scholarly journals 230TRANSCRIPTOMICS ANALYSIS OF CHANGES IN GENE EXPRESSION IN BOVINE OVIDUCT EPITHELIAL CELLS DURING THE ESTROUS CYCLE

2004 ◽  
Vol 16 (2) ◽  
pp. 236
Author(s):  
S. Bauersachs ◽  
S. Rehfeld ◽  
S. Koelle ◽  
S. Mallok ◽  
K. Prelle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Estrous Cycle ◽  
Cdna Array ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Ciliated Cells ◽  
Expression Levels ◽  
Functional Changes ◽  
Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs;; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n=42) were expressed at a higher level at estrus;; for the other (n=42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell–cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle.

scholarly journals Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle

2004 ◽  
Vol 32 (2) ◽  
pp. 449-466 ◽  
Author(s):  
S Bauersachs ◽  
S Rehfeld ◽  
SE Ulbrich ◽  
S Mallok ◽  
K Prelle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Expression Profiles ◽  
Cdna Array ◽  
Oestrous Cycle ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Expression Levels ◽  
Starting Point ◽  
Oviduct Epithelial Cells

The oviduct epithelium undergoes marked morphological and functional changes during the oestrous cycle. To study these changes at the level of the transcriptome we did a systematic gene expression analysis of bovine oviduct epithelial cells at oestrus and dioestrus using a combination of subtracted cDNA libraries and cDNA array hybridisation. A total of 3072 cDNA clones of two subtracted libraries were analysed by array hybridisation with cDNA probes derived from six cyclic heifers, three of them slaughtered at oestrus and three at dioestrus. Sequencing of cDNAs showing significant differences in their expression levels revealed 77 different cDNAs. Thirty-seven were expressed at a higher level at oestrus, for the other 40 genes expression levels were higher at dioestrus. The identified genes represented a variety of functional classes. During oestrus especially genes involved in the regulation of protein secretion and protein modification, and mRNAs of secreted proteins, were up-regulated, whereas during dioestrus particularly transcripts of genes involved in transcription regulation showed a slight up-regulation. The concentrations of seven selected transcripts were quantified by real-time RT-PCR to validate the cDNA array hybridisation data. For all seven transcripts, RT-PCR results were in excellent correlation (r>0.92) with the results obtained by array hybridisation. Our study is the first to analyse changes in gene expression profiles of bovine oviduct epithelial cells during different stages of the oestrous cycle, providing a starting point for the clarification of the key transcriptome changes in these cells.

181 MESSENGER RNA PROFILING OF BOVINE ENDOMETRIUM DURING THE ESTROUS CYCLE USING A CUSTOM-MADE cDNA ARRAY

2008 ◽  
Vol 20 (1) ◽  
pp. 170
Author(s):  
K. Mitko ◽  
H. Blum ◽  
E. Wolf ◽  
S. Bauersachs
Keyword(s):  
Estrous Cycle ◽  
Messenger Rna ◽  
Expression Profiles ◽  
Cdna Array ◽  
Differentially Expressed ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Tissue Samples ◽  
Functional Changes ◽  
Custom Made

