Characterization of the Interrenal Gland and Sexual Traits Development in cyp17a2-Deficient Zebrafish

Abstract

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2-null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2-deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a (pomca) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2-deficient fish. Intriguingly, the cyp17a2-deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2-deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2-/- females and cyp17a2-/- males, respectively. By examining the ovaries development of cyp17a2-deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2-deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2-deficient females.

Keywords: cortisol; cyp17a2; oocyte maturation; sexual trait; zebrafish.

Figure 1

Deletion of cyp17a2 in zebrafish by CRISPR/Cas9. (A) The gRNA target site of cyp17a2 in the second exon. cyp17a2-null zebrafish with 7 base pairs deletion was obtained. (B) The representative chromatograms of the DNA sequences of control fish and cyp17a2-/- zebrafish, as evidenced by DNA sequencing of the PCR amplified DNA fragments using the genome DNA or cDNA as the template. The red squares in panel A and B indicate the 7 base pairs which have been deleted in cyp17a2-/- fish. (C) The predicted Cyp17a2 protein and mutant protein of cyp17a2-/- fish. The peptides of mutant Cyp17a2 identical to control Cyp17a2 protein are shown in green, the frameshift mutation is shown in gray. The premature stop codon is indicated with purple asterisk in panel (C) aa, amino acid. The amino acids before and after the start site of frameshift mutation in cyp17a2-/- fish and the corresponding area in the control fish is highlighted in dotted square and the detailed information is shown in panel (D). (D) The detailed amino acid sequence of the dotted square in panel (C).

Figure 2

cyp17a2-deficiency caused pituitary pomca upregulation and interrenal gland enlargement in zebrafish larvae. WISH analyses with probes of pomca (A1, A2), cyp11a1 (B1, B2), cyp21a2 (C1, C2) and hsd3b1 (D1, D2) in cyp17a2+/+ and cyp17a2-/- fish at 5 dpf. WISH analyses with probes of pomca (E1–E4), cyp11a1 (F1–F4), cyp21a2 (G1–G4), and hsd3b1 (H1–H4) in fish at 5 dpf. (E1, F1, G1, H1) cyp17a2+/+;pomca+/+ fish. (E2, F2, G2, H2) cyp17a2-/-;pomca+/+ fish. (E3, F3, G3, H3) cyp17a2+/+;pomca-/- fish. (E4, F4, G4, H4) cyp17a2-/-;pomca-/- fish. Black, red, and green arrows indicate normal, enlarged, and decreased interrenal gland, respectively. (I) Bar chart represents the quantification and statistical analyses of WISH signals in pituitary and interrenal gland of fish at 5 dpf. (J1, J2, K1, K2) WISH analysis using the probe of cyp21a2 in cyp17a2+/+ and cyp17a2-/- fish administrated with DMSO or HC from 1 dpf to 5 dpf. (J1, J2) DMSO. (K1, K2) HC. (L) Bar chart represents the quantification and statistical analyses of WISH signals in interrenal gland of fish administrated with DMSO or HC. HC, hydrocortisone. DMSO, dimethyl sulfoxide. n.s., no significant difference. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 3

The steroid measurements. Whole-body levels of cortisol (A), DHP (B), P4 (C), 11-KT (D), T (E), and E2 (F) in cyp17a2+/+ and cyp17a2-/- zebrafish at 3 mpf (n = 6). n.s., no significant difference. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.

Figure 4

cyp17a2-deficiency resulted in impaired locomotion activity and stress response to light in zebrafish. (A, B) The locomotor trajectories of cyp17a2+/+ and cyp17a2-/- adult fish within 5 minutes (n = 10). (C) The distance fish covered at large, slow-mild, and inactive speed. The locomotor trajectories and distance covered at large (> 5 cm/sec), slow-mild (2-5 cm/sec) and inactive (< 2 cm/sec) speed in panel A-C are highlighted in red, green, and grey, respectively. (D) The movement distance curve analyzed in every 30 seconds under the light-stimulated stress within 20 minutes (n = 5). (E) All distance under the light-stimulated stress within 20 minutes (n = 5). (F) The oxygen consumption of cyp17a2+/+ and cyp17a2-/- fish after 2 days starvation (n = 3/group, 3 groups per genotype). n.s., no significant difference. *P < 0.05. **P < 0.01. ****P < 0.0001.

Figure 5

The analyses of primary and secondary sex characters and fertility assessment. (A–D) Gross appearance of cyp17a2+/+ male, cyp17a2-/- male, cyp17a2+/+ female, cyp17a2-/- female (n = 10). (E–H) The genital papilla. (I–L) The pectoral fin. (M–P) The anatomical observations. (Q–T) The histological analysis of gonads. Black and green arrow indicates normal and diminished genital papilla, respectively. (U) GSI of cyp17a2+/+ fish and cyp17a2-/- fish at 3 mpf (n > 10). (V) The fertility analysis of cyp17a2-/- males and cyp17a2-/- females mated with WT females and males, respectively (n = 10). (W) The statistical analyses of ovulated eggs of cyp17a2+/+, cyp17a2+/- and cyp17a2-/- females mated with WT males (n = 10). (X) The statistical analyses of fertilization ratio of the ovulated eggs from WT females when mated with cyp17a2+/+ males and cyp17a2-/- males, respectively (n = 10). n.s., no significant difference. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 6

The over-activated oocytes in cyp17a2-/- females. (A–D) Under visual microscopy, the oocytes from control females unpaired and paired with WT males were not transparent and transparent, respectively (n = 5). (E–H) The oocytes from cyp17a2-/- females unpaired and paired with WT males were both transparent (n = 5). (I–L) The 20 nM hydrocortisone treatment in cyp17a2-/- females did not promotes oocyte maturation as observed under visual microscopy (n = 5).

Figure 7

Expression profiles of pituitary hormones. Expressions of pomca, lhβ, fshβ, gh1, prl and tshβ in cyp17a2+/+ and cyp17a2-/- zebrafish at 3 mpf were analyzed with qPCR. All mRNA levels were calculated as the fold expression relative to the housekeeping gene β-actin. (A) Control females and cyp17a2-/- females. (B) Control males and cyp17a2-/- males. n.s., no significant difference. *P < 0.05. ****P < 0.0001.