Human KGN Research Articles

Structural determinants of parabens in inhibiting human and rat gonadal 3β-hydroxysteroid dehydrogenase

This study delved into the impacts of 10 parabens on the activity of human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) within human KGN cell and rat testicular microsomes, as well as on the secretion of progesterone in KGN cells and the inhibitory potency was compared between human and rats. Intriguingly, the outcomes revealed that ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl parabens displayed varying IC50 values for human 3β-HSD2, from 4.15 to 139.96 μM, demonstrating characteristics of mixed inhibitors. Notably, within KGN cells, all examined parabens, excluding nonyl and phenyl parabens, significantly inhibited progesterone secretion at 5–50 μM. In the case of rats, the IC50 values for these parabens on gonadal 3β-HSD1 fluctuated between 7.15 and 110.76 μM, likewise functioning as mixed inhibitors. Through docking analysis, it was proposed that most parabens effectively bind to NAD+ and/or steroid binding site. Moreover, bivariate correlation analysis unveiled an inverse correlation between IC50 values and structural characteristics such as LogP, molecular weight, heavy atom number, and carbon number within the alcohol moiety of parabens. 3D-QSAR elucidated pivotal regions, comprising hydrogen bond donor, hydrogen bond acceptor, and hydrophobic region, with the most potent inhibitor nonyl paraben engaging with all regions, while the weakest inhibitor ethyl paraben interacted with the regions except for the hydrophobic region. In conclusion, this investigation underscored the inhibitory effects imparted by several parabens on both human and rat gonadal 3β-HSD activity, with their inhibitory potency being modulated by aspects of hydrophobicity and carbon chain length.

Klotho accelerates the progression of polycystic ovary syndrome through promoting granulosa cell apoptosis and inflammation.

The morbidity of polycystic ovary syndrome (PCOS) is in highly increasing rate nowadays. PCOS not only affects the fertility in women, but also threatens the health of whole life. Hence, to find the prognostic risk factors is of great value. However, the effective predictors in clinical practice of PCOS are still in blackness. In this study, we found Klotho was increased in FF (Follicular Fluid) and primary luteinized granulosa cells (GCs) from PCOS patients with hyperandrogenism. Furthermore, we found follicular Klotho was negatively correlated with numbers of mature oocytes, and positively correlated with serum testosterone, LH, and LH/FSH levels menstrual cycle and number of total antral follicles in PCOS patients. In primary luteinized GCs, the increased Klotho was accompanied with upregulation of cell apoptosis and inflammation-related genes. In ovaries of PCOS mice and cultured human KGN cell line, Klotho was up-regulated and accompanied by apoptosis, inflammation and mitochondrial dysfunction. Therefore, our findings suggest new mechanisms for granulosa cell injury and revealed to target inhibit Klotho maybe a new therapeutic strategy for treatment of PCOS.

Dysregulated RNA editing of EIF2AK2 in polycystic ovary syndrome: clinical relevance and functional implications

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear.MethodsTen RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression.ResultsRNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway.ConclusionsOur study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.

Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3β-hydroxysteroid dehydrogenases

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3β-HSD2 with IC50 values of 114.79, 106.98, and 5.40 μM, respectively. For pig 3β-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 μM, respectively. Similarly, for rat 3β-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 μM, respectively. They were mixed inhibitors of pig and rat 3β-HSD, while triphenyltin was identified as a competitive inhibitor of human 3β-HSD2. The mechanism underlying the inhibition of organotins on 3β-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3β-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Progestin and adipoQ receptor 7 (PAQR7) mediate the anti-apoptotic effect of P4 on human granulosa cells and its deficiency reduces ovarian function in female mice

