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 (0.001-100 μM). Cell images were analyzed to assess cytotoxicity and effects on phenotypic endpoints. Only MEHP (100 μM) 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.

The Expanding Role of Mitochondria, Autophagy and Lipophagy in Steroidogenesis

The fundamental framework of steroidogenesis is similar across steroidogenic cells, especially in initial mitochondrial steps. For instance, the START domain containing protein-mediated cholesterol transport to the mitochondria, and its conversion to pregnenolone by the enzyme P450scc, is conserved across steroidogenic cells. The enzyme P450scc localizes to the inner mitochondrial membrane, which makes the mitochondria essential for steroidogenesis. Despite this commonality, mitochondrial structure, number, and dynamics vary substantially between different steroidogenic cell types, indicating implications beyond pregnenolone biosynthesis. This review aims to focus on the growing roles of mitochondria, autophagy and lipophagy in cholesterol uptake, trafficking and homeostasis in steroidogenic cells and consequently in steroidogenesis. We will focus on these aspects in the context of the physiological need for different steroid hormones and cell-intrinsic inherent features in different steroidogenic cell types beyond mitochondria as a mere site for the beginning of steroidogenesis. The overall goal is to provide an authentic and comprehensive review on the expanding role of steroidogenic cell-intrinsic processes in cholesterol homeostasis and steroidogenesis, and to bring attention to the scientific community working in this field on these promising advancements. Moreover, we will discuss a novel mitochondrial player, prohibitin, and its potential role in steroidogenic mitochondria and cells, and consequently, in steroidogenesis.

Gonadal Sex Differentiation: Supporting Versus Steroidogenic Cell Lineage Specification in Mammals and Birds

The gonads of vertebrate embryos are unique among organs because they have a developmental choice; ovary or testis formation. Given the importance of proper gonad formation for sexual development and reproduction, considerable research has been conducted over the years to elucidate the genetic and cellular mechanisms of gonad formation and sexual differentiation. While the molecular trigger for gonadal sex differentiation into ovary of testis can vary among vertebrates, from egg temperature to sex-chromosome linked master genes, the downstream molecular pathways are largely conserved. The cell biology of gonadal formation and differentiation has long thought to also be conserved. However, recent discoveries point to divergent mechanisms of gonad formation, at least among birds and mammals. In this mini-review, we provide an overview of cell lineage allocation during gonadal sex differentiation in the mouse model, focusing on the key supporting and steroidogenic cells and drawing on recent insights provided by single cell RNA-sequencing. We compare this data with emerging information in the chicken model. We highlight surprising differences in cell lineage specification between species and identify gaps in our current understanding of the cell biology underlying gonadogenesis.

Dissecting gonadal cell lineage specification and sex determination during human development using a single-cell transcriptomics approach

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series scRNA-seq strategy, we analyzed the fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that DLK1+ cells may be the progenitors of steroidogenic cell lineages in both sexes and that TAC1+ cells may be the progenitors of granulosa cells in females. Intriguingly, the testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a step-wise manner. Moreover, interactions between gonadal somatic cells were systematically explored and verified in our study. In detail, we observed that Sertoli cells interacted with Leydig cells through DHH-PTCH1 and PDGFA-PDGFRA/PDGFRB ligand-receptor gene pairs. More importantly, we identified cell type-specific developmental defects of both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development and interactions of human FGCs and gonadal microenvironment cells.

Mouse Gonad Development in the Absence of the Pro-Ovary Factor WNT4 and the Pro-Testis Factor SOX9

The transcription factors SRY and SOX9 and RSPO1/WNT4/β-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/β-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/β-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.

Inactivation of Wt1 causes pre-granulosa cell to steroidogenic cell transformation and defect of ovary development†

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1−/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3β-HSD-positive steroidogenic cells were detected in ovaries of Wt1−/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3β-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1−/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1−/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.

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.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Insulin Resistance, Hyperinsulinemia, and LH: Relative Roles in Peripubertal Obesity-Associated Hyperandrogenemia

Context Peripubertal obesity is associated with variable hyperandrogenemia, but precise mechanisms remain unclear. Objective To assess insulin resistance, hyperinsulinemia, and LH roles in peripubertal obesity–associated hyperandrogenemia. Design Cross-sectional analysis. Setting Academic clinical research unit. Participants Eleven obese (body mass index for age ≥95%) peripubertal girls. Intervention Blood samples were taken during a mixed-meal tolerance test (1900 to 2100), overnight (2100 to 0700), while fasting (0700 to 0900), and during an 80 mU/m2/min hyperinsulinemic-euglycemic clamp (0900 to 1100). Main Outcome Measures The dependent variable was morning free testosterone level; independent variables were insulin sensitivity index (ISI), estimated 24-hour insulin, and estimated 24-hour LH levels. Results All participants demonstrated insulin resistance and hyperinsulinemia. ISI, but not estimated 24-hour insulin level, correlated positively with morning free testosterone level when correcting for estimated 24-hour LH level and Tanner stage (rs = 0.68, P = 0.046). The correlation between estimated 24-hour LH and free testosterone levels approached significance after adjusting for estimated 24-hour insulin level and Tanner stage (rs = 0.63, P = 0.067). Estimated 24-hour insulin level did not correlate with free testosterone level after adjusting for estimated 24-hour LH level and Tanner stage (rs = 0.47, P = 0.20). Conclusion In insulin-resistant obese girls with hyperinsulinemia, free testosterone levels correlated positively with insulin sensitivity and, likely, circulating LH concentrations but not with circulating insulin levels. In the setting of relatively uniform hyperinsulinemia, variable steroidogenic-cell insulin sensitivity may correlate with metabolic insulin sensitivity and contribute to variable free testosterone concentrations.