# Transcriptional Activation of Estrogen Receptor-Alpha and Estrogen Receptor-Beta from Elephant Shark (Callorhynchus milii)

## TL;DR

This study explores how estrogen activates receptors in elephant sharks, revealing similarities to human estrogen signaling despite 425 million years of evolution.

## Contribution

The discovery of three estrogen-responsive ERa genes and one estrogen-unresponsive ERa gene in the elephant shark.

## Key findings

- Elephant shark has three estrogen-responsive ERa genes (ERa1, ERa2, ERa3) and one estrogen-unresponsive ERa4 gene.
- Estrogen activation of C. milii ERa and ERb is similar to human ERs, showing evolutionary conservation.
- E2 had the lowest EC50 for all four ERs, with similar fold-activation by E2 and E3 across ERa and ERb.

## Abstract

Background/Objectives: Humans and other vertebrates contain two estrogen receptors (ERs), ERa and ERb. Among cartilaginous fish (sharks, rays, skates), which are chondrichthyans that evolved about 425 million years ago, only activation by steroids of ERb orthologs has been characterized. To remedy this gap in understanding estrogen signaling in chondrichthyans, we studied estrogen activation of orthologs of human ERa and ERb from elephant shark (Callorhynchus milii). Methods/Results: Unexpectedly, we found that C. milii contained three estrogen-responsive ERa genes: ERa1 (596 amino acids), ERa2 (600 amino acids), and ERa3 (599 amino acids) with strong sequence similarity to each other. We also found an estrogen-unresponsive gene, ERa4 (561 amino acids), with a 39 amino acid deletion in the DNA-binding domain. An estrogen-responsive ERb ortholog (580 amino acids) was also present in C. milii. The three active C. milii ERas are of similar length to human ERa (595 amino acids); however, C. milii ERb is longer than human ERb (530 amino acids). We studied transcriptional activation of ERa and ERb by estradiol (E2), the main reproductive estrogen in humans. We also studied estrone (E1), the main postmenopausal estrogen, and estriol (E3), which is synthesized during pregnancy. We determined the half-maximal response (EC50) and fold-activation to E2, E1, and E3 of C. milii ERa1, ERa2, ERa3, and ERb. Among these estrogens, E2 had the lowest EC50 for all four ERs. Fold-activation by E2 and E3 was similar for ERa1, ERa2, ERa3, and ERb. Conclusions: Overall, estrogen activation of C. milii ERa and ERb was similar to that for human ERa and ERb, indicating substantial conservation of the vertebrate ER during the 425 million years since the divergence of cartilaginous fish and humans from a common ancestor.