137 Effects of lysophosphatidic acid on follicle development in mouse ovaries

Our previous studies demonstrated that ovary fragmentation induces follicle development by increasing actin polymerization, leading to disruption of Hippo signaling and nuclear localization of the Hippo signaling effector Yes-associated protein (YAP), followed by an increase in downstream connective tissue growth factors. Lysophosphatidic acid (LPA) is known to exhibit actin polymerization activity. LPA is an important bioactive lipid involved in physiological processes such as cell proliferation, cell migration, cell differentiation, inflammatory reactions, cell death, and immune responses in vivo. LPA interacts with LPA receptors to induce intracellular signal transduction. LPA receptors are transmembrane proteins belonging to the G protein-coupled receptor family, and by binding to LPA, they regulate the production of second messengers and intracellular signal transduction. Six subtypes of LPA receptors have been identified, each with different affinities for LPA, suggesting different physiological functions. However, the mechanism of LPA affecting follicular development is poorly understood. In this study, we investigated the effects of LPA on actin polymerization in the ovary and the promotion of follicle development in mouse ovarian tissues. To test the effects of LPA, ovaries from 10-day-old ICR mice were incubated with 10 µM LPA or medium alone (control). The ratio of F-actin to G-actin in the ovaries was determined using an F-actin/G-actin in vivo assay kit. The conversion of G-actin to F-actin was time-dependent, and treatment with LPA for 15–30 min significantly increased the ovarian F-/G-actin ratios compared with the controls (Student’s t-test; P < 0.05). After ovarian incubation with LPA for 2 h, nuclear staining of YAP was observed in granulosa cells of primary and secondary follicles. In control ovaries, immunohistochemical staining indicated that YAP was localized in the cytoplasm of granulosa cells in most follicles during the primary and secondary stages. Real-time RT-PCR analyses showed LPA treatment induced the expression of CCN2 in ovarian tissue. Peak increases in CCN2 transcript levels were detected 2 h after culturing with LPA (ANOVA, post-hoc Bonferroni; P < 0.05). In contrast, Ki16425, a lysophosphatidic acid receptor 3 (LPAR3) antagonist, blocked LPA-induced CCN2 mRNA expression in a concentration-dependent manner. To investigate the effect of LPA treatment on follicle growth, ovaries were cultured for 4 days with LPA. After histological analyses and follicle counting of ovarian sections, an increase in the percentage of secondary follicles was found in the LPA-treated group compared with the controls (Student’s t-test; P < 0.05). From these results, we confirmed that the LPA-LPAR3 signaling pathway in ovaries promotes follicle development mediated by Hippo signaling disruption as well as ovary fragmentation treatment.