RF22 | PMON06 The Contraceptive Synthetic-Progesterone Levonorgestrel (LNG) Significantly Accelerates Fetal Hypothalamic Neuronal Development (Neuroendogenesis) in the Zebrafish (Danio rerio).

Abstract

Abstract The fetal brain has a known vulnerability to exogenous influences due to its plasticity and high volume of replicating and differentiating cells. It is particularly susceptible to a group of environmental molecules classified as endocrine disrupting chemicals (EDCs). Previous vertebrate studies have provided supportive evidence that steroid mimicking EDCs might stimulate hypothalamic progenitor cell differentiation to neurons (neuroendogenesis). Changing the normal timing of neuroendogenesis could play a role in establishing an alternative developmental timeline leading to disruptions in key endocrine regulated process, like pubertal onset. Levonorgestrel (LNG), a synthetic progestin used in contraceptive pills, has been found at physiologically relevant levels in water sources worldwide. Therefore, we investigated the effect LNG had on neuroendogenesis in zebrafish (Danio rerio), an established comparative neurobiological animal model. Fertilized zebrafish embryos were collected (in-house breeding) and individually placed in 96-well plates. Starting at 3 hours post fertilization (hpf), embryos were exposed to either 5 ng of LNG (previously established physiologically relevant dose), 100ng of estradiol (E2, positive control) or a vehicle control for 5 days (5 dpf); covering the duration of neurogenesis. LNG had no effect on fertilization ratios, hatching or morphology compared to controls. On 5 dpf, larvae were paraffin-processed, slides were serially sectioned at (3-4 um) and prepped for immunohistochemistry (IHC). Brain tissue s were stained with hematoxylin and eosin (H&E) to identify regions of the brain including the hypothalamus and thalamus. Brain sections were also stained for the immunofluorescent (IF) neuronal marker α-HUC and/or estrogen receptor (ER) and imaged on a confocal microscope. The number of total cells, positive stained cells and negative stained cells were counted in the preoptic area (POA), rostral hypothalamus (RH) and thalamus, comparisons were made between groups. Interestingly, LNG significantly (p<0.001) increased the number of positive cells expressing α-HUC in the hypothalamus compared to controls. Similar results were observed in the POA (p< 0.01) and RH (p <0.001) compared to their respective controls. Furthermore, we observed no significant changes in α-HUC expression in the thalamus due to LNG treatment comparted to controls. Our data suggests that LNG selectively targets hypothalamic neuronal development, accelerating neuroendogenesis increasing the number of endocrine-regulating neurons in the brain. This is the first study to show that exposure to low environmentally relevant levels of LNG increases neurons in the hypothalamus, thus, highlighting the possibility that LNG may target the brain to potentiate subsequent health issues later in life. Presentation: No date and time listed