Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice

Abstract

Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

Figure 1.

Epab^−/− oocytes fail to promote cumulus expansion and Epab^−/− CCs fail to undergo expansion in the presence of WT oocytes. Oocytes and OOXs were obtained from COCs of 12-week-old WT or Epab^−/− mice and cultured in combinations, as indicated, for 16 hours in the presence (+) or absence (−) of 10-ng/mL EGF. A, Representative images of OOX/oocyte coculture combinations in the presence or absence of 10-ng/mL EGF. WT OOXs cultured with WT oocytes showed cumulus expansion in the presence of EGF. Conversely, WT OOXs cultured with KO oocytes or KO OOXs cultured with WT oocytes did not undergo expansion despite EGF treatment. Scale bar, 10 μm. B, Cumulus expansion of OOXs was scored after 16 hours of coculture with WT or Epab^−/− DOs in the presence of EGF. The grade of cumulus expansion was assessed as previously described (55). A score of 0/1 indicates no detectable response (0) or the minimum observable response (1). A score of 4 indicates the maximum degree of expansion in which the cumulus oophorus and corona radiata have undergone expansion. A score of 2 or 3 indicate intermediate levels of expansion between 0/1 and 4. Data are presented as mean ± SEM from 3 independent experiments. Bars with different letters are significantly different (P < .05). Significance was determined by one-way ANOVA.

Figure 2.

The expression of transcripts encoding proteins that mediate cumulus expansion do not increase in response to EGF in CCs from WT OOX/KO oocyte and KO OOX/WT oocyte groups. Oocytes (Oo) and OOX's were obtained from COCs of 12-week-old WT or Epab^−/− mice 44–48 hours after PMSG injection. The expression of Ptgs2 (A), Btc (B), and Tnfaip6 (C) in CCs after 16 hours of coculture in the presence or absence of 10-ng/mL EGF was assessed using qRT-PCR. Expression of target genes was normalized to β-actin levels, and results are shown as the fold change in gene expression between EGF stimulation (+) and no EGF stimulation (−). Data are presented as mean ± SEM from 3 independent experiments. *, significance between transcript levels with or without EGF treatment (P < .05). Significance was determined by t test.

Figure 3.

The expression of GDF9 and BMP15 is not altered in the oocytes of Epab^−/− mice. A, GDF9 and BMP15 expression was detected by Western blot analysis using 300 GV oocytes from WT or Epab^−/− mice. GCs from WT mice were used as a negative control. B, Band intensities were analyzed using densitometry and normalized to GAPDH. Data are represented as the mean ± SEM from 3 independent experiments. There was no significant difference in GDF9 or BMP15 expression between WT and Epab^−/− oocytes. Arrow designates WT GC bar.

Figure 4.

The response to LH stimulation is impaired in GCs of Epab^−/− mice. A–D, Western blot analysis was performed to compare LH-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK in GCs from WT and Epab^−/− mice. GCs were collected from WT and Epab^−/− mice at 12 weeks of age 44–48 hours after 5-IU PMSG injection, cultured, serum starved, and treated with LH (1 μg/mL) for 5 and 10 minutes. Representative Western blottings of total (T) and phosphorylated (p) proteins are shown in A. The intensity of Western blot bands was analyzed using densitometry. The ratio of p-MEK1/2 (B), p-ERK1/2 (C), and p-p90RSK (D) to total protein was normalized to untreated WT control. Data are presented as mean ± SEM from 3 separate experiments. *, significant difference between groups (*, P < .05; **, P < .01; ***, P < .001). Significance was determined by two-way ANOVA followed by Tukey's multiple comparisons test.

Figure 5.

The response to EGF stimulation is impaired in GCs of Epab^−/− mice. A–D, Western blotting was performed to compare EGF-induced phosphorylation of MEK1/2, ERK1/2, and p90RSK in GCs from WT and Epab^−/− mice. GCs were collected from WT and Epab^−/− mice at 12 weeks of age 44–48 hours after 5-IU PMSG injection, cultured, serum starved, and treated with EGF (10 ng/mL) for 5 and 10 minutes. Representative Western blottings of total (T) and phosphorylated (p) proteins are shown in A. The intensity of Western blot bands was analyzed using densitometry. The ratio of p-MEK1/2 (B), p-ERK1/2 (C), and p-p90RSK (D) to total protein was normalized to untreated WT control. Data are presented as mean ± SEM from 3 separate experiments. *, significant difference between groups (*, P < .05; **, P < .01; ***, P < .001). Significance was determined by two-way ANOVA followed by Tukey's multiple comparisons test.

Figure 6.

The activation of EGFR is impaired in GCs of Epab^−/− mice. A–C, Western blot analysis was performed to compare EGF-induced phosphorylation of EGFR in GCs from WT and Epab^−/− mice. GCs were collected from WT and Epab^−/− mice at 12 weeks of age after 44–48 hours of 5-IU PMSG injection, cultured, serum starved, and treated with EGF (10 ng/mL) for 5 and 10 minutes. Representative Western blottings are shown in A. The intensity of Western blot bands corresponding to total (T) EGFR (B) and phosphorylated (p) EGFR (C) were analyzed using densitometry. The ratio of p-EGFR to total EGFR was normalized to untreated WT. Data are presented as mean ± SEM from 3 separate experiments. *, significant difference between groups (*, P < .05; ***, P < .001; ***, P < .0001). Significance was determined by two-way ANOVA followed by Tukey's multiple comparisons test.

Figure 7.

CREB activation does not increase in response to short-term LH treatment in GCs from WT or Epab^−/− mice. Western bot analysis was performed to compare LH-induced phosphorylation of CREB in GCs from WT and Epab^−/− mice. GCs were collected from WT and Epab^−/− mice at 12 weeks of age 44–48 hours after 5-IU PMSG injection, cultured, serum starved, and treated with LH (1 μg/mL) for 5 and 10 minutes. Representative Western blottings of total (T) and phosphorylated (p) CREB are shown in A. The intensity of Western blot bands was analyzed using densitometry (B). The ratio of p-CREB to total CREB was normalized to untreated WT control. Data are presented as mean ± SEM from 3 separate experiments. Phosphorylation of CREB was not significantly different in WT or Epab^−/− GCs after 5 or 10 minutes of LH treatment.