Regulation of 17α-Hydroxyprogesterone Production during Induced Oocyte Maturation and Ovulation in Amur Sturgeon (Acipenser schrenckii)

In several teleosts, 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) has been identified as a maturation-inducing steroid. DHP is synthesized from 17α-hydroxyprogesterone (17OHP) by 17β-hydroxysteroid dehydrogenase type 12-like (hsd17b12L). Along with 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3β-HSD), 17α-hydroxylase and C17-20 lyase are associated with 17OHP production. This study aimed to determine the roles of Amur sturgeon hsd3b, P450c17-I (cyp17a1), and P450c17-II (cyp17a2) in 17OHP production and to examine their enzyme activity and mRNA expression pattern during oocyte maturation. In the sturgeons used in this study, hsd3b encoded 3β-HSD, cyp17a1 catalyzed 17α-hydroxylase production with C17-20 lyase activity, and cyp17a2 processed 17α-hydroxylase activity alone. In the ovarian follicles of individuals that underwent induced ovulation, hsd3b mRNA levels increased rapidly, cyp17a1 expression was downregulated, and cyp17a2 expression was upregulated during oocyte maturation. Finally, an in vitro study revealed that salmon pituitary extract (SPE) stimulation rapidly induced hsd3b expression, whereas cyp17a1 expression was downregulated. In vitro, cyp17a2 expression did not rapidly increase with SPE stimulation. This rapid upregulation of hsd3b during oocyte maturation was first observed in teleosts. It was suggested that hsd17b12L expression is upregulated after 17OHP production, which is regulated by hsd3b, cyp17a1, and cyp17a2, resulting in DHP production.

Phenylalanine 4-Hydroxylase Contributes to Endophytic Bacterium Pseudomonas fluorescens’ Melatonin Biosynthesis

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series

Context 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. Objective To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. Design Case series. Patients and results We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. Conclusion Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. Significance statement Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

X-Linked Hypophosphatemic Rickets: Multisystemic Disorder in Children Requiring Multidisciplinary Management

X-linked hypophosphatemic rickets (XLH) is the commonest inherited form of rickets. It is caused by an impaired regulation of fibroblast growth factor 23 (FGF23) due to a PHEX gene mutation, which leads to reduced tubular reabsorption of phosphate and renal 1α-hydroxylase activity and increased renal 24-hydroxylase activity. Hypophosphatemia associated with renal phosphate wasting, normal serum levels of calcium, parathyroid hormone, and 25-hydroxyvitamin D represents the main biochemical sign in affected patients. Patients with XLH show rickets and osteomalacia, severe deformities of the lower limbs, bone and muscular pain, stunted growth, and reduced quality of life. However, XLH is a multisystemic disorder requiring multidisciplinary approaches in specialized subdisciplines. Severe complications may occur in patients with XLH including craniosynostosis, hearing loss, progressive bone deformities, dental and periodontal recurrent lesions, and psychosocial distress. Moreover, long-term conventional treatment with active vitamin D metabolites and oral inorganic phosphate salts may cause endocrinological complications such as secondary or tertiary hyperparathyroidism, and adverse events in kidney as hypercalciuria, nephrocalcinosis, and nephrolithiasis. However, conventional treatment does not improve phosphate metabolism and it shows poor and slow effects in improving rickets lesions and linear growth. Recently, some trials of treatment with recombinant human IgG1 monoclonal antibody that targets FGF23 (burosumab) showed significant improvement of serum phosphate concentration and renal tubular reabsorption of phosphate that were associated with a rapid healing of radiologic signs of rickets, reduced muscular and osteoarticular pain, and improved physical function, being more effective for the treatment of patients with XLH in comparison with conventional therapy. Therefore, a global management of patients with XLH is strongly recommended and patients should be seen regularly by a multidisciplinary team of experts.

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.

