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Alignment of the amino acid sequences of P450(11b) from tilapia and other vertebrates. P450 enzymes characteristic regions are boxed: (A) Steroid binding; (B) oxygen-binding; (C) Ozol'; (D) aromatic and (E) heme-binding regions. Refer to Section 2 for GenBank accession numbers. BOXSHADE (http://www.ch.embnet.org/software/ BOX_form.html) was used to make this figure.
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P450 11beta-hydroxylase, encoded by P450(11beta) gene, is a key mitochondrial enzyme to produce 11beta-hydroxy testosterone, substrate for the production of 11-ketotestosterone (11-KT), which has been shown to be potent androgen in several fish species. In the present work, two alternative splicing isoforms i.e. P450(11beta)-1 and P450(11beta)-2 cD...
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... alignment of the amino acids sequences of the tila- pia P450(11b)-1 and those from other vertebrates revealed that P450(11b) was conserved through evolution (Fig. 2), especially at the characteristic regions of the P450 enzymes, i.e. (A) ste- roid binding site, (B) oxygen-binding site, (C) Ozols', (D) aro- matic, and (E) heme-binding site. Regions A, B and E were highly conservative, 92-100% homology with those of other fish P450(11b), $84% with amphibian and $58% with mammalian P450(11b). In ...
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... lowest homology with those of other vertebrates, 58-83% homology with fish, $58% and $41% with its amphibian and mammalian counter- parts, respectively, while, region C shared the moderately con- served homology with those of other vertebrates, 79-89% homology with fish, 73% and 57% with its amphibian and mam- malian counterparts, respectively (Fig. 2). Based on an align- ment of 19 P450(11b) sequences, a phylogenetic tree was constructed using the zebrafish p450 aromatase as the out- group. Tilapia P450(11b) showed the highest similarity (80.5% in nucleotide sequence and 84.2% in aa sequence) to that of medaka. The high homology of this protein between tilapia and medaka, as shown ...
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The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, a...
Citations
... Li et al. [72] reported that disruption of Cyp19a1a and FOXL2 expression led to female-to-male sex reversal in tilapia. A previous investigation by Zhang et al. [70,73] revealed that transgenic overexpression of DMRT1 in XX tilapia led to an inhibition of Cyp19a1a expression, decreased E2 levels, and eventually resulted in sex reversal. This aligns with observations in other vertebrate groups where masculinization has been induced via inhibition of endogenous estrogen synthesis using an aromatase inhibitor [74][75][76][77]. ...
... Li et al. [72] reported that disruption of Cyp19a1a and FOXL2 expression led to female-to-male sex reversal in tilapia. A previous investigation by Zhang et al. [70,73] revealed that transgenic overexpression of DMRT1 in XX tilapia led to an inhibition of Cyp19a1a expression, decreased E2 levels, and eventually resulted in sex reversal. This aligns with observations in other vertebrate groups where masculinization has been induced via inhibition of endogenous estrogen synthesis using an aromatase inhibitor [74][75][76][77]. ...
... Ovaries processed for histology were embedded in paraffin after fixation in Bouin's solution (24 h, room temperature). Embedded samples were then cut serially into slices of 5 μm for H&E staining as described previously (Zhang et al., 2010;mid-section). All sections were imaged using an Olympus BX51 optical microscope (Olympus, Japan). ...
... IHC, IF and TUNEL assays were performed as described previously Zhang et al., 2010). All sections from fish with different genotypes were imaged using a Olympus BX51 optical microscope (Olympus) with the same parameters. ...
The role of glucocorticoids in oogenesis remains to be elucidated. cyp11c1 encodes the key enzyme involved in the synthesis of cortisol, the major glucocorticoid in teleosts. In our previous study, we mutated cyp11c1 in tilapia and analyzed its role in spermatogenesis. In this study, we analyzed its role in oogenesis. cyp11c1+/- XX tilapia showed normal ovarian morphology but poor egg quality, as indicated by the mortality of embryos before 3 d post fertilization, which could be partially rescued by the supplement of exogenous cortisol to the mother fish. Transcriptome analyses revealed reduced expression of maternal genes in the eggs of the cyp11c1+/- XX fish. The cyp11c1-/- females showed impaired vitellogenesis and arrested oogenesis due to significantly decreased serum cortisol. Further analyses revealed decreased serum E2 level and expression of amh, an important regulator of follicular cell development, and increased follicular cell apoptosis in the ovaries of cyp11c1-/- XX fish, which could be rescued by supplement of either exogenous cortisol or E2. Luciferase assays revealed a direct regulation of cortisol and E2 on amh transcription via GRs or ESRs. Taken together, our results demonstrate that cortisol safeguards oogenesis by promoting follicular cell survival probably via Amh signaling.
