Cortisol effects on GnRH neuron development

We investigate the effects of chronic stress on the development of GnRH neurons in the early zebrafish (Danio rerio) embryo. Embryonic exposure to stress has been shown to cause problems in the development of GnRH neurons in other fish species most notably salmon and carp. Exposure to stress in zebrafish had been shown to cause problems in reproductive behavior and development. Prior research has also linked abnormal development of the GnRH system in zebrafish to problems in reproduction and reproductive behavior. However, little research has been done on GnRH neuron development under stress in zebrafish. We primarily focused on the extrahypothalamic GnRH1 neurons located in the terminal nerve (TN), with a putative role in neruomodulation and social regulation of reproduction. This study investigates effects of early, chronic cortisol exposure on the TN-GnRH1 neurons of embryonic zebrafish at 2 and 3 days post fertilization (dpf).
Transgenic zebrafish embryos with GnRH1 neurons tagged with green fluorescent protein (GFP) were used to easily visualize embryonic changes to the developing reproductive neuroendocrine system. Embryos were exposed to a chronic dose of the stress hormone cortisol (hydrocortisone 10μM) by way of waterborne exposure in their embryo medium from 2 hours post fertilization (hpf) through 72 hpf. Embryos were fixed at 48 and 72 hpf, mounted in agar and their TN-GnRH1 neurons were examined using an upright epifluorescent microscope. The perimeters of individual neurons were marked in ImageJ and neuron area (in μm2) and the short-diameter of the fitted ellipse (in μm) were calculated blind.

Blind analysis of the images showed that embryos exposed to cortisol through 48 hpf (n=37 from 6 fish) showed a significant 23.50% reduction in the area of the individual neurons and a significant 13.54% reduction in the radius of the TN-GnRH1neurons as compared to the control embryos (n=22 from 5 fish). Embryos exposed to cortisol through 3 dpf (n=36 from 7 fish) also showed a significant reduction, but to a lesser degree, of 10.80% in the area and 8.58% in the radius of individual GnRH1 neurons as compared to the control embryos (n=27 from 5 fish). Our study suggests that chronic exposure to stress in the early embryo delays or disrupts the development of TN-GnRH1 neurons in zebrafish (Supported by NSF CAREER Award 1253126 to SR and Univ Puget Sound UEC Grant SR1563 to DS).