Vitamin D Metabolism Is Significantly Impaired in COVID-19 Inpatients

Objective: to assess the state of vitamin D metabolism in patients hospitalized with COVID-19 infection. Materials and methods: We examined 49 patients, which were hospitalized for inpatient treatment of COVID-19 infection from May to June 2020. Study group included 24 men (49%) and 25 women (51%), median age 58 years [48; 70], BMI 26.4 kg/m2 [24.3; 30.5]. All patients were diagnosed with pneumonia due to SARS-CoV-2 with median percent of lung involvement equal to 29% [14; 37], 22 patients (45%) required oxygen support upon admission. Median SpO2 was equal to 95% (92; 97), median NEWS score was equal to 3 [2; 6]. Participants were tested for vitamin D metabolites (25(OH)D3, 1,25(OH)2D3, 3-epi-25(OH)D3, 24,25(OH)2D3 and D3) by UPLC-MS/MS, free 25(OH)D and vitamin D-binding protein by ELISA, as well as PTH by electrochemiluminescence immunoassay and routine biochemical parameters of blood serum (calcium, phosphorus, albumin) at the time of admission. Results: patients had in general very low 25()D3 levels - median 10.9 ng/mL [6.9; 15.6], corresponding to a pronounced vitamin D deficiency in half of the patients. Levels of 24,25(OH)2D3 were also low – 0.5 ng/mL [0.2; 0.9], and resulting vitamin D metabolite ratios (25(OH)D3/24,25(OH)2D3) were high-normal or elevated in most patients – 24.1 [19.0; 39.2], indicating decreased activity of 24-hydroxylase. Levels of 1,25(OH)2D3, on the contrary, were high-normal or elevated - 57 pg/mL [46; 79], which, in accordance with 25(OH)D3/1,25(OH)2D3 ratio (219 [134; 266]) suggests an increase in 1α-hydroxylase activity. Median level of 3-epi-25(OH)D3 was 0.7 ng/mL [0.4; 1.0] and D3 metabolite was detectable only in 6 patients. Median DBP level was 432 mg/L [382; 498], median free 25(OH)D was 5.6 pg/mL [3.3; 6.7], median calculated free 25(OH)D was 2.0 pg/mL [1.4; 3.3]. Most patients had albumin-adjusted serum calcium level in the lower half of reference range (median 2.24 mmol/L [2.14; 2.34]). Seven patients had secondary hyperparathyroidism and one patient had primary hyperparathyroidism, the rest of the patients had PTH levels within the normal range.25(OH)D3 levels showed significant negative correlation with percent of lung involvement (r = -0.36, p&lt;0.05) and positive correlation with SpO2 (r = 0.4, p&lt;0.05). 1,25(OH)2D3 levels correlated positively with 25(OH)D3 levels (r = 0.38, p&lt;0.05) and did not correlate significantly with PTH levels (p&gt;0.05). Conclusion: Our data suggests that hospitalized patients with COVID-19 infection have significant impairment of vitamin D metabolism, in particular, an increase in 1α-hydroxylase activity, which cannot be fully explained by pre-existing conditions such as vitamin D deficiency and secondary hyperparathyroidism. The observed profound vitamin D deficiency and association of vitamin D levels with markers of disease severity indicate the importance of vitamin D supplementation in these patients.

Vitamin D Metabolism Alteration in Acromegaly and Its Impact on Calcium-Phosphorus Metabolism

Objective: to evaluate metabolism of vitamin D and calcium-phosphorus metabolism in patients with an active phase of acromegaly in comparison with healthy individuals. Materials and Methods: The study included 44 patients with an active acromegaly (IGF-1 788 [521; 963] ng/mL), as well as 49 conditionally healthy individuals. There were more men in the Acromegaly group (41% vs. 20%, p &lt;0.05), patients were older (median age 42.7 [35.5; 26.5] vs. 26.3 [25; 33.5] years, p &lt;0.05) and had a higher BMI (28.4 [25.2; 30.2] vs. 22.2 [20.1; 26.1] kg/m2, p &lt;0.05) in a minor way compared with the control group. All participants were tested for vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3) by UPLC-MS/MS, free 25(OH)D and vitamin D-binding protein by ELISA, PTH by electrochemiluminescence immunoassay, as well as routine biochemical parameters of blood serum (calcium, phosphorus, creatinine, albumin, magnesium) and urine (calcium and phosphorus-creatinine ratio in spot urine). Results: In the Acromegaly group, we observed significantly higher levels of serum total calcium (2.46 [2.37; 2.56] vs. 2.38 [2.33; 2.45] mmol/L, p &lt;0.05), albumin-corrected calcium (2.33 [2.28; 2.42] vs. 2.26 [2.21; 2.31] mmol/L, p &lt;0.05) and phosphorus (1.39 [1.25; 1.55] vs. 1.15 [1.06; 1.23] mmol/L, p &lt;0.05) as well as lower levels of serum albumin (45 [44; 47] vs. 46 [45; 48] g/L, p &lt;0.05). The rest of the studied biochemical parameters and PTH levels did not differ significantly between the groups. The IGF-1 level in patients with acromegaly positively correlated with the level of total calcium (r = 0.49, p &lt;0.05), albumin-corrected calcium (r = 0.49, p &lt;0.05) and phosphorus (r = 0.55, p &lt;0.05). The Acromegaly group showed lower levels of 25(OH)D3 (14.8 [11.8; 20.5] vs. 20.5 [14.8; 24.6] ng/mL, p &lt;0.05), 3-epi-25(OH)D3 (1.0 [0.7; 1.4] vs. 1.4 [0.9; 1.8] ng/mL, p &lt;0.05), 24,25(OH)2D3 (0.8 [0.4; 1.2] vs. 1.7 [0.9; 2.6] ng/ml, p &lt;0.05) and free 25(OH)D (4.6 [3.7; 5.6] vs. 5.9 [4.0; 7.5] pg/mL, p &lt;0.05), higher levels of 1,25(OH)2D3 (50 [42; 63] vs. 39 [34; 45] pg/mL, p &lt;0.05), a lower 25(OH)D3/1,25(OH)2D3 ratio (289 [226; 443] vs. 517 [340; 641], p &lt;0.05) and a higher 25(OH)D3/24,25(OH)2D3 ratio (19.3 [15.4; 27.7] vs. 11.9 [9.6; 15.2], p &lt;0.05). Conclusion: Our data suggest that high levels of the active vitamin D metabolite (1,25(OH)2D3) resulting from an increase in 1α-hydroxylase activity may contribute to the elevation of calcium and phosphorus serum levels in patients with acromegaly. Our results also indicate a decrease in 24-hydroxylase activity in patients with acromegaly, which may be due to lower levels of 25(OH)D3 in these patients. The results obtained should be evaluated taking into account the observed differences in age, gender and BMI between groups.

Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia

Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.