Steroid Secretion Research Articles

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1).Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring.Sensitivity ranged within 0.012–38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4–122.0, 55.6–104.1 and 76.4–106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2–102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2–27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids.In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.

Does kisspeptin exert a local modulatory effect on bovine ovarian steroidogenesis?

Kisspeptin, a hypothalamic neuropeptide encoded by the KISS1 gene, has a pivotal role in promoting GnRH secretion in mammals. Kisspeptin and its receptor (KISS1R) are also expressed in certain peripheral tissues including gonads, suggesting intra-gonadal roles. Such actions at the level of the bovine ovary have not been explored previously. The current aims were to determine whether KISS1 and its receptor (KISS1R) are expressed in the bovine ovary and whether kisspeptin or a kisspeptin antagonist can modulate ovarian steroid production by cultured ovarian cells. Granulosa (GC) and theca interna (TC) were collected from antral follicles (3-18 mm) categorized into five class sizes. Early, mid and regressing corpora lutea (CL) were also collected for RT-qPCR analysis of KISS1 and KISS1R expression. Bovine TC and GC cultured under both non-luteinizing (serum-free) and luteinizing (serum-supplemented) conditions were treated for 4 days with kisspeptin-10 (10-10-10-6M) or kisspeptin antagonist (p234; 10-10-10-6M), alone and in combination with either FSH (GC), LH (TC) or forskolin (luteinized GC/TC). Steroid secretion (GC: oestradiol, progesterone; TC: androstenedione, progesterone; luteinized GC/TC: progesterone) was measured by ELISA and viable cell number determined by neutral red uptake assay. KISS1 and KISS1R transcripts were detected in TC, GC and CL with significant differences between follicle categories and CL stages. However, neither kisspeptin-10 nor kisspeptin antagonist affected steroid secretion or viable cell number in any of the four ovarian cell culture models. As such, the hypothesis that kisspeptin has a direct intra-ovarian role to modulate follicular or luteal steroidogenesis, or cell proliferation/survival, is not supported.

Studying paraben-induced estrogen receptor- and steroid hormone-related endocrine disruption effects via multi-level approaches

Increasing concerns have been raised on the health risks of parabens in the regard of their widespread applications and potential endocrine disrupting activities. In this study, four typical parabens, including methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), and butyl paraben (BuP) were systematically investigated for their estrogen receptor- and steroid hormone-related endocrine disruptions using multi-level approaches. Paraben exposure promoted the proliferation of MCF-7 cells, increased the luciferase activity in MVLN cells, and induced the vitellogenin (vtg) expression in zebrafish larvae, showing the typical estrogenic effects. The in vitro protein assays further revealed that PrP and BuP could bind with two isoforms of estrogen receptors (ERs). The estrogenic activities of parabens were predicted to be positively correlated with their chemical structure complexity by using molecular docking analysis. Furthermore, the synthesis and secretion of estradiol (E2) and testosterone (T) were significantly disturbed in H295R cells and zebrafish larvae, which could be regulated by paraben-induced transcriptional disturbance in both in vitro steroidogenesis and in vivo hypothalamic-pituitary-gonadal (HPG) axis. Parabens could disturb the endocrine system by activating the ERs and disrupting the steroid hormone synthesis and secretion, suggesting their potential deleterious risks to the environment and human health.

Metamifop as an estrogen-like chemical affects the pituitary-hypothalamic-gonadal (HPG) axis of female rice field eels (Monopterus albus).

