FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence

Abstract

A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality.

Figure 1.

TPX2 and TEX19.1 accumulation and IL-7 secretion during oocyte maturation. A, CEOs were cultured for 17 hours in milrinone (GV) or in the absence (IVM) or presence of FSH (IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and extracts used for Western blot. Representative immunoblots of TPX2, TEX19.1, and α-tubulin (TUB) are reported. The bar graph represents the ratio TPX2 to α-tubulin or TEX19.1 to α-tubulin (mean ± SEM) in the different treatments (n ≥ 3). ***, Significant differences between IVM and IVM+FSH (paired t test, P < .001). B, CEOs were cultured for 24 hours in the absence (IVM) or presence of FSH (IVM+FSH). At the end of the incubation, spent media were collected and the IL7 content measured by an ELISA. The bar graph represents IL-7 concentration (mean ± SEM) in the two treatments (n ≥ 3). *, Significant differences (Mann-Whitney test, P < .05).

Figure 2.

FSH promotes an increase in translation of Tpx2 and Il7 but not Tex19.1 reporters. A–C, cRNA was injected in oocytes still enclosed in the cumulus cells and cultured for 16 hours in milrinone (GV) or different IVM treatments (IVM, IVM+AREG, IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and the luciferase activity measured in the oocyte extract. The bar graphs represent the ratio (mean ± SEM) between the renilla luciferase activity, translated under the control of the Tpx2 3′ UTR (n = 5), Il7 3′ UTR (n ≥ 3), or Tex19.1 3′ UTR (n = 3), and the FL activity used for injection normalization. *, **, Significant differences (P < .05 and P < .01, respectively) in the luciferase activity between IVM+AREG and IVM+FSH compared with IVM (one way ANOVA followed by Tukey's multiple comparison test). NS, not significant.

Figure 3.

Oocyte AKT is phosphorylated in response to FSH treatment. CEOs were cultured for 2.5 hours in milrinone (GV) or different IVM treatments (IVM, IVM+AREG, IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and extracts used for Western blot. Representative immunoblots of phosphorylated (p-AKT) and total AKT (AKT) are reported. The bar graph represents the ratio p-AKT to AKT (mean ± SEM) in the different treatments (n = 6). *, Significant differences between treatments (Kruskal-Wallis test followed by Dunn's multiple comparison test, P < .05).

Figure 4.

The knockdown of the EGFR in the somatic compartment prevents both AREG- and FSH-induced AKT activation and Tpx2 reporter translation. A, CEOs were collected from Egfr^fl/fl or Egfr^δ/fl:Cyp19cre mice, homogenized, and extracts used for Western blots. Representative immunoblots for EGFR and α-tubulin (TUB) are reported. B, CEOs were collected from Egfr^fl/+ or Egfr^δ/fl:Cyp19cre mice and cultured for 2.5 hours in different IVM conditions (IVM, IVM+AREG, IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and extracts used for Western blots. Representative immunoblots of phosphorylated (p-AKT) and total AKT (AKT) are reported. The bar graph represents the ratio of p-AKT to AKT (mean ± SEM) in the different treatment/genotype (n = 6). ** and *, Significant differences (P < .01 and P < .05) between treatments in Egfr^fl/+ mice, whereas no differences were observed in Egfr^δ/fl:Cyp19cre mice (Kruskal-Wallis test followed by Dunn's multiple comparison test). C, CEOs were collected from Egfr^fl/+ or Egfr^δ/fl:Cyp19cre mice. Oocytes still enclosed in the cumulus cells were injected with Tpx2 reporter and cultured for 16 hours in milrinone (GV) or in in different IVM conditions (IVM, IVM+AREG, IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and the luciferase activity measured in the oocyte extract. The bar graph represents the ratio (mean ± SEM) between the RL activity, translated under the control of the Tpx2 3′ UTR and the FL activity used for injection normalization (n = 7). *, Significant differences (P < .05) in the luciferase activity between IVM+AREG and IVM+FSH compared with the IVM in Egfr^fl/+ mice, whereas no differences were observed in Egfr^δ/fl:Cyp19cre mice (two way ANOVA followed by Tukey's multiple comparison test).

Figure 5.

