Steroidogenic Cell Lines Research Articles

12203 FDXR Variants Cause Adrenal Insufficiency And Atypical Sexual Development

Abstract Disclosure: E. Pignatti: None. J. Slone: None. M. Gomez-Cano: None. T.M. Campbell: None. J. Vu: None. K. Sauter: None. A.V. Pandey: None. M. Alonso-Riaño: None. F. Martínez-Azorín: None. D. Neilson: None. N. Longo: None. T. Du Toit: None. C.D. Voegel: None. T. Huang: None. C.E. Flueck: Consulting Fee; Self; Novo Nordisk. Grant Recipient; Self; Novo Nordisk, Sandoz, Pfizer, Inc., Merck. Speaker; Self; Sandoz. Background. Biallelic variants in the mitochondrial flavoprotein ferredoxin—NADP(+)-reductase (FDXR) have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM). FRM patients often experience worsening or demise following stress, but the cause is unclear. Ambiguous genitalia at birth have now been observed in some FRM patients. As FDXR is an essential partner for several steroid enzymes, we explored the impact of FDXR variants on steroid biosynthesis. Methods. To investigate whether the abnormal stress response of many FRM patients is caused by glucocorticoid deficiency, and whether observed ambiguous genitalia is related to androgen excess, we investigated the impact of FDXR variants on adrenal steroidogenesis. Clinical, genetic, biochemical, and functional studies were performed in index patients, steroidogenic cell lines reprogrammed from patient fibroblasts, and a novel disease mouse model. Results. Two paradigmatic female FRM patients carrying novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life had cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. Steroidogenic FDXR-variant cells lines reprogrammed from three patients’ fibroblasts also displayed deficient mineralocorticoid and glucocorticoid production. Likewise, Fdxr-variant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Conclusions. Our comprehensive studies show that FDXR variants in FRM patients may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance. Presentation: 6/1/2024

Cordycepin suppresses steroidogenic acute regulatory protein expression by reducing SP1 in human granulosa-lutein cells.

Cordycepin (COR), a compound derived from Cordyceps, is recognized as an adenosine analog with numerous beneficial effects on human health. However, its impact on steroidogenic acute regulatory protein (STAR) expression in ovarian granulosa cells is not well understood. This study demonstrates that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPβ pathway

Ethnopharmacological relevanceZishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated. Aim of the studyTo verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinβ (C/EBPβ) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. Materials and methodsBased on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPβ in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPβ using siRNAs, the relationship between KLF4 and C/EBPβ in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPβ pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPβ pathway. ConclusionsZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPβ were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPβ pathway. Furthermore, we confirmed that KLF4 and C/EBPβ regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.

TGF-β1 upregulates secreted protein acidic and rich in cysteine expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome

BackgroundOvarian hyperstimulation syndrome (OHSS) is a serious complication during in vitro fertilization (IVF) treatment. The upregulation of ovarian transforming growth factor-beta 1 (TGF-β1) is involved in the development of OHSS. The secreted protein acidic and rich in cysteine (SPARC) is a secreted multifunctional matricellular glycoprotein. Although the regulatory effects of TGF-β1 on SPARC expression have been reported, whether TGF-β1 regulates SPARC expression in the human ovary remains unknown. In addition, the role of SPARC in the pathogenesis of OHSS is unclear.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing IVF treatment were used as experimental models. OHSS was induced in rats, and ovaries were collected. Follicular fluid samples were collected from 39 OHSS and 35 non-OHSS patients during oocyte retrieval. The underlying molecular mechanisms mediating the effect of TGF-β1 on SPARC expression were explored by a series of in vitro experiments.ResultsTGF-β1 upregulated SPARC expression in both KGN and hGL cells. The stimulatory effect of TGF-β1 on SPARC expression was mediated by SMAD3 but not SMAD2. The transcription factors, Snail and Slug, were induced in response to the TGF-β1 treatment. However, only Slug was required for the TGF-β1-induced SPARC expression. Conversely, we found that the knockdown of SPARC decreased Slug expression. Our results also revealed that SPARC was upregulated in the OHSS rat ovaries and in the follicular fluid of OHSS patients. Knockdown of SPARC attenuated the TGF-β1-stimulated expression of vascular endothelial growth factor (VEGF) and aromatase, two markers of OHSS. Moreover, the knockdown of SPARC reduced TGF-β1 signaling by downregulating SMAD4 expression.ConclusionsBy illustrating the potential physiological and pathological roles of TGF-β1 in the regulation of SPARC in hGL cells, our results may serve to improve current strategies used to treat clinical infertility and OHSS.66JbE8YWJdrsTB-WmCj_-wVideo

