Steroid Production Research Articles

Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide

BackgroundThe conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process.ResultsWe substantiated, using the production of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) as a model, that increasing intracellular FAD supply could effectively increase the conversion of phytosterols into 9-OHAD. Overexpressing ribB and ribC, two key genes involving in FAD synthesis, could significantly enhance the amount of intracellular FAD by 167.4% and the production of 9-OHAD by 25.6%. Subsequently, styrene monooxygenase NfStyA2B from Nocardia farcinica was employed to promote the cyclic regeneration of FAD by coupling the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+, and the production of 9-OHAD was further enhanced by 9.4%. However, the viable cell numbers decreased by 20.1%, which was attributed to sharply increased levels of H2O2 because of the regeneration of FAD from FADH2. Thus, we tried to resolve the conflict between FAD regeneration and cell growth by the overexpression of catalase and promotor replacement. Finally, a robust strain NF-P2 was obtained, which could produce 9.02 g/L 9-OHAD after adding 15 g/L phytosterols with productivity of 0.075 g/(L h), which was 66.7% higher than that produced by the original strain.ConclusionsThis study highlighted that the cofactor engineering, including the supply and recycling of FAD and NAD+ in Mycolicibacterium, should be adopted as a parallel strategy with pathway engineering to improve the productivity of the industrial strains in the conversion of phytosterols into steroid synthons.

Time- and glucose-dependent differentiation of 3T3-L1 adipocytes mimics dysfunctional adiposity

The 3T3-L1 murine adipocyte cell line remains one of the most widely used models to study the mechanisms of obesity and related pathologies. Most studies investigate such mechanisms using mature adipocytes that have been chemically induced to differentiate for 7 days in media containing 25 mM glucose. However, the dysfunctional characteristics commonly observed in obesity including adipocyte hypertrophy, increased expression of inflammatory markers, enhanced production of reactive oxygen species (ROS), increased steroidogenic enzyme expression/activity and production of steroid hormones, are not necessarily mimicked in these cells. The aim of this study was to provide an inexpensive model which represents the well-known characteristics of obesity by manipulating the time of adipocyte differentiation and increasing the concentration of glucose in the cell media. Our results showed a glucose- and time-dependent increase in adipocyte hypertrophy, ROS production and gene expression of the pro-inflammatory cytokine interleukin-6 (IL-6), as well as a time-dependent increase in lipolysis and in the gene expression of the chemokine monocyte chemoattractant protein 1 (MCP1). We also showed that gene expression of the steroidogenic enzymes 11-beta-hydroxysteroid dehydrogenase type 1 (11βHSD1), 17βHSD type 7 and 12, as well as CYP19A1 (aromatase), were significantly higher in the hypertrophic model relative to the control adipocytes differentiated using the conventional method. The increase in 11βHSD1 and 17βHSD12 expression was consistent with the enhanced conversion of cortisone and androstenedione to cortisol and testosterone, respectively. As these characteristics reflect those commonly observed in obesity, hypertrophic 3T3-L1 adipocytes are an appropriate in vitro model to study mechanisms of adipocyte dysfunction in an era where the rise in obesity incidence is a global health concern, and where access to adipose tissue from obese patients are limited.