Metamifop (MET) is a widely used herbicide. It is likely for it to enter water environment when utilized, thus potential impacts may be produced on aquatic animals. Little information is available about its effects on the endocrine system of fish to date. In the current study, female rice field eels (Monopterus albus) were exposed to different MET concentrations (0, 0.2, 0.4, 0.6, 0.8mgL -1) for 96h to examine the effect of MET on the hypothalamic-pituitary-gonadal (HPG) axis and sexual reversal. The results showed that high concentrations of MET exposure increased vitellogenin (VTG) levels in liver and plasma, but plasma sex hormone levels were not affected by MET exposure. MET exposure increased the expression of CYP19A1b and CYP17 that regulate sex hormone production in the brain, but the expression of genes (CYP19A1a, CYP17, FSHR, LHCGR, hsd11b2, 3β-HSD) associated with sex hormone secretion in the ovary and the estrogen receptor genes (esr1, esr2a, esr2b) in the liver were all suppressed. In addition, the expression of sex-related gene (Dmrt1) was suppressed. This study revealed for the first time that MET has estrogen-like effects and has a strong interference with the expression of HPG axis genes. MET did not show the ability to promote the sexual reversal in M. albus, on the contrary, the genes expression showed that the occurrence of male pathway was inhibited.

Liver function in transgender persons: Challenges in the COVID-19 era.

Transgender persons constitute a non-negligible percentage of the general population. Physical gender-transitioning in trans persons is mainly achieved with hormonal cross-sex therapy and sex reassignment surgeries that aim to align bodily appearance with gender identity. Hormonal treatment acts via suppressing the secretion of the endogenous sex hormones and replacing them with the hormones of the desired sex. The administration of testosterone is the typical masculinizing treatment in trans men, whilst trans women are routinely treated with estradiol agents in combination with anti-androgens or gonadotrophin-releasing hormone agonists if testes are present. Exogenous androgenic steroids, estradiol agents, and anti-androgens have been implicated in a series of hepatotoxic effects. Thus, liver integrity is a major concern with the long-term administration of cross-sex therapy. Hepatic tissue is susceptible to coronavirus disease 19 (COVID-19) through various pathophysiological mechanisms. Special consideration should be paid to minimize the risk of hepatic damage from the potential cumulative effect of COVID-19 and gender-affirming treatment in transgender patients. Appropriate care is significant, with continuous laboratory monitoring, clinical observation and, if needed, specific treatment, especially in severe cases of infection and in persons with additional liver pathologies. The pandemic can be an opportunity to provide equal access to care for all and increase the resilience of the transgender population.

Morphometric study of sexual dimorphism in sacrum- A review paper

The accurate observations and measurable investigations to identify the sex is the intrinsic step in biological profile development of the sample. In such scenario, sacrum identifies unknown sex and associated sex related functional differences more precisely. In the past, studies have shown that the morphological features of the sacrum are influenced by the sex hormone secretion and its associated reproductive functions. There are several parameters that can be considered when the sacral dimorphism is considered. Along with the sacrum measurements, the subpubic angle of the individual can also be measured. Among all the parameters used, the breadth and curvature of the sacrum were reliable indicators of sex among these measures. Additionally, it was also reported that none of the criteria could completely determine the sex of the bones. Therefore, it can be said that in order to determine the sex of the sacrum with 100 percent accuracy, consideration of all the parameters should be done. Further, other factors associated with the environment, physical stress, and genetic factors also affects this measurement. Hence, a groundwork about the previous research, is required while evaluating the sexual dimorphism in sacrum.

Integrated analysis of lncRNA and mRNA for the apoptosis of porcine ovarian granulosa cells after polyphenol resveratrol treatment.

Resveratrol (RES) is a non-flavonoid polyphenol compound that can be involved in follicular development and ovulation. However, the mechanism by which resveratrol regulates the apoptosis of porcine ovarian granulosa cells (POGCs) through long non-coding RNA (lncRNA) is poorly understood. We generated POGCs models of different doses of RES (0, 25, 50, 75, and 100 μM). It was observed that the cell viability was the highest in the 50 μM group, and the highest apoptosis rates were recorded in the 100 μM group. Therefore, a control group (n = 3, 0 μM RES group), a low RES group (n = 3, 50 μM RES group), and a high RES group (n = 3, 100 μM RES group) of POGCs were created for next RNA sequencing. Gene Ontology (GO) indicated that differentially expressed lncRNAs associated with apoptotic process were highly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of lncRNA target genes found that the Wnt signaling pathway and PI3K-Akt signaling pathway were both enriched. Furthermore, we constructed lncRNA-mRNA networks related to Metabolic and Cell Apoptosis, respectively. In the networks, five key-lncRNAs were screened, which may play a significant role in the process of POGCs metabolism and apoptosis. Furthermore, we focused on the function of a lnc-GAM (lncRNA associated with Granulosa cells Apoptosis and Metabolism) and verified that lnc-GAM could influence cell apoptosis in POGCs development by affecting the mRNA expression of apoptosis-related markers, and also affects the secretion of steroid hormones and related genes expression in POGCs cultured in vitro. Our study provides seminal data and important new insights into the regulation of reproductive mechanisms in porcine and other female mammals.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