Ferredoxin 1 regulates granulosa cell apoptosis and autophagy in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a common reproductive endocrine disorder in women of reproductive age, causes anovulatory infertility. Increased apoptosis of granulosa cells has been identified as one of the key factors contributing to abnormal follicular development. Ferredoxin 1 (FDX1) encodes a small ferredoxin that is involved in the reduction in mitochondrial cytochromes and the synthesis of various steroid hormones and has the potential to influence the function of granulosa cells. In the present study, we aimed to determine the relationship between FDX1 and follicular granulosa cell function. To this end, we investigated the difference between FDX1 expression in the granulosa cells of 50 patients with PCOS and that of the controls. Furthermore, we sought to elucidate the role and mechanism of FDX1 in PCOS granulosa cells by establishing a mouse PCOS model with dehydroepiandrosterone and KGN (a steroidogenic human granulosa cell-like tumor cell line). The results indicated significant up-regulation of FDX1 in the granulosa cells after androgen stimulation. Knockdown of FDX1 promoted the proliferation of KGN and inhibited apoptosis. Moreover, FDX1 could regulate autophagy by influencing the autophagy proteins ATG3 and ATG7. Our results demonstrated that FDX1 plays a critical role in female folliculogenesis by mediating apoptosis, autophagy, and proliferation. Therefore, FDX1 may be a potential prognostic factor for female infertility.

Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells

BackgroundHeparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments.ResultsWestern blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway.ConclusionThis study increases the understanding of the physiological role of HB-EGF in human luteal functions.7G2SU9-j2App9A69cYCW26Video

The Intracellular Cholesterol Pool in Steroidogenic Cells Plays a Role in Autophagy, Basal Steroidogenesis and Mitochondrial Dynamics

In steroidogenesis, cholesterol‐ the precursor substrate molecule for all steroid hormones is central and the framework of steroidogenesis across steroidogenic cells is constructed around it— including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria where cholesterol is converted to pregnenolone by the P450 side chain cleavage enzyme. Thus, cholesterol and the mitochondria are highly interconnected in all steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone‐induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in the regulation of steroidogenic events and factors remain unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell‐intrinsic factors and events involving steroidogenesis. We used an intermediary approach to investigate the role of cholesterol in the regulation of steroidogenesis in different steroidogenic cell types such as MA‐10 cells (a murine Leydig cell line), BeWo cells (placental cell line) producing different steroid hormones. Steroidogenic cells were cultured in a lipoprotein‐depleted medium that leads to the depletion of the intracellular cholesterol pool and lipid droplets. Furthermore, cholesterol deprivation induced steroidogenic events. To get a snapshot of cholesterol deprived (CD)‐induced changes in lipid droplets, autophagy, and mitochondrial dynamics at an ultrastructural level we analyzed steroidogenic cells using transmission electron microscopy. As expected, cholesterol deprivation resulted in the depletion or loss of intracellular lipid droplets and the cholesterol pool across steroidogenic cell lines. Moreover, a difference in autophagy‐related structural changes was observed between cells cultured under the normal and CD conditions. In general, the structural characteristics of autophagy were more apparent in the CD group when compared with the normal control group across different steroidogenic cell types. Moreover, a change in mitochondrial shape was observed between the cells from two groups. For instance, the elongated or tubular mitochondria showing signs of mitochondrial dynamics (fusion or fission) were more common in the CD experimental group. Importantly, an increase in lysosome size and numbers were consistently found in cells under CD conditions which inversely correlated with the depletion of lipid droplets and the cholesterol pool, suggesting a compensatory increase in cholesterol mobilization. Taken together, this data suggests that cholesterol indeed plays a role in the regulation of autophagy and mitochondrial dynamics across steroidogenic cells in a context‐dependent manner. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non‐steroidogenic cells such as HepG2 (human hepatoma cells) and H4IIE (rat hepatoma cells), indicating that cholesterol insufficiency‐induced changes in steroidogenic cells are specific to steroidogenesis. Thus, we unraveled this previously unknown role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone‐dependent and independent steroidogenesis during development, as well as in health and disease.

