VEGFA165 can rescue excess steroid secretion, inflammatory markers and follicle arrest in the ovarian cortex of high A4 cows

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and Vascular Endothelial Growth Factor A (VEGFA) would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later stage follicles compared to Controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, Anti-Mullerian Hormone (AMH) and A4 secretion compared to Controls. In trial 2, we tested if VEGFA isoforms could rescue the High A4 phenotype. High A4 (n = 5) and Control (n = 5) ovarian cortex was cultured with: 1) PBS; 2) VEGFA165 (50 ng/ml); 3) VEGFA165B (50 ng/ml); or 4) VEGFA165 + VEGFA165B (50 ng/ml each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than Controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids, and had greater indicators of inflammation compared to Controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFβ secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and promote folliculogenesis.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11β-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11β enzyme is expressed in preterm adrenal glands, so we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P=0.0004), cortisol production (P<0.0001) and 11β-hydroxylase activity index (P<0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs. 0.60 nmol/L, P<0.0001). ADM-overexpression mice revealed lower 11β-hydroxylase activity index (P<0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs. 0.46 ng/mL, P<0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge towards an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Expression and role of resistin on steroid secretion in the porcine corpus luteum

Resistin plays an important role in adipogenesis, obesity, insulin resistance and reproduction. Previous studies showed resistin action on ovarian follicular cells; however, whether resistin regulates steroid secretion in luteal cells is still unknown. Our aim was first to determine the expression of resistin and its potential receptors (tyrosine kinase-like orphan receptor 1 [ROR1] and Toll-like receptor 4 [TLR4]) in the porcine corpus luteum (CL), regulation of its expression, effect on kinases phosphorylation and luteal steroidogenesis. Our results showed that the expression of resistin and its receptors was dependent on the luteal phase and this was at the mRNA level higher in the late compared with the early and middle luteal phase. At the opposite, resistin protein expression was higher in the middle and late compared with the early luteal phase, while ROR1 and TLR4 expression was highest in the early luteal phase. Additionally, we observed cytoplasmic localisation of resistin, ROR1 and TLR4 in small and large luteal cells. We found that luteinising hormone, progesterone (P4), insulin and insulin-like growth factor 1 regulated the protein level of resistin, ROR1 and TLR4. Resistin decreased P4 and increased oestradiol (E2) secretion via changing in steroidogenic enzymes expression and via the activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAP3/1), increased the expression of receptors LHCGR and ESR2 and decreased the expression of PGR. Moreover, resistin decreased PKA phosphorylation and enhanced MAP3/1 phosphorylation. Taken together, resistin could act directly on steroid synthesis and serve as an important factor in in vivo luteal cell function.

Incomplete Pattern of Steroidogenic Protein Expression in Functioning Adrenocortical Carcinomas

Autonomous steroid secretion is a common feature of adrenocortical carcinomas (ACC), although not always clinically evident owing to inefficient steroidogenesis with increased release of steroid precursors. Our study aim was to analyze the expression profile of four key proteins involved in the steroidogenesis cascade, in different adrenocortical tumors. Expression of proteins involved in steroidogenesis, namely steroidogenic acute regulatory protein (StAR), 11β-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and 17α-hydroxylase (CYP17A1), were analyzed by immunohistochemistry in ACC (n = 14), adenomas presenting with Cushing’s syndrome (ACAc) (n = 11) and clinically non-functioning adenomas (ACAn) (n = 15). A percentage of the stained area for each protein was analyzed using ImageJ software for computerized morphometric quantification. CYP11B1, StAR and CYP17A1 expression were significantly lower in ACC when compared to ACAc. In addition, ACC presented co-staining cells for CYP11B1 and CYP11B2. CYP11B1 was the steroidogenic enzyme with the most discriminative power to distinguish ACC from ACAc, with a sensitivity of 100%, specificity of 92%, and an expression higher than 4.44%, indicating the presence of a cortisol secreting adenoma. ACC depicts an incomplete pattern of steroidogenic protein expression, with decreased CYP11B1 and CYP17A1, which could explain the predominant secretion of steroid precursors.

A Novel Antigonadotropic Role of Thyroid Stimulating Hormone on Leydig Cell-Derived Mouse Leydig Tumor Cells-1 Line

Subclinical hypothyroid men characterized by a rise in only thyroid stimulating hormone (TSH) levels, and normal thyroid hormone levels showed a fall in their serum progesterone and testosterone levels. This suggested a role of TSH in regulating Leydig cell steroidogenesis. Therefore, we investigated the direct role of TSH on steroid production and secretion using a mouse Leydig tumor cell line-1 (MLTC-1). MLTC-1 cells were treated with different doses of TSH isolated from porcine pituitary as well as recombinant TSH. Steroid secretion was measured by radioimmunoassay (RIA). The mRNA levels of steroidogenic enzymes were quantitated by real-time polymerase chain reaction (RT-PCR), whereas the corresponding protein levels were determined by western blot. In MLTC-1 cells, pituitary TSH as well as recombinant TSH inhibited progesterone and testosterone secretion in a dose-dependent manner. The inhibitory action of TSH on steroid secretion was unique and not mimicked by other anterior pituitary hormones including follicle stimulating hormone and adrenocorticotropic hormone. Recombinant TSH showed no effect on steroidogenic acute regulatory protein and CYP11A1, the enzymes catalyzing the nonsteroidogenic and steroidogenic rate-limiting steps of steroid synthesis, respectively. Recombinant TSH was shown to inhibit steroidogenesis in MLTC-1 cells by inhibiting the 3-β hydroxy steroid dehydrogenase mRNA and protein levels, the enzyme that catalyzes the conversion of pregnenolone to progesterone. This inhibitory effect of TSH is probably direct as both mRNA and protein of the TSH receptor were shown to be present in the MLTC-1 cells.

Testicular Vein Sampling Can Reveal Gonadotropin-Independent Unilateral Steroidogenesis Supporting Spermatogenesis

Suppressed gonadotropins combined with high-normal serum testosterone concentrations in oligozoospermic men suggest either use of exogenous testosterone or presence of a testosterone-producing tumor. We describe the case of a 31-year-old man referred for primary infertility. Gonadotropins were undetectably low, but testosterone and estradiol were in the high-normal range. Semen analysis showed oligoasthenospermia. He denied using exogenous testosterone. Scrotal ultrasound showed microlithiasis and millimetric hypolucent lesions in the left testis but no intratesticular mass. Human chorionic gonadotropin was low. To investigate unilateral hormone secretion, selective testicular venous sampling was performed. Testosterone and estradiol were clearly higher on the left side than on the right (130 vs 26 nmol/L and 1388 vs 62 pmol/L, respectively), with a left spermatic vein–to-periphery gradient of 4.3 for testosterone and 13 for estradiol; there were no similar gradients on the right side. This finding confirms that all sex steroid secretion came from the left testis. The patient was therefore referred for left orchidectomy. Histopathology revealed multifocal seminoma, germ cell neoplasia in situ, and Leydig cell hyperplasia but no choriocarcinoma. However, gonadotrophin levels increased after orchidectomy, indicating that the source of gonadotropin-independent sex steroid secretion was removed. Testosterone and estradiol decreased to the mid-normal range. Sperm concentration improved. This report thus shows that endogenous testosterone secretion in one testicle supports spermatogenesis without measurable levels of gonadotropins. Selective testicular venous sampling is useful to identify the site of unilateral secretion when the clinical picture is inconclusive. However, histopathology could not reveal the factor that stimulated Leydig cell steroidogenesis.