Diagnostic of cryptorchidism in horses.

Cryptorchidism in horses is the most common congenital malformation, caused by a failure in the descent of testicles from the abdominal cavity into the scrotum. As a result, there is a partial or complete loss of reproductive potential although the production and secretion of steroid sex hormones and anti-Mueller hormone, as well as behavioral characteristics of the stallion, are maintained. The exact location of the undescended testis is established through a thorough clinical examination of the interior (of the cryptorchid horse), supported by an ultrasound examination of the inguinal region, and the transrectal ultrasound visualization of the bladder area and the inner ring of the inguinal canal. The results of several genetic studies suggest a genetic basis for cryptorchidism. In particularly difficult cases of suspected cryptorchidism in horses (hemi-castrated animals without a known clinical history), it is necessary to determine androgenic hormone levels and the anti-Mueller hormone level and to perform a testosterone production stimulation test with hCG induction (Cox test). The exact location of the retained testicle needs to be known before surgical removal so that optimal decisions can be made regarding the technique of the procedure and selection of appropriate anaesthesia.

Effect of maternal dietary 25OHD3 on the gonadal development of offspring mice

Context Studies have widely reported that a vitamin D3 metabolite 25OHD3 improves the growth performance of animals and offspring, but few studies have focused on offspring sex hormone secretion and gonadal development in the offspring. Aims This study focused on the effects of gestational feeding with 25OHD3 on the growth, serum calcium (Ca2+) and phosphorus (Pi) concentrations, and ovarian and testicular development in offspring. Methods From the beginning of gestation until the end of labour, maternal mice were gavaged daily with 25 ug of 25OHD3. We weighed offspring on Days 1, 10, and 21 postnatally and collected offspring serum and gonadal samples on Day 21. Key results The results showed that 25OHD3 significantly increased maternal serum Ca2+ and Pi concentrations, and increased the growth rate and serum Ca2+ and Pi concentrations of offspring, significantly increased the serum concentrations of luteinising hormone (LH) and follicle-stimulating hormone (FSH), and accelerated the follicle development in the female offspring. Male offspring showed significantly increased LH, FSH, and testosterone serum concentrations, and 3β-HSD and 17β-HSD mRNA expressions, and a significantly decreased CYP17A1 mRNA expression. Conclusions In conclusion, the present findings indicated that maternal feeding with 25OHD3 during pregnancy positively affects offspring hormone secretion and gonadal development. Implications These insights have shed light on the potential significance of maternal nutrition in ensuring healthier animal populations and offer valuable considerations for animal husbandry practices.

Effects of melatonin on development and hormone secretion of sheep theca cells in vitro