The Effects of Organophosphate Esters Used as Flame Retardants and Plasticizers on Granulosa, Leydig, and Spermatogonial Cells Analyzed Using High-Content Imaging.

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making.

Phthalates and alternative plasticizers differentially affect phenotypic parameters in gonadal somatic and germ cell lines†.

The developmental and reproductive toxicity associated with exposure to phthalates has motivated a search for alternatives. However, there is limited knowledge regarding the adverse effects of some of these chemicals. We used high-content imaging to compare the effects of mono (2-ethylhexyl) phthalate (MEHP) with six alternative plasticizers: di-2-ethylhexyl terephthalate (DEHTP); diisononyl-phthalate (DINP); di-isononylcyclohexane-1,2-dicarboxylate (DINCH); 2-ethylhexyl adipate (DEHA); 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TXIB) and di-iso-decyl-adipate (DIDA). A male germ spermatogonial cell line (C18-4), a Sertoli cell line (TM4) and two steroidogenic cell lines (MA-10 Leydig and KGN granulosa) were exposed for 48h to each chemical (0.001-100μM). Cell images were analyzed to assess cytotoxicity and effects on phenotypic endpoints. Only MEHP (100μM) was cytotoxic and only in C18-4 cells. However, several plasticizers had distinct phenotypic effects in all four cell lines. DINP increased Calcein intensity in C18-4 cells, whereas DIDA induced oxidative stress. In TM4 cells, MEHP, and DINCH affected lipid droplet numbers, while DEHTP and DINCH increased oxidative stress. In MA-10 cells, MEHP increased lipid droplet areas and oxidative stress; DINP decreased the number of lysosomes, while DINP, DEHA, and DIDA altered mitochondrial activity. In KGN cells, MEHP, DINP and DINCH increased the number of lipid droplets, whereas DINP decreased the number of lysosomes, increased oxidative stress and affected mitochondria. The Toxicological Priority Index (ToxPi) provided a visual illustration of the cell line specificity of the effects on phenotypic parameters. The lowest administered equivalent doses were observed for MEHP. We propose that this approach may assist in screening alternative plasticizers.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AngII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding prosteroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a prosteroidogenic expression program that actively resolves to prevent aldosterone overproduction.

RNA Decay Controls the Kinetics of the Angiotensin II Gene Expression Response

Complex cellular resonses require the temporal coordination of stimulus-induced gene expression programs. Angiotensin II (AngII) is the active 8 amino acid peptide in the renin-angiotensin-aldosterone system that controls blood pressure and fluid balance. AngII binds to type I angiotensin receptor in the adrenal cortex to initiate a cascade of temporally coordinated events leading to the production of aldosterone, a master regulator of blood pressure and volume. We stimulated a steroidogenic human cell line (H295R) with AngII and performed RNA-seq at twelve points. We identified twelve distict temporally distinct groups of gene expression responses each encoding functionally related proteins important for various steps of aldosterone productionl. Interstingly, the shape of the impulse response suggested a key role for RNA decay. Indeed, RNA decay rates in unstimulated H295R cells strongly correlated with the amplitude and peakiness of the gene expression response for each group of genes. We also found evidence for increases in RNA decay during the AngII response. Next, we selected candidate RBPs based on motif finding, adrenal specific expression, and AngII responsiveness. We performed an siRNA knockdown screen on these 22 candidates to identify RBPs that regulate aldosterone levels. Eight of these RBPs exhibited statistically significant changes in aldosterone for at least two independent siRNAs. Interestingly, multiple RBPs that promote RNA decay were found to suppress aldosterone production and induced in response to AngII-stimulation. These RBPs could be responsible for our observed increases in RNA decay. Altogether, these data support a model in which RNA decay is a critical regulator of the timing and strength of AngII-induced gene expression and ultimately aldosterone production.