PurposePAQR7 plays a key role in cell apoptosis as a progesterone membrane receptor. The physiological mechanism of PAQR7 in ovarian function and its anti-apoptotic action in mammals remain poorly understood.MethodsWe first added 0.2 µM aminoglutethimide (AG), an inhibitor of endogenous progesterone (P4) secretion, and transfected siPAQR7 co-incubated with P4 in human KGN cells to identify granulosa cell apoptosis, respectively. Additionally, we used Paqr7 knockout (PAQR7 KO) mice to assess the role of PAQR7 in the ovary.ResultsThe PAQR7 deficiency significantly increased apoptosis of KGN cells, and this significant difference disappeared following P4 supplementation. The Paqr7−/− female mice showed a prolonged estrous cycle, reduced follicular growth, increased the number of atresia follicles, and decreased the concentrations of E2 and AMH. The litters, litter sizes, and spontaneous ovulation in the Paqr7−/− mice were significantly decreased compared with the Paqr7+/+ mice. In addition, we also found low expression of PAQR7 in GCs from human follicular fluids of patients diagnosed with decreased ovarian reserve (DOR) and ovaries of mice with a DOR-like phenotype, respectively.ConclusionsThe present study has identified that PAQR7 is involved in mouse ovarian function and fertilization potential. One possible mechanism is mediating the anti-apoptotic effect of P4 on GC apoptosis via the BCL-2/BAX/CASPASE-3 signaling pathway. The mechanism underlying the effect of PAQR7 on ovarian development and aging remains to be identified.

PHLPP1 inhibits the growth and aerobic glycolysis activity of human ovarian granular cells through inactivating AKT pathway

BackgroundPolycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features, and PCOS is associated with infertility. PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) has been shown to regulate AKT. The aim of present study is to investigate the role of PHLPP1 in PCOS.MethodsThe expression levels of PHLPP1 in dihydrotestosterone (DHT)-treated human ovarian granular KGN cells were determined by qRT-PCR and Western blot. PHLPP1 was silenced or overexpressed using lentivirus. Cell proliferation was detected by CCK-8. Apoptosis and ROS generation were analyzed by flow cytometry. Glycolysis was analyzed by measuring extracellular acidification rate (ECAR).ResultsDHT treatment suppressed proliferation, promoted apoptosis, enhanced ROS, and inhibited glycolysis in KGN cells. PHLPP1 silencing alleviated the DHT-induced suppression of proliferation and glycolysis, and promotion of apoptosis and ROS in KGN cells. PHLPP1 regulated cell proliferation and glycolysis in human KGN cells via the AKT signaling pathway.ConclusionsOur results showed that PHLPP1 mediates the proliferation and aerobic glycolysis activity of human ovarian granular cells through regulating AKT signaling.

The epoxiconazole and tebuconazole fungicides impair granulosa cells functions partly through the aryl hydrocarbon receptor (AHR) signalling with contrasted effects in obese, normo-weight and polycystic ovarian syndrome (PCOS) patients

Polycystic ovarian syndrome (PCOS), frequently associated to obesity, is the main reproductive disorder in women in age to procreate. Some evidence suggests that pesticides can result in alterations of the female reproductive system, including polycystic ovary syndrome (PCOS). Here, we detected two fungicides, Tebuconazole (Tb) and Epoxiconazole (Epox) in the soils and waters of French area. Our hypothesis is that these two triazoles could be associated to the etiology of PCOS. We used the human KGN cell line and primary human granulosa cells (hGCs) from different group of patients: normal weight non PCOS (NW), normal weight PCOS (PCOS NW), obese (obese) and obese PCOS (PCOS obese). We exposed in vitro these cells to Tb and Epox from 0 up to 10 mM for 24 and 48 h and analysed cell viability and steroidogenesis. In hGCs NW, cell viability was reduced from 12.5 µM for Tb and 75 µM for Epox. In hGCs NW, Epox decreased progesterone (Pg) and estradiol (E2) secretions and inhibited STAR, HSD3B and CYP19A1 mRNA expressions from 25 µM and increased AHR mRNA expression from 75 µM. Tb exposure also reduced steroid secretion and STAR and CYP19A1 mRNA expressions and increased AHR mRNA expression but at cytotoxic concentrations. Silencing of AHR in KGN cells reduced inhibitory effects of Tb and Epox on steroid secretion. Tb and Epox exposure decreased more steroid secretion in hGCs from obese, PCOS NW and PCOS obese groups than in NW group. Moreover, we found a higher gene expression of AHR within these three groups. Taken together, both Epox and Tb reduced steroidogenesis in hGCs through partly AHR and Tb was more cytotoxic than Epox. These triazoles alter more strongly PCOS and/or obese hGCs suggesting that human with reproductive disorders are more sensitive to triazoles exposure.

