After 24?h problem, a high-throughput RNA-seq technique was utilized to compare mRNA expression information between control and E2-treatment group. could induce luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and mRNA appearance in prepubertal lawn carp pituitary and and (5C7). Very similar estrogenic activities had been also within various other teleosts, such as croaker (8), Japanese eel (9), and goldfish (10). Except for LH, however, little is known about other E2-regulated genes in teleost pituitary. Physiological effects of estrogens are mediated by the classical nuclear estrogen receptors [nERs, estrogen receptor alpha (ER) and ER], which belong to the nuclear receptor superfamily users that act as nuclear transcription factors, binding to estrogen response elements within specific genes to alter their rate of transcription (11). Previous studies have reported that high levels of ER and ER were both expressed in human pituitary (12, 13). In the mean time, pituitary-specific knockout of ER could cause defects in both positive and negative estrogen feedback regulation of LH in mouse (4). In zebrafish, the three nER isoforms [ER, estrogen receptor beta 1 (ER1), and estrogen receptor beta 2 (ER2)] are all detected highly in the pituitary (7). Consistently, recent studies also reported that loss of the ER and ER could lead to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male (14). Further studies showed that E2 could bind with ER to induce LH secretion and synthesis at the pituitary level in prepubertal zebrafish (5, 6). These studies, as a whole, suggested that ERs played an important role in the teleost pituitary. In addition to the nERs, it has become obvious that estrogens also exert quick, non-genomic effects by altering different signaling pathways in both central nervous system and peripheral tissues (15). These non-genomic effects could mainly be mediated by non-classical membrane bound receptors such as G protein-coupled estrogen receptor (GPER) (16). In mammals, GPER has been recognized in the rat brain and pituitary, using immunohistochemistry and hybridization (17, 18). In addition, Rudolf and Kadokawa (19) found that GPER was recognized in bovine pituitary and might partially contribute to quick negative estradiol opinions of GnRH-induced LH secretion. In teleost, however, little is known about the functional role of GPER in the pituitary. To examine the pituitary actions of E2 in grass carp, the cDNAs of grass carp nERs and GPERs were cloned and their expression profile were characterized in brainCpituitary axis. Using primary culture of grass carp pituitary cells as a model, the effects of E2 on pituitary genes expression were examined by high-throughput RNA-seq technique. Then, using real-time PCR and fluorescence immunoassay (FIA), we further examined the direct effects of E2 on pituitary LH, FSH, and growth regulation by estrogen in breast malignancy 1 (GREB1) expression Rabbit Polyclonal to Cytochrome P450 2U1 in grass carp and and low quality reads from natural data. These high-quality clean reads were mapped to the grass carp genome3 using TopHat v2.0. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Prostaglandin E1 (PGE1) Gene expression levels were estimated by fragments per kilobase of transcript per million fragments (FPKM) mapped during different samples. Differentially expressed genes (DEGs) were recognized using the DESeq R package (1.10.1), which provided statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The values were adjusted using the Benjamini and Hochbergs approach for controlling the false discovery rate (FDR? ?0.01). Gene expressions with fold switch (FC)? ?1.5 and an adjusted value? ?0.05 found by DESeq were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the GOseq R packages based Wallenius non-central hyper-geometric distribution for adjusting gene length bias in DEGs (24). Real-Time Quantitative PCR Validation Grass carp pituitary cells were seeded in poly-d-lysin coated 24-well culture plates at a.After that, unbound second antibody was removed by decanting and a 100-l volume of QuantaBlu? Fluorogenic Peroxidase Substrate (Thermo Fisher Scientific) was then added into individual wells for transmission development. grass carp pituitary cells as model, high-throughput RNA-seq was used to examine the E2-induced differentially expressed genes (DEGs). Transcriptomic analysis showed that E2 could significantly upregulate the expression of 28 genes in grass carp pituitary cells, which were characterized into different functions including reproduction, gonad development, and central nervous system development. Further studies confirmed that E2 could induce luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and mRNA expression in prepubertal grass carp pituitary and and (5C7). Comparable estrogenic actions were also found in other teleosts, such as croaker (8), Japanese eel (9), and goldfish (10). Except for LH, however, little is known about other E2-regulated genes in teleost pituitary. Physiological effects of estrogens are mediated by the classical nuclear estrogen receptors [nERs, estrogen receptor alpha (ER) and ER], which belong to the nuclear receptor superfamily users that act as nuclear transcription factors, binding to estrogen response elements within specific genes to alter their rate of transcription (11). Previous studies have reported that high levels of ER and ER were both expressed in human pituitary (12, 13). In the mean time, pituitary-specific knockout of ER could cause defects in both positive and negative estrogen feedback regulation of LH in mouse (4). In zebrafish, the three nER isoforms [ER, estrogen receptor beta 1 (ER1), and estrogen receptor beta 2 (ER2)] are all detected highly in the pituitary (7). Consistently, recent studies also reported that loss of the ER and ER could lead to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male (14). Further studies showed that E2 could bind with ER to induce LH secretion and synthesis at the pituitary level in prepubertal zebrafish (5, 6). These