Elucidation of the Effects of Bisphenol A and Structural Analogs on Germ and Steroidogenic Cells Using Single Cell High-Content Imaging.

Concerns about the potential adverse effects of bisphenol A (BPA) have led to an increase in the use of replacements, yet the toxicity data for several of these chemicals are limited. Using high-content imaging, we compared the effects of BPA, BPAF, BPF, BPS, BPM, and BPTMC in germ (C18-4 spermatogonial) and steroidogenic (MA-10 Leydig and KGN granulosa) cell lines. Effects on cell viability and phenotypic markers were analyzed to determine benchmark concentrations (BMCs) and estimate administered equivalent doses (AEDs). In all 3 cell lines, BPA was one of the least cytotoxic bisphenol compounds tested, whereas BPM and BPTMC were the most cytotoxic. Interestingly, BPF and BPS were cytotoxic only in MA-10 cells. Effects on phenotypic parameters, including mitochondria, lysosomes, lipid droplets, and oxidative stress, were both bisphenol- and cell-line specific. BPA exposure affected mitochondria (BMC: 1.2 μM; AED: 0.09 mg/kg/day) in C18-4 cells. Lysosome numbers were increased in MA-10 cells exposed to BPA or BPAF but decreased in KGN cells exposed to BPAF or BPM. Lipid droplets were decreased in C18-4 cells exposed to BPF and in MA-10 cells exposed to BPTMC but increased in BPF, BPM, and BPTMC-exposed KGN cells. BPA and BPM exposure induced oxidative stress in MA-10 and KGN cells, respectively. In summary, structurally similar bisphenols displayed clear cell-line-specific differences in BMC and AED values for effects on cell viability and phenotypic endpoints. This approach, together with additional data on human exposure, may aid in the selection and prioritization of responsible replacements for BPA. .

The AMPK-mTOR axis requires increased MALAT1 expression for promoting granulosa cell proliferation in endometriosis.

Endometriosis is a common reproductive disorder in women, with a global prevalence of 10-15%. Long noncoding RNAs (lncRNAs) are critical to gene transcription, cell cycle modulation and immune response. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) reportedly mediates autophagy of endometrial stromal cells in endometriosis. The present study aimed to evaluate the role and mechanism of MALAT1 in granulosa cells (GCs) in endometriosis. Consequently, MALAT1 expression was upregulated in GCs obtained from patients with endometriosis and in the steroidogenic human granulosa-like tumor cell line KGN. However, MALAT1 knockdown consequently decreased the proliferation and viability of these cells, as determined by MTT and 5-ethynyl-2'-deoxyuridine staining assays. Both Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry and western blotting performed to detect proapoptotic factors indicated that MALAT1 depletion might promote KGN cell apoptosis. Furthermore, MALAT1 knockdown increased GC autophagy, as evidenced by microtubule-associated protein 1A/1B-light chain 3 (LC3) cleavage upregulation and p62 degradation. In addition, although 5'-AMP-activated protein kinase (AMPK) mRNA expression and protein levels decreased in GCs obtained from patients with endometriosis and KGN cells, MALAT1 knockdown restored AMPK levels. However, addition of BML-275 (MALAT1 inhibitor) to MALAT1-knockdown KGN cells recovered their viability and proliferative capacity and simultaneously reduced their apoptotic and autophagic capacity. Therefore, MALAT1 may regulate GC proliferation via AMPK-mTOR-mediated cell apoptosis and autophagy.

JIL-O: Establishment of a new steroidogenic human adrenocortical carcinoma cell line

JIL-O: Establishment of a new steroidogenic human adrenocortical carcinoma cell line

SUN-211 Active Steroid Hormone Synthesis Renders Adrenocortical Cells Highly Susceptible to Type II Ferroptosis Induction