Structure-activity relationship and docking analysis of nature flavonoids as inhibitors of human and rat gonadal 3β-hydroxysteroid dehydrogenases for therapeutic purposes

The potential inhibitory effects of flavonoids on gonadal steroid biosynthesis have gained attention due to their widespread presence in natural plant sources. Specifically, our study focused on evaluating the inhibitory efficacy of these compounds on human 3β-hydroxysteroid dehydrogenase 2 (h3β-HSD2) and rat homolog r3β-HSD1, enzymes responsible for the conversion of pregnenolone to progesterone. Through our investigations, we observed that the potency of flavonoids was silymarin (IC50, 1.31 μM) > luteolin (4.63 μM) > tectorigenin > (5.86 μM), and rutin (44.12 μM) in inhibiting human KGN cell microsomal h3β-HSD2. Similarly, the potency of flavonoids was silymarin (9.50 μM) > luteolin (11.49 μM) > tectorigenin (14.06 μM), and rutin (145.71 μM) in inhibiting rat testicular r3β-HSD1. Silymarin, luteolin, and tectorigenin acted as mixed inhibitors of both human and rat 3β-HSDs. Luteolin and tectorigenin were able to penetrate human KGN cells to inhibit progesterone secretion. Furthermore, docking analysis and structure-activity relationship analysis highlighted the importance of hydrogen bond formation for the inhibitory efficacy of these compounds against h3β-HSD2 and r3β-HSD1. Overall, this study demonstrates that silymarin exhibits the most potent inhibition of human and rat gonadal 3β-HSDs, and significant SAR differences exist among the tested compounds.

FRI392 Functional Interrelationship Between Oxytocin And Bone Morphogenetic Proteins In Ovarian Steroidogenesis By Granulosa Cells

Abstract Disclosure: K. Yamamoto: None. Y. Nakano: None. N. Iwata: None. Y. Soejima: None. A. Suyama: None. T. Hasegawa: None. F. Otsuka: None. Oxytocin (OXT) is a neuropeptide that is produced in the hypothalamus and is secreted from the posterior pituitary. OXT has diverse physiological roles mediated by the OXT receptor (OXTR), including not only stimulation of uterine contractions during labor and peripheral functions of milk ejection during lactation but also functions in pair bonding, maternal nurturing, and social behavior related to the action of OXT on the brain. It has been shown that OXT and OXTR are expressed in ovarian granulosa cells (GCs) in several species including humans. Secretion of OXT by bovine GCs is reported to be increased after the preovulatory luteinizing hormone surge, which is coincident with an increase in progesterone secretion and a decrease in secretion of estradiol and androgen, suggesting a functional interrelationship between OXT and ovarian steroidogenesis. It has been demonstrated that application of OXT to cultures of GCs from preovulatory follicles in bovines, marmoset monkeys, and humans led to increases in progesterone production. However, the precise molecular mechanism by which OXT modulates ovarian steroidogenesis in GCs remains unknown. Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) superfamily, play important roles in steroidogenesis and cellular proliferation in ovarian follicles via autocrine/paracrine fashion. BMP ligands and receptors are expressed in a cell-dependent manner in the ovary and have a pivotal role in female fertility. Throughout folliculogenesis, BMPs commonly inhibit follicle-stimulating hormone (FSH)-induced progesterone production, and oocyte-derived factors BMP-6 and BMP-15 are known to act on GCs through BMP receptors and suppress FSH-induced progesterone synthesis. To clarify the reproductive role of OXT in ovarian steroidogenesis and its functional interaction with BMPs, the effects of OXT on ovarian steroidogenesis were investigated by using primary culture of rat GCs and human granulosa KGN cells. Here we found that the OXT receptor was expressed in both rat GCs and human KGN cells and that OXT treatment significantly increased FSH- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat GCs and KGN cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA levels of CYP11A1 and HSD3B2 induced by FSK in KGN cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the mRNA level of ID1 induced by BMP-15, but not those induced by BMP-6, in KGN cells. It was also revealed that OXT application upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by KGN cells. Taken together, it was shown that OXT enhances ovarian progesterone production with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis. Presentation: Friday, June 16, 2023

