Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia

Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L.; Storbeck, Karl-Heinz; Cunliffe, Vincent T.; Müller, Ferenc; Krone, Nils

doi:10.1210/en.2017-00549

Cited by 28 publications

(22 citation statements)

References 35 publications

(46 reference statements)

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“…Cytochrome P450 family 17 polypeptide 2 (cyp17a2), created as previously described (Eachus et al, 2017), both prolyl hydroxylase 3 (phd3; BC066699), and lactate dehydrogenase A (ldha1; BC067188) probes, generated as previously described (van Rooijen et al, 2009;Santhakumar et al, 2012).…”

Section: Whole-mount In Situ Hybridisationmentioning

confidence: 99%

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Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

Marchi

Santhakumar

Markham

et al. 2019

Preprint

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AbstractIn the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.Author summaryHypoxia is a common pathophysiological condition to which cells must rapidly respond in order to prevent metabolic shutdown and subsequent death. This is achieved via the activity of Hypoxia-Inducible Factors (HIFs), which are key oxygen sensors that mediate the ability of the cell to cope with decreased oxygen levels.Although it aims to restore tissue oxygenation and perfusion, it can sometimes be maladaptive and contributes to a variety of pathological conditions including inflammation, tissue ischemia, stroke and growth of solid tumours. In this regard, synthetic glucocorticoids which are analogous to naturally occurring steroid hormones, have been used for decades as anti-inflammatory drugs for treating pathological conditions which are linked to hypoxia (i.e. asthma, rheumatoid arthritis, ischemic injury). Indeed, previous in vitro studies highlighted the presence of a crosstalk between HIF and glucocorticoids. However, how this interplay precisely occurs in an organism and what the molecular mechanism is behind it are questions that still remain unanswered. Here, we provide a thorough in vivo genetic analysis, which allowed us to propose a logical model of interaction between glucocorticoid and HIF signalling. In addition, our results are important because they suggest a new route to downregulate HIF for clinical purposes.

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Section: Whole-mount In Situ Hybridisationmentioning

confidence: 99%

“…Previous work also suggested that HIF promotes POMC activity in the mouse hypothalamic region(Zhang et al, 2011). On the other hand, Cyp17a2 is an enzyme involved in steroid hormone biosynthesis at the level of interrenal gland, which is activated upon ACTH stimulation(Ramamoorthy and Cidlowski, 2016;Eachus et al, 2017;Weger et al, 2018).…”

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confidence: 97%

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

Marchi

Santhakumar

Markham

et al. 2019

Preprint

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“…The short-term exposure to cortisol also induced changes in the expression of glucocorticoid responsive gene (fkbp5) and glucocorticoid receptors (grα and grβ) in the zebrafish testicular explants. FK506 binding protein 5 (Fkbp5) is a glucocorticoid receptor chaperone protein, also known to be a robust glucocorticoid responsive gene in zebrafish [41,53,58]. This observation was confirmed in Fdx1b-deficient zebrafish, where males showed a decreased expression of fkbp5 in response to the lower plasma cortisol levels [53].…”

Section: Discussionmentioning

confidence: 86%

Cortisol Directly Stimulates Spermatogonial Differentiation, Meiosis, and Spermiogenesis in Zebrafish (Danio rerio) Testicular Explants

et al. 2020

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Cortisol is the major endocrine factor mediating the inhibitory effects of stress on vertebrate reproduction. It is well known that cortisol affects reproduction by interacting with the hypothalamic–pituitary–gonads axis, leading to downstream inhibitory and stimulatory effects on gonads. However, the mechanisms are not fully understood. In this study, we provide novel data demonstrating the stimulatory effects of cortisol on spermatogenesis using an ex vivo organ culture system. The results revealed that cortisol treatment did not modulate basal androgen production, but it influenced transcript levels of a selected number of genes involved in the zebrafish testicular function ar (androgen receptor), star (steroidogenic acute regulatory), cyp17a1 (17α-hydroxylase/17,20 lyase/17,20 desmolase), cyp11a2 (cytochrome P450, family 11, subfamily A, polypeptide 2), hsd11b2 (11-beta hydroxysteroid dehydrogenase), cyp2k22 (cytochrome P450, family 2, subfamily K, polypeptide 22), fkbp5 (FKBP prolyl isomerase 5), grα (glucocorticoid receptor alpha), and grβ (glucocorticoid receptor beta) in a short-term culture. We also showed that cortisol stimulates spermatogonial proliferation and differentiation in an androgen independent manner as well as promoting meiosis and spermiogenesis by increasing the number of spermatozoa in the testes. Moreover, we demonstrated that concomitant treatment with RU 486, a potent glucocorticoid receptor (Gr) antagonist, did not affect the cortisol effects on spermatogonial differentiation but blocked the induced effects on meiosis and spermiogenesis. Supporting the Gr-mediated effects, RU 486 nullified the cortisol-induced expression of sycp3l (synaptonemal complex protein 3), a marker for the meiotic prophase that encodes a component of the synaptonemal complex. This is consistent with in silico analysis that found 10 putative GREs (glucocorticoid response elements) upstream of the zebrafish sycp3l. Finally, we also showed that grα mRNA is expressed in Sertoli and Leydig cells, but also in several types of germ cells, including spermatogonia and spermatocytes. Altogether, this evidence indicates that cortisol exerts paracrine roles in the zebrafish testicular function and spermatogenesis, highlighting its effects on spermatogonial differentiation, meiosis, and spermiogenesis.

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“…Indeed, recent studies have demonstrated that cyp11a2 , fdx1b and hsd3b1 , but not their corresponding paralogues, are involved in cortisol synthesis in zebrafish . In addition, we have recently shown a key role for cyp21a2 in glucocorticoid biosynthesis in zebrafish …”

Section: Discussionmentioning

confidence: 88%

“…Therefore, it is highly likely that cyp17a2 is involved in the biosynthesis of glucocorticoids during larval stages, whereas cyp17a1 might be important for steroidogenesis later during development, when the gonads are developed and sex steroid synthesis in fish is also required. Support for this assumption is provided by observations showing that androgen precursor androstenedione cannot be detected in 120 hpf zebrafish larvae and that both cyp17 genes can be detected in the kidney head of adult zebrafish with much higher expression levels for cyp17a1 (28 fpkm) than for cyp17a2 (0.3 fpkm) (ArrayExpress database; http://www.ebi.ac.uk/arrayexpress, accession number E‐MTAB‐460).…”

Section: Discussionmentioning

confidence: 99%

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co‐factors in zebrafish

Weger

Diotel

Weger

et al. 2018

J Neuroendocrinology

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The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function.

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Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia

Cited by 28 publications

References 35 publications

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

Cortisol Directly Stimulates Spermatogonial Differentiation, Meiosis, and Spermiogenesis in Zebrafish (Danio rerio) Testicular Explants

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co‐factors in zebrafish

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