Fibroblast growth factor 2 regulates cumulus differentiation under the control of the oocyte

Abstract

Purpose: We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM.

Methods: In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA.

Results: FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6.

Conclusions: This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.

Keywords: Apoptosis; Cumulus expansion; FGF2; IVM; Meiosis; Oocyte-secreted factors.

Fig. 1

Effects of time in culture (A; FSH at 10 ng/mL, equivalent to 2 × 10⁻⁵ UI/mL); and FSH graded doses (B) on relative values of FGF2 mRNA abundance in bovine cumulus cells undergoing IVM. Data are presented as mean values (± S.E.M.) relative to a calibrator sample calculated by the ΔΔCt method with efficiency correction. Different calibrator samples were utilized in the time-course (A) and dose-response (B) experiments. Bars with different letters are significantly different (P < 0.05)

Fig. 2

Effects of oocyte removal on relative values of FGF2 mRNA expression. Intact (COC), oocytectomized (OOX), and OOX plus denuded oocytes (OOX + OD; 1 oocyte/μL) were subjected to IVM for 4 h (n = 4 replicates) and 22 h (n = 5 replicates). Data are presented as mean values (± S.E.M.) relative to a calibrator sample calculated by the ΔΔCt method with efficiency correction. Different calibrator samples were utilized in experiments assessing the effect of oocyte removal at 4 and 22 h of IVM. Bars with different letters are significantly different (P < 0.05)

Fig. 3

Effects of FGF2 on progression of meiosis. COC were subjected to IVM with graded doses of FGF2 for 6 h to assess germinal vesicle status (A; GVBD = germinal vesicle breakdown; n = 5 replicates) and for 22 h to assess meiosis II completion (B; MII = meiosis II/includes oocytes from telophase I to metaphase II; n = 5 replicates). Bars with different letters are significantly different (P < 0.05)

Fig. 4

Effects of FGF2 on the percentage of COC exhibiting different degrees of cumulus expansion (A; Grade 1: fully expanded, Grade 2: partially expanded, Grade 3: weakly or not expanded; n = 5 replicates) and on dissociation of cumulus cells (B; n = 5 replicates). Bars with different letters are significantly different (P < 0.05)

Fig. 5

Effects of FGF2 on mRNA abundance of genes regulating cumulus expansion. COC were subjected to IVM with graded doses of FGF2 for 6 h (n = 5 replicates). Data are presented as mean values (± S.E.M.) relative to a calibrator sample by the ΔΔCt method with efficiency correction. Bars with different letters are significantly different (P < 0.05)

Fig. 6

Effects of FGF2 on the percentage of viable cumulus cells (cells marked with hoechst 33342 but not with propidium iodide), of cumulus cells with activated caspase 3/7, and of cumulus cells stained for Annexin V (n = 6 replicates). Data are presented as means (± S.E.M.) and bars with different letters are significantly different (P < 0.05)