Supplementary Material for: Expression of apelin and apelin receptor protein in the hypothalamo–pituitary–ovarian axis during estrous cycle of mice

Introduction: Apelin is an endogenous neuropeptide expressed in the hypothalamus, pituitary and ovary; binds to apelin receptor for various functions. Hypothalamus, pituitary and ovarian axis is tightly regulated during estrous cycle. The status of apelin and its receptor has not been investigated in HPO axis during estrous cycle. Methods: To explore the expression of apelin and APJ in HPO axis of mice during estrous cycle, mice were divided into four groups as proestrus, estrus, metestrus and diestrus, and apelin and apelin receptor were checked. Further, to explore the role of apelin in the gonadotropin secretion, in vitro study of pituitary was performed at proestrus and estrus stages. Result: The expression apelin and APJ in the hypothalamus showed elevation during estrous cycle of post-ovulatory phases, metestrus and diestrus. The immunolocalization of apelin and APJ in the anterior pituitary showed more abundance in the estrus and diestrus. In vitro results showed that GnRH agonist simulated LH secretion was suppressed by the apelin13 peptide from pituitary of proestrus and estrus phases. This suggests an inhibitory role of apelin on the gonadotropin secretion. The ovary also showed conspicuous changes in the presence of apelin and APJ during estrous cycle. The expression of apelin and APJ coincides with folliculogenesis and corpus luteum formation and expression of apelin system in the different cell types of ovary suggest its cell specific role. Apelin has shown stimulatory role in the ovarian steroid secretion, proliferation and corpus luteum. Conclusion: Overall our results showed that apelin system changes along HPO axis during estrous cycle and might have an inhibitory at level of hypothalamus and pituitary and stimulatory at ovarian level.