Effect of anti-GnRH vaccine on testicular tissues in stallions

Anti-GnRH Vaccine is known as an alternative to surgical castration. The main advantage is the reversibility of immunization in 90% of the cases (Bruyas et al. Reprod Dom Anim, p.55: https://doi.org/10.1111/rda.12800). Anti-GnRH immunization reduces fertility by inhibition of gonadotropin synthesis involved in steroid production and spermatogenesis. Only few papers report information on the recovery of sperm production after prime immunization (twice 4 weeks apart) without booster in some stallions. We aimed to assess the effect of the vaccination protocol (Primo immunization or/and boosters) and the delay between the last injection of anti-GnRH vaccine and surgical castration on testicular histology. Twelve unvaccinated (control) and 28 immunized Lusitanian stallions were castrated at different time points after the last immunization (range 0.5-14 months). Vaccination protocol was a primo vaccination by intramuscular injection of 1 mL IMPROVAC® twice 4 weeks apart, followed by booster(s) when stallion behavior returned or scrotal size re-increased (range 1-10 injections). Testes were fixed in formaldehyde (10%) and stained by HES (Hematoxylin-Eosin-Saffron) and Androgen Receptor immunohistochemistry. Testicular size and spermatogenesis were evaluated by 7 parameters: Johnsen scores, seminiferous tubule diameter, number and density of Sertoli cells, number and density of germ cells, and the ratio of germ to Sertoli cells.Statistical analysis was performed by ANOVA. Values are given as mean ±standard deviation. Testis size was larger for unvaccinated controls (88±5 mm; P<0.001) compared to vaccinated stallions (70 ±8 mm). The Johnsen scores were higher in control (9.3 ±0.3; P<0.0001) compared to vaccinated stallions (8.0 ±1.2). Seminiferous tubule diameters, number and density of germ cells, and the ratio of germ to Sertoli cells were larger in control stallions: respectively 173 ±10 µm (P<0.001), 180 ±34 germ cells per tubule (P<0.001), 6330 ±897 germ cells per mm² of tubule (P<0.001), and 8.2 ±1.5 (P<0.001) germ cells per Sertoli cell compared to vaccinated stallions: 135 ±22 µm, 83 ±47 germ cells per tubule, 4404 ±1454 germ cells per mm² of tubule, and 3.3 ±1.9 germ cells per Sertoli cell. Conversely, Sertoli cell numbers and density were larger in vaccinated: 27 ±4 Sertoli cells per tubule (P=0.005) and 1720 ±582 Sertoli cells per mm² of tubule (P<0.001) compared to 23 ±4 Sertoli cells per tubule and 817 ±165 Sertoli cells per mm² of tubule in controls. There was no significant difference among the vaccination protocols (e.g., number of boosters, delay between last injection and gonadectomy) on these parameters. In conclusion, multiple rounds of anti-GnRH immunization in stallions: (1) induced testicular hypoplasia is more dependent on stallion rather the vaccination protocol, and (2) had no reversible effects on testes (according to Johnsen scores).

An in vitro model for equine placental research

A fundamental understanding of placental development and its associated pathologies is crucial to mitigating the losses associated with placental disorders. Organoids are widely used to investigate the developmental biology of organs and in disease modeling. In humans, placental organoids have been successfully developed using trophoblast cells. In this study, we aimed to 1) develop placental organoids using pre-term and term equine placentae, 2) evaluate the organoid structures, 3) compare the organoid transcriptome with their respective chorioallantois (CA), and 4) evaluate organoids based on their starting material (pre-term vs term). The CA was collected from healthy placentae following elective abortion (80-100d of gestation) and following spontaneous term parturition. CA samples were processed for culture, fixed for histology, and stored in RNAlater. For culture, each CA sample was enzymatically digested to a single cell suspension and was seeded into 50µL Matrigel Matrix droplets surrounded by medium. Passage-3 organoids (P3) were used for histology (H&E), immunohistochemistry (IHC), and transcriptomic analysis. Steroid production and conversion were measured by mass spectrometry (MS) of culture media. After optimization of culture conditions, we produced 3D-organoids (Pre-Term, n=6 and Term, n=3). Organoids were passaged up to 12 times over ±3 months and were successfully frozen and re-cultured post-thaw. Total extracted RNA from initial CA samples and organoids was sequenced (150PE; ∼43 million reads/sample). The genes expressed in the organoidsclosely resemble those of the tissue of origin, 19.4% of the expressed genes were differentially expressed (DEGs) between the initial CA and P3 (n=3,949 genes; FDR<0.001). H&E staining of both CA and the organoids indicates the epithelial origin of the cells within the organoids. The enriched expression of trophoblast-specific genes PLAC8B, GATA3, KRT7, VGLL1, and EGFR, and the presence of the trophoblast-specific genes TFAP2A and TFAP2C, and GATA2 in the organoids confirm their trophoblast identity. The presence of SOX2 and OCT4, as demonstrated by IHC, confirms the pluripotency of the organoid cells. The SRD5A1 and SRD5A3 genes were expressed in all organoids and our preliminary MS analysis demonstrated the conversion of progesterone to 5α-DHP, further suggesting functional integrity. Comparison between Pre-Term and Term organoids revealed 552 DEGs (FDR<0.001), with downregulation of immune/inflammatory/defense responses and blood vessel development, and upregulation of molecule transport, development, and adhesion in the Pre-Term group. To conclude, this is the first report of placental organoids in domestic animals. The organoids show structural, transcriptomic, and functional similarities to the equine placenta in vivo and consequently could be used as an in vitro model to study placental development and associated pathologies. Funded by The Foundation for The Horse, GJCRF, UGent BOF, BAEF, L'Oréal-UNESCO FWO, and the Center for Equine Health at UC Davis.