Theca cells (TCs) play a unique role in the structure and function of the ovary. They are not only the structural basis of the follicle but also the androgen-secreting cells in female mammals, which can affect the normal development and atresia of the follicle. The results showed that melatonin receptor (MTR) MT1 and MT2 were expressed on sheep TCs. In the present study, the effects of different concentrations of MT at 0, 10−10, 10−8, 10−6 and 10−4 M/L on sheep TCs with regards to the antioxidant levels, proliferation, apoptosis and steroid hormone secretion were investigated. The results showed that in sheep TCs, all concentrations of MT significantly decreased reactive oxygen species (ROS) concentration and BAX expression; increased Cat, Sod1, and BCL-2 expression. The proliferation viability of TCs was significantly inhibited in all groups except for 10−10 M/L MT, and the expression of cyclin D1 and CDK4 was significantly reduced. MT significantly increased StAR expression and progesterone secretion in TCs, but there was no significant effect on androgen secretion and CYP11A1, CYP17A1 and 3β-HSD expression in all groups. MT-induced progesterone secretion was completely inhibited by Luzindole (a nonspecific MT1 and MT2 inhibitor) and partially inhibited by 4p-PDOT (specific MT2 inhibitor). MT-induced progesterone secretion can be inhibited by LY294002 (PI3K/AKT pathway inhibitor). This study indicated that MT inhibits apoptosis and proliferation of in vitro cultured sheep TCs, which has implications for slowing ovarian atresia and aging. MT activates the PI3K/Akt pathway to mediate the synthesis and secretion of progesterone by TCs. This study provides a basis for further exploration of the role of TCs on follicle development and ovarian steroid hormone secretion.

Endocrine disrupting effect and reproductive toxicity of the separate exposure and co-exposure of nano-polystyrene and diethylstilbestrol to zebrafish

The co-occurrence of nanoplastics and other pollutants in the environment has gotten a lot of attention, but information on the biological toxicity of their co-exposure was limited. This study aims to reveal the endocrine disrupting effect and reproductive toxicity of nano-polystyrene (NPS) and diethylstilbestrol (DES) to zebrafish under separate and combined exposure. Results indicated that NPS and DES exposure in isolation reduced the hepatosomatic index and gonadosomatic index, and altered the cell maturity in gonads in both cases. Even worse, the co-exposure of NPS and DES exacerbated the damage to the liver and gonads of fish. The two pollutants individually inhibited the secretion of sex hormones and vitellogenin. The inhibition effect of DES was especially dose-dependent, while NPS had weaker effect than DES. Their combined action on the secretion of sex hormones and vitellogenin exhibited additive effect. However, NPS did not affect the content of thyroid hormones in fish, and also had no significant effect on the reduction of thyroid hormone caused by DES exposure. Furthermore, their co-exposure decreased the cumulative eggs from 1031 to 306, and the spawning number from 12 to 8. The fertilization rate and hatchability rete of eggs were reduced by 30.9% and 40.4%, respectively. The abnormality rate of embryos was 65.0%, significantly higher than in separate DES and NPS groups (55.7% and 30.8% respectively). The abnormal development of offspring was mainly pericardial cyst, spinal curvature, and growth retardation.

Effects of Nd(2)O(3) exposure of rare earth particles on C57 BL/6J male mice sex hormone secretion and CYP11A1/PLZF/STRA8 protein expression

