Growth hormone-induced STAT5B regulates Star gene expression through a cooperation with cJUN in mouse MA-10 Leydig cells

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star mRNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (EMSA and supershift) and in vivo (ChIP) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3–5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

The effect of Alpha-lipoic acid in glyphosate treated granulosa cells on the human folliculogenesis genes

Pesticides have a wide range of infertility in female reproductive. GLP is the most common herbicide used worldwide. The present study tended to evaluate the effects of alpha-lipoic acid (ALA) on expression of folliculogenesis genes in human granulosa cells (GCs) treated with glyphosate. In this study, GC samples were taken from infertile male patients who had tubal factors and received ICSI or IVF treatment for the first time. The medium used to culture cells was DMEM-F12 containing FBS 10%, penicillin 1%, and streptomycin 1%. The cells were treated with glyphosate (GLP) (250 µg/ml), ALA (50 µg/ml), and their mixture (250 GLP + 50 ALA) for 24 hr. The FoxO1, NOX4, Vnn1, and STAR gene expression in GCs was determined by real-time PCR. Exposure to GLP decreased gene expression Vnn1 and STAR in the GCs, while in comparison to control group, FOXO1 and NOX4 expression insignificantly increased. Further, ALA treatment decreased FOXO1, NOX4, in the GCs, and increased VNN1 and STAR gene expression. Transcriptional expression of FoxO1, NOX4 and STAR in GCs also decreased in their combinations (GLY + ALA). At the same time, the level of Vnn1 mRNA in GCs was increased. The findings showed that exposure to GLP alters the morphological structure of GCs and the expression of follicogenic genes, leading to dysfunction of the reproductive system. Moreover, the results showed that cell characteristics were preserved efficiently in human GCs exposed to ALA as an antioxidant.

Variations in the Initial Presentation of a Rare Congenital Adrenal Hyperplasia: Steroidogenic Acute Regulatory Deficiency

Background: Steroidogenic Acute Regulatory (StAR) deficiency is a rare form of congenital adrenal hyperplasia characterized by dysregulated cholesterol transport mediated by StAR enzyme across mitochondrial membranes. Adrenal dysfunction is due to the two-hit hypothesis: 1) defective StAR protein and 2) cholesterol accumulation in the adrenals and gonads. With variable cellular damage, adrenal crisis can occur early or late. Clinical cases: We present two cases of StAR deficiency with contrasting presentations. Case 1: A 9-day old ex full term female from a nonconsanguineous union presented to a rural hospital with hypothermia, lethargy, and poor feeding. She had hypoglycemia 41 mg/dL (60–105), hyponatremia 120 mEq/L (135–145), hyperkalemia 7.7 mEq/L (3.5–5.5) and cortisol < 0.4 ug/dL (4.5–23). Baby was started on hydrocortisone (HCT) 100 mg/m2 and one-time fludrocortisone (FCT). She decompensated requiring chest compressions, intubation and pressors. She was transferred to our institution. Newborn screen was normal; she had typical female external genitalia. US demonstrated a uterus; ovaries and adrenals were not identified. Upon extubation and clinical improvement, her HCT was weaned to physiologic doses. She became hyponatremic requiring FCT and salt supplements. Post-HCT wean, ACTH level was 304 pg/mL (7–63) with aldosterone < 4.0 ng/dL (6.5–86). Karyotype was 46,XX. Genetic analysis revealed a novel heterozygous likely pathogenic variant in the STAR gene, (STAR c.65-12_68del variant) without defect in the other STAR gene. Case 2: A 9-month-old ex full-term female of Iraqi descent from a nonconsanguineous union presented with fatigue, poor oral intake and weight loss from 50%-ile to 3%-ile. She had hyponatremia 122 mEq/L, hyperkalemia 8.0 mEq/L, but was normoglycemic. She was normotensive; EKG was normal. Parents noted progressive hyperpigmentation including her gums, palmar and plantar creases. She had typical external female genitalia with a hypoplastic clitoris (2 mm x 2 mm). ACTH stimulation test showed low cortisol (0.5 ug/dL) at 60 minutes. She was treated with HCT 100 mg/m2 for 5 days, then tapered to maintenance dosing, with FCT and salt supplements. Her ACTH level returned > 5000 pg/ml. Aldosterone, 17-OH-Progesterone, 17-OH-Pregnenolone, 11-Deoxycortisol and androstenedione were undetectable. Pelvic US did not identify uterus or ovaries. Pelvic MRI identified bilateral inguinal testes with enlarged adrenal glands. Karyotype was 46, XY. We suspected StAR deficiency with sex-reversal. Genetic analysis revealed a known homozygous mutation in STAR (c.545G>A). Conclusion: StAR deficiency is clinically indistinguishable from P450scc deficiency and genetic testing is needed. Both entities can present with early or delayed adrenal crisis. While classic for StAR deficiency, adrenal enlargement is inconsistent. Karyotype is vital to identify sex reversal.

