Identification of metaphase II-specific gene transcripts in porcine oocytes and their expression in early stage embryos

2005 ◽  
Vol 17 (6) ◽  
pp. 625 ◽  
Author(s):  
Xiang-Shun Cui ◽  
Hyuk Song ◽  
Nam-Hyung Kim
Keyword(s):  
Oocyte Maturation ◽  
Early Stage ◽  
Sequence Similarity ◽  
Germinal Vesicle ◽  
Specific Gene ◽  
Early Cleavage ◽  
Reverse Transcription Pcr ◽  
Gene Transcripts ◽  
Polymerase Chain ◽  
Inducible Protein

Annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) was used to identify the genes that are specifically or prominently expressed in porcine oocytes at the metaphase II (MII) and germinal vesicle (GV) stages. By using 60 ACPs, 13 differentially expressed genes (DEGs) were identified. The cloned genes or expressed sequence tags (ESTs) showed sequence similarity with known genes or ESTs of other species in GenBank. The mRNA expression during oocyte maturation and early embryonic development in both pigs and mice of four of these genes (namely transcription factor TZP, annexin A2, hypoxia-inducible protein 2, and ATPase 6) was further characterised by real-time quantitative reverse transcription–PCR. All four genes were markedly upregulated in pig and mouse MII oocytes compared with GV-stage oocytes. The expression levels of the four genes decreased gradually during early cleavage. Thus, these genes may play important roles during oocyte maturation and/or early cleavage in mammals. Although the detailed functions of these genes remain to be determined, their identification in the present study provides insights into meiotic maturation and fertilisation.

pha-4 is Ce-fkh-1, a fork head/HNF-3alpha, beta, gamma homolog that functions in organogenesis of the C. elegans pharynx

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2171-2180 ◽  
Author(s):  
J.M. Kalb ◽  
K.K. Lau ◽  
B. Goszczynski ◽  
T. Fukushige ◽  
D. Moons ◽  
...  
Keyword(s):  
Early Stage ◽  
Sequence Similarity ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Specific Gene ◽  
Gata Factor ◽  
Enhancer Element ◽  
C Elegans ◽  
Fork Head ◽  
Organ Identity

The C. elegans Ce-fkh-1 gene has been cloned on the basis of its sequence similarity to the winged-helix DNA binding domain of the Drosophila fork head and mammalian HNF-3alpha, beta, gamma genes, and mutations in the zygotically active pha-4 gene have been shown to block formation of the pharynx (and rectum) at an early stage in embryogenesis. In the present paper, we show that Ce-fkh-1 and pha-4 are the same gene. We show that PHA-4 protein is present in nuclei of essentially all pharyngeal cells, of all five cell types. PHA-4 protein first appears close to the point at which a cell lineage will produce only pharyngeal cells, independently of cell type. We show that PHA-4 binds directly to a ‘pan-pharyngeal enhancer element’ previously identified in the promoter of the pharyngeal myosin myo-2 gene; in transgenic embryos, ectopic PHA-4 activates ectopic myo-2 expression. We also show that ectopic PHA-4 can activate ectopic expression of the ceh-22 gene, a pharyngeal-specific NK-2-type homeodomain protein previously shown to bind a muscle-specific enhancer near the PHA-4 binding site in the myo-2 promoter. We propose that it is the combination of pha-4 and regulatory molecules such as ceh-22 that produces the specific gene expression patterns during pharynx development. Overall, pha-4 can be described as an ‘organ identity factor’, completely necessary for organ formation, present in all cells of the organ from the earliest stages, capable of integrating upstream developmental pathways (in this case, the two distinct pathways that produce the anterior and posterior pharynx) and participating directly in the transcriptional regulation of organ specific genes. Finally, we note that the distribution of PHA-4 protein in C. elegans embryos is remarkably similar to the distribution of the fork head protein in Drosophila embryos: high levels in the foregut/pharynx and hindgut/rectum; low levels in the gut proper. Moreover, we show that pha-4 expression in the C. elegans gut is regulated by elt-2, a C. elegans gut-specific GATA-factor and possible homolog of the Drosophila gene serpent, which influences fork head expression in the fly gut. Overall, our results provide evidence for a highly conserved pathway regulating formation of the digestive tract in all (triploblastic) metazoa.

