Stress decreases spermatozoa quality and induces molecular alterations in zebrafish progeny.

BACKGROUND: Chronic stress can produce a severe negative impact on health not only in the exposed individuals but also in their offspring. Indeed, chronic stress may be contributing to the current worldwide scenario of increasing infertility and decreasing gamete quality in human populations. Here, we evaluate the effect of chronic stress on behavior and male reproductive parameters in zebrafish. Our goal is to provide information on the impact that chronic stress has at molecular, histological, and physiological level in a vertebrate model species. RESULTS: We evaluated the effects of a 21-day chronic stress protocol covering around three full waves of spermatogenesis in Danio rerio adult males. The induction of chronic stress produced anxiety-like behavior in stressed males as assessed by a novel tank test. At a molecular level, the induction of chronic stress consistently resulted in the overexpression of two genes related to endoplasmic reticulum (ER) stress in the brain. Gene set enrichment analysis (GSEA) of testes suggested a dysregulation of the nonsense-mediated decay (NMD) pathway, which was also confirmed on qPCR analysis. Histological analysis of the testicle did not show significant differences in terms of the relative proportions of each germ-cell type; however, the quality of sperm from stressed males was compromised in terms of motility. RNA-seq analysis in stress-derived larval progenies revealed molecular alterations, including those predicted to affect translation initiation, DNA repair, cell cycle control, and response to stress. CONCLUSIONS: Induction of chronic stress during a few cycles of spermatogenesis in the vertebrate zebrafish model affects behavior, gonadal gene expression, final gamete quality, and progeny. The NMD surveillance pathway (a key cellular mechanism that regulates the stability of both normal and mutant transcripts) is severely affected in the testes by chronic stress and therefore the control and regulation of RNAs during spermatogenesis may be affected altering the molecular status in the progeny.

Impact of different levels of handling on Solea senegalensis culture: effects on growth and molecular markers of stress.

Aquaculture routine practices may cause stress induction on the fish and compromise their welfare affecting the production. This experiment aimed to evaluate the potential links between handling during culture with stress responses and growth on Senegalese sole (Solea senegalensis). We worked with two fish cohorts in terms of initial body weight and culture stage: Trial 1 included specimens in the fattening stage (226 ± 4.96 g) and Trial 2 animals in the pre-fattening stage (27.20 ± 0.44 g). The tested culture protocol, which lasted 6 and 4 months for Trial 1 and 2, respectively, mainly reduced handling-derived stressors in the experimental tanks via lowering routine samplings to a minimum. This decrease of the handling-derived stress was reflected in both trials with lower concentration of circulating cortisol in blood plasma from the experimental fish when compared to controls. Moreover, the proposed protocol promoted higher growth in the fish cultured in the less disturbing protocol in Trial 2. Higher specific growth rates and mean body weight and length were reported. In order to further explore the potential beneficial effects of our protocol, we studied the musculoskeletal from Trial 2 gene expression of key genes regulating glucocorticoid signaling pathway and apoptosis: glucocorticoid receptors 1 and 2 (gr1, gr2), heat shock protein 90 AA (hsp90aa), and caspase 6 (casp6). In line with the cortisol reduced level in this trial, gr1, hsp90aa, and casp6 genes showed lower expression in the samples coming from the experimental group. The findings of this study provide valuable information to the aquaculture industry for the management of Solea senegalensis stress and welfare.

Male reproductive dysfunction in Solea senegalensis: new insights into an unsolved question.

Senegalese sole (Solea senegalensis) is a species with a high commercial value that exhibits a reproductive dysfunction in males born and raised in captivity (F1) that hinders their sustainable culture. The present study evaluates the sperm quality and dopaminergic pathway of males born in the wild environment and of F1 males. Traditional sperm analyses were performed, finding only significant differences in curvilinear velocity (VCL) and no significant differences in viability and total motility. No differences in global sperm methylation were observed either in spermatozoa or brain between the two groups (F1 and wild-born males). However, our results point to a different sperm molecular signature between wild fish and fish born in captivity, specifically the differential expression in miR-let7-d and miR-200a-5p between these two groups. miR-let7-d has been correlated with spermatogenesis and sex preferences, whereas the miR-200 family is implied in target innervation of dopaminergic neurons in zebrafish. When we analysed the dopaminergic pathway, no differences were found in terms of different mRNA expression of dopaminergic markers. However, some differences were detected in terms of tyrosine hydroxylase protein expression by western blot analysis, thus suggesting an altered post-transcriptional regulation in F1 males. The results of this study suggest that an altered sperm miRNA signature in F1 males could be one possible mode of transmission of reproductive dysfunction to the progeny.

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

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.