... In this study, we characterized the gene structure, amino acid sequence and phylogenetic relationship of CYP11A and CYP11B in black rockfish, examined the tissue-specific expression in adult, cell localization in gonads, and the effects of exogenous hormones on its expression to catch more function information of these two genes. Previous studies describe that cyp11a and cyp11b are conserved in teleosts according their expression analysis in tissues (Kazeto et al., 2006;Rajakumar & Senthilkumaran, 2014;Zhang et al., 2010). ) male, ( Tissues used were testis for male and ovary for female in (d) and (e). ...
... were not less required to participate in the synthesis of sex hormones temporarily before the sexual differentiation, while they were necessary for the development of germ cells such as oocytes and spermatocytes (Liang et al., 2018;Zhang et al., 2010). The ISH results show that cyp11a and cyp11b in black rockfish are both located in ovarian oocytes and spermatocytes of the testisa We speculate that cyp11a and cyp11b may be related to gonadal development and sex hormone synthesis. ...
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.
... In barramundi, dmrt1 and cyp11b were found to be upregulated during early testicular differentiation (Banh et al., 2017). Male specific expression of cyp11b, one of the key J o u r n a l P r e -p r o o f steroidogenic enzymes, which catalyzes biosynthesis of the potent androgen 11ketotestosterone (Kime, 1993;Kusakabe et al., 2002), was documented in Nile tilapia (Zhang et al., 2010a), sparid sharpsnout seabream (Manousaki et al., 2014), bluehead wrasse (Thalassoma bifasciatum) and barramundi (Ravi et al., 2014). Sexually dimorphic patterns of esr1 (i.e. higher expression in testis than ovary) were also reported in Nile tilapia (Ijiri et al., 2008;Tao et al., 2013), rainbow trout (Baron et al., 2008;Delalande et al., 2015), European sea bass (Blázquez et al., 2008), and barramundi (Ravi et al., 2014); yet, the opposite expression of esr1 in other species (Davis et al., 2008;Lynn et al., 2008), and the regulation of other possible estrogen receptor subtypes, such as esr2 in teleosts (Nelson and Habibi, 2013), suggests there needs to be further study on the mechanism of estrogen hormones and their receptors in sex change/differentiation of barramundi. ...
Sex control is vital for the efficient breeding of aquaculture species. Barramundi (Lates calcarifer) is a protandrous sequential hermaphrodite that naturally sex change from male to female over its lifespan. The induction of precocious female barramundi will permit breeding of males and females of the same-generation individuals in selective breeding programs, increasing the rates of genetic gain, while reducing infrastructure costs. Accordingly, the efficacy of two dosages of 17β-estradiol (E2) delivered via implants to induce the precocious female barramundi were evaluated. Six-month-old male barramundi (405 ± 50 g body weight (BW)) were given a single cholesterol-based pellet implant containing either 0 mg E2 kg⁻¹ BW (untreated control), 4 mg E2 kg⁻¹ BW (‘low dose’), or 8 mg E2 kg⁻¹ BW (‘high dose’). Changes in gonadal morphology and liver condition of implanted males, along with RT-qPCR and bisulfite amplicon sequencing to quantify expression profiles and DNA methylation of key male-female sex-related genes were then examined after 9 weeks post-implantation. Results showed that at 9 weeks post-implantation, in the ‘high dose’ E2 treatment group, 78% (7/9) of fish sex-changed completely to female, signified by gonads containing oocytes (20–30 μm) and no observed residual sperm. Comparably, 44% (4/9) of fish in the ‘low dose’ E2 treatment group had sex-changed, while remaining fish showed complete testicular regression with gonads containing only undifferentiated germ cells. In the ‘high dose’ E2 treatment, upregulation of female-biased genes (cyp19a1a and foxl2) and downregulation of male-biased genes (dmrt1, cyp11b and esr1) were observed. Increased gene expression was accompanied by decreased DNA methylation in cyp19a1a, but no significant changes in DNA methylation of foxl2 or esr1 were observed. The success of artificially-induced sex change in barramundi provides an important tool that is critical to improving selective breeding of this species.