Cardiac Arrest in a Child with Non-classic Lipoid Congenital Adrenal Hyperplasia Associated with a New STAR Gene Mutation

Introduction: Steroidogenic acute regulatory (STAR) protein regulates steroid hormone synthesis by transporting cholesterol into mitochondria. STAR gene mutations lead to lipoid congenital adrenal hyperplasia (LCAH), the rare but most severe form of congenital adrenal hyperplasia in children. We present an unusual case with an episode of cardiac arrest in a young girl during an acute febrile illness and later she was diagnosed with adrenal insufficiency secondary to a non-classic LCAH. Case: 2-year 11-month-old previously healthy white female was brought to an urgent care clinic due to severe lethargy and a seizure-like activity during a fever illness. She was found to have an undetectable blood glucose level and went into cardiac arrest shortly after arrival. CPR was performed for approximately 11 minutes. She then developed sever respiratory distress and was intubated. She was transferred to the PICU with IV sodium bicarbonate given en route. On admission, her body weight was 13.26 kg (36.80th percentile), height 90 cm (17.56th percentile), and BMI 16.17 (62.88th percentile). Her physical exam revealed normal external female genitalia and normal skin pigmentation. Lab evaluation revealed normal sodium and potassium but elevated anion gap, hyperuricemia, elevated creatinine kinase, abnormal liver function tests and abnormal coagulation profile. Brain MRI revealed findings consistent with hypoxic-ischemic encephalopathy. Renal function improved within 24 hours and hepatic function returned to normal after 20 days. Due to her severe hypoglycemic event, a high-dose ACTH stimulation test was performed. The results were consistent with adrenal insufficiency: baseline cortisol level, 7.3 μg/dL; 30 minutes cortisol, 7.8 μg/dL; 60 minutes cortisol, 9 μg/dL (normal response, ≥18 mcg/mL at 30 or 60 minutes). The baseline ACTH level was significantly elevated, 1688 pg/mL (0–46) as well as the renin activity, 24.3 ng/hour (1.7–11.2). Genetic testing revealed a 46 XX karyotype. STAR gene analysis identified compound heterozygosity; a novel deletion (c.811delC, p.Leu271Cysfs*50) and a previously reported missense mutation (c.661G>A, p.Gly221Ser). The girl is now 11 years old and exhibits normal growth, normal cognitive development, and she has developed early signs of puberty (Tanner stage 2 for breast). She takes daily hydrocortisone, fludrocortisone and stress dose hydrocortisone as needed. Conclusion: In non-classic LACH, the onset is generally late or not acute. Initial clinical features are variable and nonspecific. For this reason, non-classic LCAH may be overlooked. Adrenal crisis is a life-threatening complication, and it is important that clinicians are aware of the clinical features of non-classic LCAH and consider it in the differential diagnoses. Genetic testing for STAR should be considered in individuals with non-autoimmune primary adrenocortical insufficiency.

