**3. Indirect actions of BPA in the ovary through adipokines**

Leptin, apelin, chemerin and adiponectin are adipokines that are mainly produced by adipose tissues, but also by other tissues. Adipokines and their receptors are expressed by cells of both the normal and cancerous ovary in humans and other mammals. They play important roles in metabolic processes, such as in the regulation of insulin sensitivity, food intake, adipogenesis and inflammation. Adipokines also regulate ovarian function, including steroidogenesis and oocyte maturation. They also affect ovarian cancer cell proliferation, apoptosis, tumour inva‐ sion and angiogenesis.

The first discovered adipokine is **leptin**, a 167‐amino acid protein encoded by the *ob* gene. The leptin receptor [LEPR, also referred to as the obesity receptor (Ob‐R)] is a single mem‐ brane‐spanning receptor with six isoforms (Ob‐Ra, b, c, d, e and f) resulting from alternative RNA splicing [48]. However, only full‐length Ob‐Rb can transduce signals into cells. Leptin regulates food intake, energy balance and body weight [49]. For example, there is a strong cor‐ relation between the serum leptin level and body fat content; the serum leptin level is higher in obese individuals than in those who are non‐obese [50].

Granulosa and theca cells in mammalian ovaries express both leptin and LEPR. Leptin stimu‐ lates the production of ovarian steroid hormones by affecting insulin, insulin‐like growth fac‐ tor 1 (IGF‐1) and different gonadotrophins in the cow [51–53], pig [54, 55], rodent [56, 57] and human [58–60]. There is a correlation between the serum leptin level and the P4 concentration during the menstrual cycle in humans, as well as between E<sup>2</sup> and human chorionic gonado‐ trophin (hCG) levels throughout pregnancy [61].

Additionally, a previous study showed an association between Ob‐Rb overexpression and survival in 59.2% of ovarian epithelial cell cancers [62]. OVCAR‐3 cells express both long (Ob‐ Rb) and short (ObRt) leptin isoforms [63, 64], which associate with the progression of ovarian epithelial cell cancers. In vitro studies show that leptin promotes BG‐1 and OVCAR‐3 cell proliferation [34, 63] and inhibits SKOV3, MDAH2774 and OVCAR‐3 cell apoptosis [34, 62]. Moreover, leptin stimulates OVCAR‐3 cell migration, which is mediated via the activation of ERK1/2, AKT and STAT3 signalling [65]. Leptin also acts on ovarian cancer cells in endocrine manner because they do not produce leptin [35].

characterised by the upregulation of mesenchymal proteins such as N‐cadherin, downregu‐ lation of epithelial cell‐associated proteins such as E‐cadherin and overexpression of matrix metalloproteinases (MMPs). MMP‐2 and MMP‐9 are the key enzymes required for the ini‐ tial steps of ovarian cancer metastasis [36, 37]. In OVCAR‐3 cells, BPA upregulates MMP‐2, MMP‐9 and N‐cadherin expression by activating ERK1/2 and AKT signalling, which pro‐

**Figure 4.** BPA action on epithelial ovarian cancer progression. Stars indicate the sites of BPA action. The arrow facing up indicates a stimulation, and the arrow facing down indicates an inhibition by BPA. Matrix metalloproteinase‐2 (MMP‐2), matrix metalloproteinase‐9 (MMP‐9), vascular endothelial growth factor‐A (VEGF‐A), vascular endothelial growth factor receptor 2 (VEGF‐R2), extracellular signal‐regulated kinases (ERK1/2), signal transducer and activator of

Vascular endothelial growth factor‐A (VEGF‐A), which is upregulated in most solid tumours, including ovarian cancers, correlates with tumour progression and poor prognosis [39, 40]. Several studies show that the serum VEGF‐A level is higher in patients with ovarian cancer than in healthy individuals [41–43]. In addition, the expression of VEGF‐A and its receptor (VEGF‐R2) is higher in cancerous ovarian tissues than in benign or normal ovarian tissue [44]. BPA upregulates VEGF‐A expression in reproductive organs, such as the uterus and vagina in the rat [45] and the ovary in the pig [46]. Moreover, BPA markedly increases VEGF‐A and VEGF‐R2 expression in OVCAR‐3 and SKOV‐3 cells [47], indicating a possible intensification

These findings indicate that BPA promotes the progression of epithelial ovarian cancer by stimulating epithelial cell proliferation and migration and inhibiting apoptosis. However, there is no evidence to indicate that BPA affects stromal‐ and germinal‐derived ovarian

motes cell migration [38] (**Figure 4**).

transcription 3 (STAT3) and protein kinase B (Akt).

62 Bisphenol A Exposure and Health Risks

cancers.

of pro‐angiogenic activity in ovarian cancer cells (**Figure 4**).

