Sperm-mediated effects of caloric restriction (CR) on offspring phenotype and epigenotype
Final Report Abstract
The project was conducted to analyze metabolic and molecular effects of paternal food deprivation on the parental and filial generation in Danio rerio (zebrafish). Our study revealed that the vertebrate model is not optimal to address this fundamental research question, because the animals does not react very sensitive to experimental food deprivation. We found that a starvation period of ≥ 17 days is necessary to substantially reduce the body weight (15%) and the fat content of adult males. The starvation regime decreases both the sperm number (44%) and the ejaculate volume (31%). A starvation period of 10 or 17 days, respectively, increases most of the sperm velocity parameters (VCL, VSL, VAP and BCF). The effects were more pronounced after 17 days compared to the 10 days period. Both conditions increase the fraction of static sperms. A TUNEL and a Tubulin staining assay revealed that the effects of starvation on sperm behaviour is neither caused by DNA damage nor by sperm tail morphology structures. Under natural breeding conditions, we found that paternal starvation decelerates the pace of embryonic development up to hatching. This transgenerational effect was more pronounced after 17 days of starvation compared to the 10 days period. In vitro fertilization (IVF) experiments could not mimic the identified transgenerational effect. Up to 5 dpf (days post fertilization), fat content of larvae depends mainly on the amount of maternal provided yolk protein. Using a BODIPY 493/503 lipid staining method, we could not find any differences between 5 dpf larvae derived from starved father compared to ad libitum fed males. At 8 dpf, the body length differed slightly between offspring of 17 days starved vs. ad libitum fed fathers (4.33 ± 0.02 vs. 4.28 ± 0.03). Because of technical issues (e. g. gallbladder, pigmentation), this dataset is not robust enough to draw confidential conclusions. At 10 dpf, the inter-individual variance in fish length and size was too high for further phenotypic analyses. In another set of laborious experiments under natural breeding conditions, we found that the oviposition of females crossed with a 17 days starved male was only half when compared to ad libitum fed males (50.28 ± 10.53 vs. 103.11 ± 12.59). This effect was not found after 10 days starvation regime. Unfortunately, the available egg number was too small for continuing analyses and a male-induced maternal effect could not be excluded. Retrospective, IVF experiments might be the appropriate approach to study transgenerational effects in more detail. In conclusion, the basic challenge of this project was the establishment of a starvation regime inducing a reproducible and drastic phenotype of the fathers as well as of the respective offspring. Another challenge was the antagonism between the number of samples and drastic phenotypes. However, finally we could establish reproducible starvation-induced phenotypes in males, whereby the effects on sperm parameters seem to be the most interesting findings for further analyses. Due to the small effects observed in offspring derived from drastically starved males, other model organisms (e. g. rodents) than zebrafish might be more suitable to analyze diet-induced paternal effects.