Azole fungicides inhibit human and rat gonadal 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Azole fungicides are widely used in the agricultural industry to control fungal infections in crops. However, recent studies have shown that some azole fungicides inhibit the activity of 3β-hydroxysteroid dehydrogenases (3β-HSDs) in the gonads. Out of the 16 azole fungicides tested, 8 were found to inhibit human KGN cell 3β-HSD2 with IC50 values of less than 100μM. The strongest inhibitor was difenoconazole, with an IC50 value of 1.88μM. In contrast, only 3 of the azole fungicides inhibited rat testicular 3β-HSD1, which was less sensitive to inhibition. Azole fungicides potently inhibited progesterone secretion by KGN cells under basal and forskolin stimulated conditions at ≥ 5μM. The inhibitory strength of azole fungicides was determined by their lipophilicity (LogP), molecular weight, pKa, and binding energy. A pharmacophore analysis revealed that the hydrogen bond acceptor-lipid group was a critical feature required for inhibition. Overall, these findings suggest that the use of azole fungicides have unintended consequences on reproductive health due to their inhibition of gonadal 3β-HSDs. Key words: Azole fungicides; steroid hormones; 3β-hydroxysteroid dehydrogenase; docking analysis; lipophilicity.

Benzophenone-1 and -2 UV-filters potently inhibit human, rat, and mouse gonadal 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis

Benzophenone (BP) ultraviolet (UV) -filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD) catalyse the conversion of pregnenolone to progesterone. This study explored the effect of 12 BPs on human, rat, and mouse 3β-HSD isoforms, and analysed the structure-activity relationship (SAR) and underlying mechanisms. The inhibitory potency was BP-1 (IC50, 5.66 ± 0.95 μM) > BP-2 (5.84 ± 2.22 μM) > BP-6 (185.8 ± 115.2 μM) > BP3-BP12 on human KGN 3β-HSD2, BP-2 (5.90 ± 1.02 μM) > BP-1 (7.55 ± 1.26 μM) > BP3-B12 on rat testicular 3β-HSD1, and BP-1 (15.04 ± 5.20 μM) > BP-2 (22.64 ± 11.81 μM) > BP-6（125.1 ± 34.65 μM）> BP-7 (161.1 ± 102.4 μM) > other BPs on mouse testicular 3β-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3β-HSDs, and BP-2 is a mixed inhibitor of human and rat 3β-HSDs and a noncompetitive inhibitor of mouse 3β-HSD6. 4-Hydroxyl substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, and mouse gonadal 3β-HSDs. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at ≥ 10 μM. Docking analysis revealed that the 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bonds with residue Ser123 of human 3β-HSD2 and residue Asp127 of rat 3β-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, and mouse gonadal 3β-HSDs and that there is a significant SAR difference.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

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.

