Embryogenic staging of fugu, Takifugu rubripes, and expression profiles of aldh1a2, aldh1a3 and cyp26a1

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis – the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.     

Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a teleost fish, the medaka, Oryzias latipes

The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.     

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.     

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.     

Molecular cloning and expression of a novel CYP26 gene (cyp26d1) during zebrafish early development

Proper restriction of retinoid signaling by Cyp26s is essential for development of vertebrate embryos while inappropriate retinoid signaling can cause teratogenesis. Here, we report cloning and expression analysis of a novel cyp26 gene (cyp26d1) isolated from zebrafish. The predicted protein encoded by cyp26d1 consists of 554 amino acids. It exhibits 54% amino acid identity with human Cyp26C1, 50% with zebrafish Cyp26B1 and 38% with zebrafish Cyp26A1. Whole-mount in situ hybridization shows that cyp26d1 is first expressed in sphere stage, then disappears at 50% epiboly and resumes its expression at 75% epiboly. During segmentation period, cyp26d1 message is found at presumptive hindbrain. Double in situ hybridization with krox20 and cyp26d1 reveals that cyp26d1 is expressed in presumptive rhombomere 2-4 (r2-r4) at 2-somite stage. At 3-somite stage, cyp26d1 gene is expressed in r6 and pharyngeal arch (pa) one in addition to its expression at r2 and r4. At 6-somite stage, cyp26d1 message is present in continuous bands at r2-r6 and in pa1. This expression pattern is maintained from 10-somite stage through 21-somite stage except that the expression level is greatly reduced at r2 and r4. At 21-somite stage, cyp26d1 is also found in a group of cells in telencephalon and diencephalons. At 25-31h post-fertilization (hpf), the zebrafish cyp26d1 expression domain is extended to eyes, otic vesicles and midbrain in addition to its expression in hindbrain, telencephalon, diencephalons, and pharyngeal arches. At 35-48hpf, the expression of cyp26d1 is mainly restricted to otic vesicles, pharyngeal arches and pectoral fins and the expression level is greatly reduced.     

应用TaqMan定量方法研究巴马香猪CYP1A1、2A19、2E1 mRNA表达

目的巴马香猪是我国具有特色和优势的实验用小型猪资源品系,用于药物评价具有广阔前景。方法 以β-actin作校正,利用TaqMan定量技术对巴马香猪肝、肾、肾上腺、小肠、皮肤、脑、肺、睾丸、前列腺、子宫和卵巢等组织中CYP1A1、2A19和2E1 mRNA的表达水平进行检测,检测结果与报道的人体对应酶CYP1A2、2A6、2E1进行比较。结果巴马香猪CYP1A1、2A19、2E1 mRNA均以肝脏中最高,肝外组织明显较低,并且巴马香猪肝脏CYP1A1、2A19、2E1 mRNA均低于报道的人肝对应酶。结论巴马香猪CYP1A1、2A19、2E1与人体对应酶CYP1A2、2A6、2E1的mRNA组织表达存在一定差异,提示在其作为相应CYP亚型代谢的药物评价时应考虑这种种属差异对实验结果推广到人的影响。     

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.     

Differential expression of the glutamate transporter GLT-1 in pancreas

The glutamate uptake transporter GLT-1 is best understood for its critical role in preventing brain seizures. Increasing evidence argues that GLT-1 also modulates, and is modulated by, metabolic processes that influence glucose homeostasis. To investigate further the potential role of GLT-1 in these regards, the authors examined GLT-1 expression in pancreas and found that mature multimeric GLT-1 protein is stably expressed in the pancreas of wild-type, but not GLT-1 knockout, mice. There are three primary functional carboxyl-terminus GLT-1 splice variants, called GLT-1a, b, and c. Brain and liver express all three variants; however, the pancreas expresses GLT-1a and GLT-1b but not GLT-1c. Quantitative real time-PCR further revealed that while GLT-1a is the predominant GLT-1 splice variant in brain and liver, GLT-1b is the most abundant splice variant expressed in pancreas. Confocal microscopy and immunohistochemistry showed that GLT-1a and GLT-1b are expressed in both islet β- and α-cells. GLT-1b was also expressed in exocrine ductal domains. Finally, glutamine synthetase was coexpressed with GLT-1 in islets, which suggests that, as with liver and brain, one possible role of GLT-1 in the pancreas is to support glutamine synthesis.     

