**2. Estrogen receptor types**

Estrogen modulates cellular function *via* activation of one of four receptor subtypes: estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), G-protein coupled estrogen receptor (GPER), and ER-X. ERα was first described in the 1950s as a ligand-activated receptor for estrogen, while ERβ was discovered more recently in 1996 [4]. ERs are located throughout the cell [5]. Two distinct genes encode ligand-activated ERα and ERβ. These genes and their products are subject to epigenetic modifications and alternative RNA splicing [6–11]. Nuclear ERα and ERβ can modulate gene transcription, while localization of these receptors on the cell membrane results in the rapid activation of signaling cascades *via* non-genomic mechanisms. Studies utilizing ERα and ERβ knockout (KO) mice have provided insight into the function of each receptor type. Male and female ERα KO mice are infertile, while ERβ KO mice are fertile but produce small litters [12–14]. These studies highlight the importance of estrogen in the development of reproductive systems of both sexes [14, 15].

ERα and ERβ are also found on the membranes of cellular organelles, including the endoplasmic reticulum and the mitochondrion, where they mediate various cellular functions. Localization to mitochondria was first confirmed using radioligand binding methods in mitochondria isolated from rat uterus and later by immunocytochemistry in rat pancreatic acinar cells [16]. MALDI-TOF mass spectrometry studies have shown that mitochondrial ERs are identical to ERs located in the nucleus [17]. ERβ is the predominant mitochondrial receptor in most tissues: for example ovary, uterus, spermatocytes, cerebral and hippocampal neurons, cardiomyocytes, and endothelial cells [17, 18]. In contrast, the identification of mitochondrial ERα has been limited to the uterus, ovary, and the MCF-7 breast cancer cell line [18]. The presence of ERs on mitochondria and estrogen responsive elements on the mitochondrial DNA suggests a role for estrogen in regulating the structure and/or function of the organelle [19].

Estrogen also binds to a GPER on the plasma membrane. GPER specifically binds to estradiol and mediates numerous responses including cell proliferation, vasodilation, and regulation of glucose metabolism by non-genomic mechanisms [20]. GPER has also been localized to intracellular sites. In the endoplasmic reticulum, GPER activation induces calcium release and activation of the phosphoinositide 3-kinase (PI3K)-*Akt* pathway, which induces cell proliferation [21, 22]. While GPER is not associated with the mitochondria, its regulation of cellular calcium handling indirectly impacts mitochondrial function and mitochondrial-induced cell death [22, 23]. Calcium uptake by mitochondria results in the opening of the mitochondrial permeability transition pore (mPTP) and induction of the intrinsic cell death pathway. GPERspecific agonist G1 binding to GPER has been shown to attenuate these responses in a rodent model of ischemia/reperfusion (I/R) by preventing endoplasmic reticulum calcium release [23]. ER-X is an additional estrogen receptor type that is associated with the cell membrane. This novel receptor shares sequence homology with ERα and ERβ, which is expressed primarily in the brain during development and becomes re-expressed in response to ischemic brain injury [24]. While little is known regarding the function of ER-X, some data suggest that it exerts a cytoprotective role in the brain [24].
