Fig. 1 Comparison of the rLH/hCG effects on cGMP levels in mouse ovary in vivo and cultured POFs in vitro. A) Comparison of the rLH/hCG effects on cGMP.

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Fig. 1 Comparison of the rLH/hCG effects on cGMP levels in mouse ovary in vivo and cultured POFs in vitro. A) Comparison of the rLH/hCG effects on cGMP levels in Pde3a<sup>−/−</sup> and control ovaries. The 22- to 23-day-old mice were stimulated with eCG for 44–48 h, followed by a single hCG injection. Ovaries were collected at different times after the injection and immediately processed for cGMP measurement. The levels of cGMP were normalized by protein in the extract. Data are the mean ± SEM, n = 5. B) In vitro time course of cGMP levels in isolated follicles after rLH stimulation. POFs were isolated from ovaries stimulated with eCG for 44–48 h, and then stimulated in vitro with rLH (5 IU/ml) for the times indicated in the abscissa. Pools of 5–20 POFs were used for each measurement. Data are the mean ± SEM for follicles from three to five mice at each time point (n = 10). Data were analyzed using an unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 2 Nppc mRNA levels in POFs after rLH/hCG treatment Fig. 2 Nppc mRNA levels in POFs after rLH/hCG treatment. A) Nppc mRNA in eCG-primed mouse ovaries treated with hCG. Ovaries were collected at the indicated times after hCG injection and processed by microarray analysis. Data are the mean ± SEM (n = 4). Inset: quantification of CNP protein content in ovaries following hCG stimulation. Data are the mean ± SEM (n = 5). B) Nppc mRNA content in follicles after rLH stimulation. POFs were isolated from mice primed with eCG for 44–48 h. A total of 3–10 POFs were used for each time point after rLH (5 IU/ml) treatment. Quantitative RT-PCR was performed on the RNA extracted at the end of the incubation. Data are the mean ± SEM (n = 4). Data were analyzed using an unpaired t-test; *P < 0.05; ***P < 0.001; ****P < 0.0001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 3 Role of EGFR signaling on the LH-dependent suppression of Nppc mRNA. POFs were isolated from eCG-treated mice. They were then incubated in the absence or presence of rLH (5 IU/ml) or Areg (100 nM) for 2 h pretreated with or without AG1478 (500 nM) for 30 min. The Nppc mRNA levels were measured by quantitative RT-PCR after culture. Data are the mean ± SEM; the number of POFs analyzed (n) includes data from at least three experiments. Data were analyzed using an unpaired t-test; ***P < 0.001; ****P < 0.0001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 4 Comparison of the rLH effects on Nppc mRNA in wild-type mice and in mice with different mutations in the EGFR signaling pathway. POFs were isolated from mice stimulated with eCG for 44–48 h and then incubated with rLH for 3 h. At the end of the incubation, mRNA was extracted from the follicles and Nppc mRNA levels were measured by quantitative RT-PCR. Data are the mean ± SEM (n = 5). Data were analyzed using an unpaired t-test; *P < 0.05; **P < 0.01; ****P < 0.0001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 6 Time course of cGMP levels in Egfr<sup>Δ/fl</sup>Cyp19-Cre mice after rLH treatment. A) POFs were isolated from Egfr<sup>Δ/fl</sup>Cyp19-Cre mice stimulated with eCG for 44–48 h. Each measurement used pools of 5–20 POFs. Data are the mean ± SEM from three to five mice at each time point, and the number of experiment is indicated above every time point. B) Summary of cGMP levels in the POFs isolated from eCG-primed Egfr<sup>Δ/fl</sup>Cyp19-Cre and control mice without treatment. Controls were Egfr<sup> fl/+</sup>Cyp19<sup>+/+</sup>, Egfr<sup> fl/fl</sup>Cyp19<sup>+/+</sup>, or Egfr<sup> fl/+</sup>Cyp19-Cre mice from the same litter. Data are shown as 10th–90th percentile. C) Cyclic-GMP levels in the POFs isolated from eCG-primed Egfr<sup>Δ/fl</sup>Cyp19-Cre and control mice stimulated with rLH for 1 h. Data are shown as 10th–90th percentile. D) POFs isolated from Egfr<sup>Δ/fl</sup>Cyp19-Cre and control mice in the same litter were cultured with rLH for 4 h in vitro. The percentage of GVBD oocytes was assessed