Regulation of expression of the retinoic acid-synthesising enzymes retinaldehyde dehydrogenases in the uteri of ovariectomised mice after treatment with oestrogen, gestagen and their combination

2006 ◽  
Vol 18 (3) ◽  
pp. 339 ◽  
Author(s):  
Ralph Rühl ◽  
Britta Fritzsche ◽  
Julien Vermot ◽  
Karen Niederreither ◽  
Ulrike Neumann ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Early Pregnancy ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Oestrous Cycle ◽  
Regulation Of Expression ◽  
Mouse Uterus ◽  
Blastocyst Implantation ◽  
Polymerase Chain

The active metabolite of vitamin A, retinoic acid (RA), plays an important role in the female reproductive system. The synthesis of RA is tightly regulated by the activity of retinaldehyde dehydrogenases (Raldh). Among these, Raldh1 and Raldh2 exhibit specific temporal and spatial expression patterns in the mouse uterus, both during the oestrous cycle and early pregnancy. In the present study, we have assessed whether oestradiol and progesterone directly influence the uterine expression of Raldh1 and Raldh2 in ovariectomised mice. We investigated the effect of gestagen (promegestone 0.3 mg kg−1 bodyweight), oestrogen (oestradiol 3 µg kg−1 bodyweight) and their combination on the uterine expression of Raldh2. Expression was analysed using in situ hybridisation and quantified using real-time detection reverse transcription–polymerase chain reaction. The results show that the expression of Raldh2 is rapidly (within 1–4 h) induced in stromal cells by oestrogen, but not by gestagen, treatment, whereas combined oestrogen + gestagen treatment leads to a more prolonged (48 h) response. In contrast, oestrogen, but not progesterone, treatment downregulates (within 4–24 h) Raldh1 expression in the uterine glandular epithelium. We conclude that the uterine RA concentrations are regulated by oestrogens via an effect on the expression of the Raldh synthesising enzymes. Such a regulation is consistent with the natural fluctuations of Raldh expression during the oestrous cycle, early pregnancy and blastocyst implantation.

scholarly journals The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow

1999 ◽  
Vol 160 (1) ◽  
pp. 21-33 ◽  
Author(s):  
RS Robinson ◽  
GE Mann ◽  
GE Lamming ◽  
DC Wathes
Keyword(s):  
Cross Sections ◽  
Early Pregnancy ◽  
In Situ Hybridisation ◽  
Progesterone Receptors ◽  
Prostaglandin F2alpha ◽  
Interferon Tau ◽  
Uterine Endometrium ◽  
Low Concentrations ◽  
Oxytocin Antagonist

The expression of oxytocin receptor (OTR) in the uterine endometrium plays an important role in the initiation of luteolysis. During early pregnancy, the conceptus secretes interferon tau (IFN|gt) which inhibits OTR up-regulation and luteolysis. In this study, uterine horn cross sections were collected on day 16 from 15 pregnant cows (PREG), 9 uninseminated controls and 5 inseminated cows with no embryo present. The latter two groups had similar results and were combined to form a single non-pregnant (NP) group. The animals were given an oxytocin challenge shortly before tissue collection to assess prostaglandin F2alpha (PGF2alpha) release through the measurement of the metabolite 13,14-dihydro-15-keto PGF2alpha (PGFM). The mRNAs for OTR, oestrogen receptor (ER) and progesterone receptor (PR) were localised by in situ hybridisation. The results were quantified by optical density (OD) measurements from autoradiographs using image analysis. OTR protein was measured by autoradiography with iodinated oxytocin antagonist and ER and PR protein was detected by immunocytochemistry. The release of PGFM after the oxytocin challenge was significantly higher in the 14 NP cows (187%+/-15%) compared with the PREG group (131%+/-11%) (P<0.01). Low concentrations of OTR mRNA were localised to the luminal epithelium (LE) in 6 out of the 14 NP cows, of which 2 also expressed OTR protein, while OTR mRNA and protein were undetectable in all the pregnant animals. These results indicated that the sampling time coincided with the onset of the luteolytic mechanism in the NP cows. On day 16 ER mRNA was detectable in both the LE and glands of both PREG and NP animals. There were no differences in either ER mRNA or protein between NP and PREG samples. PR mRNA was moderately expressed in the caruncular stroma, with lower levels in the dense caruncular-like stroma and glands. There were no differences between PREG and NP animals. The expression of PR mRNA and protein in the deep glands was variable between animals. These results suggested that, in cows, the presence of an embryo suppressed the expression of OTR, but had no effect on the expression of the transcriptionally regulated ER on day 16.