Novel STAR gene variant in a patient with classic lipoid congenital adrenal hyperplasia and combined pituitary hormone deficiency

We report the first case of classic lipoid congenital adrenal hyperplasia and combined pituitary hormone deficiency. We identified pathogenic variants in the STAR gene: a novel variant of c.126_127delCCinsG, namely, p.Thr44Profs*2 and an already reported variant of c.634C>T, namely, p.Gln212*. The association with combined pituitary hormone deficiency might be just a coincidence.

Non-classical lipoid adrenal hyperplasia presenting as hypoglycemic seizures

IntroductionPrimary adrenal insufficiency is a potentially life-threatening condition that can have many underlying causes. Mutations in the steroidogenic acute regulatory protein (StAR) gene produce lipoid congenital adrenal hyperplasia (LCAH) which usually presents in the infantile period with severe symptoms of adrenal insufficiency. Less commonly, a non-classical form is identified which may present at a later age in affected individuals. Till date, around 30 individuals with the non-classical form have been described.Case presentationWe describe a 4-year-old 46, XX Indian girl who presented with hypoglycemic seizures and was subsequently diagnosed as non-classical LCAH on genetic analysis, with homozygous R188C mutation in the StAR gene.ConclusionsStAR mutations may have a variety of clinical presentations and are likely under-diagnosed. Genetic diagnosis is important for treatment as well as monitoring of reproductive function.

Abstract 14148: Local Aldosterone Production via StAR in PAH and in Human IPS Cell-derived Cardiomyocytes: Efficacy of Spironolactone in Cardiac Remodeling and Mitochondrial Dynamics Under Hypoxic Stress

Objective: Pulmonary arterial hypertension (PAH) is a progressive disease that frequently leads to right ventricular (RV) remodeling. Increased plasma aldosterone levels have been reported in patients with PAH. The aim of this study was to examine the expression of steroidogenic acute regulatory protein (StAR) in patients and animal model of PAH, and in the human iPS cell-derived cardiomyocytes (hiPSC-CMs). Furthermore, we evaluated therapeutic effects of mineralocorticoid receptor (MR) antagonist, spironolactone (SPL) on mitochondrial dynamics in the RV myocardium. Methods: Autopsied heart samples (n=20) were obtained from patients with or without PAH. PAH model rats (n=46) were injected with Sugen5416 (20 mg/kg) and then exposed to hypoxia and reoxygenation for 4 weeks with/without treatment with SPL (25 mg/kg/day). Human umbilical vein endothelial cells (HUVECs) and hiPSC-CMs were cultured under hypoxic conditions (37 °C, 1% O 2 , 5% CO 2 ) with/without treatment with SPL (10 μM). In addition, we applied the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) technology and repressed StAR gene expression in hiPSC-CMs under hypoxic conditions. Results: In PAH patients, the expression of StAR and MR was significantly increased in cardiomyocytes and endothelial cells in the RV myocardium. In PAH rats, RV systolic pressure elevated over 60 mm Hg with deteriorated RV function. Under electron microscopy, severe degeneration of mitochondria and capillaries with myofibrillar lysis in the RV myocardium were observed, which were compatible with those findings in PAH patients. Hypoxic stress significantly increased the StAR and phosphorylated JNK, ERK-1/2, and NF-kB proteins in hiPSC-CMs as well as in HUVECs. Furthermore, we established the CRISPRi-hiPSC cell lines where StAR gene was successfully knockdown. Treatment with SPL decreased the phosphorylation of JNK, ERK-1/2, NF-kB, and Smad3 in HUVECs and hiPSC-CMs. Strikingly, SPL attenuated mitochondrial degeneration and increased mitophagy, suppressing the RV remodeling under hypoxic stress. Conclusion: SPL might regulate mitochondrial dynamics and attenuates RV remodeling in PAH via StAR/JNK signaling pathway.