11ß-Hydroxylase loss disrupts steroidogenesis and reproductive function in zebrafish.

The roles of androgens in male reproductive development and function in zebrafish are poorly understood. To investigate this topic, we employed CRISPR/Cas9 to generate cyp11c1 (11ß-hydroxylase) mutant zebrafish lines. Our study confirms recently published findings from a different cyp11c1-/- mutant zebrafish line, and also reports novel aspects of the phenotype caused by loss of Cyp11c1 function. We report that Cyp11c1-deficient zebrafish display predominantly female secondary sex characteristics, but may possess either ovaries or testes. Moreover, we observed that cyp11c1-/- mutant male zebrafish are profoundly androgen- and cortisol-deficient. These results provide further evidence that androgens are dispensable for testis formation in zebrafish, as has been demonstrated previously in androgen-deficient and androgen-resistant zebrafish. Herein, we show that the testes of cyp11c1-/- mutant zebrafish exhibit a disorganised tubular structure; and for the first time demonstrate that the spermatic ducts, which connect the testes to the urogenital orifice, are severely hypoplastic in androgen-deficient zebrafish. Furthermore, we show that spermatogenesis and characteristic breeding behaviours are impaired in cyp11c1-/- mutant zebrafish. Expression of nanos2, a type A spermatogonia marker, was significantly increased in the testes of Cyp11c1-deficient zebrafish, whereas expression of markers for later stages of spermatogenesis was significantly decreased. These observations indicate that in zebrafish, production of type A spermatogonia is androgen-independent, but differentiation of type A spermatogonia is an androgen-dependent process. Overall, our results demonstrate that whilst androgens are not required for testis formation, they play important roles in determining secondary sexual characteristics, proper organisation of seminiferous tubules, and differentiation of male germ cells.

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

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.

Ferredoxin 1b Deficiency Leads to Testis Disorganization, Impaired Spermatogenesis, and Feminization in Zebrafish.

The roles of steroids in zebrafish sex differentiation, gonadal development, and function of the adult gonad are poorly understood. Herein, we used ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults in which concentrations of androgens and cortisol are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish exhibit reduced characteristic breeding behaviors and impaired sperm production, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by in vitro fertilization. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several promale and spermatogenic genes is decreased in the testes of fdx1b-/- mutant males, including promale transcription factor sox9a and spermatogenic genes igf3 and insl3. This study establishes an androgen- and cortisol-deficient fdx1b zebrafish mutant as a model for understanding the effects of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development, and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.

Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

TBK1: a new player in ALS linking autophagy and neuroinflammation.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder affecting motor neurons, resulting in progressive muscle weakness and death by respiratory failure. Protein and RNA aggregates are a hallmark of ALS pathology and are thought to contribute to ALS by impairing axonal transport. Mutations in several genes known to contribute to ALS result in deposition of their protein products as aggregates; these include TARDBP, C9ORF72, and SOD1. In motor neurons, this can disrupt transport of mitochondria to areas of metabolic need, resulting in damage to cells and can elicit a neuroinflammatory response leading to further neuronal damage. Recently, eight independent human genetics studies have uncovered a link between TANK-binding kinase 1 (TBK1) mutations and ALS. TBK1 belongs to the IKK-kinase family of kinases that are involved in innate immunity signaling pathways; specifically, TBK1 is an inducer of type-1 interferons. TBK1 also has a major role in autophagy and mitophagy, chiefly the phosphorylation of autophagy adaptors. Several other ALS genes are also involved in autophagy, including p62 and OPTN. TBK1 is required for efficient cargo recruitment in autophagy; mutations in TBK1 may result in impaired autophagy and contribute to the accumulation of protein aggregates and ALS pathology. In this review, we focus on the role of TBK1 in autophagy and the contributions of this process to the pathophysiology of ALS.