Development of the spatial pattern of retinoic acid receptor-beta transcripts in embryonic chick facial primordia

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 805-813
Author(s):  
A. Rowe ◽  
J.M. Richman ◽  
P.M. Brickell
Keyword(s):  
Retinoic Acid ◽  
Neural Crest ◽  
Retinoic Acid Receptor ◽  
In Situ Hybridisation ◽  
Acid Receptor ◽  
Retinoic Acid Receptor Beta ◽  
Low Levels ◽  
Cranial Neural Crest Cells ◽  
Receptor Beta

Retinoic acid causes a range of embryonic defects, including craniofacial abnormalities, in both birds and mammals and is believed to have a number of roles in normal development. We have previously shown that the distribution of retinoic acid receptor-beta (RAR-beta) transcripts is spatially restricted within the neural-crest-derived upper beak primordia of the chick embryo. We have now used in situ hybridisation to trace the distribution of RAR-beta transcripts during the migration of cranial neural crest cells and during formation of these primordia. RAR-beta transcripts were present in a subset of migrating neural-crest-derived cells in the head of the stage 10 embryo. These cells were situated in pathways followed by cells that migrate from the neural crest overlying the posterior prosencephalic/anterior mesencephalic region of the developing brain. Cells containing RAR-beta transcripts accumulated around the developing eyes and in the regions of the ventral head from which the upper beak primordia later develop. We mapped the distribution of RAR-beta transcripts as the facial primordia were forming, with particular reference to the development of the maxillary primordia. We found that these form in a region of the ventral head that includes the boundary between regions of high and low levels of RAR-beta transcripts. The boundary between these two groups of cells persisted as the maxillary primordia developed.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbial quantification in activated sludge: the hits and misses

2003 ◽  
Vol 48 (3) ◽  
pp. 121-126 ◽  
Author(s):  
S.J. Hall ◽  
J. Keller ◽  
L.L. Blackall
Keyword(s):  
Activated Sludge ◽  
Molecular Genetic ◽  
In Situ Hybridisation ◽  
Biological Nutrient Removal ◽  
Physical Parameters ◽  
Fluorescence In Situ Hybridisation ◽  
Batch Tests ◽  
Polymerase Chain ◽  
Key Microorganisms

Since the implementation of the activated sludge process for treating wastewater, there has been a reliance on chemical and physical parameters to monitor the system. However, in biological nutrient removal (BNR) processes, the microorganisms responsible for some of the transformations should be used to monitor the processes with the overall goal to achieve better treatment performance. The development of in situ identification and rapid quantification techniques for key microorganisms involved in BNR are required to achieve this goal. This study explored the quantification of Nitrospira, a key organism in the oxidation of nitrite to nitrate in BNR. Two molecular genetic microbial quantification techniques were evaluated: real-time polymerase chain reaction (PCR) and fluorescence in situ hybridisation (FISH) followed by digital image analysis. A correlation between the Nitrospira quantitative data and the nitrate production rate, determined in batch tests, was attempted. The disadvantages and advantages of both methods will be discussed.

scholarly journals In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

2001 ◽  
Vol 22 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Mario Menschikowski ◽  
Margot Vogel ◽  
Rolf Eckey ◽  
Gerd Dinnebier ◽  
Werner Jaross
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Nested Pcr ◽  
In Situ Hybridisation ◽  
Target Sequence ◽  
Chain Reaction ◽  
Multiple Control ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

