scholarly journals Redefining the Initiation and Maintenance of Zebrafish Interrenal Steroidogenesis by Characterizing the Key Enzyme Cyp11a2

Endocrinology ◽  
2013 ◽  
Vol 154 (8) ◽  
pp. 2702-2711 ◽  
Author(s):  
Silvia Parajes ◽  
Aliesha Griffin ◽  
Angela E. Taylor ◽  
Ian T. Rose ◽  
Irene Miguel-Escalada ◽  
...  
Keyword(s):  
Early Development ◽  
Model Organism ◽  
Chain Cleavage ◽  
Morpholino Knockdown ◽  
Cleavage Enzyme ◽  
Key Enzyme ◽  
Interrenal Gland ◽  
Functional Equivalent

Abstract Zebrafish are emerging as a model to study steroid hormone action and associated disease. However, steroidogenesis in zebrafish is not well characterized. Mammalian P450 side-chain cleavage enzyme (CYP11A1) catalyzes the first step of steroidogenesis, the conversion of cholesterol to pregnenolone. Previous studies describe an essential role for zebrafish Cyp11a1 during early development. Cyp11a1 has been suggested to be the functional equivalent of mammalian CYP11A1 in the zebrafish interrenal gland (equivalent to the mammalian adrenal), gonad, and brain. However, reported cyp11a1 expression is inconsistent in zebrafish larvae, after active cortisol synthesis commences. Recently a duplicated cyp11a gene, cyp11a2, has been described, which shares an 85% identity with cyp11a1. We aimed to elucidate the specific role of the two cyp11a paralogs. cyp11a1 was expressed from 0 to 48 hours post-fertilization (hpf), whereas cyp11a2 expression started after the development of the interrenal primordium (32 hpf) and was the only paralog in larvae. cyp11a2 is expressed in adult steroidogenic tissues, such as the interrenal, gonads, and brain. In contrast, cyp11a1 was mainly restricted to the gonads. Antisense morpholino knockdown studies confirmed abnormal gastrulation in cyp11a1 morphants. cyp11a2 morphants showed impaired steroidogenesis and a phenotype indicative of metabolic abnormalities. The phenotype was rescued by pregnenolone replacement in cyp11a2 morphants. Thus, we conclude that cyp11a1 is required for early development, whereas cyp11a2 is essential for the initiation and maintenance of zebrafish interrenal steroidogenesis. Importantly, this study highlights the need for a comprehensive characterization of steroidogenesis in zebrafish prior to its implementation as a model organism in translational research of adrenal disease.

scholarly journals The P450 side-chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish

2020 ◽  
Vol 244 (2) ◽  
pp. 309-321 ◽  
Author(s):  
Nan Li ◽  
James A Oakes ◽  
Karl-Heinz Storbeck ◽  
Vincent T Cunliffe ◽  
Nils P Krone
Keyword(s):  
Cell Types ◽  
Side Chain ◽  
Sex Characteristics ◽  
Side Chain Cleavage ◽  
Genomic Engineering ◽  
Zebrafish Larvae ◽  
Chain Cleavage ◽  
Morpholino Knockdown ◽  
Cleavage Enzyme

Cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino-knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous cyp11a2 mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2 −/− mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

scholarly journals The role of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins essential for adrenal steroidogenesis in the late-gestation ovine fetus

2001 ◽  
Vol 168 (3) ◽  
pp. 475-485 ◽  
Author(s):  
PJ Simmonds ◽  
ID Phillips ◽  
KR Poore ◽  
ID Coghill ◽  
IR Young ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Late Gestation ◽  
Mrna Levels ◽  
Steroidogenic Factor 1 ◽  
Chain Cleavage ◽  
Adrenal Steroidogenesis ◽  
Acth Receptor ◽  
Cleavage Enzyme ◽  
Ovine Fetus

To further understand the relative roles of the pituitary gland and ACTH in the regulation of mRNAs encoding proteins that are essential for adrenal development, we investigated the effects of, first, an ACTH infusion and labour in intact fetuses and, secondly, the effect of an ACTH infusion to fetuses with and without a pituitary gland, on the relative abundance of the mRNA encoding for the ACTH receptor (MC2R), steroidogenic factor 1 (SF-1), cholesterol side-chain cleavage enzyme (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase (P450(C17)) in the fetal adrenal gland. ACTH(1-24) infusion (14.7 pmol/kg per h) to intact fetuses was without effect on the abundance of mRNA encoding MC2R and SF-1, irrespective of whether the infusion was given for 18 (115-132 days of gestation) or 32 days (115 days to term (147 days of gestation)). Hypophysectomy (HX) did not alter the expression of MC2R mRNA; however, the abundance of SF-1 mRNA fell by approximately 50% following the removal of the pituitary gland. ACTH(1-24) infusion to HX fetuses failed to restore levels of SF-1 mRNA to that seen in intact animals. P450(scc) and 3betaHSD mRNAs were increased by ACTH(1-24) infusion for 18 days in intact animals, although no effects of the infusion were seen on P450(C17) mRNA levels. For all three of these mRNAs, there was a significant increase in their abundance between 132 days of gestation and term in intact fetuses. By term, ACTH(1-24) infusion was without any additional effect on their abundance. HX decreased the expression of P450(scc), 3betaHSD and P450(C17) mRNAs, while ACTH(1-24) infusion to HX fetuses increased the expression of these mRNAs to levels seen in intact animals. There were significant correlations between the abundance of the mRNA for P450(scc), 3betaHSD and P450(C17), but not MC2R and SF-1, and premortem plasma cortisol concentrations. These results emphasise the importance of the pituitary gland and ACTH in the regulation of the enzymes involved in adrenal steroidogenesis. Factors in addition to ACTH may also play some role, as the infusion was not always effective in increasing the abundance of the mRNAs. Surprisingly, the mRNA for MC2R and SF-1 did not appear to be regulated by ACTH in the late-gestation ovine fetus, though a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance.