The inhibition of steroid hormones determines the fate of IPC-366 tumor cells, highlighting the crucial role of androgen production in tumor processes

Inflammatory mammary cancer (IMC) is a disease that affects female dogs. It is characterized by poor treatment options and no efficient targets. However, anti-androgenic and anti-estrogenic therapies could be effective because IMC has a great endocrine influence, affecting tumor progression. IPC-366 is a triple negative IMC cell line that has been postulated as a useful model to study this disease. Therefore, the aim of this study was to inhibit steroid hormones production at different points of the steroid pathway in order to determine its effect in cell viability and migration in vitro and tumor growth in vivo. For this purpose, Dutasteride (anti-5αReductase), Anastrozole (anti-aromatase) and ASP9521 (anti-17βHSD) and their combinations have been used. Results revealed that this cell line is positive to estrogen receptor β (ERβ) and androgen receptor (AR) and endocrine therapies reduce cell viability. Our results enforced the hypothesis that estrogens promote cell viability and migration in vitro due to the function of E1SO4 as an estrogen reservoir for E2 production that promotes the IMC cells proliferation. Also, an increase in androgen secretion was associated with a reduction in cell viability. Finally, in vivo assays showed large tumor reduction. Hormone assays determined that high estrogen levels and the reduction of androgen levels promote tumor growth in Balb/SCID IMC mice. In conclusion, estrogen levels reduction may be associated with a good prognosis. Also, activation of AR by increasing androgen production could result in effective therapy for IMC because their anti-proliferative effect.

Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. Previous studies documented the role of autophagy in sex steroid hormone biosynthesis in different animal models and human testis. Here we demonstrate in this study that sex steroid hormones estrogen and progesterone are produced through the same autophagy-mediated mechanism in the human ovary in addition to the human testis. In brief, pharmacological inhibition and genetic interruption of autophagy through silencing of autophagy genes (Beclin1 and ATG5) via siRNA and shRNA technologies significantly reduced basal and gonadotropin-stimulated estradiol (E2), progesterone (P4) and testosterone (T) production in the ex vivo explant tissue culture of ovary and testis and primary and immortalized granulosa cells. Consistent with the findings of the previous works, we observed that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LD)s with lysosome to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis. Gonadotropin hormones are likely to augment the production of sex steroid hormones by upregulating the expression of autophagy genes, accelerating autophagic flux and promoting the association of LDs with autophagosome and lysosome. Moreover, we detected some aberrations at different steps of lipophagy-mediated P4 production in the luteinized GCs of women with defective ovarian luteal function. The progression of autophagy and the fusion of the LDs with lysosome are markedly defective, along with reduced P4 production in these patients. Our data, together with the findings of the previous works, may have significant clinical implications by opening a new avenue in understanding and treatment of a wide range of diseases, from reproductive disorders to sex steroid-producing neoplasms, sex steroid-dependent malignancies (breast, endometrium, prostate) and benign disorders (endometriosis).

Ketoconazole: A Promising Drug for Fungal Infections

Ketoconazole is a synthetic imidazole antifungal agent that can be used to treat surface fungal infections, genital, chronic mucocutaneous candidosis, and candidosis. Ketoconazole is a synthetic imidazole derivative that is effective after oral administration, unlike older imidazole antifungals like miconazole and clotrimazole. It works well for fungal infections on the skin, such as dermatophytosis and genital candidiasis. It has become useful for the treatment of persistent mucocutaneous candidiasis. In immunocompromised individuals, ketoconazole is also used to prevent fungal infections. It has lately been utilised as a therapy for advanced prostatic cancer because it inhibits the production of adrenal sex hormone. Although oral ketoconazole has been subjected to labelling modifications and market withdrawal because of substantial adverse effects, topical ketoconazole is widely regarded to be efficacious, safe, and best for treating superficial fungal infections. Onychomycosis, blepharitis, and hair loss are some of the new dermatologic indications for topical ketoconazole usage.

