Journal: Protein & Cell

Article Title: Follicle stimulating hormone controls granulosa cell glutamine synthesis to regulate ovulation

doi: 10.1093/procel/pwad065

Figure Lengend Snippet: Glutamine inhibits GC apoptosis via ASK1 apoptotic signaling pathway. (A) Glutamine starvation specifically induces apoptosis of mice granulosa cells. Primary mouse TCs and GCs were cultured in McCoy’s 5A medium and then changed to a glutamine-free 1640 medium. Cell apoptotic ratios were measured at the indicated time points using flow cytometry ( n = 3). (B) Glutamine starvation induces apoptosis in granulosa cells. Flow cytometry analysis ( n = 3) of the apoptotic ratio of granulosa cells at 0, 2, 4, 6, 8, 12, and 24 h after glutamine deprivation in COV434 cells. (C–H) Glutamine deprivation induces GC apoptosis by activating ASK1 signaling. ASK1 and its downstream molecular pathways, JNK and P38, were activated during glutamine deprivation in a time-dependent manner (C), and inhibited by glutamine supplementation (D), as well as in the FF floating cells of PCOS patients (E). ASK1 knockdown reversed apoptotic signaling induced by glutamine deficiency (F), whereas overexpression of ASK1 promoted apoptotic signaling in COV434 cells (G and H). (I) JNK activation in mice follicles. Immunofluorescence staining of p-JNK (red) and cleaved Caspase-3 (green) with DAPI (blue) in mouse ovaries. Scale bar: 50 μm. (J–M) Fas-signaling rather than TNFR signaling mediates granulosa cell apoptosis. Inhibition of Fas signaling by KR33493, but not TNF-α signaling by R7050, attenuates JNK and P38 phosphorylation (J) and apoptosis (K) induced by glutamine deprivation. Consistent with this, Fas-knockdown, but not TNFR-knockdown, COV434 cells inhibited JNK and P38 phosphorylation (L) and resistance to apoptosis (M) induced by glutamine deprivation ( n = 3).

Article Snippet: The reagents employed in this study were purchased from the following companies: Glutamine (Sigma-Aldrich, Cat. No. E6627), FSH (Sigma, Cat. No. F4021 and Solarbio, Cat. No. F8470), LH (Sigma, Cat. No. L6420 and Solarbio, Cat. No. L8040), β-Estradiol (Sigma, Cat. No. E8875), KR33493 (MCE, Cat. No. HY-100755), R7050 (MCE, Cat. No. HY-110203), AT-101 (Selleck, Cat. No. S2812), Gefitinib (MCE, Cat. No. HY-50895), Erlotinib (MCE, Cat. No. HY-50896), hFSH-β-(33-53) (MCE, Cat. No. HY-P3343A), and 13 C 6 -Glucose (Sigma, Cat. No. 389374).

Techniques: Cell Culture, Flow Cytometry, Knockdown, Over Expression, Activation Assay, Immunofluorescence, Staining, Inhibition

Journal: Protein & Cell

Article Title: Follicle stimulating hormone controls granulosa cell glutamine synthesis to regulate ovulation

doi: 10.1093/procel/pwad065

Figure Lengend Snippet: ASK1 signaling activation alleviates PCOS traits in mice PCOS models. (A) Inhibition of Fas-ASK1 death signaling decreases the apoptosis in GCs of mice ovaries. Immunofluorescence staining of cleaved caspase-3 (red) with DAPI (blue) staining of ovaries from mice treated with GS4997, KR33493, and R7050. Scale Bar: 100 µm. (B) Inhibition of Fas-ASK1 death signaling induces polycystic ovary morphology in mice. H&E staining of ovaries from mice treated with saline (CON), GS4997, KR33493, and R7050. CL: corpus luteum. Scale Bar: 100 µm. (C–E) AT-101 increases granulosa cell-specific apoptosis and ovulation in mice. TUNEL staining (C, left), immunofluorescence staining (C, right), and hematoxylin and eosin staining (D) of ovaries from GLN or EsR1-KO mice fed via oral gavage of saline (Mock) or AT-101 (AT-101). Scale bars: 100 µm. (E) Quantitative analysis of early antral follicles, antral follicles, pre-ovulatory follicles, and corpus luteum per ovary section in GLN, GLN-AT-101, EsR1-KO, and EsR1-KO-AT-101 ovaries ( n = 6 per group). (F and G) AT-101 partially reverses PCOS mice model estrous cycles. Representative estrous cycles of GLN, GLN-AT-101, EsR1-KO, and EsR1-KO-AT-101 females (F) and days taken for GLN, GLN-AT-101, EsR1-KO, and EsR1-KO-AT-101 mice to complete one estrous cycle (G). (H) AT-101 decreases serum testosterone levels in GLN mice and EsR1-KO mice. Serum testosterone levels in CON, AT-101, GLN, GLN-AT-101, EsR1-KO, and EsR1-KO-AT-101 mice ( n = 6–12 per group). (I and J) AT-101 treatment increases glucose tolerance and insulin sensitivity in GLN mice. GTT (I) and ITT (J) of CON, GLN, and GLN mice injected with AT-101 (CON and GLN: n = 6; GLN-AT-101: n = 5). (K) AT-101 treatment increases the number of pups in GLN mice. Litter pup numbers in GLN and GLN mice treated with AT-101 ( n = 8–9). (L and M) AT-101 treatment increases glucose tolerance and insulin sensitivity in EsR1-KO mice. GTT (L) and ITT (M) of CON, EsR1-KO, and EsR1-KO mice injected with AT-101 (CON: n = 6; EsR1-KO and EsR1-AT-101: n = 5).

Article Snippet: The reagents employed in this study were purchased from the following companies: Glutamine (Sigma-Aldrich, Cat. No. E6627), FSH (Sigma, Cat. No. F4021 and Solarbio, Cat. No. F8470), LH (Sigma, Cat. No. L6420 and Solarbio, Cat. No. L8040), β-Estradiol (Sigma, Cat. No. E8875), KR33493 (MCE, Cat. No. HY-100755), R7050 (MCE, Cat. No. HY-110203), AT-101 (Selleck, Cat. No. S2812), Gefitinib (MCE, Cat. No. HY-50895), Erlotinib (MCE, Cat. No. HY-50896), hFSH-β-(33-53) (MCE, Cat. No. HY-P3343A), and 13 C 6 -Glucose (Sigma, Cat. No. 389374).

Techniques: Activation Assay, Inhibition, Immunofluorescence, Staining, Saline, TUNEL Assay, Injection