The endometrium provides the optimal conditions for the transport of sperm to the oviduct, to the site of fertilization, and later on for the reception of the embryo. This is reflected by specific morphological and functional changes during the estrous cycle, which are mainly regulated by the hormones progesterone, estradiol, and oxytocin. To study these changes on the level of messenger RNA, a microarray analysis of endometrial tissue samples was performed. Tissue samples were collected from 20 cyclic heifers (Deutsches Fleckvieh, between 17 and 31 months of age) after slaughter at Days 0, 3.5, 12, and 18 of the estrous cycle. The Day 18 group split into two subgroups, one with high and one with low progesterone levels in peripheral blood. Altogether there were 4 heifers in each experimental group. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array (Bauersachs et al. 2007 J. Dairy Sci. 90, in press). The cDNAs present on the array were derived from several previously conducted differential gene expression studies of bovine endometrium between different stages of the estrous cycle, during early pregnancy, and from studies of bovine oviduct epithelial cells. In all of these studies, cDNAs of differentially expressed genes were identified using a combination of subtracted cDNA libraries and cDNA array hybridization. Redundant cDNA clones were removed, resulting in 1440 cDNA fragments on the array, representing 950 different genes. Twenty radioactively (33P) labeled cDNA samples (n = 4 for each cycle stage) were hybridized with the BOE array. After normalization of raw data (using BioConductor open source software) and significance analysis (SAM, FDR 1%), 272 mRNAs were identified that showed alterations of their concentration during the estrous cycle. Expression data from cDNAs with significant changes during the estrous cycle were used for cluster analyses with MultiExperiment Viewer 4.0 (Saeed et al. 2003 Biotechniques 34, 374–378). The main clusters represented genes upregulated either during the estrus or during the diestrus phase. Quantitatively enriched Gene Ontology (GO) categories were identified to find relevant functional groups and prominent biological processes. At estrus, e.g., GO categories extracellular matrix, cytoskeleton organization, and growth factor activity indicate changes in the composition of the endometrium during the estrous cycle. At diestrus, only a few overrepresented GO categories were found, mostly related to immune response and metabolism. The genes of known function were further analyzed in the context of interaction and regulatory networks. One of a number of central factors was TGF-β, which controls the expression of 12 genes upregulated at estrus and 8 at diestrus. In conclusion, the present study extended the results of our previously conducted analysis of bovine endometrium between the estrus and diestrus stages (Bauersachs et al. 2005 J. Mol. Endocrinol. 32, 449–466), revealed distinct temporal expression profiles, and identified additional genes differentially expressed during the estrous cycle. This work was supported by Grant BMBF FUGATO-Fertilink.

Effect of Estrous Cycle Phases on Gene Expression of Bovine Oviduct Epithelial Cells

2021 ◽  
Author(s):  
Ricaurte Lopera Vasquez ◽  
Fabián Uribe-García ◽  
Iang Rondón-Barragán
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Estrous Cycle ◽  
Oviduct Epithelial Cells

scholarly journals Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes

2005 ◽  
Vol 34 (3) ◽  
pp. 889-908 ◽  
Author(s):  
S Bauersachs ◽  
S E Ulbrich ◽  
K Gross ◽  
S E M Schmidt ◽  
H H D Meyer ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Cdna Array ◽  
Transforming Growth Factor Β ◽  
Early Embryo ◽  
Oestrous Cycle ◽  
Cdna Libraries ◽  
Functional Changes ◽  
Retinoic Acid Signalling ◽  
Subtracted Cdna Libraries

The endometrium plays a central role among the reproductive tissues in the context of early embryo–maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle – end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-β signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

scholarly journals Activation of the JAK1 / STAT1 Signaling Pathway is Associated with Prdx6 Expression Levels in Human Epididymis Epithelial Cells

2021 ◽  
Author(s):  
Jianyuan Li ◽  
Hui Shi ◽  
Xiaoyu Liu ◽  
Yanwei Wang ◽  
Haiyan Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Signaling Pathway ◽  
Expression Patterns ◽  
Digital Gene Expression ◽  
Protective Role ◽  
Expression Levels ◽  
Human Epididymis ◽  
Total Antioxidant ◽  
Stat1 Signaling

Abstract I. Background: Peroxiredoxin 6 (Prdx6) is widely expressed in mammalian tissues. Our previous study demonstrated that Prdx6 was expressed in human epididymis and spermatozoa, and the protective role of Prdx6 in human spermatozoa was also reported. In this study, we demonstrate the potential role and mechanism of Prdx6 in human epididymis epithelial cells (HEECs).II. Methods and Results: Western blotting was used to measure expression levels of key proteins in the JAK / STAT signaling pathway. Digital gene expression analysis (DGE) was used to identify gene expression patterns in control HECs and in HECs after Prdx6-RNA interference (P6-RNAi). The DGE analysis identified 589 up-regulated and 314 down-regulated genes (including Prdx6) in Prdx6-RNAi (P6-RNAi) HEECs. Thirteen significantly different pathways were identified between the two groups, with the majority different expressed genes belonging to the CCL, CXCL, IL, and IFIT families. In particular, the expression levels of IL6, IL6ST, and eighteen IFN related genes were significantly increased in the condition of the down-regulated expression of Prdx6. Compared to control HEECs, the expression levels of JAK1, STAT1, phosphorylated JAK1 and STAT1 were significantly increased, while the expression levels of SOCS3 was significantly decreased in P6-RNAi HEECs. The Malondialdehyde (MDA) level and total antioxidant capacity in P6-RNAi HEECs were significantly increased and decreased compared to that of control, respectively. III. Conclusions: We speculated that knockdown of Prdx6 resulted in higher levels of ROS in HEECs, which in turn, activated the JAK1 / STAT1 signaling pathway induced by IL-6 receptor and IFN.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality

2000 ◽  
Vol 279 (4) ◽  
pp. R1239-R1250 ◽  
Author(s):  
Eric Glasgow ◽  
Takashi Murase ◽  
Bingjun Zhang ◽  
Joseph G. Verbalis ◽  
Harold Gainer
Keyword(s):  
Gene Expression ◽  
Supraoptic Nucleus ◽  
Cdna Libraries ◽  
Global Changes ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Magnocellular Neurons ◽  
In Situ Hybridization Histochemistry ◽  
Functional Classes

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10–20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na+-K+-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

51 AGGREGATION REDUCES THE VARIATION OF GENE EXPRESSION LEVELS IN BOVINE CLONES

2006 ◽  
Vol 18 (2) ◽  
pp. 134
Author(s):  
S. Kurosaka ◽  
N. A. Leu ◽  
K. J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Coefficient Of Variation ◽  
Blastocyst Stage ◽  
Average Level ◽  
Cdna Libraries ◽  
Cell Stage ◽  
Glucose Transporter 1 ◽  
Expression Levels ◽  
Gene Expression Levels

Mammalian somatic cell clones frequently exhibit abnormal gene expression that presumably results from errors in reprogramming of the transplanted genome. In the mouse, aggregation of 4-cell stage clones with each other improves reprogramming with respect to Oct-4 expression in blastocysts and an increase in term development (Boiani et al. 2003 EMBO J. 22, 5304-5312). To determine if clone-clone aggregation has a similar beneficial effect in the bovine, we aggregated 8-16 cell bovine clones with each other and profiled gene expression levels in bovine clones and clone-clone aggregates at the blastocyst stage. Clone embryos were produced from fibroblasts and cultured in vitro in SOF supplemented with fetal bovine serum at 39�C in an atmosphere of 5% CO2, 5% O2, and 90% N2. For aggregation of embryos, we first removed the zonae pepellucidae by treatment with 0.5% pronase at the 8-16 cell stage and then placed two zona-free embryos per well into deep microwells produced on the bottom of a culture dish by pressing a heated darning needle onto the surface. Seven to 10 microwells in close proximity were covered by a culture 50-�L drop of culture medium, and embryos were cultured until Day 7. Real-time RT-PCR analysis for Oct-4, DNA methyltransferase 1 (Dnmt1), Dnmt3, glucose transporter 1 (Glut1), Glut3, and Poly(A) polymerase (PolyA) was performed on reusable Dynabead Oligo (dT)25-cDNA libraries synthesized from individual blastocysts at Day 7. In vitro-fertilized embryos were used as controls. To compare the variation of gene expression in each embryo within the group, the coefficient of variation (COV; standard deviation/mean) was calculated. Although spatial distribution of Oct-4 transcript is normal in bovine blastocyst stage clones (Kurosaka et al. 2004 Reprod. Fertil. Dev. 16, 147), we detected disturbances in the level of Oct-4 expression in clones: 44.4% (8 of 18) of clones expressed Oct-4 within a range of 0.5- and 1.5-fold of the average level of expression in IVF embryos, compared to 81.8% (9 of 11) of IVF embryos. Only 22.2% (4 of 18) of clones expressed all genes examined within a range of 0.5- and 2.0-fold of the average level of IVF embryos, versus 45.5% (5 of 11) of IVF embryos. Clone-clone aggregation did not increase the proportion of clones with normal expression levels but did reduce the coefficient of variation of gene expression levels between individual clones for the genes Oct-4, Dnmt1, Dnmt3a and PolyA, but not for Glut1 and Glut3. Interestingly, bovine clone-clone aggregates (n = 25) had less variation between individual embryos compared to IVF aggregates (n = 11) for all genes except Glut1 and Glut3, although variation of single clones was larger than that of single IVF embryos. Analysis of Oct-4 and �-Actin transcripts in mouse clone blastocysts indicated a similar decrease in gene expression variation subsequent to aggregation of mouse clones. These results demonstrate that bovine pre-implantation stage clones exhibit a high degree of variation in gene expression levels and suggest that aggregation of clones is beneficial in reducing the variation in expression of some genes.