Comparative developmental genomics of sex-biased gene expression in early embryogenesis across mammals

BackgroundMammalian gonadal sex is determined by the presence or absence of a Y chromosome and the subsequent production of sex hormones contributes to secondary sexual differentiation. However, sex chromosome-linked genes encoding dosage-sensitive transcription and epigenetic factors are expressed well before gonad formation and have the potential to establish sex-biased expression that persists beyond the appearance of gonadal hormones. Here, we apply a comparative bioinformatics analysis on a pair of published single-cell datasets from mouse and human during very early embryogenesis—from two-cell to pre-implantation stages—to characterize sex-specific signals and to assess the degree of conservation among early acting sex-specific genes and pathways.ResultsClustering and regression analyses of gene expression across samples reveal that sex initially plays a significant role in overall gene expression patterns at the earliest stages of embryogenesis which potentially may be the byproduct of signals from male and female gametes during fertilization. Although these transcriptional sex effects rapidly diminish, sex-biased genes appear to form sex-specific protein–protein interaction networks across pre-implantation stages in both mammals providing evidence that sex-biased expression of epigenetic enzymes may establish sex-specific patterns that persist beyond pre-implantation. Non-negative matrix factorization (NMF) on male and female transcriptomes generated clusters of genes with similar expression patterns across sex and developmental stages, including post-fertilization, epigenetic, and pre-implantation ontologies conserved between mouse and human. While the fraction of sex-differentially expressed genes (sexDEGs) in early embryonic stages is similar and functional ontologies are conserved, the genes involved are generally different in mouse and human.ConclusionsThis comparative study uncovers much earlier than expected sex-specific signals in mouse and human embryos that pre-date hormonal signaling from the gonads. These early signals are diverged with respect to orthologs yet conserved in terms of function with important implications in the use of genetic models for sex-specific disease.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, withkoffrates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14αC-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Stimulatory effects of vasopressin on progesterone production and BMP signaling by ovarian granulosa cells

The aim of the present study was to clarify the effects of arginine vasopressin (AVP) on ovarian steroid production and its functional relationship to the ovarian bone morphogenetic protein (BMP) system. The results showed that AVP treatment significantly increased gonadotropin- and forskolin-induced progesterone synthesis by primary culture of rat granulosa cells and human granulosa cells, respectively. In contrast, estradiol production was not significantly affected by AVP. Treatment with AVP significantly increased forskolin-induced cAMP synthesis by human granulosa cells and mRNA levels of the progesterogenic enzymes CYP11A1 and HSD3B2 in the cells. On the other hand, AVP also enhanced BMP-15-induced phosphorylation of SMAD1/5/9 and ID1 transcription. It was further revealed that the expression levels of BMP receptors, including ALK3, ALK6 and BMPR2, were upregulated by AVP. Collectively, the results indicate that AVP stimulates progesterone production via the cAMP-PKA pathway with upregulation of BMP signaling that inhibits progesterone production, which may lead to fine adjustment of progesterone biosynthesis by granulosa cells.

The puzzle of extensive tinea incognito with a young woman: a case report a tinea incognito with Cushing’s disease

Introduction: Tinea incognito is a rare condition commonly found in abused topical or systemic steroids. Considering their immunosuppression status, Cushing’s syndrome predisposes patients to infectious diseases. Most patients of Cushing’s syndrome are as a result of adrenocorticotropic hormone production from a pituitary adenoma, which is referred to as Cushing’s disease. Here, we reported a rare co-occurrence of tinea incognito and Cushing’s syndrome secondary to a giant pituitary tumor. Case presentation: Extensive cutaneous and nail lesions were diagnosed with the tinea incognito and tinea unguium, and endocrine or rheumatic disorders were considered as underlying disease-causing extensive tinea incognito. A series of laboratory and imaging tests were performed. We found that a giant pituitary tumor was the primary etiology of multiple cutaneous appearances and secondary disorders. There was an exceptionally unusual co-occurrence of extensive tinea incognito and pituitary adenoma in our case. Discussion: We observed a rare co-occurrence of extensive tinea incognito and pituitary adenoma, which should be taken into consideration in the urgent diagnostic criterion of tinea incognito given the potential underlying condition. Conclusion: Tinea incognito is not only associated with a history of topical or systemic steroid use, but also the association that underlying diseases related to excessive endogenous steroid production are supported to be taken into account.

