Conservation of estrogen receptor function in invertebrate reproduction

Abstract

Background: Rotifers are microscopic aquatic invertebrates that reproduce both sexually and asexually. Though rotifers are phylogenetically distant from humans, and have specialized reproductive physiology, this work identifies a surprising conservation in the control of reproduction between humans and rotifers through the estrogen receptor. Until recently, steroid signaling has been observed in only a few invertebrate taxa and its role in regulating invertebrate reproduction has not been clearly demonstrated. Insights into the evolution of sex signaling pathways can be gained by clarifying how receptors function in invertebrate reproduction.

Results: In this paper, we show that a ligand-activated estrogen-like receptor in rotifers binds human estradiol and regulates reproductive output in females. In other invertebrates characterized thus far, ER ligand binding domains have occluded ligand-binding sites and the ERs are not ligand activated. We have used a suite of computational, biochemical and biological techniques to determine that the rotifer ER binding site is not occluded and can bind human estradiol.

Conclusions: Our results demonstrate that this mammalian hormone receptor plays a key role in reproduction of the ancient microinvertebrate Brachinous manjavacas. The presence and activity of the ER within the phylum Rotifera indicates that the ER structure and function is highly conserved throughout animal evolution.

Keywords: Estrogen receptor; Receptor; Rotifera.

Fig. 1

A homology model of the 3D structure of the B. manjavacas estrogen-like receptor LBD (yellow). a Superimposition of the rotifer (model) and human (X-ray) ER LBDs (pink). The model suggests that the structure of the binding pocket is conserved between human and rotifer. b Close up view of the rotifer ER LBD ligand binding site occupied by estradiol. The amino acids that form the ligand binding site are indicated. Hydrogen bonds are dashed black lines. van der Waals contacts are diffuse red lines. The 3D structure of the human ER LBD was used as a template to construct the 3D model of the rotifer estrogen-like receptor LBD

Fig. 2

Synthetic fluorescent ligands bind selectively within B. manjavacas neonates. Bright field confocal, and merged microscopic images of rotifers treated with ER binding small molecules. The compounds bind primarily to the reproductive tissues, the vitellarium, and the mastax. Estradiol and compound AB-89 exhibited greater binding in the vitellarium than in other tissues

Fig. 3

Chemical Complementation is a binding assay. This assay couples yeast survival to the presence of a small molecule ligand (red). The yeast contains the GAL4 response element that controls the expression of HIS3 biosynthesis genes. The rotifer putative ER LBD is fused to a GAL4 DNA binding domain (GBD). This fusion protein binds to the GAL4 response element. Ligand binding by the ER LBD leads to recruitment of the SRC-1 coactivator, which is fused to the GAL4 activation domain (GAD). Transcription of the histidine biosynthesis genes, upon ligand binding, allows the yeast to survive in media lacking histidine [–40]. The results show that estradiol and the synthetic fluorescent ligands bind to the rotifer the ER-like LBD in vivo. The negative control (with no added ligand) exhibited minimal growth. The natural ligand, estradiol, increases growth on the same order as the synthetic ligands. Neither estradiol, nor the synthetic fluorescent compounds have any effect on yeast growth in the absence of Gal4-ER [35]

Fig. 4

a Survivorship of rotifers treated with estrogen-like receptor binding small molecules. b manjavacas neonates were treated with 10 μM of each compound for the duration of their life. No significant changes in lifespan were observed in the treated animals when compared to animals grown without ligands. b AMI ligands do not negatively affect the reproductive rate of rotifers. The effects of ER binding AMI molecules on rotifer reproduction are shown above. AB-89 significantly increased reproduction compared to the control group. Exposure to ER binding AMI molecule AB-89 at 10 μM significantly increased by 38%. The average reproductive rate for B. manjavacas reproduction increased from 18 to 24 offspring per female

Fig. 5

Error estimate for the rotifer ER-like LBD homology model. RMSD of atomic positions (Å) were calculated by superimposing the homology model for each organism against the real crystal structure. RMSD values vs. percentage sequence similarity are plotted. We can estimate the RMSD of the rotifer ER-like LBD model based on its sequence similarity to the human ER LBD. The estimated RMS error for the rotifer model is 1.7 Å. R² = 0.95