scholarly journals Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 455-472 ◽  
Author(s):  
Tracy M Clement ◽  
Matthew D Anway ◽  
Mehmet Uzumcu ◽  
Michael K Skinner
Keyword(s):  
Gene Expression ◽  
Sex Determination ◽  
Candidate Genes ◽  
Expression Profiles ◽  
Gonadal Development ◽  
Developmental Period ◽  
Fold Change ◽  
Ovary Development ◽  
Testis Development

Gene expression profiles during sex determination and gonadal differentiation were investigated to identify new potential regulatory factors. Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cordsin vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13–E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13–E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant.

scholarly journals IFIH1 Contributes to M1 Macrophage Polarization in ARDS

2021 ◽  
Vol 11 ◽  
Author(s):  
Shi Zhang ◽  
Cuilin Chu ◽  
Zongsheng Wu ◽  
Feng Liu ◽  
Jianfeng Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Expression Profiles ◽  
Macrophage Polarization ◽  
Gene Expression Profiles ◽  
Gene Set Enrichment Analysis ◽  
Expression Vectors ◽  
Molecular Control ◽  
M1 Macrophage

Accumulated evidence has demonstrated that the macrophage phenotypic switch from M0 to M1 is crucial in the initiation of the inflammatory process of acute respiratory distress syndrome (ARDS). Better insight into the molecular control of M1 macrophages in ARDS may identify potential therapeutic targets. In the current study, 36 candidate genes associated with the severity of ARDS and simultaneously involved in M1-polarized macrophages were first screened through a weighted network algorithm on all gene expression profiles from the 26 ARDS patients and empirical Bayes analysis on the gene expression profiles of macrophages. STAT1, IFIH1, GBP1, IFIT3, and IRF1 were subsequently identified as hub genes according to connectivity degree analysis and multiple external validations. Among these candidate genes, IFIH1 had the strongest connection with ARDS through the RobustRankAggreg algorithm. It was selected as a crucial gene for further investigation. For in vitro validation, the RAW264.7 cell line and BMDMs were transfected with shIFIH1 lentivirus and plasmid expression vectors of IFIH1. Cellular experimental studies further confirmed that IFIH1 was a novel regulator for promoting M1 macrophage polarization. Moreover, gene set enrichment analysis (GSEA) and in vitro validations indicated that IFIH1 regulated M1 polarization by activating IRF3. In addition, previous studies demonstrated that activation of IFIH1-IRF3 was stimulated by viral RNAs or RNA mimics. Surprisingly, the current study found that LPS could also induce IFIH1-IRF3 activation via a MyD88-dependent mechanism. We also found that only IFIH1 expression without LPS or RNA mimic stimulation could not affect IRF3 activation and M1 macrophage polarization. These findings were validated on two types of macrophages, RAW264.7 cells and BMDMs, which expanded the knowledge on the inflammatory roles of IFIH1 and IRF3, suggesting IFIH1 as a potential target for ARDS treatment.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

Εξέλιξη και βελτίωση της τεχνικής βιοψίας βλαστοκύστεων για μοριακές αναλύσεις και εκτίμηση της επιτυχίας εμφύτευσης και εγκυμοσύνης κατά την εξωσωματική γονιμοποίηση σε σχέση με το μοριακό υπόστρωμα

2013 ◽  
Author(s):  
Γεωργία Κόκκαλη
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Clinical Practice ◽  
Expression Profiles ◽  
Chain Reaction ◽  
Trophectoderm Cells ◽  
Monogenic Diseases ◽  
Blastocyst Biopsy ◽  
Polymerase Chain