The Relationship Between Menstrual Cycle Disorders In Obese Adolescents

Menstrual cycle disorders are the main symptom of anovulation, a phenomenon accompanied by decreased ovarian steroid secretion and production. Weight gain and an increase in adipose tissue in obese women, especially in the midsection, can disrupt the balance of steroid hormones such as androgens, estrogens, and sex hormone-binding globulins, which can affect the menstrual cycle in adolescents. The mechanism of obesity that is most often associated with menstrual cycle disorders. On that basis, this study aims to assess the relationship between insulin resistance and menstrual cycle disorders in obese adolescents. Method : This study used a cross-sectional design with comparisons where in this study the researchers compared the levels of the Homeostatic Model obese adolescent women who had normal menstrual cycles and abnormal menstrual cycles. Result : the group with abnormal menstrual cycles were found to be lower than those in the normal menstrual cycle group, but no significant difference was found. In addition, the abnormal menstrual cycle group were found to be lower but not significant. Conclusion : Obesity no significant relationship to the menstrual cycle in obese adolescent girls

New insights into signal transduction pathways in adrenal steroidogenesis: role of mitochondrial fusion, lipid mediators, and MAPK phosphatases.

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

The genetics of autism and steroid-related traits in prenatal and postnatal life.

Autism likelihood is a largely heritable trait. Autism prevalence has a skewed sex ratio, with males being diagnosed more often than females. Steroid hormones play a mediating role in this, as indicated by studies of both prenatal biology and postnatal medical conditions in autistic men and women. It is currently unclear if the genetics of steroid regulation or production interact with the genetic liability for autism. To address this, two studies were conducted using publicly available datasets, which focused respectively on rare genetic variants linked to autism and neurodevelopmental conditions (study 1) and common genetic variants (study 2) for autism. In Study 1 an enrichment analysis was conducted, between autism-related genes (SFARI database) and genes that are differentially expressed (FDR<0.1) between male and female placentas, in 1st trimester chorionic villi samples of viable pregnancies (n=39). In Study 2 summary statistics of genome wide association studies (GWAS) were used to investigate the genetic correlation between autism and bioactive testosterone, estradiol and postnatal PlGF levels, as well as steroid-related conditions such as polycystic ovaries syndrome (PCOS), age of menarche, and androgenic alopecia. Genetic correlation was calculated based on LD Score regression and results were corrected for multiple testing with FDR. In Study 1, there was significant enrichment of X-linked autism genes in male-biased placental genes, independently of gene length (n=5 genes, p<0.001). In Study 2, common genetic variance associated with autism did not correlate to the genetics for the postnatal levels of testosterone, estradiol or PlGF, but was associated with the genotypes associated with early age of menarche in females (b=-0.109, FDR-q=0.004) and protection from androgenic alopecia for males (b=-0.135, FDR-q=0.007). The rare genetic variants associated with autism appear to interact with placental sex differences, while the common genetic variants associated with autism appear to be involved in the regulation of steroid-related traits. These lines of evidence indicate that the likelihood for autism is partly linked to factors mediating physiological sex differences throughout development.