LncRNA DLEU1 is overexpressed in premature ovarian failure and sponges miR-146b-5p to increase granulosa cell apoptosis

BackgroundmiR-146b-5p has been reported to participate in premature ovarian failure (POF) in mice. However, its role in POF patients is unclear. We predicted that miR-146b-5p might interact with lncRNA DLEU1, a crucial player in ovarian cancer. We then explored the interaction between DLEU1 and miR-146b-5p.MethodsExpression of DLEU1 and miR-146b-5p in POF and control ovary tissues was determined by RT-qPCR. The subcellular location of DLEU1 in human KGN cells was analyzed using subcellular fractionation assays. The direct interaction between DLEU1 and miR-146b-5p was analyzed using RNA pull-down assays. The role of DLEU1 in miR-146a expression was analyzed using overexpression assay. Cell proliferation was analyzed using cell apoptosis assay.ResultsIncreased DLEU1 expression and decreased miR-146b-5p expression were observed in POF. DLEU1 directly interacted with MiR-146b-5p and was expressed in both nuclear and cytoplasm samples of KGN cells. In KGN cells, DLEU1 and miR-146b-5p failed to regulate the expression of each other. However, DLEU1 promoted cell apoptosis and reduced the inhibitory effects of miR-146b-5p on cell apoptosis.ConclusionsDLEU1 is overexpressed in POF and sponges miR-146b-5p to increase KGN cell apoptosis.

The protective effect of small peptides from Periplaneta americana on hydrogen peroxide-induced apoptosis of granular cells.

This study investigates the protective effect of small peptides from Periplaneta americana (SPPA) on hydrogen peroxide (H2O2)-induced apoptosis of ovarian granular cells. H2O2 was applied to human ovarian granular cells (KGN cell strains). Cell viability was tested by cell counting Kit-8 (CCK-8). Cell apoptosis was tested by flow cytometry, and a cell apoptosis model was established. The model cells were treated with SPPA, and the cell survival rate was monitored using the CCK-8 method. The oxidative stress state of cells was examined using SOD, ROS, MDA, and NO kits. The protein expression levels of SIRT1, p53, and the apoptosis-related gene Caspase3 were measured using Western Blot methodology. Relative to the control group, cell viability declined significantly after the H2O2 treatment only (P < 0.01), while the apoptosis rate increased significantly (P < 0.01). The activity of SOD was weakened significantly (P < 0.01), while the cell levels of ROS, MDA, and NO increased dramatically (P < 0.01). Cell viability dramatically recovered (P < 0.01), and the SOD activity is hugely increased (P < 0.01) after SPPA treatment. In contrast, contents of ROS, MDA, and NO decreased sharply (P < 0.01), and significant dose-response relationships are characterized. Moreover, the H2O2 treatment group showed significantly downregulated expression of SIRT1 (P < 0.01) but significantly upregulated expressions of p53 and Caspase3 (P < 0.01) compared to the control group. Following the SPPA treatment of apoptosis cells, expression of SIRT1 increased significantly, while expressions of p53 and Caspase3 declined significantly (P < 0.01). This study suggests that SPPA inhibits H2O2-induced human KGN cell apoptosis through antioxidation, and the SIRT1/p53 signal pathway mediates the antioxidation.

Haploinsufficiency in non-homologous end joining factor 1 induces ovarian dysfunction in humans and mice

BackgroundPremature ovarian insufficiency (POI) is a common disease in women that leads to a reduced reproductive lifespan. The aetiology of POI is genetically heterogeneous, with certain double-strand break (DSB) repair...

Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.

CXCL12 and its receptors regulate granulosa cell apoptosis in PCOS rats and human KGN tumor cells.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder accompanied by chronic low-grade inflammation; its etiology is still undefined. This study investigated the expression of CXCL12, CXCR4, and CXCR7 in PCOS rats and their role in regulation of apoptosis. To accomplish this, we established an in vivo PCOS rat model and studied KGN cells (human ovarian granulosa cell line) in vitro. In PCOS rats, the ovarian expression of CXCL12, CXCR4, and CXCR7 was reduced, and the apoptosis rate of granulosa cells was increased, accompanied by decreased expression of BCL2 and increased expression of BAX and cleaved CASPASE3 (CASP3). We further showed that recombinant human CXCL12 treatment upregulated BCL2, downregulated BAX, and cleaved CASP3 in KGN cells to inhibit their apoptosis in a concentration-dependent manner; moreover, the effect of CXCL12 was weakened by CXCR4 antagonist AMD3100 and anti-CXCR7 neutralizing antibody. In conclusion, PCOS rats showed decreased CXCL12, CXCR4, and CXCR7 expression and increased apoptosis rate of ovarian granulosa cells. Further, in human KGN cells, CXCL12 regulated the expression of BAX, BCL2, and cleaved CASP3 to inhibit apoptosis through CXCR4- and CXCR7-mediated signal transmission. These findings may provide a theoretical and practical basis for illuminating the role of proinflammatory cytokines in the pathogenesis of PCOS.