Objective: To explore the effects of Nd(2)O(3) exposure to rare earth particles on the secretion of sex hormones, cytochrome P450 family member 11A1 (CYP11A1) , spermatogenesis markers promyelocytic leukemia zinc finger protein (PLZF) and retinoic acid stimulating gene 8 (STRA8) protein in C57 BL/6J male mice. Methods: In March 2021, Forty-eight male C57 BL/6J mice aged 6-8 weeks divided into control group and Nd(2)O(3) exposure low, medium and high dose groups (exposing doses of 62.5, 125.0, 250.0 mg/ml Nd(2)O(3)) , 12 per group. The mice in the Nd(2)O(3) groups were perfused with different doses of Nd(2)O(3) suspension by a one-time non-exposing tracheal instillation method, and the control group was perfused with an equal volume of normal saline, with a volume of 0.1 ml, to establish a mouse reproductive function injury model. After 28 days of exposure, the mice's body weight, testes and epididymis were weighed, and the organ coefficients were calculated; the two epididymis were taken to make a sperm suspension to determine the sperm count, survival rate, and deformity rate; inductively coupled plasma mass spectrometry (ICP-MS) method was used to detect the content of Nd in mouse testis tissue; HE staining was used to detect testicular tissue pathological changes and quantitative analysis; enzyme-linked immunosorbent assay (ELISA) method was used to detect serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) and testosterone (T) content; western blot was used to detect the protein levels of CYP11A1, PLZF and STRA8 in testicular tissues. Results: Compared with the control group, with the increase of the exposure dose, the Nd content in the testis of the mice showed an increasing trend, the sperm survival rate and LH showed a decreasing trend, and the sperm deformity rate showed an increasing trend (P<0.05) ; Pathological showed that the number of sperm in the seminiferous tubules of the testicular tissue in the Nd(2)O(3) medium and high dose groups was significantly reduced, and the germinal epithelial disintegration, intraepithelial vacuolization, and exfoliation of spermatogenic cells and supporting cells occurred; The height of germinal epithelium was significantly reduced, and the percentage of damaged seminiferous tubules showed an increasing trend (P<0.05) ; FSH and T levels in serum in the middle and high dose groups of Nd(2)O(3), and CYP11A1, PLZF and STRA8 proteins in testicular tissues showed a downward trend with increasing dose (P<0.05) . Conclusion: The rare earth particulate Nd(2)O(3) may interfere with the expression of CYP11A1, PLZF and STRA8 protein, thereby causing the disorder of sex hormone secretion in the body, the maintenance of spermatogonia and the obstruction of the process of meiosis, causing reproductive function damage.

Circadian clock genes Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion in razor clam Sinonovacula constricta

The circadian clock is an endogenous regulation mechanism that coordinates biological processes with daily changes, which are regulated by circadian clock genes. Bmal1 and Period are key circadian clock genes and their roles in reproductive development have been widely studied. The spawning time of Sinonovacula constricta is limited to the night even under external artificial stimulation, and it might be regulated by the internal circadian clock. In this study, the heart rate of S. constricta was higher between 20:00-04:00 at night and lower between 12:00-16:00 during the day, and the sex hormone contents were the highest at 00:00 and the lowest at 18:00 (P &amp;lt; 0.01). Therefore, these obvious changes in the circadian rhythm indicate that S. constricta is a nocturnal animal. The open reading frame (ORF) of Bmal1 comprises 1944 bp encoding 647 aa, while the ORF of Period comprises 3111 bp encoding 1036 aa. Bmal1 and Period were both expressed in four tissues, but they had opposite rhythmic expression patterns. Bmal1 expression was higher at 00:00-06:00 and lower at 12:00-18:00, and Period expression was opposite, thereby suggesting that Bmal1 and Period are involved in positive and negative pathways regulated by the circadian clock, respectively. Strong protein fluorescence signals of Bmal1 and Period proteins were observed in mature oocytes, spermatids, hepatocytes, and epithelial cells of siphons. After siRNA interference, the expression of both Bmal1 and Period significantly decreased (P &amp;lt; 0.01), and the sex hormone contents decreased significantly from 3 to 7 days in the siRNA treatment groups (P &amp;lt; 0.01). Therefore Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion. These findings provide a theoretical basis for understanding the molecular mechanism related to spawning, and may facilitate the artificial propagation of mollusks.

Alteration in glucocorticoids secretion and metabolism in patients affected by cystic fibrosis.