Perfluorooctane sulfonic acid modulates expression of placental steroidogenesis-associated genes and hormone levels in pregnant rats

Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. Reports show that PFOS is a potential endocrine disruptor; however, the possible effects of PFOS on placental endocrine function are unclear. This study aimed to investigate the endocrine-disrupting effects of PFOS on the placenta in pregnant rats and its potential mechanism. Pregnant rats from gestational days 4–20 were exposed to 0, 10, and 50 μg/mL PFOS through drinking water followed by analysis of various biochemical parameters. PFOS dose-dependently decreased fetal and placental weight in both sexes, with a specific decrease in weight of labyrinth but not junctional layer. Plasma progesterone (↑166%), aldosterone (↑201%), corticosterone (↑205%), testosterone (↑45%), luteinizing hormone (↑49%) levels were significantly increased, while estradiol (↓27%), prolactin (↓28%) and hCG (↓62%) levels were reduced in groups exposed to higher doses of PFOS. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis revealed a significant increase in mRNA levels of placental steroid biosynthesis enzymes, including Cyp11A1 and 3β-HSD1 in male placenta and StAR, Cyp11A1, 17β-HSD1 and 17β-HSD3 in female placenta of PFOS dams. Cyp19A1 expression in ovaries was significantly decreased in PFOS dams. mRNA levels for placental steroid metabolism enzyme UGT1A1 increased in male but not in female placenta of PFOS dams. These results suggest that the placenta is a target tissue of PFOS and PFOS-induced dysregulation in steroid hormone production might be related to the altered expression of hormone biosynthesis and metabolism enzyme genes in the placenta. This hormone disruption might affect maternal health and fetal growth.

Pathogen odors elicit distinct neurosteroid and behavioral responses in Rainbow trout (Oncorhynchus mykiss)

Rainbow trout can smell virus and bacteria cues that in turn activate an immune response in the brain in absence of a systemic response (i.e. the immune response is activated by the nervous system instead of the pathogen inside tissues or bloodstream). However, little is known if pathogen odors can elicit different physiological responses (i.e. behavioral and immune) and how are pathogen odors integrated into different responses in the brain. We hypothesize that pathogen odors not only activate immune responses but also induce avoidance behavior. Moreover, we hypothesized that pathogen olfactory responses are modulated by neurosteroids at different times in different parts of the brain. We used a two-choice maze test to determine whether inactivated Yersinia ruckeri elicits aversive behavior. Furthermore, to check if behavioral and immune responses are modulated by neurosteroids, we performed a second experiment where we nasally administered inactivated Y. ruckeri to rainbow trout. After that, the nose, olfactory bulb, cerebellum, and optic tectum were collected at four different time points (15 min, 4 hours, 24 hours, and 7 days) and samples were extracted to measure gene expression of several steroidogenic enzymes ( cyp19a1, 3βHSD, 11HSD, and 17HSD) and fish production of neurosteroids (e.g. cortisol, estradiol, and progesterone). We found that rainbow trout actively avoids pathogen odors. Moreover, we found a different expression of neuro steroidogenic enzymes at different brain areas and times post-exposure. Fish exposed to pathogen odor express a significant increase (ANOVA 2 factor, P&lt;0.05) of cyp19a1 (involved in estradiol synthesis) as soon as 15 min in the nose and olfactory bulb, and 4 hours post-exposure in the cerebellum and optic tectum. This gene expression was parallel to a significant increase of estradiol in the nose, bulb, and rest of the brain between 15 min and 4 hours. Moreover, there was a significant increase of 11HSD and 17HSD (related to the production of testosterone, cortisol, and corticosterone) 4 hours after exposure in the nose and cerebellum but a significant decrease in the olfactory bulb and optic tectum. These gene expression patterns were parallel o an increase of cortisol in the nose and olfactory bulb. Finally, we found a significant increase of 3βHSD (involved in the first steps of steroidogenesis) after 24 hours in the nose and olfactory bulb, although we didn’t find an increase in steroid production in our preliminary assays. Our results showed that exposure to pathogen odors activates different neuro steroidogenic pathways at different times after exposure, and at different areas of the brain. These findings pinpoint the use of the sense of smell by fish to control behavioral and immune responses to pathogens, with neurosteroids playing a key role in modulating and integrating these olfactory responses. This material is based upon work supported by the National Science Foundation under Grant No. 1755348 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Identification of a novel class of cortisol biosynthesis inhibitors and its implications in a therapeutic strategy for hypercortisolism