BPA can affect the expression of adipokines. BPA increases leptin mRNA expression in the preadipocyte 3T3‐L1 cell line [66] and LEPR mRNA and protein expression in OVCAR‐3 cells, which creates more binding sites for leptin [34] (**Figure 5**). BPA and leptin also inhibit the apoptosis of cancerous ovarian cells, indicating that BPA can potentiate leptin action in OVCAR‐3 cells [35]. These results suggest that BPA increases leptin activity in cancerous ovarian cells.

**Apelin** is a bioactive peptide that was originally identified in bovine stomach extracts as the endogenous ligand of the orphan G protein‐coupled apelin receptor (APJ) [67]. The apelin level is elevated in obese and insulin‐resistant individuals and in those with high insulin lev‐ els. Apelin functions in a broad range of physiological processes, including fluid homeostasis, food intake, energy metabolism, cardiovascular function and angiogenesis.

**Figure 5.** BPA action on adipokines and their receptors expression in the epithelial ovarian cancer cells. Stars indicate the sites of BPA action. The arrow facing up indicates a stimulation, and the arrow facing down indicates an inhibition. Leptin receptor (LEPR), orphan G protein‐coupled apelin receptor (APJ), chemokine‐like receptor 1 (CMKLR1), adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2).

The APJ is expressed by granulosa cells, and both apelin and its receptor are expressed by theca cells in the bovine ovary [68]. Apelin and APJ expression in theca cells are induced by LH, whereas increased APJ expression in granulosa cells associates with follicular atresia [68]. Apelin and APJ expression in mature follicles indicate that the apelin‐APJ system is important for follicle selection and dominance in cows [69]. Apelin and APJ immunoexpression have been reported in granulosa and theca cells, as well as in oocytes in human follicles, at different stages of development [70]. Furthermore, apelin promotes ovarian steroid hormone secretion, in particular, P<sup>4</sup> , and cell proliferation in pigs [71] and E<sup>2</sup> synthesis in humans [70], indicating that apelin has a direct role in folliculogenesis. A recent in vitro study showed that apelin stimulates rat granulosa cell proliferation; however, apelin inhibits granulosa cell apoptosis via PI3K/AKT signalling [72].

The human KGN cell line, which is derived from granulosa‐like tumours, expresses apelin and APJ mRNA and protein [70]. Apelin and its receptor are also expressed by cancerous ovarian epithelial cell lines (OVCAR‐3, SKOV‐3 and Caov‐3), the cancerous granulosa cell line (COV434) and the non‐cancerous ovarian epithelial cell line (HOSEpiC). Moreover, the basal apelin concentration in both epithelial and granulosa cancer is 0.4–0.6 ng/ml. At these concentrations, apelin acts as a mitogen in these cells. However, BPA increases apelin expres‐ sion and secretion only in epithelial cancer cells (**Figure 5**). BPA activates the peroxisome proliferator‐activated receptor gamma (PPARγ) and not ERα and ERβ, because the PPARγ antagonist (GW9662) abolished the effects of this environmental toxicant on apelin ovarian expression [73].

**Chemerin**, also referred to as RARRES2 or TIG2, is secreted as prochemerin, an inactive pre‐ cursor that is processed into biologically active chemerin [74]. Several isoforms of biologi‐ cally active chemerin with variable C‐terminal amino acids have been characterised by their abilities to bind and activate the chemokine‐like receptor 1 (CMKLR1). Chemerin regulates adipogenesis, lipolysis and glucose metabolism.

Human granulosa and theca cells express chemerin and its receptor, CMKLR1. Chemerin reduces IGF‐1–induced thymidine incorporation, as well as E<sup>2</sup> and P4 synthesis, by decreasing the phosphorylation of the IGF‐1R beta subunit and MAPK ERK1/2 in cultured human gran‐ ulosa cells [75]. Similarly, chemerin decreases steroid hormone production and MAPK3/1 phosphorylation, probably through CMKLR1, in cultured bovine granulosa cells. In cumu‐ lus‐oocyte complexes, chemerin blocks meiotic progression at the germinal vesicle stage and inhibits MAPK3/1 phosphorylation in both oocytes and cumulus cells during in vitro matura‐ tion [76]. Chemerin also induces rat granulosa cell apoptosis and suppresses basal, and FSH‐ and growth differentiation factor‐9‐stimulated, follicular growth in vitro [77].

Chemerin and its receptor are expressed by KGN cells, where chemerin markedly reduces IGF‐1–induced cell proliferation and P<sup>4</sup> and E<sup>2</sup> synthesis [75]. Human cancerous ovarian epi‐ thelial cell lines (OVCAR‐3 and SKOV‐3), cancerous granulosa cell lines (COV434 and KGN) and the non‐cancerous ovarian epithelial cell line (HOSEpiC) also express chemerin and its receptor. Moreover, chemerin expression decreases in BPA‐treated GCTs (unpublished data). However, there is no information on the roles of chemerin in the development and progres‐ sion of ovarian cancer and no data on the serum chemerin level in patients with ovarian cancer.