Frataxin inactivation leads to steroid deficiency in flies and human ovarian cells

Friedreich ataxia (FA), the most common inherited autosomal-recessive ataxia in Caucasians, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and increased incidence of diabetes. FA is caused by a GAA repeat expansion in the first intron of the gene encoding frataxin, an evolutionarily conserved mitochondrial protein, which results in decreased gene expression. Ubiquitous inactivation of the fly frataxin ortholog dfh blocks the transition from larval to pupal stages. In this study, we show that this phenotype is due to ecdysteroid deficiency and that feeding larvae with the 20-hydroxyecdysone steroid hormone rescues this developmental blockage. In mammals, adrenodoxin, the ferredoxin FDX1, is an Fe-S-containing protein essential for the synthesis of various steroid hormones. We show here that the two fly ferredoxins, Fdxh and Fdxh2 (encoded by CG1319), are also involved in steroidogenesis. This provides a potent mechanism by which frataxin, known to be involved in Fe-S cluster biosynthesis, could affect steroidogenesis through reduced ferredoxin activity. Finally, we show that frataxin inactivation decreases progesterone synthesis in human KGN ovarian granulosa cells. Thus, the involvement of frataxin in steroid synthesis appears to be a conserved function of the protein from flies to human and our data suggest that steroidogenesis could be affected in FA patients.

Up-regulated expression of WNT5a increases inflammation and oxidative stress via PI3K/AKT/NF-κB signaling in the granulosa cells of PCOS patients.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder accompanied by chronic low-grade inflammation, but the molecular mechanism remains unclear. We investigated the action of WNT5a in the development of chronic inflammation in PCOS and the related molecular signaling pathways. This was a prospective study conducted at the Division of Reproduction Center, Peking University Third Hospital. A total of 35 PCOS patients and 87 control women who reported to the clinic for the in vitro procedure and the cause of marital infertility was male azoospermia were included. Mural granulosa cells (GCs) of 35 PCOS patients and 37 controls were collected during oocyte retrieval and gene expression was analyzed. The human KGN cells and mural GCs from 50 control subjects (six to eight samples were pooled together for each experiment) were cultured in vitro. The regulation of inflammation and oxidative stress was confirmed by quantitative PCR, flow-cytometric assay, and dual-luciferase reporter assay after inflammatory stimuli or WNT5a overexpression. Relevant signaling pathways were identified using specific inhibitors. Our data demonstrate significantly elevated WNT5a expression in the mural GCs of PCOS patients compared with the controls. Lipopolysaccharide stimulation increased WNT5a expression in KGN cells and mural GCs, and BAY-117082 and pyrrolidinedithiocarbamic acid [nuclear factor-κB (NF-κB) inhibitor] treatments suppressed WNT5a mRNA below the control level. WNT5a overexpression also enhanced the expression of inflammation-related genes and increased intracellular reactive oxygen species, whereas both BAY-117082 and LY-294002 (phosphatidylinositol 3-kinase inhibitor) significantly inhibited WNT5a-induced inflammation and oxidative stress. WNT5a acts as a proinflammatory factor in human ovarian GCs. The up-regulated expression of WNT5a in PCOS increases inflammation and oxidative stress predominantly via the phosphatidylinositol 3-kinase/AKT/NF-κB signaling pathway. The proinflammatory cytokines induced might further enhance WNT5a expression via NF-κB-dependent regulation, indicating a novel regulatory system for chronic inflammation in PCOS.