scholarly journals Cloning and Functional Characterization of CsUGD2 in Cucumber (Cucumis sativus L.)

2018 ◽  
Vol 47 (2) ◽  
pp. 288-293
Author(s):  
Yitong DUAN ◽  
Shuocheng ZENG
Keyword(s):  
Glucose Dehydrogenase ◽  
Functional Characterization ◽  
Protein Alignment ◽  
Cucumis Sativus L ◽  
Irreversible Oxidation ◽  
Cucumber Fruit ◽  
Key Enzyme ◽  
Cucumber Roots

UGD gene encodes UDP-glucose dehydrogenase (UGD) which is a key enzyme in the biosynthesis of cell wall, and it catalyses the irreversible oxidation of UDP-glucose (UDP-Glc) into UDP-glucuronic acid (UDP-GlcA). In cucumber, the expression level of CsUGD2 genes was higher in phloem tissues of pedicel and fruit than that in stalk. This study investigated the function of CsUGD2 in cucumber by different methods. Structure analysis indicated that CsUGD2 gene only has an exon with a length of 1,443 bp. Protein alignment suggested that UGD protein was highly conservative in different species. Phylogenetic analysis showed that CsUGD2 protein and CmoUGD2 protein form a same clade which is far away from UGDs in Arabidopsis. Real-time fluorescence quantitative analysis of CsUGD2 in different tissues of cucumber in the same period showed that CsUGD2 expressed highest in the root of cucumber. When we transformed CsUGD2 into wild type Arabidopsis, the roots of transgenic plants were shorter and the flowering time was delayed.  These results suggested that CsUGD2 may play an important role in the development of cucumber roots and only act on the development of cucumber fruit when compared with other plants fruits. However, the role of CsUGD2 in regulating the growth and development process of cucumber need to further study.

scholarly journals The role of the StAR protein in steroidogenesis: challenges for the future

2000 ◽  
Vol 164 (3) ◽  
pp. 247-253 ◽  
Author(s):  
DM Stocco
Keyword(s):  
Side Chain ◽  
Inner Mitochondrial Membrane ◽  
Star Protein ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Hormone Biosynthesis ◽  
Steroidogenic Acute Regulatory ◽  
The Brain ◽  
Star Gene

The steroidogenic acute regulatory or StAR protein has been shown to be instrumental in the acute regulation of steroid hormone biosynthesis through its action in mediating cholesterol transfer to the inner mitochondrial membrane and the cholesterol side chain cleavage enzyme system. Since the time of its cloning in 1994, a number of studies have been performed which underscore the important role that this protein plays in steroidogenesis. While it is now quite apparent that StAR fulfills the criteria for the acute regulator as proposed by early studies, several crucial areas remain poorly understood. This list is topped by the so far intractable nature of the mechanism of action of StAR in transferring cholesterol to the P450scc enzyme. A second area which should prove to be of great interest is that of further understanding the regulation of the StAR gene which, like many genes, is quite complex. Lastly, with the recent demonstration of StAR being present in the brain, determining if StAR has a role in the synthesis of neurosteroids should prove to be of great importance.

Inducible expression of protein kinase Cα suppresses steroidogenesis in Y-1 adrenocortical cells

1998 ◽  
Vol 275 (3) ◽  
pp. C780-C789 ◽  
Author(s):  
Mary E. Reyland ◽  
David L. Williams ◽  
Elizabeth K. White
Keyword(s):  
Protein Kinase ◽  
Cell Lines ◽  
Adrenocortical Cells ◽  
Chain Cleavage ◽  
Protein Kinase Cα ◽  
Cleavage Enzyme ◽  
Camp Induction ◽  
First Time ◽  
The Relationship

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCα isoform mediates this suppression, we have developed Y-1 cell lines in which PKCα is expressed from a tetracycline-regulated promoter. Induction of PKCα expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCα expression is increased. However, expression of PKCα has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCα but not PKCε or PKCζ. These studies define for the first time the role of a specific PKC isoform (PKCα) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.

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