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

Abstract

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.

Augmentation of progestin signaling rescues testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling

“…The administration of DHP effectively restored the phenotypes of GSI and the number of spermatozoa in ar -/- males ( Figure 3 ). In addition, compared with the cyp17a1 -/-; ar -/- fish that showed the hypertrophic testis and enhanced spermatogenesis, the cyp17a1 -/-; ar -/-; npgr -/- fish exhibited phenotypes of defective spermatogenesis and testis organization, which are also seen in the ar -, cyp11a2 -, or cyp11c1 -deficient male zebrafish ( Li et al, 2020 ; Oakes et al, 2020 ; Tang et al, 2018 ; Yu et al, 2018 ). These suggest that the accumulated progestin may play an alternative signaling role in promoting testis organization and spermatogenesis, which is independent of androgen signaling (in cyp17a1 -/- fish and cyp17a1 -/-; ar -/- fish) ( Figures 4 and 5 ).…”

“…To our knowledge, the cyp17a1 -/- zebrafish generated by us is a unique model exhibiting deteriorated androgen signaling but enhanced testicular development and spermatogenesis ( Li et al, 2020 ; Zhai et al, 2018 ). In our newly generated cyp17a1 -/-; ar -/- fish, the phenotype with testicular development and spermatogenesis similar to cyp17a1 -/- fish was observed.…”

“…Androgens and estrogens exert considerable influence on the formation of primary sex characteristic (PSC) and secondary sex characteristic (SSC) in fish. In the zebrafish model, the steroidogenic pathway in reproduction has been extensively investigated as zebrafish possess several advantages for research, including the ease of germ cells observation, gonadal dissection, fertility capacity assessment, knockout strategy ( Crowder et al, 2018 ; Lau et al, 2016 ; Li et al, 2020 ; Lu et al, 2017 ; Tang et al, 2018 ; Tang et al, 2016 ; Yin et al, 2017 ; Yu et al, 2018 ; Zhai et al, 2018 ; Zhu et al, 2015 ), as well as chemical reagent administration, including estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT), fadrozole, flutamide, and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) ( Chen et al, 2013 ; Fenske and Segner, 2004 ; Shu et al, 2020 ; Zhai et al, 2018 ; Zhai et al, 2017 ). Based on observations of ar - or npgr -deficient zebrafish, androgen signaling is reported to be essential for sex determination, testis organization, male-typical SSCs, and fertility ( Crowder et al, 2018 ; Li et al, 2020 ; Tang et al, 2018 ; Yu et al, 2018 ; Zhai et al, 2018 ), while progestin signaling has been suggested to be important for female ovulation ( Tang et al, 2016 ; Wu and Zhu, 2020 ).…”

“…In mice, Cyp17a1 knockout leads to infertility and loss of sexual behaviors ( Liu et al, 2005 ). In zebrafish, the knockout of ar , cyp11c1 (encoding 11β-hydroxylase) or cyp11a2 (encoding cytochrome P450 side-chain cleavage enzyme, the first and rate-limiting enzyme for steroid hormone biosynthesis) results in impaired spermatogenesis and disorganized testes, which is accompanied by androgen signaling insufficiency ( Crowder et al, 2018 ; Li et al, 2020 ; Oakes et al, 2020 ; Tang et al, 2018 ; Yu et al, 2018 ; Zhang et al, 2020 ). However, as evidenced by a recent study reported from our laboratory, a phenotype of all-male cyp17a1 -deficient fish that exhibited reductions of T and 11-KT in plasma and brain samples and loss of male-typical SSCs and mating behaviors with enhanced testicular development and spermatogenesis was observed ( Shu et al, 2020 ; Zhai et al, 2018 ; Zhai et al, 2017 ).…”

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

“…In the current study, stressed levels of cortisol did not affect the transcript levels for most steroidogenic enzymes (star, cyp17a1, hsd11b2, cyp19a1a, and cyp11c1) and had no effect on 11-KT production. However, mRNA levels of cyp11a2, which encodes a crucial enzyme for cholesterol side-chain cleavage in zebrafish steroidogenesis [74], were increased in the zebrafish testicular explants following cortisol treatment (7 days). Moreover, treatment with cortisol reduced the transcript levels of androgen receptor (ar) after 7 days of culture.…”