Down-regulation of long non-coding RNA MALAT1 inhibits granulosa cell proliferation in endometriosis by up-regulating P21 via activation of the ERK/MAPK pathway.

Li Y ，Liu YD ，Chen SL ，Chen X ，Ye DS ，Zhou XY ，Zhe J ，Zhang J ... -  《-》

NGF promotes mouse granulosa cell proliferation by inhibiting ESR2 mediated down-regulation of CDKN1A.

Nerve growth factor (NGF) is known to play key roles in ovarian follicular development, such as the assembly of early follicles and follicular ovulation through its high-affinity receptor, tyrosine kinase receptor A (trkA). Herein, the molecular mechanism controlling NGF-induced granulosa cell (GC) proliferation was not clear. In this study, we found that NGF is abundant in preantral GCs and knockdown of trkA in GCs attenuated NGF-induced GC proliferation and further decreased the levels of phosphorylated extracellular regulated protein kinases 1/2 (ERK1/2). Cyclin-dependent kinase inhibitor 1A (CDKN1A), also named p21, a factor which could be either a negative or a positive regulator via transformation related protein 53 (TRP53, also named p53)-dependent or independent pathways in cell proliferation, was up-regulated during the process of NGF-induced GC proliferation. Blockade of trkA (K252α) and ERK1/2 (U0126) in GCs decreased NGF-induced expression of CDKN1A and did not alter the expression of TRP53, indicating that NGF stimulates CDKN1A expression via the trkA-ERK1/2 pathway in a TRP53-independent manner. Meanwhile, ESR2, a tumor suppressor which is exclusively expressed in GCs, was suppressed in NGF-induced GC proliferation, and this effect was abrogated by U0126. Blockade of ESR2 (ICI182,780) caused the promotion of GC proliferation and CDKN1A expression, indicating that ESR2 may be downstream of the ERK1/2 pathway in mediating the effect of CDKN1A on NGF-induced GC proliferation. Therefore, ESR2 may be involved in the integration of intracellular signal cascades and cell cycle proteins in affecting GC proliferation. Here, we provide mechanistic insights into the roles of CDKN1A in NGF-induced GC proliferation. Understanding potential cross-points between CDKN1A and ESR2 affecting GC proliferation will help in the discovery of new therapeutic targets in some female infertility disorders.

Wang Y ，Liu W ，Du J ，Yu Y ，Liang N ，Liang M ，Yao G ，Cui S ，Huang H ，Sun F ... -  《-》

The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.

What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries? Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS. In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women. Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability. AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis. Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7 M (P < 0.05; P < 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P = 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P = 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P < 0.05, one-way ANOVA) and FSHR (P < 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n = 7, PCOM n = 5, PCOS n = 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P < 0.05, normal n = 6, PCOS n = 3). AMH at 20 and 50 ng/ml decreased KGN-GC cell proliferation but not viability after 8 days of treatment (P < 0.005, two-way ANOVA). N/A. Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT. Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'. This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose. N/A.

Dilaver N ，Pellatt L ，Jameson E ，Ogunjimi M ，Bano G ，Homburg R ，D Mason H ，Rice S ... -  《-》

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3β-hydroxysteroid dehydrogenase (3βHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3βHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY » funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. N/A.

Froment P ，Plotton I ，Giulivi C ，Fabre S ，Khoueiry R ，Mourad NI ，Horman S ，Ramé C ，Rouillon C ，Grandhaye J ，Bigot Y ，Chevaleyre C ，Le Guevel R ，Mallegol P ，Andriantsitohaina R ，Guerif F ，Tamburini J ，Viollet B ，Foretz M ，Dupont J ... -  《-》

NME1 suppression promotes growth, adhesion and implantation of endometrial stromal cells via Akt and MAPK/Erk1/2 signal pathways in the endometriotic milieu.

Li MQ ，Shao J ，Meng YH ，Mei J ，Wang Y ，Li H ，Zhang L ，Chang KK ，Wang XQ ，Zhu XY ，Li DJ ... -  《-》