Nlrp4g is an oocyte-specific gene but is not required for oocyte maturation in the mouse

2014 ◽  
Vol 26 (5) ◽  
pp. 758 ◽  
Author(s):  
Hui Peng ◽  
Wenchang Zhang ◽  
Tianfang Xiao ◽  
Yong Zhang
Keyword(s):  
Electron Microscopy ◽  
Oocyte Maturation ◽  
In Situ Hybridisation ◽  
Gene Expression Pattern ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Immunogold Electron Microscopy ◽  
Specific Gene ◽  
Protein Distribution

The Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. The aim of the present study was to analyse the Nlrp4g gene expression pattern, protein distribution and function in mouse oocyte maturation. Quantitative real-time polymerase chain reaction and in situ hybridisation were performed on Nlrp4g mRNA. Western blotting, immunohistochemistry and immunofluorescence were used to assess expression at the protein level. Confocal and immunogold electron microscopy analyses and RNA interference approach were used to determine the location of the NLRP4G protein and inhibit Nlrp4g function specifically in mouse germinal vesicle oocytes, respectively. Nlrp4g transcripts and proteins (~85 kDa) are specifically expressed in mouse ovaries, restricted to the oocytes at various follicular stages and decline with oocyte aging. There is a marked decline in transcript levels in preimplantation embryos before zygotic genome activation, but the protein remains present through to the blastocyst stage. Confocal microscopy demonstrated that this protein is localised in the cytoplasm. Immunogold electron microscopy further confirmed that NLRP4G protein was present in the cytosol rather than in oocyte cytoplasmic organelles. Furthermore, knockdown of Nlrp4g in germinal vesicle oocytes did not affect oocyte maturation. These results provide the first evidence that Nlrp4g is an oocyte-specific gene but dispensable for oocyte maturation, suggesting that this gene may play roles in mouse oogenesis and/or preimplantation development.

Maternally derived transcripts: identification and characterisation during oocyte maturation and early cleavage

2007 ◽  
Vol 19 (1) ◽  
pp. 25 ◽  
Author(s):  
Xiang-Shun Cui ◽  
Nam-Hyung Kim
Keyword(s):  
Oocyte Maturation ◽  
Gene Knockout ◽  
Tail Length ◽  
Specific Gene ◽  
Early Cleavage ◽  
Suppression Subtractive Hybridisation ◽  
Early Embryos ◽  
Gene Functions ◽  
Recent Developments ◽  
Subtractive Hybridisation

The identification and characterisation of differentially regulated genes in oocytes and early embryos are required to understand the mechanisms involved in maturation, fertilisation, early cleavage and even long-term development. Several methods, including reverse transcription–polymerase chain reaction-based suppression subtractive hybridisation, differential display and cDNA microarray, have been applied to identify maternally derived genes in mammalian oocytes. However, conventional gene-knockout experiments to determine specific gene functions are labour intensive and inefficient. Recent developments include the use of RNA interference techniques to establish specific gene functions in mammalian oocytes and early embryos. Regulation of the poly(A) tail length is a major factor in controlling the activities of maternal transcripts in mammals. Further studies are required to clarify the mechanisms by which expression levels of maternally derived transcripts are regulated. In the present review, we focus on the identification and functions of the differentially expressed transcripts during oocyte maturation, fertilisation and early cleavage.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

GB Virus C/Hepatitis G Virus Isolates in Japanese Haemophiliacs and their Origins

1998 ◽  
Vol 80 (08) ◽  
pp. 242-245 ◽  
Author(s):  
Yoshihide Fukuda ◽  
Tetsuo Hayakawa ◽  
Junki Takamatsu ◽  
Hidehiko Saito ◽  
Hiroaki Okamoto ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
West African ◽  
Blood Products ◽  
Hepatitis G Virus ◽  
Gb Virus C ◽  
Route Of Transmission ◽  
Polymerase Chain ◽  
Hepatitis G ◽  
Virus C ◽  
Virus Isolates