Context: Cell death in the adrenal cortex is ill understood but of high clinical relevance. Resistance of adrenocortical carcinoma (ACC) to current treatment with mitotane and chemotherapy calls for an improved understanding of adrenal cortical cell death processes. Ferroptosis is an iron-dependent form of regulated cell death which is characterized by polyunsaturated lipids adrenic (AdA) and arachidonic acid (AA) peroxidation. Aim: To address the potential role of ferroptosis in the adrenal gland as a potential treatment target of ACC. Methods: Human ACC cells H295R, CU-ACC1 and 2 were used. Protein expression of key enzymes was determined by western blotting. Lipid peroxidation was quantified with BODIPY 581/591 and cell viability with CellTiterGlo after treatment with known inducers and inhibitors of ferroptosis and steroidogenesis, respectively. Results: Adrenocortical tissues are enriched in AdA and AA and express high levels of genes relevant to ferroptosis, such as glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4). Inhibition of GPX4 with RSL3 led to cell death in H295R, CU-ACC1 and 2 cells at EC50 values of 2.4x10-7, 8.1x10-7 and 1.5x10-8 M, respectively. The steroidogenesis inhibitor ketoconazole completely reversed RSL3 cytotoxicity in all three steroidogenic cell lines by reducing lipid peroxidation. Mitotane induced lipid peroxidation but inhibition of ferroptosis with liproxstatin did not protect mitotane-induced cell death. Conclusion: Adrenocortical cells are highly sensitive to ferroptosis due to active steroidogenesis. Triggering this form of cell death could present future novel treatment options against ACC.

Resveratrol protects mitochondrial quantity by activating SIRT1/PGC-1α expression during ovarian hypoxia.

PurposeResveratrol is a well‐known potent activator of sirtuin‐1 (SIRT1). We investigated the direct effects of hypoxia and resveratrol on SIRT1/ peroxisome proliferator‐activated receptor‐gamma coactivator 1α (PGC‐1α) pathways, vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α, and mitochondrial quantity in a steroidogenic human ovarian granulosa‐like tumor cell line (KGN) cells.MethodsKGN cells were cultured with cobalt chloride (CoCl2; a hypoxia‐mimicking agent) and/or resveratrol. The mRNA and protein levels, protein secretion, and intracellular localization were assessed by real‐time PCR, Western blot analysis, ELISA, and immunofluorescence staining, respectively. Mitochondrial quantity was measured based on the mitochondrial DNA (mtDNA) copy number.ResultsCoCl2 simultaneously attenuated the levels of SIRT1 and mtDNA expression, and induced the levels of VEGF protein production. In contrast, resveratrol significantly increased the levels of SIRT1 and mtDNA copy number, but reduced VEGF production in normoxia. Resveratrol could recover CoCl2‐suppressed SIRT1 and mtDNA expression and antagonize CoCl2‐induced VEGF production. CoCl2 treatment resulted in a downregulation of PGC‐1α expression, and this effect was recovered by resveratrol. Resveratrol significantly suppressed the production of the CoCl2‐induced HIF‐1α and VEGF proteins.ConclusionThese results suggest that resveratrol improves mitochondrial quantity by activating the SIRT1/PGC‐1α pathway and inhibits VEGF induction through HIF‐1α under hypoxic conditions.

The effects of di(2-ethylhexyl) phthalate exposure in women with polycystic ovary syndrome undergoing in vitro fertilization.

ObjectivesDi(2-ethylhexyl) phthalate (DEHP) is a common endocrine-disrupting chemical, which has potential reproductive toxicity. This study aimed to explore the effects of DEHP exposure in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization.MethodsIn this case-control study, DEHP levels in follicular fluid (FF) of women with PCOS (n = 56) and controls (n = 51) were measured. The in vitro effects of DEHP exposure on primary-cultured human granulosa cells (GCs) and a steroidogenic human granulosa-like tumor cell line (KGN cells) were analyzed.ResultsConcentrations of DEHP in FF were significantly higher in women with PCOS than in controls. The clinical pregnancy rate was significantly lower in women with PCOS with high levels of DEHP than in controls. The levels of androgens produced by human GCs were significantly increased following DEHP exposure. Compared with controls, DEHP-treated human GCs and KGN cells showed significantly lower viability, cell cycle arrest, higher apoptosis, and altered expression of apoptosis-related genes.ConclusionWomen with PCOS are exposed to increased levels of DEHP in follicles, which may be associated with pregnancy loss following in vitro fertilization. DEHP may disrupt steroid production, balance in cellular proliferation, and apoptosis in human granulosa cells.