Cystic fibrosis (CF) is an inherited syndrome associated with a mutation in a cystic fibrosis transmembrane conductance regulator gene, composed of exocrine gland dysfunction involving multiple systems that may result in chronic respiratory infections, pancreatic enzyme deficiency, and developmental disorders. Our study describes for the first time the urinary profile of glucocorticoid metabolites and the activity of the enzymes involved in the development and metabolism of cortisol in patients with CF, using a gas chromatography/mass spectrometry method. Data were obtained from 25 affected patients and 70 sex- and age- matched healthy volunteers. We have shown a general decrease in the activity of enzymes involved in the peripheral metabolism of cortisol, such as 11β-hydroxysteroid dehydrogenase type 2, 5α- and 5β-reductases. In contrast, the activity of 11β-hydroxysteroid dehydrogenase type 1, the enzyme that converts cortisone to cortisol, increased. Furthermore, our study found a significant decrease in glucocorticoid excretion in patients with CF. This may suggest adrenal insufficiency or dysregulation of the HPA axis and the development of peripheral mechanisms to counteract cortisol degradation in the case of reduced synthesis of glucocorticoids by the adrenal glands. Furthermore, the activity of 5α-reductase seems to be enhanced only through the backdoor pathway, especially when we taking into consideration 11β-hydroxyandrosterone/11β-hydroxyetiocholanolone ratio which has been shown to be the best differential marker for enzyme activity. CF impairs nutritional effects and energetic balance in patients; thus, our findings suggest the existence of adaptive mechanisms due to limited secretion of adrenal steroids and subsequent diminished amounts of their metabolites in urine. On the other hand, local control of cortisol availability is maintained by enhanced 11βHSD1 activity and its recovery from cortisone in organs and tissues which need this. Steroid hormone dysregulation might be another important factor in the course of CF that should be taken into account when planning an effective and comprehensive therapy.

Steroid profiling using liquid chromatography mass spectrometry during adrenal vein sampling in patients with primary bilateral macronodular adrenocortical hyperplasia.

Adrenal vein sampling (AVS) is not a routine procedure in patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH), but has been used to determine lateralization of cortisol secretion in order to guide decision of unilateral adrenalectomy. Our aim was to characterize the steroid fingerprints in AVS samples of patients with PBMAH and hypercortisolism and to identify a reference hormone for AVS interpretation. Retrospectively, we included 17 patients with PBMAH from the German Cushing's registry who underwent AVS. 15 steroids were quantified in AVS and peripheral blood samples using LC-MS/MS. We calculated lateralization indices and conversion ratios indicative of steroidogenic enzyme activity to elucidate differences between individual adrenal steroidomes and in steroidogenic pathways. Adrenal volume was negatively correlated with peripheral cortisone (r=0.62, p<0.05). 24-hour urinary free cortisol correlated positively with peripheral androgens (rDHEA=0.57, rDHEAS=0.82, rA=0.73, rT=0.54, p<0.05). DHEA was found to be a powerful reference hormone with high selectivity index, which did not correlate with serume cortisol and has a short half-life. All investigated steroids showed lateralization in single patients indicating the heterogenous steroid secretion pattern in patients with PBMAH. The ratios of corticosterone/aldosterone (catalyzed by CYP11B2), androstenedione/dehydroepiandrosterone (catalyzed by HSD3B2) and cortisone/cortisol (catalyzed by HSD11B2) in adrenal vein samples were higher in smaller adrenals (p<0.05). ARMC5 mutation carriers (n=6) showed lower androstenedione/17-hydroxyprogesterone and higher testosterone/androstenedione (p<0.05) ratios in peripheral blood, in line with lower peripheral androstenedione concentrations (p<0.05). Steroid profiling by LC-MS/MS led us to select DHEA as a candidate reference hormone for cortisol secretion. Lateralization and different steroid ratios showed that each steroid and all three steroidogenic pathways may be affected in PBMAH patients. In patients with germline ARMC5 mutations, the androgen pathway was particularly dysregulated.

Effects of zearalenone on vulva area, liver function, serum immunoglobulin, antioxidant capability and sex hormone secretion of prepubertal gilts

