Molecular characterization of estrogen receptors 1, 2a, and 2b and their tissue and ontogenic expression profiles in fathead minnow (Pimephales promelas)

There are two estrogen receptor (ER) subtypes in fish, Esr1 and Esr2 (formerly ERalpha and ERbeta), and in some species the Esr2 subtype has two forms, Esr2b (formerly ERbeta1) and Esr2a (formerly ERbeta2 or ERgamma). There is little information, however, on the different characteristics and functional significance of the two receptor subtypes in fish, and this is especially relevant for understanding the disruption of ER signaling by chemicals with estrogenic activity. In this study, the full-length cDNAs for esr1 (3167 base pairs [bp]) and esr2b (2318 bp), and a partial-length (267 bp) cDNA for esr2a, were cloned and characterized in fathead minnow (fhm; Pimephales promelas), and their patterns of expression established during development and in adults. Real-time polymerase chain reaction revealed some clear distinctions in the ontogenic and tissue expression of fhm esr1, esr2b, and esr2a, suggesting different functions for each ER subtype. Fhm ERs were expressed in brain, pituitary, liver, gonad, intestine, and gill of male and female fish, esr2b and esr2a were also expressed in muscle. Fhm esr1 and esr2b were expressed predominantly in the liver, whereas fhm esr2a was expressed predominantly in intestine and was lowest expressed in liver. Responses of the different hepatic ERs in male fathead minnow exposed to 100 ng estradiol/L differed, with a significant induction (5-fold) of fhm esr1 but no effect on esr2b or esr2a expression, suggesting different mechanisms of regulation for the different ERs. The detailed characterization of ERs in fathead minnow provides the foundation for understanding the molecular basis of estrogenic disruption in fish.     

Seasonal and Sex-specific mRNA Levels of Key Endocrine Genes in Adult Yellow Perch (Perca flavescens) from Lake Erie

To better understand the endocrine mechanisms that underlie sexually dimorphic growth (females grow faster) in yellow perch (Perca flavescens), real-time quantitative polymerase chain reaction (qPCR) was used to measure pituitary, liver, and ovary mRNA levels of genes related to growth and reproduction-sex in this species. Adult perch were collected from Lake Erie and body mass, age, gonadosomatic index (I (G)), hepatosomatic index (I (H)), and gene expression for growth hormone (GH), prolactin, somatolactin, insulin-like growth factor Ib (IGF-Ib), estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a), and aromatase (cyp19a1a) were measured. Females had higher body mass, I (H), and liver esr1 mRNA level than males, while males had higher liver IGF-Ib, liver esr2a, and liver cyp19a1a mRNA levels. In both sexes, season had a significant effect on GH and liver IGF-Ib mRNAs with higher levels occurring in spring, which also corresponded with higher liver cyp19a1a mRNA levels. For females, I (G), liver esr1, and ovary cyp19a1a mRNA levels were higher in autumn than the spring, and ovary cyp19a1a mRNA levels showed a significant negative correlation with pituitary GH and liver IGF-Ib mRNA levels. The most significant (p 相似文献   

Establishment of estrogen receptor 1 (ESR1)‐knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka,Oryzias latipes

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss‐of‐function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen‐induced vitellogenin gene expression, an estrogen‐responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.     

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.     

From molecule to behavior: Brain aromatase (cyp19a1b) characterization,expression analysis and its relation with social status and male agonistic behavior in a Neotropical cichlid fish

The enzyme aromatase, responsible for the conversion of C19 androgens to C18 estrogens, exists as two paralogue copies in teleost fish: Cyp19a1a mostly expressed in the gonads, referred as gonadal aromatase, and Cyp19a1b, mostly expressed in the brain, accordingly known as brain aromatase. The neural localization of Cyp19a1b is greatly contained within the social behavior network and mesolimbic reward system in fish, suggesting a strong role of estrogen synthesis in the regulation of social behavior. In this work we aimed to analyze the variation in cyp19a1b expression in brain and pituitary of males of a highly social cichlid, Cichlasoma dimerus (locally known as chanchita), and its relation with inter-individual variability in agonistic behavior in a communal social environment. We first characterized chanchita's cyp19a1b mRNA and deduced amino acid sequence, which showed a high degree of conservation when compared to other teleost brain aromatase sequences, and its tissue expression patterns. Within the brain, Cyp19a1b was solely detected at putative radial glial cells of the forebrain, close to the brain ventricles. We then studied the relative expression levels of cyp19a1b by Real Time PCR in the brain and pituitary of males of different social status, territorial vs. non-territorial, and its relationship with an index of agonistic behavior. We found that even though, brain aromatase expression did not differ between types of males, pituitary cyp19a1b expression levels positively correlated with the index of agonistic behavior. This suggests a novel role of the pituitary in the regulation of social behavior by local estrogen synthesis.     

