Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3ß-hydroxysteroid dehydrogenase (3ß-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3ß-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3ß-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11ß1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Pharmacogenetic study of ACE, AGT, CYP11B1, CYP11B2 and eNOS gene variants in hypertensive patients from Faisalabad, Pakistan.

OBJECTIVE: To investigate the association of genetic variants of renin angiotensin aldosterone system, endothelial nitric oxide synthase and 11-beta-hydroxylase genes, and the drug efficacy of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker. METHODS: This two time-point study was conducted from April to November 2016 at Allied Hospital, Faisalabad and National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, and comprised of hypertensive patients taking angiotensin-converting enzyme inhibitor and angiotensin receptor blocker who were followed up for 12 weeks. Baseline and follow-up clinical and biochemical parameters were measured for all patients. Total 11 polymorphisms were genotyped by polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism and amplification-refractory mutation system-polymerase chain reaction assays. Data was divided into baseline and follow-up groups, while the latter group was further divided into responding and non-responding subgroups on the basis of patient response to angiotensin-converting enzyme inhibitor and angiotensin receptor blocker drugs. Data was analysed using SPSS 20. RESULTS: Of the 45 patients, 25(55.5%) were females and 20(44.5%) were males. There was a significant reduction in the systolic blood pressure (p=0.004) and low-density lipoprotein cholesterol (p<0.001) from the baseline to the follow-up. Systolic blood pressure was significantly reduced in the responding group (p=0.003), while diastolic blood pressure (p=0.121) was not significantly different. There was no effect of angiotensin-converting enzyme, angiotensinogen, 11-beta-hydroxylase, aldosterone synthase and endothelial nitric oxide synthase gene polymorphisms on angiotensin converting enzyme inhibitor and angiotensin receptor blocker efficacy. CONCLUSIONS: Inter-individual response to angiotensin converting enzyme inhibitor and angiotensin receptor blocker was found to be independent of genetic polymorphisms in renin angiotensin aldosterone system, endothelial nitric oxide synthase and 11-beta-hydroxylase genes.

Induction of oocyte development in previtellogenic eel, Anguilla australis.

The role of gonadotropins during early ovarian development in fish remains little understood. Concentrations of gonadotropins were therefore experimentally elevated in vivo by administration of recombinant follicle-stimulating hormone (rec-Fsh) or human chorionic gonadotropin (hCG) and the effects on ovarian morphology, sex steroid levels and mRNA levels of genes expressed in pituitary and ovary examined. Hormones were injected thrice at weekly intervals in different doses (20, 100 or 500 µg/kg BW for rec-Fsh and 20, 100 or 500 IU/kg BW for hCG). All treatments, especially at the highest doses of either rec-Fsh or hCG, induced ovarian development, reflected in increased oocyte size and lipid uptake. Both gonadotropins up-regulated follicle-stimulating hormone receptor (fshr) mRNA levels and plasma levels of estradiol-17ß (E2). Exogenous gonadotropins largely decreased the expression of follicle-stimulating hormone ß-subunit (fshb) and had little effect on those of luteinizing hormone ß-subunit (lhb) in the pituitary. It is proposed that the effects of hCG on ovarian development in previtellogenic eels could be indirect as a significant increase in plasma levels of 11-ketotestosterone (11-KT) was found in eels treated with hCG. Using rec-Fsh and hCG has potential for inducing puberty in eels in captivity, and indeed, in teleost fish at large.

Detrimental effects of uterine disease and lipopolysaccharide on luteal angiogenesis.

