Expression of apelin and apelin receptor (APJ) in porcine ovarian follicles and in vitro effect of apelin on steroidogenesis and proliferation through APJ activation and different signaling pathways

Highlights

• Expression of apelin and APJ increased with ovarian follicular growth.

• Apelin stimulation basal, but decrease IGF1- and FSH-induced steroidogenesis.

• Apelin increased cell proliferation and AMPK, Akt and ERK1/2 activation.

Abstract

Apelin was thought to be an adipocyte-specific hormone, but recent studies indicate a link between apelin and female reproductive function. Using real-time PCR, immunoblotting, immunohistochemistry and ELISA, we demonstrated expression of apelin and its receptor (APJ) in ovarian follicles of different sizes from mature pigs. Apelin concentration in the follicular fluid, and expression of both apelin and APJ, increased with follicular growth; greatest values were found in large follicles. Immunohistochemistry revealed the positive staining for apelin and APJ in membranes of granulosa, than theca cells. Furthermore, we observed strong expression of apelin in oocytes and APJ in the zona pellucida. The effect of apelin (0.02, 0.2, 2 and 20 ng/ml) on basal and IGF1- and FSH-induced steroid hormone (progesterone [P4], and estradiol [E2]) secretion, steroidogenic enzyme (3βHSD and CYP19A1) expression and cell proliferation (Alamar blue) was determined. Apelin was found to increase basal steroid secretion, but decrease IGF1- and FSH-induced steroid secretion, and 3βHSD and CYP19 expression. Apelin also increased cell proliferation and the phosphorylation level of 5′-monophosphate-activated protein kinase (AMPK), phosphatidyl inositol 3′ kinase/Akt (Akt) and extracellular signal-regulated kinases (ERK1/2). AMPKα was involved in the action of apelin in P4 production, and MAPK/ERK and Akt/PI3 mediated the proliferative effect of apelin. However, these effects on steroid secretion and cell proliferation were abolished when cultured in the presence of ML221, an APJ antagonist. In conclusion, apelin appears to regulate ovarian follicular functions such as steroidogenesis and proliferation via APJ activation and different signaling pathways.

Introduction

Apelin is a bioactive peptide that was originally identified in bovine stomach extracts as the endogenous ligand of the orphan G protein-coupled receptor APJ [1]. It is derived from a 77-amino-acid prepropeptide that is cleaved into a 55-amino-acid fragment and then into shorter forms. The physiologically active form is thought to be apelin-36, although the pyroglutamylated form of apelin-13, which is also produced endogenously, is more potent. Boucher et al. [2] found that apelin is produced and secreted by mature human and murine adipocytes, and that the apelin mRNA levels found in these cells are similar to those found in the stroma-vascular fraction (which contains other cell types present in adipose tissue) and in organs such as the kidney and heart [2]. In obese humans, plasma apelin levels are significantly higher than in lean controls [2]. Apelin is involved in a broad range of physiological functions, e.g., fluid homeostasis [3], regulation of food intake [4], energy metabolism [5], the cardiovascular system [6] and angiogenesis [7].

Recent data has suggested that apelin, like other adipokines such as resistin [8], [9], adiponectin [10], chemerin [11] and visfatin [12], may affect male and female fertility. Indeed, mRNA and protein expression of apelin and APJ has been reported in several reproductive tissues including the hypothalamus [13], [14], [15], pituitary [14], [15], [16], testis, uterus [17] and ovary [18], [19], [20], [21]. Intracerebroventricular injection of apelin-13 significantly suppressed luteinizing hormone (LH) [22] and follicle-stimulating hormone (FSH) [23] in male rats, and led to decreases in serum testosterone (T) levels [22], [23]. However, the role of apelin in ovarian steroid secretion has only been partly investigated. Recently published data by Roche et al. [24] showed that apelin stimulated progesterone (P4) secretion in cultured human luteinized granulosa cells. The expression of apelin and APJ has been demonstrated in bovine ovarian follicles and corpus luteum (CL) [19], [20], [21], as well as in human ovarian follicles [24]. In ovarian follicles, P4 increases expression of APJ in cultured bovine granulosa cells, while LH induces the expression of apelin and APJ in cultured theca cells, suggesting that apelin has a role in ovarian follicle physiology, e.g., follicle selection and dominance [20]. Published literature led us to hypothesize that apelin is expressed in the porcine ovary and modulates ovarian function by directly affecting cell steroidogenesis and proliferation. Pigs are becoming a valid alternative to traditional non-rodent species in pharmacological, physiological and toxicological studies because many of their physiological characteristics resemble those of humans.

The objectives of this study were to investigate 1) the mRNA and protein expression of apelin and APJ in porcine ovarian follicles of different sizes, and their immunolocalization and concentrations in follicular fluid and ovarian follicle, and 2) the direct effect of recombinant human apelin-13 on basal and IGF1- and FSH-stimulated steroidogenesis and cell proliferation in co-culture of granulosa (Gc) and theca (Tc) cells. As a molecular mechanism of apelin action on ovarian steroidogenesis, we propose the activation of adenosine 5′-monophosphate-activated protein kinase (AMPK), and the promotion of cell proliferation by the activation of phosphatidylinositol 3′-kinase/Akt and the extracellular-signal-regulated kinase (ERK1/2) signaling pathway. Studies have provided evidence that AMPK is involved in the regulation of ovarian steroid production in various species [25], [26], [27], while ERK1/2 and Akt signaling pathways are widely expressed in ovarian cells and are involved in cell survival and proliferation in the ovary [28], [29], [30].