2005 ◽  
Vol 56 (8) ◽  
pp. 1127 ◽  
Author(s):  
D. G. Bourne ◽  
R. L. Blakeley ◽  
P. Riddles ◽  
G. J. Jones
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
In Situ Hybridisation ◽  
Cyanobacterial Blooms ◽  
Chain Reaction ◽  
Fluorescent In Situ Hybridisation ◽  
Cyanobacterial Toxin ◽  
Specific Amplification ◽  
Polymerase Chain

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

scholarly journals The expression of the IGF system in the bovine uterus throughout the oestrous cycle and early pregnancy

2000 ◽  
Vol 165 (2) ◽  
pp. 231-243 ◽  
Author(s):  
RS Robinson ◽  
GE Mann ◽  
TS Gadd ◽  
GE Lamming ◽  
DC Wathes
Keyword(s):  
Cross Sections ◽  
Early Pregnancy ◽  
In Situ Hybridisation ◽  
Endometrial Biopsy ◽  
Oestrous Cycle ◽  
Luminal Epithelium ◽  
Mrna Levels ◽  
Igf Binding Proteins ◽  
Igf System ◽  
Igf I

The IGF system is expressed in the uterus during the oestrous cycle and early pregnancy and is likely to play an important role in regulating the development of the embryo and uterus. The IGF peptides (IGF-I and -II) mediate their effects through the type 1 IGF receptor (IGF-1R), while the IGF-binding proteins (IGFBP-1 to -6) modulate their interaction with the receptor. In this study, the expression of the IGF system in the bovine uterus was determined throughout the oestrous cycle and on day 16 of pregnancy. Endometrial biopsy samples were collected from four cows over three cycles such that there were samples for every 2 days from day 0 (oestrus) to day 14 and then every day until day 21. To assess the effect of pregnancy, uterine horn cross-sections were collected on day 16 from 15 pregnant (PREG), five inseminated non-pregnant (INP) and nine uninseminated cyclic controls (CONT). The expression of mRNA for the IGFs, IGF-1R and IGFBP-1 to -5 was determined by in situ hybridisation and the results were quantified by measuring the optical density units from autoradiographs. The main region of IGF-I mRNA expression was the sub-epithelial stroma underlying the luminal epithelium. The expression of IGF-I mRNA was highest at oestrus and lowest during the early and late luteal phases. On day 16, IGF-I mRNA levels were low in all groups, with pregnancy having no effect on the IGF-I mRNA concentrations. The strongest expression of IGF-II mRNA was in the caruncular stroma, with pregnancy having no significant effect in this region. IGF-1R mRNA was also present in the caruncles and was strongly expressed in all epithelial cells both throughout the oestrous cycle and during early pregnancy. The expression of IGFBP-1 mRNA was confined to the luminal epithelium, with the strongest expression seen on day 14 of the cycle. On day 16 the expression of IGFBP-1 mRNA was higher in the PREG group compared with the CONT group. The expression of IGFBP-2 mRNA was localised to the sub-epithelial stroma with more INP than PREG cows showing detectable levels of IGFBP-2. The strongest expression of IGFBP-3 mRNA was in the caruncular stroma; expression in the endometrial stroma was similarly decreased during early pregnancy. IGFBP-5 mRNA was mainly expressed in the inner ring of myometrium and was not affected by pregnancy on day 16. In conclusion, these results show that many components of the uterine IGF system are differentially regulated during the oestrous cycle and early pregnancy and suggest that modulation of the IGF system may influence uterine activity during this period.

scholarly journals Cloning and regulation of the rat activin betaE subunit

2000 ◽  
Vol 24 (3) ◽  
pp. 409-418 ◽  
Author(s):  
MK O'Bryan ◽  
KL Sebire ◽  
O Gerdprasert ◽  
MP Hedger ◽  
MT Hearn ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Alternative Polyadenylation ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Mrna Transcripts ◽  
Polymerase Chain

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

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