**3. Mitochondrial function**

by conversion to pregnenolone by cytochrome P450 side chain cleavage (CYP11A1). Pregnenolone then diffuses to the theca cells where it is converted to androstenedione and then re-routed to the granulosa cells for the aromatase-mediated conversion to estrogen. Androstenedione can also be converted to testosterone. Thus, mitochondria play an impor-

Estrogen serves a major role in determining female secondary sex characteristics during development and in regulating the estrous cycle. During puberty and throughout the female reproductive cycle, estrogen levels fluctuate, and as women age, sex steroid production decreases [1]. Estrogen levels oscillate during the estrous cycle. They are lowest during menstruation, steadily rise during the follicular stage and reach a maximal level during ovulation. If a woman becomes pregnant, estrogen levels will remain high, but if fertilization does not occur, hormone levels decline during the luteal phase. Following the luteal phase, menstruation occurs, and the cycle resumes. As estrogen levels fluctuate during the estrous cycle, mito-

Estrogen is a pleiotropic hormone that exerts its effects *via* both transcriptional and nongenomic mechanisms. In addition to regulating reproductive function, estrogen exerts numerous cytoprotective effects. With respect to atherosclerosis, estrogen regulates levels of circulating lipids by stimulating the formation of high density lipoprotein (HDL) and decreasing expression of low density lipoprotein (LDL) [2]. It exerts antioxidant and anti-inflammatory effects by preventing the oxidation of LDL, inhibiting the expression of endothelial cell adhesion molecules and stimulating nitric oxide formation [3]. As discussed in this chapter, differential responses to estrogen are due to activation of different receptor subtypes. Recent studies also suggest that many of the protective responses to estrogen are related to the ability of the hormone to maintain normal mitochondrial function. Mitochondrial localization of estrogen receptors has been shown to regulate mitochondrial gene expression. In this manner, estrogen plays an important role in the supporting mitochondrial respiration and adenosine 5′ triphosphate (ATP) production, reducing reactive oxygen species (ROS) formation and inhibiting activation of mitochondrial cell death pathways. The goal of this chapter is to discuss mechanisms by which estrogen regulates mitochondrial signaling and function under

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.

tant role in estrogen biosynthesis.

464 Mitochondrial Diseases

chondria alter the production of pregnenolone accordingly.

normal physiological conditions and in the context of disease.

**2. Estrogen receptor types**

The mitochondria are classically described as the powerhouse of the cell by virtue of its ability to generate ATP. Physiological processes underlying mitochondrial bioenergetics and respiration have been previously reviewed [25]. Under aerobic conditions, mitochondria utilize electron transport and a protomotive force to produce ~36 ATP molecules for every glucose molecule. Reducing equivalents produced by the Krebs cycle (NADH and FADH<sup>2</sup> ) are accepted by the respiratory chain at Complex I (NADH Dehydrogenase) and Complex II (Succinate Dehydrogenase), respectively. Electrons are shuttled through the complexes with oxygen acting as the final electron acceptor at Complex IV. Concurrently, hydrogen ions are pumped from the electronegative mitochondrial matrix into the more positively charged inner membrane space by Complexes I, III (cytochrome C reductase), and IV (cytochrome C oxidase). The protomotive force thus generated allows hydrogen ions to flow down their concentration gradient at Complex V (ATP Synthase), resulting in the formation of ATP. This chemiosmotic process is tightly regulated and highly efficient. Although ATP is the primary (and often most studied) product of cellular respiration, ROS and thermal energy/heat are also generated by mitochondria. ROS include chemical species produced by the incomplete reduction of O<sup>2</sup> . These molecules include superoxide anion (O<sup>2</sup> •−), hydrogen peroxide (H<sup>2</sup> O2 ), and hydroxyl radical (•OH). Mitochondrial ROS are thought to perform a variety of cell signaling functions under normal physiological conditions.

