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1.
Genet Mol Res ; 15(2)2016 Apr 07.
Article in English | MEDLINE | ID: mdl-27173207

ABSTRACT

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARß common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.


Subject(s)
Fish Proteins/genetics , Perciformes/genetics , Receptors, Androgen/genetics , Animals , Cloning, Molecular , Estrogens/pharmacology , Female , Fish Proteins/chemistry , Fish Proteins/metabolism , Gonads/drug effects , Gonads/growth & development , Gonads/metabolism , Male , Methyltestosterone/pharmacology , Open Reading Frames , Perciformes/metabolism , Protein Domains , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism
2.
Genet Mol Res ; 14(4): 14056-65, 2015 Oct 30.
Article in English | MEDLINE | ID: mdl-26535720

ABSTRACT

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17ß-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 µg/L and 100 µg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.


Subject(s)
Carrier Proteins/biosynthesis , Estradiol/pharmacology , Invertebrate Hormones/biosynthesis , Penaeidae/drug effects , Phenols/pharmacology , Animals , Carrier Proteins/genetics , Estrogen Antagonists/pharmacology , Estrogens/metabolism , Female , Gene Expression/drug effects , Invertebrate Hormones/genetics , Ovary/drug effects , Ovary/metabolism , Penaeidae/genetics , Penaeidae/metabolism , Real-Time Polymerase Chain Reaction , Tamoxifen/pharmacology , Vitellogenesis/drug effects , Vitellogenins/metabolism
3.
Genet Mol Res ; 14(3): 10705-16, 2015 Sep 09.
Article in English | MEDLINE | ID: mdl-26400300

ABSTRACT

Many studies exist concerning the use of stem cells as delivery vehicles in gene therapy, expressing genes such as vascular endothelial growth factor 165 and hepatocyte growth factor. However, few reports regarding adipose tissue-derived stem cells (ADSCs) and the heme oxygenase 1 (HO-1) gene have been published. Therefore, we established a lentiviral vector encoding HO-1 and used this to infect ADSCs with the aim of producing therapeutic seed cells. In this study, ADSCs were isolated from mouse adipose tissue (AT), cultured, and identified according to the expression of antigens on their cell surface and their capacity for multilineage differentiation. A lentiviral vector encoding HO-1 was constructed, ADSCs were infected with this, and HO-1 protein expression was examined by western blotting. Our results show that ADSCs can be isolated from mouse AT, while DNA sequencing demonstrated that HO-1 was successfully transferred to the vector fused with GFP. Following 293T cell transfection, lentivirus titers were approximately 3 x 10(8) TU/mL. Fluorescence microscopy confirmed the expression of the HO-1 construct in lentivirus-infected ADSCs and the overexpression of HO-1 protein in these cells was verified by western blot. The production of ADSCs overexpressing HO-1 described in this study may aid in the development of a novel method for the treatment of asthma.


Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Genetic Vectors/chemistry , Heme Oxygenase-1/genetics , Lentivirus/genetics , Membrane Proteins/genetics , Stem Cells/metabolism , Adipocytes/cytology , Adipose Tissue/cytology , Animals , Cell Differentiation , Gene Expression , Genes, Reporter , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Heme Oxygenase-1/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Primary Cell Culture , Sequence Analysis, DNA , Stem Cells/cytology , Transfection
4.
Genet Mol Res ; 14(1): 2691-701, 2015 Mar 30.
Article in English | MEDLINE | ID: mdl-25867417

ABSTRACT

Nucleotide-binding oligomerization domain-containing protein-1 (NOD1) is a cytoplasmic pattern recognition receptor (PRR) and a key member of the NOD-like receptor (NLR) family. It has been reported that NLRs recognize a variety of microbial infections to induce the host innate immune response via modulation of NF-κB signaling. However, no reports on chicken NOD1 have been reported to date. In the current study, the full-length cDNA sequence of NOD1 was cloned. The complete open reading frame of NOD1 contains 2856 bp and encodes a 951 amino acid protein. Structurally, it is comprised of one caspase recruitment domain at the N-terminus, seven leucine-rich repeat regions at the C-terminus, and one NACHT domain between the N and C-termini. Phylogenetic analyses showed that chicken NOD1 clusters with duck and turkey. Furthermore, tissue-specific expression analyses of chicken NOD1 were performed using quantitative reverse transcription-PCR. NOD1 is widely distributed in various tissues, with the highest expression observed in testes. Finally, induced expression of chNOD1 and its associated adaptor molecule receptor-interacting protein 2, as well as the effector molecule NF-κB, was observed following S. enterica serovar Enteritidis infection. These findings highlight the important role of chicken NOD1 in response to pathogenic invasion. The present study is the first report of the cloning, expression, and functional analysis of chicken NOD1 and provides the foundation for future research on the structure and function of chicken NOD1.


Subject(s)
Avian Proteins/genetics , Chickens/genetics , Gene Expression Profiling , Nod1 Signaling Adaptor Protein/genetics , Salmonella Infections, Animal/genetics , Animals , Chickens/microbiology , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Host-Pathogen Interactions , Male , Molecular Sequence Data , NF-kappa B/genetics , Nod1 Signaling Adaptor Protein/classification , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Salmonella Infections, Animal/microbiology , Salmonella enteritidis/physiology , Sequence Analysis, DNA
5.
Genet Mol Res ; 13(2): 3275-82, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24841659

ABSTRACT

Chinese black-bone chickens are valued for the medicinal properties of their meat in traditional Chinese medicine. We investigated the genetic diversity and systematic evolution of Chinese black-bone chicken breeds. We sequenced the DNA of 520 bp of the mitochondrial cyt b gene of nine Chinese black-bone chicken breeds, including Silky chicken, Jinhu black-bone chicken, Jiangshan black-bone chicken, Yugan black-bone chicken, Wumeng black-bone chicken, Muchuan black-bone chicken, Xingwen black-bone chicken, Dehua black-bone chicken, and Yanjin black-bone chicken. We found 13 haplotypes. Haplotype and nucleotide diversity of the nine black-bone chicken breeds ranged from 0 to 0.78571 and 0.00081 to 0.00399, respectively. Genetic diversity was the richest in Jinhu black-bone chickens and the lowest in Yanjin black-bone chickens. Analysis of phylogenetic trees for all birds constructed based on hyplotypes indicated that the maternal origin of black-bone chickens is predominantly from three subspecies of red jungle fowl. These results provide basic data useful for protection of black-bone chickens and help determine the origin of domestic chickens.


Subject(s)
Breeding , Chickens/genetics , Genetic Variation , Meat , Animals , Animals, Domestic/genetics , DNA, Mitochondrial/genetics , Haplotypes , Medicine, Chinese Traditional , Phylogeny
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