Regulation of cyp26a1 on Th17 cells in mouse peri‐implantation

Cytochrome P450 26A1 (cyp26a1) is expressed in the mouse uterus during peri‐implantation. The repression of this protein is closely associated with a reduction in implantation sites, suggesting a specific role for cyp26a1 in pregnancy and prompting questions concerning how a metabolic enzyme can generate this distinct outcome. To explore the effective downstream targets of cyp26a1 and confirm if its role in peri‐implantation depends on its metabolic substrate RA (retinoic acid), we characterized the changes in the peripheral blood, spleen and uterine implantation sites using the cyp26a1 gene vaccine constructed before. Flow cytometry results showed a significant increase in CD4⁺RORγt⁺ Th17 cells in both the peripheral blood and spleen in the experimental group. The expression of RORγt and IL‐17 presented the Th17 cells reduction in uterus followed by the suppression of cyp26a1 expression. For greater certainty, cyp26a1 antibody blocking model and RNA interference model were constructed to determine the precise target immune cell group. High performance liquid chromatography results showed a significant increase in uterine at‐RA followed by the immunization of cyp26a1 gene vaccine. Both the ascertain by measuring RARα protein levels in peri‐implantation uterus after gene vaccine immunization and researches using the specific agonist and antagonist against RARα suggested that RARα may be the main RA receptor for signal transduction. These results provided more evidence for the signal messenger role of RA in cyp26a1 regulation from the other side. Here, we showed that the cyp26a1‐regulated Th17 cells are dependent on at‐RA signalling, which is delivered through RARα in mouse peri‐implantation.     

MicroRNA-19a attenuates hypoxia-induced cardiomyocyte apoptosis by downregulating NHE-1 expression and decreasing calcium overload

miR-19a has been shown to be involved in coronary microvascular obstruction injury; however, the underlying molecular mechanisms remain unknown. In our study, we tried to explore the role of miR-19a in cardiomyocyte apoptosis and calcium overload in vivo and in vitro induced by hypoxia. We established the acute myocardial infarction (AMI) rat model by ligating the left anterior descending artery. The expression of miR-19a in the infarct zone of AMI rats and myocardial tissue in the same position in sham rats was analyzed using RT-qPCR while Na⁽⁺⁾/H⁽⁺⁾ exchanger 1 (NHE-1) was detected by Western blotting. We also observed the effects of overexpressing miR-19a or administering an NHE-1 inhibitor (cariporide) on hypoxia-induced (HI) calcium overload and apoptosis in primary cardiomyocytes. In addition, dual-luciferase reporter assays were conducted to investigate the potential target of miR-19a on NHE-1. Decreased miR-19a expression, as well as increased apoptosis and NHE-1 expression, were observed in the AMI model. Furthermore, after hypoxia stimulation, miR-19a was gradually reduced as time increased in primary cardiomyocytes. Overexpressing miR-19a using mimics ameliorated the increase in NHE-1 in hypoxic cardiomyocytes and thereby reduced the HI cell calcium overload and cell apoptosis rate from 12.32% to 9.5% (P < .01). In addition, the dual-luciferase reporter gene assay results verified that NHE-1 was the direct target of miR-19a. Our findings suggest that miR-19a activation can attenuate HI cardiomyocyte apoptosis by downregulating NHE-1 expression and decreasing calcium overload.     

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX‐XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species. genesis 47:289–299, 2009. © 2009 Wiley‐Liss, Inc.     

The cloning of Dmc1 cDNAs and a comparative study of its expression in different ploidy cyprinid fishes

Dmc1 (disrupted meiotic cDNA) is a functionally specific gene, which was firstly discovered in yeast and then found to encode a protein required for homologous chromosome synapsis during the process of meiosis. In this investigation, we cloned the partial cDNAs of Dmc1 of diploid red crucian carp, Japanese crucian carp, common carp, triploid crucian carp and allotetraploid hybrids by using a pair of degenerate primers based on the conservative sequence of amino acids of the DMC1 protein in yeast, mouse and human. The full length cDNAs were then obtained by rapid amplification of cDNA ends (RACE). Our data showed that the full length cDNAs of Dmc1 in the three diploid fishes are all 1375 bp long, while it is 1383 bp long in triploids and 1379 bp long in allotetraploids. And despite of the variation in length, all the cDNAs encode a protein of 342 amino acids. A high homology of 97.3% of the DMC1 protein can be drawn by comparing the amino acid sequences in the three diploids, which is also of 86%, 86% and 95% similarity to human, mouse and zebrafish, respectively. A comparative study of the expression pattern of Dmc1 was carried out by RT-PCR using specific primers against the same sequences of coding regions in different ploidy cyprinid fishes, from which it was showed that Dmc1 was expressed only in gonads of these five kinds of fishes. The expression pattern of Dmc1 in both ovaries and testes from different ploidy fishes within breeding season was also studied by Real-time PCR, and the results showed that the expression of this gene was greatly different among the three different ploidy fishes, which was the highest of triploid and lowest of allotetraploids. The histological sections data showed matured gonads of both diploid red crucian carp and allotetraploids in breeding season, although the latter demonstrated a higher maturation, and no gonadal maturation could be observed in triploids. In conclusion, we suggest that Dmc1 is specifically expressed in the period of meiosis in all the ploidy cyprinid fishes and directly related with the development of gonad in a manner of ploidy-independent way. And further, the high expression of Dmc1 in female triploids might be associated with abnormal meiosis and sterility.