Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii)

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10⁻⁶ mol ml^–1 of 17α-MT and 10⁻⁸ mol ml^–1 of E2 in ovary and 10⁻¹⁰ mol ml^–1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.     

The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus)

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole‐genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual‐luciferase reporter assays; Posox8a overexpression‐promoted 3β‐hydroxysteroid dehydrogenase expression and Posox8b overexpression‐promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts.     

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17β‐estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low‐convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT‐PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.     

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25‐hydroxyvitamin D₃ (25D) to hormonal 1,25‐dihydroxyvitamin D₃ (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25‐hydroxyvitamin D‐1α‐hydroxylase (1α‐hydroxylase). To establish new models for assessing the physiological importance of the 1α‐hydroxylase‐25D‐axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α‐hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5–150 nM) or active 1,25D (0.1–10 nM) induced dose responsive expression (15–95‐fold) of the vitamin D‐target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full‐length zebrafish cyp27b1 cDNA was then generated using RACE and RT‐PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC‐8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25‐fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft‐shelled turtle (Pelodiscus sinensis)

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full‐length ovarian cyp19a gene from the Chinese soft‐shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17β‐estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft‐shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.     

Enantioselectivity in Developmental Toxicity of rac‐metalaxyl and R‐metalaxyl in Zebrafish (Danio rerio) Embryo

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac‐metalaxyl and R‐metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac‐metalaxyl and R‐metalaxyl, and the LC₅₀s at 96 h are 416.41 (353.91, 499.29) mg · L^‐1 and 320.650 (279.80, 363.46) mg · L^‐1, respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic–pituitary–gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic–pituitary–thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac‐metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R‐metalaxyl. And likewise, rac‐metalaxyl only caused the upregulation of dio2, while R‐metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489–494, 2016. © 2016 Wiley Periodicals, Inc.     

Seasonal changes in gene expression of steroidogenic enzymes,androgen and estrogen receptors in frog testis

The anuran amphibian Pelophylax esculentus shows an annual cycle of sexual steroid production and spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we investigated the mRNA expression of key enzymes involved in the androgenic and oestrogenic biosynthesis pathways in the testis of frogs taken in the reproductive and postreproductive period. Furthermore, we also analysed androgen and oestrogen levels and their own receptor gene expressions. Our findings showed that during the reproductive period, 3β‐hydroxysteroid dehydrogenase, 17β‐hydroxysteroid dehydrogenase and 5α‐reductase mRNA levels were higher than those during the postreproductive period. High testosterone and 5α‐dihydrotestosterone titres as well as the expression levels of androgen receptors in the reproductive testis strongly confirmed that the androgenic pathway is necessary for spermatogenesis activation. Conversely, during the postreproductive period, the highest P450 aromatase, estrogen receptor α and β mRNA levels, paralleling with oestradiol titres, indicated that the oestrogenic pathway is essential for the interruption of the reproductive processes. Our findings demonstrated, for the first time in amphibians, that testicular endocrine cyclic activity could be modulated by the up‐regulation of key steroidogenic enzyme gene expressions. This in turn determines the activation of the androgenic pathway in reproductive phase and the oestrogenic one in postreproductive phase.     

Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish,Amphiprion clarkii

To investigate the role of estrogen in the gonad of yellowtail clownfish Amphiprion clarkii, we isolated cDNA encoding cytochrome P450 aromatase (Cyp19a1a) from the adult ovary. The full-length cDNA of clownfish cyp19a1a is 1928-bp long and encodes 520 amino acids. Real-time quantitative RT-PCR analysis showed that cyp19a1a was expressed mainly in the ovary of female-phase fish. In situ hybridization and immunohistochemical observations showed that positive signals were restricted to the ovarian follicle of the female-phase fish. In contrast, Cyp19a1a signal was not detected in the ambisexual gonad of the male-phase fish. These findings suggest that Cyp19a1a is involved in oogenesis in the female-phase fish, but not in the ambisexual gonad of male-phase fish.     

Phylogeny, expression and enzyme activity of zebrafish cyp19 (P450 aromatase) genes

The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.     

Olfactory responses to steroids in an African mouth-brooding cichlid, Haplochromis burtoni(Günther)

Underwater electro‐olfactogram (EOG) recordings involving 150 steroids and eight prostaglandins were used to determine which of these potential odorants are detected by the olfactory organ of an African cichlid, Haplochromis burtoni. In initial EOG tests at 10^?9 M, H. burtoni did not respond to unconjugated steroids or prostaglandins, but did respond to 17 conjugated steroids, 11 of which (17β‐oestradiol‐17β‐glucuronide; 17β‐oestradiol‐3‐sulphate; 17β‐oestradiol‐3,17β‐disulphate; epiandrosteron‐3β‐sulphate; etiocholanolone‐3α‐glucuronide; testosterone‐17β‐sulphate; dehydroepiandrosterone‐3β‐sulphate; 5α‐pregnan‐3β‐ol‐20‐one‐3β‐sulphate; 5β‐pregnan‐3α,17‐diol‐20‐one‐3α‐glucuronide; 5β‐pregnan‐3α,17,21‐triol‐11,20‐dione‐3α‐glucuronide; pregnenolone‐3β‐sulphate) were selected for EOG concentration‐response, cross‐adaptation and binary mixture tests. The EOG detection thresholds ranged from 10^?11 to 10^?9 M in all but one instance (female threshold to pregnenolone‐3β‐sulphate; 10^?8 M), and males and females exhibited only minor differences in EOG threshold or response magnitude. Results of EOG cross‐adaptation tests, which were supported by results of binary mixture tests, indicated that the response to the 11 steroid conjugates is mediated by five putative olfactory receptor mechanisms characterized by specificity for conjugate position and type: 3‐sulphate, 17‐sulphate, 3,17‐disulphate, 3‐glucuronide, 17‐glucuronide. Although there is no evidence that H. burtoni releases, or exhibits biological response to, the steroids shown to be detected in this study, the present results are suggestive of a complex pheromone system utilizing steroid conjugates.     

Germ line control of female sex determination in zebrafish

A major transition during development of the gonad is commitment from an undifferentiated “bi-potential” state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.