Epiregulin, as inducer of nuclear maturation in in vitro grown mouse oocytes from cultured early preantral follicles

Student thesis: Doctoral Thesis


The amount of patients surviving cancer (by a more aggressive therapy) is increasing. As a result more patients are interested in preserving their fertility before undergoing a cancer treatment. Women at reproductive age, but also parents of adolescent girls and children facing cancer are offered the possibility to cryopreserve pieces of ovarian tissue and later restore their fertility when they are disease free.
Although grafting back those pieces has proven some success, the latent risk for reintroducing malignant cells, enforces the search for alternatives. The in vitro culture of ovarian tissue or follicles is an appealing alternative, however more research is needed to be able to offer such an approach to patients.
This thesis focused on the optimization of the protocol to induce meiotic resumption and mucification of cumulus cells.
In a first study the potential helping effect of a PDE3-inhibitor (PDE3-I), Org9935, in long-term culture of cumulus-corona-oocyte complexes (COC) and the efficiency of EREG in inducing oocyte maturation were assessed. COCs from cultured early antral follicles were sub-cultured for 4 days in presence of different concentrations of a PDE3A inhibitor (Org9935). COCs survival was dose dependently improved by Org9935. However, in the high Org9935 dose range meiotic resumption, regularly induced by hCG/EGF, was prohibited. When sub-culture was done in the absence of Org9935; hCG, EREG, or hCG/EREG induced low rates of polar body extrusion (0%, 17%, and 21%, respectively). Taking advantage of the improved survival but washing out the PDE3 inhibitor prior to stimulation produced on average between 63% and 66% of polar body oocytes.
The second study aimed to test the efficacy of EREG as meiotic inducer upon cultured ovarian follicles. Pre-antral mouse ovarian follicles were cultured until antral stage and final maturation was induced by administration of 0.65nM EGF or 100nM EREG without or with 1.2IU/ml hCG. Results showed that both EGF or EREG induce poor mucification/expansion of cumulus cells and low meiotic reinitiation (25% and 22% GVBD respectively; versus 97 and 90% GVBD by control hCG/EGF and hCG/EREG respectively). Moreover, EREG induced meiosis more efficiently in COCs isolated from cultured follicles. Lastly, EGF or EREG marginally produced progesterone while estradiol was not decreased. Since a similar scenario was also observed in the COC subculture of the first study, the relevance of elevated progesterone for meiotic resumption to occur was also assessed. However, progesterone was not found to be a critical factor for normal progression of meiosis in this mouse model.
Based on our previous findings, we hypothesized that LH receptor activation might play a role in oocyte meiotic resumption and cumulus cell response. In a third study we sorted out the LH - and EGF - mediated response (following LH stimulus), fully-grown in vitro cultured mouse ovarian follicles were treated with a combination of hCG plus galardin (an inhibitor of the release of endogenous EGF-like factors) or hCG plus galardin plus EGF. The results suggest that LH (hCG) by itself is insufficient to induce a maximal meiotic resumption and proves the need for concomitant EGFR activation. Analysis of transcript levels of Egfr, Ereg, Cyp19a1, Hsd3b1, Adamts1, and Has2 in cumulus cells indicates that the EGFR signalling preserves the expression profile of cumulus, which otherwise would behave as undifferentiated granulosa cells.
In conclusion, COCs used for In vitro Maturation (IVM) benefit of the presence of a PDE3A inhibitor, which improves not only survival but also meiotic resumption, on the condition that it is washed out before administering the meiosis stimulus.
Based on the facts that both LH and EGF signalling are needed for meiotic resumption to occur in cultured follicles, and EGF maintains cumulus cells functionality, it is proposed that within this in vitro mouse model, concomitant LH and EGF signalling during ovulation are required. Altogether, the knowledge reported here intends to help to understand the processes involved in in vitro ovulation and aim to be used as a guideline for further improvement of follicle culture systems.
Date of Award19 Jan 2012
Original languageEnglish
Awarding Institution
  • Vrije Universiteit Brussel
SupervisorJohan Smitz (Promotor), Christiaan Van Schravendijk (Jury), Michel De Vos (Jury), Greta Verheyen (Jury), Luc Baeyens (Jury), Soren Ziebe (Jury), Jo Leroy (Jury) & Isabelle Donnay (Jury)


  • Follicle culture
  • Gene expression
  • Epiregulin
  • EGF
  • Oocyte

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