Reproductive tract inflammatory disease (RTID) commonly occurs after the traumatic events of parturition and adversely affects follicular function. This study is the first to describe the cellular and steroidogenic characteristics of corpora lutea from cattle with RTID and the effects of pathogen-associated molecular patterns (PAMPs) on luteal angiogenesis and function in vitro. Luteal weight (P < 0.05) and progesterone content (P < 0.05) were reduced (1.2-fold) in cows with RTID, accompanied by reduced CYP11A (P < 0.05), HSD3B (P < 0.01) and STAR (P < 0.01) protein expression. Immunohistochemistry revealed that luteal vascularity (VWF) and pericyte (ACTA2) coverage were >3-fold lower in RTID cows (P < 0.05). To link these observations to bacterial infection and determine specificity of action, a physiologically relevant luteal angiogenesis culture system examined the effects of PAMPs on endothelial cell (EC) network formation and progesterone production, in the presence of pro-angiogenic factors. Luteal EC networks were reduced ≤95% (P < 0.05) by lipopolysaccharide (LPS, toll-like receptor (TLR) 4 agonist) but not by TLR2 agonists lipoteichoic acid or peptidoglycan. Conversely, progesterone production and steroidogenic protein expression were unaffected by PAMPs (P > 0.05). Moreover, the adverse effect of LPS on luteal EC networks was dose-dependent and effective from 1 ng/mL (P < 0.05), while few EC networks were present above 10 ng/mL LPS (P < 0.001). LPS reduced proliferation (P < 0.05) and increased apoptosis of EC (P < 0.001). The specific TLR4 inhibitor TAK242 reversed the effects of LPS on EC networks. In conclusion, luteal vasculature is adversely sensitive to LPS acting via TLR4, therefore ovarian exposure to LPS from any Gram-negative bacterial infection will profoundly influence subsequent reproductive potential.

Roles of two cyp11 genes in sex hormone biosynthesis in Japanese flounder (Paralichthys olivaceus).

The P450 side-chain cleavage enzymes P450scc (Cyp11a) and 11ß-hydroxylase (Cyp11b) play important roles in sex steroid and cortisol production. Here, two duplicates of cyp11 genes were identified in Japanese flounder (Paralichthys olivaceus): Pocyp11a and Pocyp11b, respectively. Phylogenetic analysis and amino acid sequence alignment revealed that Pocyp11a and Pocyp11b shared significant identity with sequences of other teleost fish species. The quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that among the studied tissues, brain tissue showed the highest expression of Pocyp11a, followed by kidney and testis tissues, whereas Pocyp11b expression was highest in the testis. The expression patterns of these two genes showed sexual dimorphism, with both genes showing higher expression in the testis than in the ovary. In-situ hybridization analysis demonstrated that Pocyp11a and Pocyp11b mRNA were both detected in oocytes, spermatocytes, and Sertoli cells, indicating that they might be involved in hormone synthesis. The expression levels of Pocyp11a and Pocyp11b were significantly downregulated by treatment with 17α-methyltestosterone (17α-MT) in the testis and ovary in both in vivo and studies. In vivo studies showed that Pocyp11a and Pocyp11b transcripts were suppressed by 17ß-estradiol (E2 ) treatment in both the testis and ovary. In addition, in vitro studies showed that the expression level of Pocyp11b was decreased by treatment with E2 , whereas that of Pocyp11a was largely unaffected. Moreover, the expression levels of Pocyp11a and Pocyp11b in the testis cell line were significantly upregulated after NR0b1 and NR5a2 (p < .05) treatment. These results indicate that Pocyp11a and Pocyp11b might play important roles in sex hormone biosynthesis. Our research can assist future studies of the mechanisms of steroid biosynthesis and functional differences between cyp11a and cyp11b in Japanese flounder.

MT-Feeding-Induced Impermanent Sex Reversal in the Orange-Spotted Grouper during Sex Differentiation.

In this study, we systematically investigated the process of sex reversal induced by 17-methyltestosterone (MT) feeding and MT-feeding withdrawal at the ovary differentiation stage in orange-spotted groupers, Epinephelus coioides. Gonadal histology showed that MT feeding induced a precocious sex reversal from immature ovaries to testes, bypassing the formation of an ovarian cavity, and MT-feeding withdrawal led to an ovarian fate. In both the MT feeding and MT-feeding withdrawal phases, cytochrome P450 family 11 subfamily B (cyp11b) gene expression and serum 11-KT levels were not significantly changed, suggesting that the MT-treated fish did not generate endogenous steroids, even though active spermatogenesis occurred. Finally, by tracing doublesex-expressing and Mab-3-related transcription factor 1 (dmrt1)-expressing cells and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) assays, we found that the efferent duct formed first, and then, the germ cells and somatic cells of the testicular tissue were generated around the efferent duct during MT-feeding-induced precocious sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex reversal induced by exogenous hormones during sex differentiation in the protogynous orange-spotted grouper.