# Estrogen Receptor 2b Is Involved in Regulating Gonadotropin-Inhibitory Hormone Expression During Early Development in Zebrafish

## TL;DR

This study shows that estrogen receptor 2b negatively regulates the expression of gonadotropin-inhibitory hormone in zebrafish during early development.

## Contribution

The novel finding is that esr2b is involved in the negative regulation of GnIH expression during zebrafish development.

## Key findings

- Estrogen treatment increases GnIH mRNA levels, with high doses triggering a feedback mechanism.
- Esr2b knockout zebrafish show significantly higher GnIH mRNA levels starting on the fourth day of development.
- Estradiol exposure increases GnIH transcription in both wild-type and esr2b knockout embryos.

## Abstract

In this study, Tg (GnIH: mCherry) and esr2b knockout zebrafish were constructed to explore the regulation between estrogen receptors and gonadotropin-inhibitory hormone (GnIH). Our work showed that GnIH neuropeptide expression location overlaps with that of Hcrt at 36 hpf and 72 hpf. Estrogen treatment experiments showed that an appropriate dose of E2 is able to induce GnIH mRNA levels. High-dose E2 will induce a feedback mechanism. Esr2b knockout led to increased GnIH mRNA levels in zebrafish embryos and it started on the fourth day. In a word, estrogen receptors are able to regulate GnIH expression, but esr2b is involved in negative regulation.

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide involved in the regulation of reproductive function in vertebrates. It is able to inhibit the synthesis and secretion of GnRH and Gth in the HPG axis. However, the regulatory role and mechanism by which current gonadal steroid hormones regulate GnIH are still unclear. In this study, transcription factor binding site analysis was performed on the promoter sequence of zebrafish GnIH. Whereafter, transgenic zebrafish (GnIH: mCherry) accurately labeled GnIH and esr2b knockout zebrafish, which were constructed previously, were used to explore the regulation between estrogen and GnIH. In vitro exposure of wild-type zebrafish embryos and transgenic zebrafish embryos to estradiol showed that 10 μM estradiol significantly increased the transcription level of GnIH. However, both 10 μM and 50 μM estradiol significantly increased the transcription level of GnIH in esr2b knockout zebrafish. We compared the expression levels of GnIH in esr2b knockout zebrafish and wild-type zebrafish at different developmental stages (48 hpf–120 hpf). The results showed that from 96 hpf, the expression level of GnIH in esr2b knockout zebrafish was significantly higher than that in wild-type zebrafish, indicating that esr2b is involved in the negative regulation of GnIH, and this regulatory relationship is established on the fourth day of zebrafish development.