Effect of low sperm quality on progeny: a study on zebrafish as model species

Abstract

Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study whether fertilization with low sperm quality could leave a molecular mark on progeny. This study explores the consequences that fertilization with low sperm quality may have on progeny, using zebrafish as a model. Good and bad breeders were established attending to sperm quality analyses and were individually tracked. Significant differences in fertilization and malformation rates were obtained in progenies between high and low quality sperm samples. Moreover an altered miR profile was found in the progenies of bad zebrafish breeders (upregulation of miR-141 and miR -122 in 24 hpf embryos) and as a consequence, some of their targets involved in male sex development such as dmrt1, suffered downregulation. Our results indicate that fertilizing with high sperm quality samples becomes relevant from a new perspective: to avoid molecular alterations in the progeny that could remain masked and therefore produce unexpected consequences in it.

Figure 1

Experimental design. Schematic representation of the experimental group determination (good and bad zebrafish breeders) analyses carried out and parameters analyzed in each type of sample.

Figure 2

Sperm quality analyses in good and bad zebrafish breeders. (A) Representation of total population distribution in terms of total motility (%). (B) Total motility (%) at different post-activation times (15 s, 30 s, 45s and 60 s) in the experimental groups (good and bad breeders). (C) Kinetic parameters (µm/s) at different post-activation times (15 s, 30 s, 45s and 60 s) in good and bad breeders. (D) Representation of total population distribution in terms of concentration (cell/ml). (E) Correlation between total motility (%) and concentration (cell/ml) in the two experimental groups analyzed. (F) Volume (µl) of good and bad zebrafish breeders. (G) Concentration (cell/ml) of the two studied groups. (H) Principal Component Analysis including all sperm quality parameters in good and bad zebrafish breeders. Representation of our experimental groups in a principal component plane. Data are the mean ± SEM. Good zebrafish breeders are represented in blue and bad breeders in white. Asterisks represent significant differences (p < 0.05) between experimental groups. Analyses were performed in sperm samples from 48 individual males.

Figure 3

Sperm molecular analyses in good and bad zebrafish breeders. (A) Global methylation status of sperm DNA. Unmethylated plasmid DNA (kit control) is digested by both enzymes, whereas methylated plasmid DNA (kit control) is susceptible only to Epi MspI digestion. All the represented samples from good and bad breeders derived from the same experiment and gel. The full length-gel image with all samples and kit controls is available in Supplementary Fig. 1. (B) Specific methylation analysis in dmrt1 promoter of good and bad zebrafish breeders. Schematic representation of zebrafish dmrt1 promoter, sequence, primer design, restriction sites, pattern of methylation and percentage of methylcytosine obtained in each experimental group. (C) Structure and expression of each microRNA normalized against that of miR-92-3p was calculated for all samples using the 2^−ΔΔCt method. The figure shows expression of each microRNA in the bad breeders relative to that in good breeders, which was set to 1. Data are expressed as the mean ± SEM of 2^−ΔΔCt values from three independent experiments of three different pools of three males per group were analyzed. Asterisks represent significant differences (p < 0.05) between experimental groups.

Figure 4

Progeny analyses from zebrafish good and bad breeders. (A) Percentage (%) of fertilization rate, embryo survival in blastula and 24 hpf stages and malformation rate in zebrafish progenies from good and bad breeders. (B) Relative expression of each microRNA calculated using the 2^−ΔΔCt method at three different early developmental stages (2–4 cells, blastula and 24 hpf). The figure shows expression of each microRNA in the bad breeders relative to that in good breeders, which was set to 1. Data are expressed as the mean ± SEM of 2^−ΔΔCt values from five independent in vitro fertilizations. Asterisks represent significant differences (p < 0.05) between experimental groups. (C) miR-200a-5p and 141-3p target analysis and the seeds matched in the 3′-UTR of each target gene. (D) Relative expression levels for each gene relative to the housekeeping genes actin beta 2 (actb2) and elongation factor 1 alpha (ef1α) were calculated for all samples using the 2^−ΔΔCt method. The figure shows expression of each gene in the progenies form bad breeders at two developmental stages (blastula and 24 hpf) relative to that progeny of good males, which was set to 1. Data are expressed as the mean ± s.e.m. of 2^−ΔΔCt values from three independent experiments with three replicates for each. Asterisks represent significant differences (p < 0.05) between experimental groups.