Oocyte-specific deletion of Pten constitutively activates AKT in the oocyte independent of the meiotic stage and culture treatment. CEOs were collected from Pten^fl/fl or Pten^fl/fl:Zp3cre mice and cultured for 2.5 hours in milrinone (GV) or in the absence (IVM) or presence of FSH (IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and extracts used for Western blots. Representative immunoblots of phosphorylated (p-AKT) and total AKT (AKT) are reported. The bar graph represents the ratio p-AKT to AKT (mean ± SEM) in the different treatment/genotype (Pten^fl/fl, n = 7, Pten^fl/fl:Zp3cre, n = 5). *** and *, Significant differences (P < .001 and P < .05, respectively) between IVM+FSH compared with GV and IVM in Pten^fl/fl mice, whereas no differences were observed in Pten^fl/fl:Zp3cre mice (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Figure 6.

The constitutive activation of AKT in the oocyte is sufficient to induce maximal translation of the Tpx2 (A) and Il7 (B) reporters during oocyte maturation. cRNA was injected in oocytes still enclosed in cumulus cells collected from Pten^fl/fl or Pten^fl/fl:Zp3cre mice and cultured for 16 hours in milrinone (GV) or in the absence (IVM) or presence of FSH (IVM+FSH). At the end of the incubation, oocytes were denuded, homogenized, and the luciferase activity measured in the oocyte extract. The bar graphs represent the ratio (mean ± SEM) between the RL activity, translated under the control of the Tpx2 3′ UTR (A, n = 3) or Il7 3′ UTR (B, n = 4) and the FL activity used for injection normalization. *, Significant differences in the luciferase activity (P < .05) between IVM and IVM+FSH treatments in Pten^fl/fl mice, whereas no differences were observed in the Pten^fl/fl:Zp3cre mice (two way ANOVA followed by Tukey's multiple comparison test).

Figure 7.

The constitutive activation of AKT in the oocyte is sufficient to improve the developmental competence after IVM in the absence of growth factors. CEOs collected from different mice strains and genotypes were matured in different conditions and in vitro fertilized. After IVF the presumptive zygotes were either fixed and the DNA stained to assess the formation of the two pronuclei (A–C) or cultured for an extra 19 hours to assess the formation of two-cell embryos (D and E) or up to E5.0 to assess the blastocyst formation (F and G). A, CEOs were collected from the ampullae of Areg null (Areg^−/−) and wild-type (WT) littermate mice 14–17 hours after the injection of hCG. The bar graph represents the fertilization rate (mean ± SEM) in the different genotypes (n = 12). ***, Significant differences (unpaired t test, P < .001). B, CEOs were in vitro matured for 17 hours in the absence (IVM) or presence of AREG (IVM+AREG). The bar graph represents the fertilization rate (mean ± SEM) in the different treatments (n = 6). **, Significant differences (paired t test, P < .01). C, CEOs were collected from Pten^fl/fl and Pten^fl/fl: Zp3cre mice and in vitro matured in the absence of any hormone/growth factor. The bar graph represents the fertilization rate (mean ± SEM) in the different genotypes (n = 5). *, Significant differences (paired t test, P < .05). D, CEOs collected from C57BL/6NxCD1 hybrids were in vitro matured in the absence (IVM) or presence of FSH (IVM+FSH). The bar graph represents the two-cell rate (mean ± SEM) in the different treatments (n = 5). *, Significant differences (paired t test, P < .05). E, CEOs were collected from Pten^fl/fl and Pten^fl/fl:Zp3cre mice and in vitro matured in absence of any hormone/growth factor. The bar graph represents the two-cell rate (mean ± SEM) in the different genotypes (n = 8). **, Significant differences (paired t test, P < .01). Note that Pten^fl/fl and Pten^fl/fl:Zp3cre mice are on a C57BL/6N pure background. This can account for the lower two-cell rate compared with the C57BL/6NxCD1 hybrids. F, CEOs collected from C57BL/6NxCD1 hybrids were in vitro matured in the absence (IVM) or presence of FSH (IVM+FSH). The bar graph represents the blastocyst rate (mean ± SEM) in the different treatments (n = 3). **, Significant differences (paired t test, P < .01). G, CEOs were collected from Pten^fl/fl and Pten^fl/fl:Zp3cre mice and in vitro matured in the absence of any hormone/growth factor. The bar graph represents the blastocyst rate (mean ± SEM) in the different genotypes (Pten^fl/fl, n = 4, and Pten^fl/fl: Zp3cre, n = 3). *, Significant differences (unpaired t test, P < .05).