The study aimed to examine the multi-organs toxicity of zearalenone on prepubertal gilts. A total of 48 (Landrace × Yorkshire) prepubertal gilts were randomly divided into 4 groups with 12 replicates in each group. The control group were fed with basal diet, the experimental groups were fed test diets supplemented with 0, 200, 800 and 1600 μg/kg zearalenone in the basal diet. The experiment lasted for 28 days. The results showed that the average daily feed intake of prepubertal gilts in each group had no significant change (p > .05). Diets supplemented with zearalenone significantly reduced the serum immunoglobulin G, immunoglobulin M, follicle stimulating hormone concentrations and total antioxidant capacity activity of prepubertal gilts (p < .05). Diets supplemented with zearalenone significantly increased the vulva area, the serum concentration of interleukin-4 and activities of alanine aminotransferase and alkaline phosphatase of prepubertal gilts (p < .05). In conclusion, dietary supplementation of zearalenone has no obvious effect on the average daily feed intake of prepubertal gilts. However, it can increase its vulva area, produce reproductive toxicity, cause liver damage, reduce the serum immunoglobulin concentrations and antioxidant capability and disrupt the secretion of sex hormones. HIGHLIGHTS Dietary supplementation of zearalenone significantly increases the vulva area of prepubertal gilts. Dietary supplementation of zearalenone significantly increases serum ALT and ALP activities of prepubertal gilts. Dietary supplementation of zearalenone reduce serum immunoglobulin levels and antioxidant capability of prepubertal gilts. Dietary supplementation of zearalenone decreased the secretion of FSH of prepubertal gilts. Zearalenone exerts its toxic effects in multiple ways simultaneously.

THE POTENTIAL EFFECTS OF TRIBULUS TERRESTRIS L. ON CELLULAR PARAMETERS AND STEROIDOGENESIS IN VITRO

Tribulus terrestris L. is an herbaceous plant native to India, widely used as a natural sexual stimulant by traditional medicine in China, India, and Greece, and also for the treatment of various diseases for example low blood pressure, cardiovascular diseases, kidney, and skin diseases, etc. Tribulus extracts are rich in numerous biologically active compounds, such as steroidal saponins, alkaloids, lignan amides, and flavonoids, which are useful for the effective treatment of sexual performance, hormonal and sexual problems. The main goal of our in vitro study was to demonstrate the potential consequences of Tribulus terrestris L. extract on TM3 cell parameters such as cell viability, membrane integrity, lysosomal activity, and steroid hormone secretion. The extract was applied to mice Leydig cells at various concentrations (ranging from 37.5 µg/mL to 600 µg/mL) for 24 h. Based on the obtained data, we may claim statistically significant inhibition of cell viability at 300 µg/mL (P &lt; 0.0001) and 600 µg/mL (P &lt; 0.0001). At the same concentrations, there was a statistically significant decline in both cell membrane integrity (P &lt; 0.05; P &lt; 0,0001) and lysosomal activity (P &lt; 0.05; P &lt; 0.001). Hormone secretion was significantly increased at 200 µg/mL (P &lt; 0.05). To better understand how Tribulus terrestris L. affects the reproductive systems of animals or humans, further research must be done.

PSUN16 Sublethal Hyperthermia Decreases Cellular Proliferation and Transiently Disrupts Steroidogenesis in Adrenal Cells