AimsDysregulation of adrenocortical steroid (corticosteroids) biosynthesis leads to pathological conditions such as Cushing's syndrome. Although several classes of steroid biosynthesis inhibitors have been developed to treat cortisol overproduction, limitations such as insufficient efficacy, adverse effects, and/or tolerability still remain. The present study aimed to develop a new class of small molecules that inhibit cortisol production, and investigated their putative modes of action. Main methodsWe screened an in-house chemical library with drug-like chemical scaffolds using human adrenocortical NCI-H295R cells. We then evaluated and validated the effects of the selected compounds at multiple regulatory steps of the adrenal steroidogenic pathway. Finally, genome-wide RNA expression analysis coupled with gene enrichment analysis was conducted to infer possible action mechanisms. Key findingsA subset of benzimidazolylurea derivatives, including a representative compound (designated as CJ28), inhibited both basal and stimulated production of cortisol and related intermediate steroids. CJ28 attenuated the mRNA expression of multiple genes involved in steroidogenesis and cholesterol biosynthesis. Furthermore, CJ28 significantly attenuated de novo cholesterol biosynthesis, which contributed to its suppression of cortisol production. SignificanceWe identified a novel chemical scaffold that exerts inhibitory effects on cortisol and cholesterol biosynthesis via coordinated transcriptional silencing of gene expression networks. Our findings also reveal an additional adrenal-directed pharmacological strategy for hypercortisolism involving a combination of inhibitors targeting steroidogenesis and de novo cholesterol biosynthesis.

Effects of chronic dichlorodiphenyldichloroethylene exposure on testosterone secretion and steroidogenic pathway in the male gonad.

Endocrine disrupting chemicals are present in the environment and/or in consumer products. These agents have the capacity to mimic and/or antagonize endogenous hormones and thus perturb the endocrine axis. The male reproductive tract expresses steroid hormone (androgen and estrogen) receptors at high levels and is a major target for endocrine disrupting chemicals. In this study, Long-Evans male rats were exposed to dichlorodiphenyldichloroethylene, a metabolite of dichlorodiphenyltrichloroethane and a chemical present in the environment, in drinking water at 0.1 and 10μg/L for 4 weeks. At the end of exposure, we measured steroid hormone secretion and analyzed steroidogenic proteins, including 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, steroidogenic acute regulatory protein, aromatase, and the LH receptor. We also analyzed Leydig cell apoptosis (poly-(ADP-ribose) polymerase) and caspase-3 in the testes. Testicular testosterone (T) and 17β-estradiol (E2) were both affected by exposure to dichlorodiphenyldichloroethylene by displaying altered steroidogenic enzyme expression. Dichlorodiphenyldichloroethylene exposure also increased the expression of enzymes mediating the pathway for programmed cell death, including caspase 3, pro-caspase 3, PARP, and cleaved PARP. Altogether, the present results demonstrate that dichlorodiphenyldichloroethylene directly and/or indirectly can target specific proteins involved in steroid hormone production in the male gonad and suggest that exposure to environmentally relevant dichlorodiphenyldichloroethylene levels has implications for male reproductive development and function.

Reproductive endocrine disruption and gonadal intersex induction in male Japanese medaka chronically exposed to betamethasone at environmentally relevant levels

The broad utilization of betamethasone in medical treatments may pose a significant ecotoxicological risk to aquatic organisms, yet its potential reproductive toxicity remains unclear. The present study examined the impacts of environmental exposure on male reproduction using Japanese medaka (Oryzias latipes). After 110 days of betamethasone exposure at environmentally relevant concentrations (0, 20 and 200 ng/L), LH/FSH synthesis and release in the pituitary was inhibited, and the production of sex hormones and their signaling pathways in the gonads of male medaka were greatly influenced. This synthetic glucocorticoid restrained testosterone (T) synthesis and gave rise to a significant increase in E2/T and E2/11-KT ratios. Furthermore, chronic betamethasone exposure (20 and 200 ng/L) led to the suppression of androgen receptor (AR) signaling and enhancement of estrogen receptors (ERs) signaling. An increase in hepatic vitellogenin contents was also detected, and testicular oocytes were observed in both 20 and 200 ng/L betamethasone-treated groups. It showed that 20 and 200 ng/L betamethasone could induce male feminization and even intersex, triggering abnormal spermatogenesis in medaka males. With its adverse effects on male fertility, betamethasone could potentially influence the fishery productivity and population dynamics in aquatic ecosystems.