**5. Mitochondrial responses to estrogen**

Both nuclear and mitochondrial genes are subject to regulation by estrogen [32]. Nuclear DNA encodes proteins that are incorporated in mitochondria and influence their function. For example, the binding of estrogen to nuclear ERα induces the expression of peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC1α) [33]. This protein plays an important role in mitochondrial biogenesis, a process by which new mitochondria are formed. The mitochondrion also contains its own maternally inherited DNA, which encodes 37 genes [25]. A recent study in MCF-7 cells showed that estrogen regulates mitochondrial RNA production under serum starvation conditions [34]. Subsequent to estrogen treatment, ERα translocated to the mitochondria and increased expression of mitochondrial tRNAs used to translate mitochondrial proteins. In GH4C1 pituitary cells, treatment with estrogen increases expression of mitochondrial-encoded RNA for subunit II of cytochrome C oxidase [35]. In female rats, levels of mitochondrial-encoded 16S RNA, a housekeeping gene used as a marker for mitochondrial number, are four times higher than males of the same age [36, 37]. Ovariectomy (OVX) in rats is characterized by an increase in liver and brain peroxide production and formation of 8-oxo-2′deoxyguanosine, a marker of mitochondrial DNA damage. These changes were associated with a reduction in the antioxidant protein GSH and MnSOD [36]. Estrogen treatment in OVX rats reversed these responses [36]. Collectively, these data suggest that increased estrogen lev-

Estrogen and Mitochondrial Function in Disease http://dx.doi.org/10.5772/intechopen.73015 467

Estrogen plays an important role in the regulation of apoptosis by stimulating Bcl-2 protein expression and translocation to the mitochondria. This is achieved *via* the Ca2+ regulated ERK pathway [38]. The regulation of both Bcl-2 and *Bax* expression by estrogen has been reported in THP-1 macrophages and human monocyte-derived macrophages. Pre-treatment with estrogen increased the Bcl-2: *Bax* ratio, thus increasing cell viability in the presence of proapoptotic stimuli. Estrogen treatment of cortical neurons has also been shown to inhibit glutamate toxicity and improve cell viability by upregulating Bcl-2 expression [39]. In SH-SY5Y neuroblastoma cells over-expressing ERβ, the receptor has been shown to interact with the pro-apoptotic protein *Bad* and prevent its binding to *Bax*, thereby inhibiting apoptosis [40]. These data suggest that both estrogen and ERs *per se* are anti-apoptotic and modulate disease pathogenesis. Estrogen also preserves cell viability by altering mitochondrial dynamics. In the myocardium of ischemia reperfusion injury rodents, OVX rodents display an increase in mitochondrial fusion after injury, which is reversed by estrogen treatment [41] Work in isolated cortical astrocytes from male and female postnatal day 1 mice shows that estrogen regulates fission and fusion genes in a gender-specific manner [42]. Further, estrogen stimulated mitochondrial biogenesis in skeletal muscle and adipocytes [43, 44]. These data suggest that estrogen can strengthen the mitochondrial network by increasing mitochondrial fusion,

Estrogen effects on mitochondrial function vary in different cell types. For example, estrogen binds specifically to the oligomycin-sensitivity conferring protein of ATP-synthase (Complex V) in brain mitochondria and inhibits ATP production [45]. In contrast, another study showed that the enzymatic activity of F0F1-ATPase, a Complex V subunit, is higher in mitochondria

els regulate mitochondrial and nuclear anti-oxidant protein expression.

thus preserving mitochondrial function and cell viability.

Mitochondria are highly dynamic organelles that are components of a constantly changing, dynamic mitochondrial network. Damaged or old mitochondria can be cleared from the cell by autophagy/mitophagy or bulk clearance and degraded by lysosomal enzymes [26]. Furthermore, mitochondria can also undergo fission and fusion. Fission is the process by which mitochondria bud off from the mitochondrial network. This is regulated by the proteins DRP-1 and FIZZ1. Fusion represents the incorporation of mitochondria in the mitochondrial network and is regulated by mitofusin1 (Mfn1), mitofusin2 (Mfn2), or optic atrophy 1 (OPA1) [27]. These regulatory proteins help to maintain the balance between fission and fusion that is required to preserve cell viability. In different disease states, however, this balance can be disrupted causing mitochondrial dysfunction and cell death.
