Expression pattern of GLUT 1, 5, 8 and citrate synthase transcripts in buffalo ( Bubalus bubalis ) preimplantation embryos produced in vitro and derived in vivo

2020 ◽  
Vol 55 (10) ◽  
pp. 1362-1370
Author(s):  
Gutulla Taru Sharma ◽  
Amar Nath ◽  
Shiv Prasad ◽  
Sumit Singhal ◽  
Vikash Chandra ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Citrate Synthase ◽  
Bubalus Bubalis ◽  
Preimplantation Embryos ◽  
Glut 1

167 EXPRESSION PATTERN OF GAP JUNCTIONAL CONNEXINS DURING IN VITRO AND IN VIVO PREIMPLANTATION EMBRYO DEVELOPMENT IN BOVINE

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
S. Balasubramanian ◽  
W. J. Son ◽  
B. Mohana Kumar ◽  
Y. I. Yang ◽  
B. J. Jeon ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Expression Pattern ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
O2 Concentration ◽  
Gene Transcripts ◽  
High O2 ◽  
Gap Junctional

During preimplantation development, several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. The present investigations were (i) to compare the expression pattern of a panel of gap junctional connexin (Cx) gene transcripts from in vitro-produced (IVP) under low (5%) and high (20%) oxygen (O2) concentrations and in vivo-derived (IVD) bovine embryos during various preimplantation stages, and (ii) to evaluate the expression of the same set of gene transcripts in blastocysts derived from IVP (low O2 concentration) and in vivo embryos following a conventional cryopreservation method using 1.5 m ethylene glycol (EG) (Hasler et al. 1997 Theriogenology 48, 563–579). Cumulus-oocyte complexes were matured in TCM199 supplemented with 10% FBS and hormones for 22 h at 39�C, 5% CO2 in air, and then inseminated and cultured in SOF medium for 7 days. IVD embryos were collected from 18 superovulated and artificially inseminated cows. In Experiment I, five pooled embryos from each developmental stage (2-, 4-, 8-, 16-cell, morula, and blastocyst) and embryo source (IVP under low and high O2 concentrations and IVD) and, in Experiment II, Day 7 IVP (low O2 concentration) and IVD blastocysts following cryopreservation and storage for at least 1 week were used for analyzing the expression pattern of gap junctional connexin (Cx30, Cx31, Cx32, Cx36, Cx43, and Cx45) gene transcripts using real-time RT-PCR (four replicates). Normalization of mRNAs at each developmental stage of bovine preimplantation embryos was performed by employing a similar amount of RNA at the reverse transcription (RT) step. This was followed by analyzing by qRT-PCR the target genes using GAPDH as a reference gene. Significant differences in gene expression were analyzed byANOVA and Student's t-test. Relative abundances (RA) of Cx30, Cx31, and Cx32 of IVD embryos were significantly (P < 0.05) higher at all stages compared to that of IVP embryos, except at the blastocyst stage for Cx32. Differences in Cx36 and Cx45 were observed at all stages, with the levels being higher (P < 0.05) in IVD than in IVP embryos. However, the differences at the 4-cell stage between the two embryo sources were not significant. The RA of Cx43 transcript in IVD embryos at 4- and 16-cell stages was higher (P < 0.05) than in IVP embryos, but did not differ at the 2-cell stage. Furthermore, there were differences at the 8-cell and blastocyst stages among IVD, and low and high O2 IVP embryos. Following cryopreservation, RA of all analyzed connexin transcripts of IVD blastocysts were significantly (P < 0.05) higher than that of the low O2 concentration. However, the expression levels in both embryo sources were lower compared to blastocysts before cryopreservation, except for Cx36. To the best of our knowledge, this is the first report on the differences in the expression pattern of a panel of gap junctional connexin gene transcripts during the key developmental stages of IVP embryos with direct comparisons of IVD counterparts.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

scholarly journals ACTN1 Supports Tumor Growth by Inhibiting Hippo Signaling in Hepatocellular Carcinoma

2020 ◽  
Author(s):  
Qian Chen ◽  
Xiao-Wei Zhou ◽  
Ai-Jun Zhang ◽  
Kang He
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Expression Pattern ◽  
Cytoskeletal Proteins ◽  
Gene Set Enrichment Analysis ◽  
Hippo Signaling ◽  
Hcc Cells

Abstract Background: Alpha actinins (ACTNs) are major cytoskeletal proteins and exhibit many non-muscle functions. Emerging evidence have uncovered the regulatory role of ACTNs in tumorigenesis, however, the expression pattern, biological functions, and underlying mechanism of ACTN1 in hepatocellular carcinoma (HCC) remain largely unexplored.Methods: Immunohistochemical analysis of a HCC tissue microarray (n = 157) was performed to determine the expression pattern and prognostic value of ACTN1 in HCC. In vitro loss-of-function study in HCC cells were carried out to investigate ACTN1 knockdown on cell proliferation. In vivo subcutaneous xenograft model and intrahepatic transplantation model were generated to decipher the contribution of ACTN1 in the tumor growth of HCC. Gene set enrichment analysis, quantitative real-time PCR, Co-immunoprecipitation, immunofluorescence and western blotting were performed to identify the underlying molecular mechanism.Results: It was found that ACTN1 was significantly upregulated in HCC tissues and closely related to llpha-fetoprotein level, tumor thrombus, tumor size, TNM stage and patient prognoses. Knockdown of ACTN1 suppressed in vitro cell proliferation and in vivo tumor growth of HCC cells. Mechanistically, knockdown of ACTN1 increased Hippo signaling pathway activity and decrease Rho GTPases activities. Mechanistically, ACTN1 could competitively interact with MOB1 and decrease the phosphorylation of LATS1 and YAP. The growth-promoting effect induced by ACTN1 was significantly abrogated by pharmacological inhibition of YAP with verteporfin or super-TDU.Conclusions: ACTN1 is highly expressed in HCC tissues and acts as a tumor promoter by suppressing Hippo signaling via physical interaction with MOB1. ACTN1 may serve as a potential prognostic marker and therapeutic target for HCC.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals Sex determining of cat embryo and some feline species