Generation of Cyp17iCre transgenic mice and their application to conditionally delete estrogen receptor alpha (Esr1) from the ovary and testis

A transgenic mouse line that expresses iCre under regulation of the Cytochrome P(450) 17alpha-hydroxylase/17, 20-lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report, we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell-specific manner.     

Characterization and evaluation of sex-specific expression of suppressors of cytokine signaling (SOCS)-1 and -3 in juvenile yellow perch (Perca flavescens) treated with lipopolysaccharide

The suppressor of cytokine signaling (SOCS) proteins are a family of intracellular proteins that are centrally involved with vertebrate growth, development and immunity via their effects as negative feed-back regulators of cytokine (and hormone) signaling. The genes for SOCS-1 & -3 were cloned, sequences analyzed and expression patterns examined in the commercially-important teleost, yellow perch (Perca flavescens). The deduced (mature) proteins for yellow perch (yp)SOCS-1 and (yp)SOCS-3 consist of 211 and 205 amino acids, respectively. Functional domains such as the Src homology-2 (SH2) and SOCS-box were present in ypSOCS-1 and ypSOCS-3 and these domains were well conserved between teleost species. Sequence analysis showed that ypSOCS-1 & -3 share highest homology (among similar teleost sequences), to the stickleback (Gasterosteus aculatus) SOCS-1 & -3 protein homologs. To investigate sex-specific expression of the ypSOCS-1 and ypSOCS-3 mRNAs, juvenile male and female yellow perch were immunologically challenged with a single injection (10?μg/g bw) of lipopolysaccharide (LPS) and tissues (gill, head kidney, kidney, liver and spleen) were sampled over a 48-h time-course. Quantitative real-time PCR analysis showed that ypSOCS-1 & -3 were expressed in all tissues examined and at all sampling time-points. LPS injection significantly induced ypSOCS-1 & -3 mRNA levels in gill, head kidney, liver, kidney and spleen, with maximal induction occurring at 6?h post-injection in each tissue. By 48-h post-injection, expression levels for ypSOCS-1 & -3 mRNAs approached, or reached, control levels in all tissues examined. While there were statistical interactions among variables (treatment, time and sex) for ypSOCS-1, we only found a main effect of sex on SOCS-3 mRNA expression in head kidney with higher copy numbers occurring in males than in females treated with LPS. Sexually-dimorphic expression of SOCS-1 or -3 mRNA has not been examined, or described, in a teleost. Our findings suggest the involvement of the SOCS genes in the yellow perch immune response and that differences among the sexes are evident and should be explored further.     

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.     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

Characterization of duplicated zebrafish cyp19 genes.

The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.     

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.     

Fish female-biased gene cyp19a1a leads to female antiviral response attenuation between sexes by autophagic degradation of MITA

From insects to mammals, both innate and adaptive immune response are usually higher in females than in males, with the sex chromosome and hormonal differences considered the main reasons. Here, we report that zebrafish cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a), an autosomal gene with female-biased expression, causes female fish to exhibit a lower antiviral response. First, we successfully constructed an infection model by intraperitoneal injection of spring viremia of carp virus (SVCV) into zebrafish (Danio rerio) and Carassius auratus herpesvirus (CaHV) in gibel carp (Carassius gibelio). Specifically, female fish were more vulnerable to viral infection than males, accompanied by a significantly weaker interferon (IFN) expression. After screening several candidates, cyp19a1a, which was highly expressed in female fish tissues, was selected for further analysis. The IFN expression and antiviral response were significantly higher in cyp19a1a^-/- than in cyp19a1a^+/+. Further investigation of the molecular mechanism revealed that Cyp19a1a targets mediator of IRF3 activation (MITA) for autophagic degradation. Interestingly, in the absence of MITA, Cyp19a1a alone could not elicit an autophagic response. Furthermore, the autophagy factor ATG14 (autophagy-related 14) was found interacted with Cyp19a1a to either promote or attenuate Cyp19a1a-mediated MITA degradation by either being overexpressed or knocked down, respectively. At the cellular level, both the normal and MITA-enhanced cellular antiviral responses were diminished by Cyp19a1a. These findings demonstrated a sex difference in the antiviral response based on a regulation mechanism controlled by a female-biased gene besides sex chromosome and hormonal differences, supplying the current understanding of sex differences in fish.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

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.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.