Abstract
BackgroundThe Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation.MethodsTo assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test or Dunnett’s post-hoc test.ResultsOur data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs.ConclusionsOur results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.
Highlights
The Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species
The findings indicate that in the absence of ovulatory signals, oocyte-secreted factors suppress Hippo signaling in cumulus cells which leads to activation of YAP1, stimulation of cell proliferation and suppression of differentiation
For co-culture experiment, the following groups were used: (1) Control: 20 intact cumulus-oocyte complex (COC) cultured for 20 h, followed by harvesting of the cumulus cells; (2) OOX: cumulus cells from 20 COCs cultured for 20 h; (3) Coculture: Cumulus cells from 20 COCs co-cultured with 40 denuded oocytes (2 oocytes/μl)
Summary
The Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. In the absence of ovulatory signals, the oocyte is maintained in meiotic arrest while the granulosa cells are highly proliferative but susceptible to. EGF causes the up-regulation of progesterone from the cumulus cells [1] which may serve as a sperm chemoattractant factor during fertilization and/or may be important for oocyte nuclear maturation [36,37,38,39]. Granulosa cells transition from highly proliferative and un-differentiated phenotype into terminally differentiated cells with little capacity to proliferate. These dramatic transformations of the somatic follicular cells in the preovulatory follicle are critical for optimal fertility and ovarian function, but the downstream intra-follicular mechanisms mediating these responses are not completely known
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