Abstract Introduction Primary Aldosteronism is the most common cause of secondary hypertension. 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. Ablation involves heating tissue &amp;gt;50°C to induce cellular necrosis. Outside the core ablation zone, the transitional zone is an area exposed to variable temperatures between 37°C – 50°C. To understand the feasibility of precision ablation in the adrenal gland, we examined the effect of applying these temperatures to adrenocortical cells to identify i) the required temperature to effectively ablate adrenal cells ii) the extent of damage that may occur to surrounding healthy adrenal cells with exposure to transitional zone temperatures. Methods Steroidogenic adrenocortical cell lines, H295R and HAC15, were treated with hyperthermia (high precision water bath) at temperatures of 37, 42, 45, 48 and 50°C and steroidogenesis was subsequently stimulated using forskolin (10µM) and angiotensin II (10nM). Cell death (Propidium iodide staining by flow cytometry), proliferation (xCELLigence real-time cell analysis), protein expression (Western blot/qRT-PCR), and steroid secretion (HPLC-Mass spectrometry) were analysed immediately and 7-days post-treatment. Results Cell death occurred at 48°C and 50°C (p&amp;lt;0.05 vs 37°C control), but not 45°C, or 42°C. Sublethal hyperthermia (42°C and 45°C for 30 minutes) induced a heat shock response demonstrated by upregulation of HSP70 and HSP90. Proliferation was subsequently reduced over 7-days alongside a decrease in aldosterone and cortisol secretion (p&amp;lt;0.05), and reduced expression of steroidogenic enzymes (CYP11B1, CYP11B2, CYP11A1) 18-hours post treatment (p&amp;lt;0.05). Following 7-days sublethal hyperthermia, steroid secretion and steroidogenic enzymatic expression returned to baseline levels. Conclusion Hyperthermia at 48°C and 50°C for 15 minutes is required for sustained cell death at 7-days post treatment. Sublethal hyperthermia, equivalent to that produced in the transitional zone during thermal ablation, produces a short-lived unsustained inhibition of steroidogenesis that recovers 7-days post treatment. Therefore, segmental adrenal sparing ablation is possible with recovery of transitional zone following ablation. This underlines the potential for precision technology development for bilateral adrenal ablation as definitive measure to treat PA caused by APA or Micronodular disease. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

OR12-1 Activity of Abiraterone Acetate in the management of Cushing syndrome associated to advanced adrenocortical carcinoma: results of the ABACUS trial.

Abstract Background More than 50% of adrenocortical carcinomas (ACC) in adults are associated with cortisol excess that makes tumor management challenging and has a negative impact on patient outcome. Abiraterone acetate (AA) is an irreversible inhibitor of the 17α-hydroxylase/C17, 20-lyase (CYP17 enzyme) that is used in patients with prostate cancer, in whom it leads to suppression of cortisol and androgens. Thus, the drug is potentially useful in the medical treatment of steroid-secreting tumors. The aim of this study was to assess the activity of AA to control cortisol excess in patients with advanced ACC and overt Cushing syndrome. Methods We designed the phase II trial ABACUS (NCT 03145285) whose primary endpoint was normalization of 24-h urinary free cortisol (UFC) excretion within 1 month from treatment start. Inclusion criteria were histologically proven ACC, locally advanced or metastatic disease, and Cushing syndrome confirmed by two 24-h UFC &amp;gt;1.5 times the upper normal limit with suppressed ACTH. No concomitant treatment with mitotane or chemotherapy was allowed for the first 4 weeks of the study. AA was given orally at the daily dose of 1000 mg. Results From 2017 to 2019, we included 17 patients with ACC (2 stage III, 15 stage IV), 13 women (76%), median age 51 years (18-76), of whom 8 have been heavily pretreated and 9 were treatment naïve. In 8 patients, multiple steroid secretion was found. Patients were treated with AA for a median of 17 days (7-163). Median 24-h UFC (measured by gas-mass spectrometry) was 368 μg/24h (121-7422) at baseline and 94 μg/24h (20-1793) at end of treatment (p=0.01). Normalization of 24-h UFC was attained in 8 patients (53%) and a &amp;gt;50% decrement in 11 patients (73%). The median time to effect was 21 days and median 24-h UFC reduction 81.8% (-97.7 - +25.9). Androgen and precursor steroids were also significantly reduced by AA treatment. The median Cushing Syndrome Score was 5.0 (2 - 8) at baseline and 3.5 (1 - 6) at the end of treatment, thus confirming clinical improvement. Blood pressure was significantly reduced and hypokalemia was not observed. In 2 patients, treatment was discontinued for toxicity. Seven patients died of ACC progression during follow-up with an overall survival of 5.4 months (0.5-39.3). Conclusions AA was able to control rapidly cortisol excess in most patients with a good safety profile. The results of this proof-of-concept study show that AA looks promising and may be viewed as an additional weapon to manage Cushing syndrome in patients with ACC. These findings pose the basis for power calculation and implementation of a prospective long-term study to establish AA efficacy in patients with a steroid-secreting ACC. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 a.m.