The APJ is expressed by granulosa cells, and both apelin and its receptor are expressed by theca cells in the bovine ovary [68]. Apelin and APJ expression in theca cells are induced by LH, whereas increased APJ expression in granulosa cells associates with follicular atresia [68]. Apelin and APJ expression in mature follicles indicate that the apelin‐APJ system is important for follicle selection and dominance in cows [69]. Apelin and APJ immunoexpression have been reported in granulosa and theca cells, as well as in oocytes in human follicles, at different stages of development [70]. Furthermore, apelin promotes ovarian steroid hormone secretion,

**Figure 5.** BPA action on adipokines and their receptors expression in the epithelial ovarian cancer cells. Stars indicate the sites of BPA action. The arrow facing up indicates a stimulation, and the arrow facing down indicates an inhibition. Leptin receptor (LEPR), orphan G protein‐coupled apelin receptor (APJ), chemokine‐like receptor 1 (CMKLR1),

that apelin has a direct role in folliculogenesis. A recent in vitro study showed that apelin stimulates rat granulosa cell proliferation; however, apelin inhibits granulosa cell apoptosis

synthesis in humans [70], indicating

, and cell proliferation in pigs [71] and E<sup>2</sup>

adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2).

in particular, P<sup>4</sup>

via PI3K/AKT signalling [72].

64 Bisphenol A Exposure and Health Risks

**Adiponectin** (APN), also referred to as ACRP30 or AdipoQ, is the most abundant secreted protein expressed exclusively by adipose tissue [78]. There are three major APN isoforms, namely, a trimeric low‐molecular‐weight (LMW) isoform, a hexameric medium‐molecular ‐weight (MMW) isoform and a multimeric high‐molecular‐weight (HMW) isoform [79]. Adiponectin binds its receptors, AdipoR1 and AdipoR2.

The expression of adiponectin and its receptors has been reported in the ovary of various species, including the rat, chicken, pig, cow and human [78]. Except for the cow, adiponectin expression is absent/low in granulosa and cumulus cells of the mouse, chicken and human. In the bovine ovary, adiponectin expression varies in different cells during development [80]. Furthermore, adiponectin receptors are expressed by oocytes and early embryos of the pig and mouse [81]. In vitro studies report adiponectin to decrease insulin‐induced androgen and P4 secretion in bovine theca cells. In rat, chicken and human cultured granulosa cells, how‐ ever, adiponectin increases P<sup>4</sup> and/or E<sup>2</sup> secretion in response to IGF‐1. Several reports in dif‐ ferent species, including humans, indicate that adiponectin can modulate not only granulosa cell steroidogenesis but also the expression of genes involved in ovulation. In the cow, adipo‐ nectin decreases insulin‐induced steroidogenesis and increases IGF‐1–induced proliferation of cultured granulosa cells. Adiponectin does not affect oocyte maturation and embryo devel‐ opment in vitro [82]; however, it stimulates oocyte meiotic maturation and embryo develop‐ ment in the pig [81].

The serum adiponectin level is markedly lower in patients with early‐stage ovarian cancer than in healthy women. Adiponectin possesses anti‐tumourigenic properties; it can suppress tumour growth and cell proliferation, arrest cell growth and induce apoptosis. AdipoR1 promotes KGN cell survival, whereas AdipoR2 regulates steroid hormone synthesis by acti‐ vating MAPK ERK1/2 [83]. Furthermore, the AdipoR1 mRNA level was lower in Leghorn chicken cancerous ovaries than in normal ovaries [84], suggesting that adiponectin signalling restricts ovarian cancer progression by suppressing tumour cell proliferation and inducing cell apoptosis.

Human cancerous ovarian epithelial cell lines (OVCAR‐3, SKOV‐3 and Caov‐3), the cancerous granulosa cell line (COV434) and the non‐cancerous ovarian epithelial cell line (HOSEpiC) express AdipoR1 and AdipoR2, but not adiponectin. Moreover, the AdipoR1 mRNA level is markedly higher in OVCAR‐3, SKOV‐3, Caov‐3 and COV434 cells than in HOSEpiC cells, whereas the AdipoR2 mRNA level is similar among all tested cell lines. BPA does not affect AdipoR1 and AdipoR2 expression (unpublished data), although it decreases the expression and secretion of adiponectin in 3T3‐L1 adipocytes [85]. In cultured porcine ovarian follicles, however, BPA markedly increases the expression and secretion of adiponectin, as well as the expression of its receptors, indicating that this environmental toxicant contributes to ovarian dysfunction in obesity‐related disorders (unpublished data).
