C‐type natriuretic peptide promotes human granulosa cell growth and estradiol production: Implications for early follicle development

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  FIGURE 1. Expression of NPPC, NPR2, and gonadotropins receptors in diverse granulosa cell lines. To identify suitable cell lines for studying the roles of CNP‐NPR2 signaling in human granulosa cells, we examined the expression of NPPC (CNP precursor protein; cleaved into CNP protein), NPR2 receptor (the cognate receptor for CNP), FSHR, and LHR in three different human granulosa cell lines: HGrC, SVOG, and KGN. The transcript levels of these genes were quantified by real‐time RT–PCR and normalized to an internal control (b‐actin) (mean ± SEM, n = 6). FSHR, follicle‐stimulating hormone receptor; HGrC, human non‐luteinizing granulosa cell line; KGN, human ovarian granulosa‐like tumor cell line; LHR, luteinizing hormone receptor; NPPC, natriuretic peptide precursor type C; NPR2, natriuretic peptide receptor 2; OV, human ovary cDNA; SVOG, human luteinizing granulosa cell line.

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  FIGURE 2. CNP‐mediated stimulation of cell proliferation. To assess the effect of CNP on human granulosa cell proliferation, HGrC cells were cultured with 3–100 nM CNP. The specific action of CNP on cell proliferation was confirmed by suppressing CNP expression using siRNA transfection. (A) Dose‐dependent effect of CNP on HGrC cell proliferation. At 48 h of culture, viable cells was quantified using a trypan blue exclusion assay (mean ± SEM, n = 6). The ratio was calculated with control set to 1. *p < 0.05, versus control (C). (B) Suppression of NPPC mRNA levels by CNP siRNA treatment. HGrC cells were transfected with siRNAs targeting CNP and a negative control. Cells were cultured for an additional 48 h before measuring NPPC mRNA levels (mean ± SEM, n = 4). (C) Suppression of CNP levels by CNP siRNA treatment. At 72 h after siRNAs transfection, CNP concentration in the culture medium supernatant was quantified by ELISA (mean ± SEM, n = 6). (D) CNP siRNA inhibition of HGrC cell proliferation. At 48 h after siRNAs transfection, viable cells were quantified using a trypan blue exclusion assay (mean ± SEM, n = 6). The ratio was calculated with control set to 1. *p < 0.05, versus control (C).…

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  FIGURE 3. Involvement of cGMP production in CNP‐induced cell proliferation. To evaluate the impact of CNP on cGMP production in HGrC cells, the cells were treated with 30 nM CNP, and zaprinast, a PDE5 inhibitor that prevented cGMP degradation. The role of guanylate cyclase in CNP‐induced HGrC cell proliferation was further investigated using LY83583, a guanylate cyclase inhibitor. (A) Increase in cGMP production by CNP treatment. At 15 min after culture, cGMP concentrations in the culture medium supernatant were quantified using an ELISA (mean ± SEM, n = 6) and expressed as pmol/well. *p < 0.05, versus control (C). (B) Suppression of CNP‐induced HGrC cell proliferation by LY83583 treatment. At 48 h after culture, viable cells were quantified using a trypan blue exclusion assay (mean ± SEM, n = 6). The ratio was calculated with control set to 1. *p < 0.05, versus control. ‘a’ is compared to the control, and ‘b’ is compared to CNP 30nM, and the former has a significant difference compared to the latter.

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  FIGURE 4. CNP stimulation of estrogen production. To assess the effect of CNP on cellular function in human granulosa cell growth, HGrC cells were cultured with 10–100 nM CNP in the presence of 10 μM androstenedione as a substrate for estradiol. As a positive control, treatment with 5 μM forskolin (an adenylyl cyclase stimulator) was applied. (A) Enhanced estradiol production by CNP treatment. At 48 h after culture, the concentration of estradiol in the culture medium supernatant was quantified by ELISA (mean ± SEM, n = 6). *p < 0.05, versus control (C). FK, forskolin. (B) Increase in the estrogen synthetic enzyme, cytochrome P450 expression by CNP treatment. At 48 h after culture, total RNA was extracted from the cells and cytochrome P450 transcript levels were quantified by real‐time RT–PCR and normalized to an internal control (b‐actin) (mean ± SEM, n = 6). *p < 0.05, versus control (C).

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  FIGURE 5. Regulation of NPR2 and CNP expressions by E2 and ODFs. To investigate the roles of a cross‐talk between oocytes and granulosa cells in regulating cell proliferation in HGrC cells, we evaluated changes in NPR2 and NPPC expressions, as well as cell proliferation, through co‐treatment of HGrC cells with fifty denuded oocytes (OC) per well. The cells were treated with 100 nM estradiol (E2), 30 nM CNP, and a combination of 100 nM estradiol (E2) + 30 nM CNP in the presence or absence of oocyte‐derived factors (ODFs), which included 500 ng/mL GDF9, BMP15, and FGF8. (A) Effect on NPR2 and NPPC expressions. At 48 h after culture, total RNA was extracted from the cells and the transcript levels of NPR2 and NPPC were quantified by real‐time RT–PCR, normalized to an internal control (b‐actin) (mean ± SEM, n = 6). In the upper left panel: *p < 0.05; right panel: a; versus control without ODFs, b; versus without ODFs, c; versus control with ODFs. In the lower right panel: a; versus control with ODFs. Letter symbols indicate significant differences (p < 0.05). (B) Effect on cell proliferation. At 48 h of culture, viable cells were quantified using a trypan blue exclusion assay (mean ± SEM, n =…