90 CHARACTERIZATION OF A PRIMARY CULTURE OF OVIDUCTAL CELLS IN THE BITCH

2014 ◽  
Vol 26 (1) ◽  
pp. 159
Author(s):  
C. Gibson ◽  
K. Reynaud ◽  
S. Thoumire ◽  
B. Grimard ◽  
M. Saint-Dizier
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Primary Culture ◽  
Culture Period ◽  
Stable Gene ◽  
Mucosal Cell ◽  
Fibroblast Cells ◽  
Mrna Levels ◽  
Ciliated Cells ◽  
Cell Functions

The oviduct is of particular importance in canine reproduction as it supports oocyte meiosis resumption, sperm capacitation and storage, fertilization and embryo development to the morula/blastocyst stage for 8 to 10 days post-ovulation. A long-time co-culture with oviducal cells could be employed to improve the yield of reproductive biotechnologies in this species, but no characterisation of canine oviduct cells in vitro has been reported to date. The objectives of this study were to (1) evaluate the viability and proportion of epithelial/fibroblast cells in a primary culture of canine oviducal cells collected around ovulation; (2) study the responsiveness of the cultured cells to steroids. Beagle bitches (n = 9) were ovariectomized between Day –1 and Day +1 around ovulation, and their oviducts were sectioned at the ampulla-isthmus junction. Mucosal cells (including stromal and epithelial cells) were collected by squeezing from the ampulla and isthmus sections and cultured separately at a concentration of 5 × 105 cells/well in 500 μL of M199 + 10% FCS at 39°C for 9 days. At Days 3 and 6, 1 × 106 cells were stimulated with 17β-oestradiol (E2, 20 pg mL–1) or progesterone (P4, 20 ng mL–1) for 6 h. At Days 3, 6, and 9 of culture, the viability of the cells was evaluated using the Live/Dead kit (Invitrogen, Carlsbad, CA, USA), and proportions of fibroblast and epithelial ciliated cells were evaluated by immuno-cytochemistry using anti-vimentin and anti-tubulin antibodies, respectively. At Days 0, 3 and 6, the total RNA was extracted from cells and mRNA levels of the oviduct-specific glycoprotein (OVGP, synthesised by nonciliated epithelial cells), E2 (ERα, ERβ) and P4 (PR) receptors were evaluated by RT-qPCR. The effects of the day of culture and of steroid exposure on mRNA levels were analysed by ANOVA followed by a Tukey test. Cell confluence was observed around Day 6 of culture and more than 90% of cells survived during the 9-day culture period. From Day 3 to Day 9, the proportion of vimentin-positive (fibroblast) cells was greater than 68% in both ampulla and isthmus cells. In contrast, the proportion of epithelial ciliated cells was low at Day 3 (9% in ampulla, 12% in isthmus) and null at Days 6 and 9 in both regions. The mRNA levels of OVGP, ER, and PR decreased significantly after 3 days of culture, and then remained stable in both ampulla and isthmus cells (P < 0.001). The steroid exposure had no effect on gene expression, except for ERα mRNA levels at Day 3, which was increased by E2 and reduced by P4 (P < 0.05). In conclusion, the method of collection did not allow us to collect a high proportion of epithelial oviducal cells. However, the relatively stable gene expression of PR and ER during the culture period provides us with a useful tool to study the steroid regulation of canine oviduct mucosal cell functions.

scholarly journals New experimental and computational approaches to the analysis of gene expression.

1998 ◽  
Vol 45 (4) ◽  
pp. 929-934 ◽  
Author(s):  
J A Rafalski ◽  
M Hanafey ◽  
G H Miao ◽  
A Ching ◽  
J M Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Data Base ◽  
Developmental Stages ◽  
Expressed Sequence Tag ◽  
Expression Profiles ◽  
Soybean Seed ◽  
Fluorescent Labeling ◽  
Cdna Libraries ◽  
Northern Analysis ◽  
Expression Levels

Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.

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