SummaryJapanese haemophiliacs have been at high risk for infection with parenterally-transmissible viruses through the use of blood products, especially imported ones. Recently, novel transfusion-transmissible virus, GB virus C (GBV-C)/hepatitis G virus (HGV) were isolated. We investigated the origin and route of transmission of GBV-C/HGV isolates in haemophiliacs in Japan. GBV-C/HGV RNA was measured by nested reverse transcription polymerase chain reaction in 91 Japanese haemophiliacs. Phylogenetic analysis and genotypic grouping of GBV-C/HGV isolates in Japanese haemophiliacs were performed based on sequences in the 5’ untranslated region, and the characteristics were compared with those of reported isolates. GBV-C/HGV infection was present in 19 of 91 haemophiliacs (20.9%). Sequence analysis showed that 15 of the 19 isolates (78.9%) showed sequence similarity to a group in which mainly West African isolates have been reported. The other 4 isolates (21.1%) showed sequence similarity to Asian isolates. None of the GBV-C/HGV isolates showed sequences similar to those generally found in isolates from USA and Europe. The majority of GBV-C/HGV isolates found in Japanese haemophiliacs who are considered to have been infected by imported blood products were similar to those detected in West Africa.

PLCz-induced Ca2+ oscillations are enhanced after germinal vesicle breakdown during mouse oocyte maturation

2016 ◽  
Author(s):  
Jessica Sanders ◽  
Ethan Bateson ◽  
Yuansong Yu ◽  
Michail Nomikos ◽  
Antony Lai ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown

scholarly journals Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 156
Author(s):  
Mohammad Al Hasan ◽  
Patricia E. Martin ◽  
Xinhua Shu ◽  
Steven Patterson ◽  
Chris Bartholomew
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Type Iii Collagen ◽  
Type Iii ◽  
Actin Stress Fibre ◽  
Matrix Gene ◽  
Gene Transcripts ◽  
Polymerase Chain ◽  
Oil Red O Staining ◽  
Oil Red O

GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.

scholarly journals Early Confirmation of Mycoplasma pneumoniae Infection by Two Short-Term Serologic IgM Examination

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 353
Author(s):  
Ha Eun Jeon ◽  
Hyun Mi Kang ◽  
Eun Ae Yang ◽  
Hye Young Han ◽  
Seung Beom Han ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Early Stage ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Short Term ◽  
Serologic Tests ◽  
Polymerase Chain ◽  
Positive Correlations ◽  
Patient Selection Bias ◽  
Mycoplasma Pneumoniae Infection

The aim of the present study is to re-evaluate the clinical application of two-times serologic immunoglobulin M (IgM) tests using microparticle agglutination assay (MAA), an enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) assay in diagnosing Mycoplasma pneumoniae (MP) infection. A retrospective analysis of 62 children with MP pneumonia during a recent epidemic (2019–2020) was conducted. The MAA and ELISA immunoglobulin M (IgM) and IgG measurements were conducted twice at admission and around discharge, and MP PCR once at presentation. Diagnostic rates in each test were calculated at presentation and at discharge. The seroconverters were 39% (24/62) of patients tested by MAA and 29% (18/62) by ELISA. At presentation, the diagnostic positive rates of MAA, ELISA, and PCR tests were 61%, 71%, and 52%, respectively. After the second examination, the rates were 100% in both serologic tests. There were positive correlations between the titers of MAA and the IgM values of ELISA. The single serologic IgM or PCR tests had limitations to select patients infected with MP in the early stage. The short-term, paired IgM serologic tests during hospitalization can reduce patient-selection bias in MP infection studies.

Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 947-957 ◽  
Author(s):  
John G Jelesko ◽  
Kristy Carter ◽  
Whitney Thompson ◽  
Yuki Kinoshita ◽  
Wilhelm Gruissem
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Meiotic Recombination ◽  
Sequence Similarity ◽  
Specific Gene ◽  
High Sequence Similarity ◽  
Paralogous Genes ◽  
Chimeric Genes ◽  
Unequal Recombination ◽  
Sister Chromatids

Abstract Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

Sign in / Sign up

Export Citation Format

Share Document