Apelin (APLN) and Apelin Receptor (APLNR) in Human Ovary: Expression, Signaling, and Regulation of Steroidogenesis in Primary Human Luteinized Granulosa Cells

Abstract

Apelin (APLN) is a recently discovered adipokine involved in the regulation of various metabolic functions. Its receptor, APLNR, is expressed in reproductive tissues, however, its role in human ovarian cells is unknown. In this study, we identified APLN and APLNR in human ovarian follicles and analyzed their expression in granulosa cells and follicular fluid obtained from obese and nonobese patients, with or without polycystic ovary syndrome (PCOS). We also investigated the effect of APLN on steroidogenesis in cultured human luteinized granulosa cells (hGCs) from nonobese patients without PCOS. Using RT-PCR and immunoblotting, we found that APLN and APLNR were expressed in hGCs and cumulus and theca cells. We confirmed these data immunohistochemically and observed that APLNR and APLN are present in human oocytes at different stages of follicular development. In patients with PCOS, we observed that follicular fluid APLN concentration and granulosa cell APLN and APLNR mRNA expression was higher than that observed in control patients. In cultured hGCs from nonobese patients without PCOS, insulin-like growth factor 1 (IGF1) increased APLNR expression, and recombinant human APLN (APLN-13 and APLN-17) increased both basal and IGF1-induced steroid secretion. These effects on steroid production were reversed when cultured in the presence of ML221, an APLNR antagonist, which was associated with an increased 3beta-hydrosteroid dehydrogenase (HSD3B) protein concentration. We showed that these effects were dependent on the activation of the AKT and MAPK3/1 pathways using pharmacological inhibitors. Our results show that APLN and APLNR are present in human ovarian cells and APLN increases IGF1-induced steroidogenesis in granulosa cells through an increase in HSD3B protein expression and activation of the MAPK3/1 and Akt pathways. Therefore, APLN and APLNR may play a role in human follicular development and the pathogenesis of PCOS.

Apelin abrogates the stimulatory effects of 17β-estradiol and insulin-like growth factor-1 on proliferation of epithelial and granulosa ovarian cancer cell lines via crosstalk between APLNR and ERα/IGF1R

“…Additionally, we previously reported that E2 stimulates migration of OVCAR-3 cells via the ERK1/2 and PI3K pathways [57]. Apelin activates PI3K/Akt in human granulosa cells [58]. Activation of the ERK1/2 signaling pathway by apelin induces proliferation and invasion of MCF-7 cells [59].…”

“…These signaling pathways were examined because binding of IGF-1 to its tyrosine kinase receptor IGF1R activates PI3K/Akt and MAPK/ERK, leading to changes in cell proliferation, differentiation, and apoptosis [65,66]. Moreover, apelin increases IGF-1-induced phosphorylation of PI3K/Akt [58] and regulates steroidogenesis in granulosa cells [67,68]. Roche et al [58] reported that the PI3K/Akt signaling pathway is involved in the effects of apelin on IGF-1-induced steroidogenesis in granulosa cells using pharmacological inhibitors.…”

The Ovary as a Target Organ for Bisphenol A Toxicity

“…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 4 , and cell proliferation in pigs [71] and E 2 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].…”

“…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 4 , and cell proliferation in pigs [71] and E 2 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).…”