... Functional studies of the two forms GtH in fish have shown that they correspond to FSH and LH in mammals ( Van der Kraak et al., 1992). Under the action of GtH, the ovary and testis produce oestrogen 17α, 20β-DHP, E2 and androgen T, 11-KT, respectively, which induce the development and maturation of spermatozoa and oocytes (Lin, 2007 (Miura et al., 1991), the level of 11-KT in blood gradually increases along with the development of testis (Assis et al., 2018;Zhang et al., 2010). However, in this study, we found in the experimental group, the testis development was inhibited after igf3 gene was interfered, but the content of 11-KT was significantly higher than that in the control group. ...
RNA interference is an important method in studying the gene function of organisms. As a novel important gene in gonadal development of teleost fish, the function of igf3 is worth of researching. In this study, the igf3b‐dsRNA of C. carpio was successfully synthesized, and long‐term igf3 interference experiments were conducted. Intraperitoneal injection of igf3b‐dsRNA could influence the serum sex hormone level and significantly reduced the expression level of insulin‐like growth factors (IGFs) in hypothalamic–pituitary–gonad (HPG) axis. After a 60‐day interference experiment, igf3 knockdown could affect the development of ovary and testis of juvenile C. carpio. These findings show that igf3 knockdown may have a negative effect on gonadal development of C. carpio through HPG axis.
... Interestingly, during pubertal development of male catfish, co-treatment of 11-KT and T abolished the stimulatory effect of 11-KT on testis growth and spermatogenesis due to T, indicated the requirement of balanced production of androgens with critical regulation during puberty (Cavaco, Bogerd, Goos, & Schulz 2001). In testis, under the stimulation of the pituitary hormone FSH, 11-KT synthesis gets triggered, which in turn induced spermatogenesis in teleost (Borg, 1994;Rinchard, Dabrowski, & Ottobre, 2001;Schulz et al., 2010;Zhang et al., 2010). In male common carp, 11-KT levels were twice as high as T during maturation when incubated with hypophyseal homogenate (Koldras, Bieniarz, & Kime, 2006). ...
Gonadal steroidogenesis is pivotal to synchronize various reproductive stages including sexual development, growth and maturation. In all vertebrates including teleost, steroidogenesis is triggered by the mobilization of cholesterol by steroidogenic acute regulatory protein from outer to inner mitochondrial membrane. Thereafter, the entire process occurs in endoplasmic reticulum or mitochondria wherein various steroidogenic enzyme genes play a crucial role. The onset of steroidogenesis during sexual development in teleost is essentially regulated by differential expression of several transcription as well as steroidogenesis-related factors. More specifically, the role of dmrt, sox9, sox3, other sox forms, ad4bp/sf-1, wt-1, foxl2, ftz-f1, gata4, gsdf, Activator protein-1, fgfs and growth factors and steroidogenic enzymes such as cytochrome P450aromatase (cyp19a1), hydroxysteroid 3β-dehydrogenase (hsd3b), hydroxysteroid 17β-dehydrogenase (hsd17b) have been well characterised. Recently, the role of pax2, THO complex (thoc), pentraxin (ptx) and few signalling molecules like wnt4/5 regulating teleostean steroidogenesis has been reported. In females, cyp19a1 appears to be critical as it converts androgens to estrogens during ovarian differentiation which suggests that estradiol-17β is indispensable for the event. Unlike females, males do not depend on testosterone for testicular determination, yet has a major role along with 11-ketotestosterone in testicular development and growth when compared to early testicular differentiation. In males, onset of sex determining or testis-related genes seems most essential. Considering these, the regulation of steroidogenesis is virtually critical at later stages. In view of this, several steroidogenic motifs pertaining to transcriptional regulation have been analysed in teleost, yet far below than the reports on mammals. In this context, the regulatory influence on HHG axis plays a critical role in teleost. Further, multiple gonadotropin-releasing hormones and duality of gonadotropins play a differential role in gonadal function. As for steroidogenic pathway, the synthesis of sex steroids predominantly uses Δ⁴, however, Δ⁵ pathway was also evident. The next aspect is shift in steroidogenesis that usually occurs in the maturing follicles during final oocyte maturation, yet such a mechanism is not clear in males. The review highlights the interactions of steroidogenic enzyme gene regulation in terms of HHG axis and other important transcription factors that are involved in the regulatory pathways as well as the influence of environmental and dietary factors by comparing both sexes to present a holistic view on steroidogenesis onset and organisation during gametogenesis in teleost.