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Francesca Ciani ◽  
Natascia Cocchia ◽  
Maria Rizzo ◽  
Patrizia Ponzio ◽  
Gennaro Tortora ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Positive Control ◽  
Domestic Cat ◽  
Preimplantation Embryos ◽  
Pcr Analysis ◽  
Domestic Cats ◽  
Hair Samples ◽  
Wild Felids

SummarySex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, β-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.

scholarly journals Birth of a Live Cria After Transfer of a Vitrified-Warmed Alpaca (Vicugna pacos) Preimplantation Embryo

2020 ◽  
Vol 7 ◽  
Author(s):  
Jennifer C. Lutz ◽  
Susan L. Johnson ◽  
Kimberly J. Duprey ◽  
Paul J. Taylor ◽  
Henry William Vivanco-Mackie ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Preimplantation Embryos ◽  
Embryo Survival ◽  
Important Species ◽  
Vicugna Pacos ◽  
Improvement Programs ◽  
The One ◽  
Humidified Air

The alpaca (Vicugna pacos) is an important species for the production of fiber and food. Genetic improvement programs for alpacas have been hindered, however, by the lack of field-practical techniques for artificial insemination and embryo transfer. In particular, successful techniques for the cryopreservation of alpaca preimplantation embryos have not been reported previously. The objective of this study was to develop a field-practical and efficacious technique for cryopreservation of alpaca preimplantation embryos using a modification of a vitrification protocol originally devised for horses and adapted for dromedary camels. Four naturally cycling non-superovulated Huacaya females serving as embryo donors were mated to males of proven fertility. Donors received 30 μg of gonadorelin at the time of breeding, and embryos were non-surgically recovered 7 days after mating. Recovered embryos (n = 4) were placed individually through a series of three vitrification solutions at 20°C (VS1: 1.4 M glycerol; VS2: 1.4 M glycerol + 3.6 M ethylene glycol; VS3: 3.4 M glycerol + 4.6 M ethylene glycol) before loading into an open-pulled straw (OPS) and plunging directly into liquid nitrogen for storage. At warming, each individual embryo was sequentially placed through warming solutions (WS1: 0.5 M galactose at 37°C; WS2: 0.25 M galactose at 20°C), and warmed embryos were incubated at 37°C in 5% CO2 in humidified air for 20–22 h in 1 ml Syngro® holding medium supplemented with 10% (v/v) alpaca serum to perform an initial in vitro assessment of post-warming viability. Embryos whose diameter increased during culture (n = 2) were transferred individually into synchronous recipients, whereas embryos that did not grow (n = 2) were transferred together into a single recipient to perform an in vivo assessment of post-warming viability. Initial pregnancy detection was performed ultrasonographically 29 days post-transfer when fetal heartbeat could be detected, and one of three recipients was pregnant (25% embryo survival rate). On November 13, 2019, the one pregnant recipient delivered what is believed to be the world's first cria produced from a vitrified-warmed alpaca embryo.

scholarly journals Selection of theIn VitroCulture Media Influences mRNA Expression of Hedgehog Genes,Il-6,and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
N. Pfeifer ◽  
D. M. Baston-Büst ◽  
J. Hirchenhain ◽  
U. Friebe-Hoffmann ◽  
D. T. Rein ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mrna Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Reactive Oxygen ◽  
Female Reproductive Tract ◽  
Preimplantation Embryos ◽  
Oxygen Species

Background. The aim of this paper was to determine the influence of differentin vitroculture media on mRNA expression of Hedgehog genes,il-6,and important genes regarding reactive oxygen species in single mouse embryos.Methods. Reverse transcription of single embryos either culturedin vitrofrom day 0.5 until 3.5 (COOK’s Cleavage medium or Vitrolife’s G-1 PLUS medium) orin vivountil day 3.5post coitum. PCR was carried out forβ-actinfollowed by nested-PCR forshh, ihh, il-6, nox, gpx4, gpx1,andprdx2.Results. The number of murine blastocysts cultured in COOK medium which expressedil-6, gpx4, gpx1,andprdx2mRNA differed significantly compared to thein vivogroup. Except fornox, the mRNA profile of the Vitrolife media group embryos varied significantly from thein vivoones regarding the number of blastocysts expressing the mRNA ofshh, ihh, il-6, gpx4, gpx1andprdx2.Conclusions. The present study shows that differentin vitroculture media lead to different mRNA expression profiles during early development. Even the newly developedin vitroculture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P.N. Moreira ◽  
R. Fernández-Gonzalez ◽  
M.A. Ramirez ◽  
M. Pérez-Crespo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Differential Effects ◽  
Glut 1 ◽  
Mouse Somatic Cell

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

scholarly journals Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

2007 ◽  
Vol 27 (8) ◽  
pp. 3123-3130 ◽  
Author(s):  
Klaus Fortschegger ◽  
Bettina Wagner ◽  
Regina Voglauer ◽  
Hermann Katinger ◽  
Maria Sibilia ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Replicative Senescence ◽  
Inner Cell Mass ◽  
Umbilical Vein ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Proliferative Potential ◽  
Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

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