5 synthases, and (c) AMPKα2, AMPKβ2, AMPKγ3, and mTOR in the spermatozoa of 40-week-old male chickens. (d) Western blot analysis of protein bands in the spermatozoa. The grouping of gels/blots cropped from different gels. All bolts are visualized with 5 min exposure time. Uncropped immunoblot scans are shown in Supplementary Fig. S2. Relative protein levels of (e) ATP5A, (f) p-AMPKα/AMPKα, and (g) p-mTOR/mTOR. For the mRNA and protein level, data are presented as the mean ± SD (n = 3) of three replicates; n represents an individual chicken. *p < 0.05 versus control; **p < 0.01 versus control, according to the one-way ANOVA with a LSD test.

Figure 3

DNA methylation levels in the spermatozoa and testis of 40-week-old male chickens. Total DNA methylation levels in the sequenced regions of (a) ATP5A1, AMPKα2, and mTOR in the spermatozoa and (b) STAR, CYP11A1, CYP17A1, and AR in the testis. Total methylation ratios were calculated by dividing the number of non-converted (methylated) cytosines by the total number of cytosines within the sequenced region; values were expressed as percentages (%). Average methylation levels for CG, CHG, and CHH in the sequenced regions of (c) ATP5A1, (d) AMPKα2, and (e) mTOR in the spermatozoa, (f) STAR, (g) CYP11A1, (h) CYP17A1, and (i) AR in the testis. Average methylation levels were expressed as percentage (%) per site for each of the three types of cytosines (CG, CHG, and CHH), and were calculated by dividing the number of non-converted cytosines by the total number of cytosines of each type. The cytosine methylation analysis results see Supplementary Fig. S3. An independent replicate on DNA methylation levels in the spermatozoa and testis of 40-week-old male chickens see Supplementary Fig. S5.

Figure 4

MicroRNA (miRNA) expression levels in 40-week-old male chicken testis. (a) Hierarchical cluster analysis of miRNA expression levels. Heat map representation of miRNAs that differed significantly between plasma treatment (P) and control (C) groups. Rows represent transcriptional units. miRNAs that share a similar trend of ascending or descending property are clustered. The yellow represents the maximum Z-score; the blue represents the minimum Z-score. (b) Scatter plot of miRNA expression levels. Red dots represent significant miRNAs; grey dots represent no significant miRNAs. (c) Bar plot of significant miRNAs. FC, fold change. miRNA with |FC| ≥ 1.5 and a p-value < 0.05 is significantly up- or down-regulated. The detailed information of significant miRNAs see Supplementary Table S1.

Figure 5

Reactive oxygen species (ROS), malondialdehyde (MDA), and antioxidant enzyme levels. Concentrations of (a) ROS and (b) MDA, and activities of antioxidant enzyme (c) superoxide dismutase (SOD), (d) catalase (CAT), and (e) glutathione peroxidase (GPx) in the serum and spermatozoa of 40-week-old male chickens. (f) Relative mRNA levels of peroxiredoxin (PRDX) 1, PRDX3, PRDX4, and PRDX6 in the spermatozoa of 40-week-old male chickens. (g) Western blot analysis of protein bands and PRDX4 relative protein level in the spermatozoa. The grouping of gels/blots cropped from different gels. All bolts are visualized with 5 min exposure time. Uncropped immunoblot scans are shown in Supplementary Fig. S2. Data are presented as the mean ± SD (n = 3) of three replicates; n represents an individual chicken. *p < 0.05 versus control; **p < 0.01 versus control, according to the one-way ANOVA with a LSD test.