... After genome duplication, the duplicated gene copies had been attributed to different or modified functions in different species or no function at all (Meyer and Schartl 1999). Several isoforms or variants of steroidogenic enzyme genes are detected in several teleosts: cyp11a1 (Parajes et al. 2013), cyp17 (Zhou et al. 2007a, b), hsd3b ), and cyp11b1 (Zhang et al. 2010;Rajakumar and Senthilkumaran 2015). Specific role for each isoform has been shown in some teleosts, yet the role of different variant forms was least understood for example hsd3b variants in tilapia ). ...
Steroid hormones modulate several important biological processes like metabolism, stress response, and reproduction. Steroidogenesis drives reproductive function wherein development and differentiation of undifferentiated gonads into testis or ovary, and their growth and maturation, are regulated. Steroidogenesis occurs in gonadal and non-gonadal tissues like head kidney, liver, intestine, and adipose tissue in teleosts. This process is regulated differently through multi-level modulation of promoter motif transcription factor regulation of steroidogenic enzyme genes to ultimately control enzyme activity and turnover. In view of this, understanding teleostean steroidogenesis provides major inputs for technological innovation of pisciculture. Unlike higher vertebrates, steroidal intermediates and shift in steroidogenesis is critical for gamete maturation in teleosts, more essentially oogenesis. Considering these characteristics, this review highlights the promoter regulation of steroidogenic enzyme genes by several transcription factors that are involved in teleostean steroidogenesis. It also addresses different methodologies involved in promoter regulation studies together with glucocorticoids and androgen relationship with reference to teleosts.
... Previously, it was reported that cyp11a and cyp11b mRNA was mainly expressed in gonads and head kidneys in teleost fish, such as Japanese eel, brown hagfish (Paramyxine atami), Nile tilapia, zebrafish, and catfish (Kazeto et al., 2006;Nishiyama et al., 2015;Rajakumar & Senthilkumaran, 2014a;Zhang et al., 2010). In our study, the tissue-specific expression patterns of cyp11a and cyp11b in flounder were similar to those reported in other teleosts. ...
The P450 side-chain cleavage enzymes P450scc (Cyp11a) and 11β-hydroxylase (Cyp11b) play important roles in sex steroid and cortisol production. Here, two duplicates of cyp11 genes were identified in Japanese flounder (Paralichthys olivaceus): Pocyp11a and Pocyp11b, respectively. Phylogenetic analysis and amino acid sequence alignment revealed that Pocyp11a and Pocyp11b shared significant identity with sequences of other teleost fish species. The quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that among the studied tissues, brain tissue showed the highest expression of Pocyp11a, followed by kidney and testis tissues, whereas Pocyp11b expression was highest in the testis. The expression patterns of these two genes showed sexual dimorphism, with both genes showing higher expression in the testis than in the ovary. In-situ hybridization analysis demonstrated that Pocyp11a and Pocyp11b mRNA were both detected in oocytes, spermatocytes, and Sertoli cells, indicating that they might be involved in hormone synthesis. The expression levels of Pocyp11a and Pocyp11b were significantly downregulated by treatment with 17α-methyltestosterone (17α-MT) in the testis and ovary in both in vivo and studies. In vivo studies showed that Pocyp11a and Pocyp11b transcripts were suppressed by 17β-estradiol (E2 ) treatment in both the testis and ovary. In addition, in vitro studies showed that the expression level of Pocyp11b was decreased by treatment with E2 , whereas that of Pocyp11a was largely unaffected. Moreover, the expression levels of Pocyp11a and Pocyp11b in the testis cell line were significantly upregulated after NR0b1 and NR5a2 (p < .05) treatment. These results indicate that Pocyp11a and Pocyp11b might play important roles in sex hormone biosynthesis. Our research can assist future studies of the mechanisms of steroid biosynthesis and functional differences between cyp11a and cyp11b in Japanese flounder.
... Then, pregnenolone is further catalyzed by other steroidogenic enzymes such as Cyp17, Hsd3β, Cyp11b1, Hsd11β and Cyp19a to form various sex steroid products [2]. The cloning of the cyp11a gene in a few fish has been reported [1,[3][4][5][6][7]. Almost similar to those in mammals [1], cyp11a in adult fish tissues is mainly expressed in gonadal and kidney tissues [5,8,9]. ...
The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17β-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 μM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 μM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.