IntroductionOne of the most difficult aspects in assisted reproductive technology (ART) is the selection of asuitable embryo for transfer to the patient’s uterus, in order to achieve implantation anddevelopment to term. This study was based on the hypothesis that preimplantation embryosmay have different gene expression profiles that characterize their ability to implant in theuterus and develop to a healthy baby at term.The main aim of this study was to investigate molecular markers associated with developmentalcompetence and successful implantation in ART. The primary aim of the study was to developand optimize a blastocyst biopsy method, suitable for application in clinical practice. Thesecondary aim of the study was to investigate the gene expression of beta Human ChorionicGonadotropin (CGβ) in blastocysts and correlate it with their morphology. Previously to thecurrent study, blastocyst biopsy was not implemented in clinical practice and no prior researchon the existence, quantification and standardization of transcripts of CGβ has been performedin blastocysts.MethodologyThe methodology for trophectoderm cell biopsy from blastocysts was developed and optimizedprimary to be a safe technique for the embryo and secondary to ensure biopsy of a sufficientnumber of cells, in order to allow the application of multiple molecular analyses. The blastocystbiopsy method involved three steps: A., opening of a hole in the zona pellucida using lowfrequency laser, B., blastocyst culture to allow trophectoderm cells to herniate from the holeand C., trophectoderm cell dissection of the blastocyst mass by laser ablation.The methodology for the investigation of CGβ gene expression in blastocysts, included RNAisolation, cDNA synthesis, amplification and quantification of CGβ transcripts. Because CGβ isencoded by a cluster of homologous genes (CGβ1, CGβ2, CGβ3, CGβ5, CGβ7, CGβ8),methodology was designed considering the homology between them into groups (A: CGβ1,CGβ2 and B: CGβ3, CGβ5, CGβ7, CGβ8). For group A, real time polymerase chain reaction (RealTime PCR, RT-PCR) was applied and then transcripts were identified using restriction enzymedigestion. For group B, nested polymerase chain reaction (Nested-PCR) was used incombination with polymerase chain reaction temperature decreasing hybridization (Touch-downPCR). Following amplification, the products were sequenced (DNA sequencing) for theiridentification.ResultsThe biopsy technique did not appear to impact on the blastocyst’s ability to reform a blastocoelecavity and continue to grow and hatch from the zona pellucida, as it was shown followingfurther in vitro culture. No blastocyst showed signs of morphological damage at the lightmicroscopic level. Blastocyst biopsy was applied in clinical practice in two steps: A., 49 couples undergoing IVF had a biopsy in 153 blastocysts. The implantation rate per blastocysttransferred was 34.3% and lead to 23 full-term pregnancies (46.9%) with 37 babies born. B.,24 couples undergoing IVF for PGD of monogenic diseases had biopsy in 144 blastocysts. Thediagnosis success rate was 93%, the implantation rate per blastocyst transferred was 40% andlead to 11 full-term pregnancies (50%) with 15 term newborns. Then, a randomized pilot studywas conducted with the aim to evaluate and compare the diagnosis and implantation successrates between patients undergoing blastomere biopsy and blastocyst transfer and those havingtrophectoderm biopsy and blastocyst transfer for the diagnosis of monogenic diseases. Theresults showed that the diagnosis success rate was superior in the blastocyst biopsy group,while implantation and pregnancy rates were not statistically significant between the twogroups.For the study of CGβ expression profiles 45 blastocysts were donated to research, of which 39generated trophectoderm cells cDNA libraries. RT-PCR revealed the presence of CGB3, CGB5,CGB7, CGB8 transcripts in 5 blastocysts. The transcripts CGB5, CGB7, CGB8 were expressed inone hatched and one hatching blastocysts (fair morphology on day 7 post insemination) and thetranscript CGβ3 was expressed in three hatched blastocysts (excellent morphology on day 5/6post insemination). The transcript CGβ1 was identified in one only blastocyst. Four blastocystswere biopsied in order to investigate whether CGβ expression can be detected at the minimallevel of few trophectoderm cells. No transcript was found in trophectoderm cell samples orbiopsied blastocyst proper.DiscussionIn recent years, many new technologies have been introduced in clinical practice of ART.Blastocyst biopsy since its first announcement in 2005, until today, has been adopted andintegrated into the application of preimplantation genetic diagnosis (Kokkali et al., 2005). Asblastocyst biopsy has the advantage of providing adequate number of cells for multipleanalyses, it has been lately used for the PGD for monogenic diseases in combination withhistocompatibility screening (HLA matching) or PGD for monogenic diseases screening forstructural or numerical chromosomal abnormalities. Besides its clinical application, blastocystbiopsy offers great opportunities for research, such as the study for the expression ofpreimplantation genetic profiles for the identification of the single most viable blastocyst amongthe cohort developing in vitro that will enable single blastocyst transfers without a concomitantreduction in pregnancy rates.In this study, we investigated whether the β HCG may be used as a predictive marker ofdevelopmental competence for human embryos. This study showed that CGβ gene expressionwas diverse and heterogeneous between blastocysts. Further studies need to be accomplishedto investigate this further.ConclusionsBlastocyst biopsy was developed and optimized to serve as powerful tool for diagnostics ofhuman diseases or to identify diagnostic markers of competence to develop to term for humanembryos.

Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts

2005 ◽  
Vol 288 (6) ◽  
pp. C1211-C1221 ◽  
Author(s):  
Steven J. Pardo ◽  
Mamta J. Patel ◽  
Michelle C. Sykes ◽  
Manu O. Platt ◽  
Nolan L. Boyd ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Bone Loss ◽  
Simulated Microgravity ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Underlying Mechanism ◽  
Bone Biology ◽  
Random Positioning Machine

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Sex differentiation in grayling (Salmonidae) goes through an all-male stage and is delayed in genetic males who instead grow faster

2017 ◽  
Author(s):  
Diane Maitre ◽  
Oliver M. Selmoni ◽  
Anshu Uppal ◽  
Lucas Marques da Cunha ◽  
Laetitia G. E. Wilkins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sex Determination ◽  
Sex Differentiation ◽  
Sex Ratios ◽  
Molecular Genetic ◽  
Specific Gene ◽  
Genetic Sexing ◽  
Experimental Conditions ◽  
Two Samples

AbstractFish can be threatened by distorted sex ratios that arise during sex differentiation. It is therefore important to understand sex determination and differentiation, especially in river-dwelling fish that are often exposed to environmental factors that may interfere with sex differentiation. However, sex differentiation is not sufficiently understood in keystone taxa such as the Thymallinae, one of the three salmonid subfamilies. Here we study a wild grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We found sex determination in the wild and in captivity to be genetic and linked to the sdY locus. We therefore studied sex-specific gene expression in embryos and early larvae that were bred and raised under different experimental conditions, and we studied gonadal morphology in five monthly samples taken after hatching. Significant sex-specific changes in gene expression (affecting about 25,000 genes) started around hatching. Gonads were still undifferentiated three weeks after hatching, but about half of the fish showed immature testes around seven weeks after hatching. Over the next few months, this phenotype was mostly replaced by the “testis-to-ovary” or “ovaries” phenotypes. The gonads of the remaining fish, i.e. approximately half of the fish in each sampling period, remained undifferentiated until six months after fertilization. Genetic sexing of the last two samples revealed that fish with undifferentiated gonads were all males, who, by that time, were on average larger than the genetic females (verified in 8-months old juveniles raised in another experiment). Only 12% of the genetic males showed testicular tissue six months after fertilization. We conclude that sex differentiation starts around hatching, goes through an all-male stage for both sexes (which represents a rare case of “undifferentiated” gonochoristic species that usually go through an all-female stage), and is delayed in males who, instead of developing their gonads, grow faster than females during these juvenile stages.Author contributionMRR and CW initiated the project. DM, OS, AU, LMC, LW, and CW sampled the adult fish, did the experimental in vitro fertilizations, and prepared the embryos for experimental rearing in the laboratory. All further manipulations on the embryos and the larvae were done by DM, OS, AU, LMC, and LW. The RNA-seq data were analyzed by OS, JR, and MRR, the histological analyses were done by DM, supervised by SK, and the molecular genetic sexing was performed by DM, OS, AU, and KBM. DM, OS, and CW performed the remaining statistical analyses and wrote the first version of the manuscript that was then critically revised by all other authors.

Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains

2019 ◽  
Vol 58 (1) ◽  
pp. 30-38
Author(s):  
Patricia Navarro-Rodríguez ◽  
Adela Martin-Vicente ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Gene Expression ◽  
Candida Glabrata ◽  
Efflux Pump ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Clear Correlation ◽  
Synonymous Mutations ◽  
First Time

AbstractCandida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.

187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

263 USE OF PORCINE PARTHENOTES AND GENE EXPRESSION PROFILING USING MICROARRAYS FOR IDENTIFICATION OF IMPRINTED GENES

2006 ◽  
Vol 18 (2) ◽  
pp. 239
Author(s):  
J. Piedrahita ◽  
S. Bischoff ◽  
J. Estrada ◽  
B. Freking ◽  
D. Nonneman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Expression Profiles ◽  
Mammalian Species ◽  
Polar Body ◽  
Gene Expression Profiles ◽  
Tissue Type ◽  
Imprinted Genes

Genomic imprinting arises from differential epigenetic markings including DNA methylation and histone modifications and results in one allele being expressed in a parent-of-origin specific manner. For further insight into the porcine epigenome, gene expression profiles of parthenogenetic (PRT; two maternally derived chromosome sets) and biparental embryos (BP; one maternal and one paternal set of chromosomes) were compared using microarrays. Comparison of the expression profiles of the two tissue types permits identification of both maternally and paternally imprinted genes and thus the degree of conservation of imprinted genes between swine and other mammalian species. Diploid porcine parthenogenetic fetuses were generated using follicular oocytes (BOMED, Madison, WI, USA). Oocytes with a visible polar body were activated using a single square pulse of direct current of 50 V/mm for 100 �s and diploidized by culture in 10 �g/mL cycloheximide for 6 h to limit extrusion of the second polar body. Following culture, BP embryos obtained by natural matings, and PRT embryos, were surgically transferred to oviducts on the first day of estrus. Fetuses recovered at 28-30 days of gestation were dissected to separate viscera including brain, liver, and placenta; the visceral tissues were then flash-frozen in liquid nitrogen. Porcine fibroblast tissue was obtained from the remaining carcass by mincing, trypsinization, and plating cells in �-MEM. Total RNA was extracted from frozen tissue or cell culture using RNA Aqueous kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. Gene expression differences between BP and PRT tissues were determined using the GeneChip� Porcine Genome Array (Affymetrix, Santa Clara, CA) containing 23 256 transcripts from Sus scrofa and representing 42 genes known to be imprinted in human and/or mice. Triplicate arrays were utilized for each tissue type, and for PRT versus BP combination. Significant differential gene expression was identified by a linear mixed model analysis using SAS 5.0 (SAS Institute, Cary, NC, USA). Storey's q-value method was used to correct for multiple testing at q d 0.05. The following genes were classified as imprinted on the basis of their expression profiles: In fibroblasts, ARHI, HTR2A, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, SGCE, SNRPN, and UBE3A; in liver, IGF2, PEG3, PLAGL1, PEG10, and SNRPN; in placenta, HTR2A, IGF2, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, and SNRPN; and in brain, none. Additionally, several genes not known to be imprinted in humans/mice were highly differentially expressed between the two tissue types. Overall, utilizing the PRT models and gene expression profiles, we have identified thirteen genes where imprinting is conserved between swine and humans/mice, and several candidate genes that represent potentially imprinted genes. Presently, our efforts are focused in the identification of single nucleotide polymorphisms (SNPs) to more carefully evaluate the behavior of these genes in normal and abnormal gestations and to test whether the candidate genes are indeed imprinted. This research was supported by USDA-CSREES grant 524383 to J. P. and B. F.

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