by light microscopy. Data are the mean ± SEM of the percentage for every mouse (n = 5). E and F) Cyclic-GMP levels were measured by EIA after rLH (5 IU), AG 1478 (500 nM), or Areg (100 nM) treatments for 1 h in POFs isolated from Egfr<sup>Δ/fl</sup>Cyp19-Cre and control mice from the same litter. Each point corresponds to measurement of pools of 5–20 POFs. Data are the mean ± SEM for follicles from two to three mice; the number of POFs analyzed (n) includes data from at least three experiments. Data were analyzed using an unpaired t-test; ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 5 Effect of pharmacological inhibition of EGFR on the LH-dependent decrease in cGMP in the ovarian follicle. A) Effect of AG1478 on cGMP levels in POFs cultured with rLH. POFs were incubated for different times with rLH in the absence or presence of 500 nM AG1478. At the end of the incubation, cGMP was measured as detailed in the Materials and Methods. Data are the mean ± SEM; the number of experiments is indicated above each time point. B) Effect of AG1478 on cGMP levels in POFs cultured with either rLH (5 IU/ml) or Areg (100 nM) for 2 h. Data are the mean ± SEM (n = 3). C) Effect of GM6001 on cGMP levels in POFs cultured with rLH for 30 min. Data are the mean ± SEM (n = 7). Data were analyzed using an unpaired t-test; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 9 Models of EGF network and CNP/NPR2 signaling in the regulation of LH-induced ovulation. In the wild-type mice, LH activates its receptor on GCs of POFs, leading to stimulation of cAMP-dependent signal cascades, which induces the expression of EGF-like factors and suppresses the CNP/NPR2 signaling. Activation of EGF network is involved at early time points (fast, 15–30 min), while CNP decrease may be involved at later time points (2–4 h). These two pathways act together or in parallel to induce meiotic resumption. In the presence of AG1478, CNP availability is primarily responsible for the reduction in cGMP. In the Egfr<sup>Δ/fl</sup>Cyp19-Cre mice, either the residual EGFR signaling or another pathway is responsible for the early decrease in cGMP. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 8 LH/hCG signaling rapidly desensitizes the effects of CNP stimulation in mice and humans. A) COCs were isolated from mice eCG-primed or hCG-injected 1 h after eCG stimulation, which were cultured in medium with or without (control) 10 nM CNP for 1 h, as indicated. COCs were collected at the end of culture, and the levels of cGMP were measured. Bars show the mean ± SEM of at least three independent experiments. Data were analyzed using an unpaired t-test; ns, not significant. **P < 0.01; ***P < 0.001. B) Luteinized human mural GCs were pretreated for 1 h with hCG at a physiologically relevant concentration of 10 mIU/ml, rapidly washed twice in M199 to remove any residual hCG, and then treated with 10 nM CNP. At the end of the indicated culture times, cGMP production was quantitated. Data are mean ± SEM of independent experiments (n = 5). Data were analyzed using one-way ANOVA. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 7 Expression and phosphorylation of EGFR in wild-type and Egfr<sup>Δ/fl</sup>Cyp19-Cre mice. A) Representative blots are shown for total and phosphorylated EGFR in wild-type and Egfr<sup>Δ/fl</sup>Cyp19-Cre POFs after treatment with rLH. B) Expression of total EGFR in wild-type, Egfr<sup> fl/+</sup>Cyp19-Cre, and Egfr<sup>Δ/fl</sup>Cyp19-Cre POFs. Data are the mean ± SEM (n = 4). C) LH-induced EGFR phosphorylation during the first 30 min of incubation. LH-stimulated EGFR phosphorylation levels were examined by Western blot in cell lysates of POFs incubated with rLH for 15 and 30 min. The ratio of phosphorylated EGFR to total EGFR was normalized to control. Results are expressed as the mean ± SEM of four separate experiments. Data were analyzed using an unpaired t-test; *P < 0.05. Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle1 Biol Reprod. 2014;91(1). doi:10.1095/biolreprod.113.116814 Biol Reprod | © 2014 by the Society for the Study of Reproduction, Inc.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com