studies, as a whole, suggested that ERs played an important role in the teleost pituitary. In addition to the nERs, it has become obvious that estrogens also exert quick, non-genomic effects by altering different signaling pathways in both central nervous system and peripheral tissues (15). These non-genomic effects could mainly be mediated by non-classical membrane bound receptors such as G protein-coupled estrogen receptor (GPER) (16). In mammals, GPER has been recognized in the rat brain and pituitary, using immunohistochemistry and hybridization (17, 18). In addition, Rudolf and Kadokawa (19) found that GPER was recognized in bovine pituitary and might partially contribute to quick negative estradiol opinions of GnRH-induced LH secretion. In teleost, however, little is known about the functional role of GPER in the pituitary. To examine the pituitary actions of E2 Prostaglandin E1 (PGE1) in grass carp, the cDNAs of grass carp nERs and GPERs were cloned and their expression profile were characterized in brainCpituitary axis. Using main culture of grass carp pituitary cells as a model, the effects of E2 on pituitary genes expression were examined by high-throughput RNA-seq technique. Then, using real-time PCR and fluorescence immunoassay (FIA), we further examined the direct effects of E2 on pituitary LH, FSH, and growth regulation by estrogen in breast malignancy 1 (GREB1) expression in grass carp and and low quality reads from natural data. These high-quality clean reads were mapped to the grass carp genome3 using TopHat v2.0. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments (FPKM) mapped during different samples. Differentially expressed Prostaglandin E1 (PGE1) genes (DEGs) were recognized using the DESeq R package (1.10.1), which provided statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The values were adjusted using the Benjamini and Hochbergs approach for controlling the false discovery.Data presented are expressed as mean??SEM (and GPER coupled with AC/cAMP/PKA, PLC/IP3, and Ca2+ cascades. highly detected in grass carp pituitary, which suggested that E2 should play an important role in grass carp pituitary. Using main cultured grass carp pituitary cells as model, high-throughput RNA-seq was used to examine the E2-induced differentially expressed genes (DEGs). Transcriptomic analysis showed that E2 could significantly upregulate the expression of 28 genes in grass carp pituitary Prostaglandin E1 (PGE1) cells, which were characterized into different functions including reproduction, gonad development, and central nervous system development. Further studies confirmed that E2 could induce luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and mRNA expression in prepubertal grass carp pituitary and and (5C7). Comparable estrogenic actions were also found in other teleosts, such as croaker (8), Japanese eel (9), and goldfish (10). Except for LH, however, little is known about other E2-regulated genes in teleost pituitary. Physiological effects of estrogens are mediated by the classical nuclear estrogen receptors [nERs, estrogen receptor alpha (ER) and ER], which belong to the nuclear receptor superfamily users that act as nuclear transcription factors, binding to estrogen response elements within specific genes to alter their rate of transcription (11). Previous studies have reported that high levels of ER and ER were both expressed in human pituitary (12, 13). Meanwhile, pituitary-specific knockout of ER could cause defects in both positive and negative estrogen feedback regulation of LH in mouse (4). In zebrafish, the three nER isoforms [ER, estrogen receptor beta 1 (ER1), and estrogen receptor beta 2 (ER2)] are all detected highly in the pituitary (7). Consistently, recent studies also reported that loss of the ER and ER could lead to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male (14). Further studies showed that E2 could bind with ER to induce LH secretion and synthesis at the pituitary level in prepubertal zebrafish (5, 6). These studies, as a whole, suggested that ERs played an important role in the teleost pituitary. In addition to the nERs, it has become clear that estrogens also exert rapid, non-genomic effects by altering different signaling pathways in both central nervous system and peripheral tissues (15). These non-genomic effects could mainly be mediated by non-classical membrane bound receptors such as G protein-coupled estrogen receptor (GPER) (16). In mammals, GPER has been identified in the rat brain and pituitary, using immunohistochemistry and hybridization (17, 18). In addition, Rudolf and Kadokawa (19) found that GPER was identified in bovine pituitary and might partially contribute to rapid negative estradiol feedback of GnRH-induced LH secretion. In teleost, however, little is known about the functional role of GPER in the pituitary. To examine the pituitary actions of E2 in grass carp, the cDNAs of grass carp nERs and GPERs were cloned and their expression profile were characterized in brainCpituitary axis. Using primary culture of grass carp pituitary cells as a model, the effects of E2 on pituitary genes expression were examined by high-throughput RNA-seq technique. Then, using real-time PCR and fluorescence immunoassay (FIA), we further examined the direct effects of E2 on pituitary LH, FSH, and growth regulation by estrogen in breast cancer 1 (GREB1) expression in grass carp and and low quality reads from raw data. These high-quality clean reads were mapped to the grass carp genome3 using TopHat v2.0. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments (FPKM) mapped during different samples. Differentially expressed genes (DEGs) were identified using the DESeq R package (1.10.1), which provided statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The values were adjusted using the Benjamini and Hochbergs approach for controlling the false discovery rate (FDR? ?0.01). Gene expressions with fold change (FC)? ?1.5 and an adjusted value? ?0.05 found by DESeq were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of the.
HDACs