Validation of an experimental strategy for studying surface-exposed proteins involved in porcine sperm - oviduct contact interactions

2005 ◽  
Vol 17 (7) ◽  
pp. 683 ◽  
Author(s):  
W. V. Holt ◽  
R. M. A. Elliott ◽  
A. Fazeli ◽  
N. Satake ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
Protein S ◽  
Ex Situ ◽  
Major Influence ◽  
Apical Plasma Membrane ◽  
Epithelial Surface ◽  
Multiple Protein ◽  
Investigative Technique

Previous experiments have shown that boar sperm survival in vitro is enhanced when co-incubated with a solubilised protein extract of porcine oviducal apical plasma membrane proteins. Here, we examine the hypothesis that the effects are mediated by direct oviduct–sperm contact and use in situ biotinylation of the oviducal epithelial surface to trace the surface-exposed biotinylated proteins through purification and solubilisation steps. We have also examined the effectiveness of mechanical scraping as a method of recovering oviducal epithelial proteins. We show that a subset of proteins originally exposed at the oviducal surface eventually bind to spermatozoa during incubation in vitro, but also show that a different protein subset is implicated if the sperm incubation is performed with proteins that had been biotinylated after (ex situ) extraction from the oviduct. Apical plasma membrane fractions biotinylated after purification contained many more biotinylated protein bands than preparations labelled before purification and multiple protein bands were eventually found to associate with spermatozoa. Although the evidence presented here supports the hypothesis that protein(s) anchored to the oviducal epithelium bind populations of spermatozoa directly and may have a role in the enhancement of sperm viability, it also shows that the choice of investigative technique exerts a major influence on experimental outcomes.

Collection and in vitro maturation of Mazama gouazoubira (brown brocket deer) oocytes obtained after ovarian stimulation

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Luciana Diniz Rola ◽  
Eveline dos Santos Zanetti ◽  
Maite del Collado ◽  
Ellen de Fátima Carvalho Peroni ◽  
José Maurício Barbanti Duarte
Keyword(s):  
Oocyte Maturation ◽  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
Wildlife Conservation ◽  
Estradiol Benzoate ◽  
Ex Situ ◽  
Follicular Aspiration ◽  
Mazama Gouazoubira

Summary In vitro production of embryos has gained prominence as a tool for use in wildlife conservation programmes in situ and ex situ. However, the development of this technique depends on steps that include ovarian stimulation, collection and oocyte maturation. The purpose of this study was to assess the feasibility of an ovarian stimulation protocol for follicular aspiration, the efficiency of videolaparoscopy for follicular aspiration and test a medium for in vitro oocyte maturation for the species Mazama gouazoubira. Five females were submitted to repeated ovarian stimulation (hormone protocol using controlled internal drug release), and estradiol benzoate on D0 and eight injections of follicle-stimulating hormone, once every 12 h, from D4 onwards at 30-day intervals. Fourteen surgical procedures were performed in superstimulated females, resulting in the collection of 94 oocytes and an average of 17.1 ± 9.1 follicles observed, 13.5 ± 6.6 follicles aspirated and 7.2 ± 3.7 oocytes collected per surgery. After collection, the oocytes were submitted to in vitro maturation for 24 h and stained with Hoechst 33342 dye to evaluate their nuclear status; 64.5% of the oocytes reached MII and 16.1% were spontaneously activated by parthenogenesis. The nuclear status of oocytes that did not undergo in vitro maturation was evaluated; 80.9% were found to be immature.

scholarly journals Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1518
Author(s):  
Maria Qatato ◽  
Vaishnavi Venugopalan ◽  
Alaa Al-Hashimi ◽  
Maren Rehders ◽  
Aaron D. Valentine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Human Thyroid ◽  
Apical Plasma Membrane ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Trace Amine ◽  
Rat Thyroid

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro

2009 ◽  
Vol 21 (3) ◽  
pp. 408 ◽  
Author(s):  
R. E. Lloyd ◽  
R. M. A. Elliott ◽  
A. Fazeli ◽  
P. F. Watson ◽  
W. V. Holt
Keyword(s):  
Plasma Membrane ◽  
Heat Shock ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Beneficial Effect ◽  
Active Component ◽  
Apical Plasma Membrane ◽  
Series Of Experiments ◽  
Dose Dependent

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39°C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

scholarly journals Ex situ conservation of genetic resources of field elm (Ulmus minor Mill) and European white elm (Ulmus laevis Pall)

Genetika ◽  
2004 ◽  
Vol 36 (3) ◽  
pp. 221-227
Author(s):  
Jelena Aleksic ◽  
Sasa Orlovic
Keyword(s):  
Genetic Resources ◽  
Ex Situ Conservation ◽  
Ex Situ ◽  
In Vitro Production ◽  
Ulmus Minor ◽  
Conservation Of Genetic Resources ◽  
Ulmus Laevis ◽  
Vitro Production

Principles of the conservation of genetic resources of elms (Ulmus spp) do not differ fundamentally from the general principles accepted for the conservation of genetic resources of other common Noble Hardwoods. Efficient conservation can best be achieved through appropriate combination of in situ and ex situ methods, which have distinct advantages. Besides that, ex situ conservation is employed when emergency measures are needed for rare endangered populations and when populations are too small to be managed in situ (e.g. risks of genetic drift and inbreeding). The aim of our research is ex situ conservation of genetic resources of field elm {Ulmus minor Mill) and European white elm (Ulmus laevis Pall) through establishment of field genebanks. Sampling was conducted in one population of field elm and one population of white elm. Plant material (buds) from 8 trees of field elm and 10 trees of white elm was used for in vitro production of clones. Obtained clones will be used for establishment of field genebanks on the experimental estate of the Institute of Lowland Forestry and Environment.

scholarly journals Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

2020 ◽  
Vol 21 (20) ◽  
pp. 7459
Author(s):  
María Elena González-Benito ◽  
Miguel Ángel Ibáñez ◽  
Michela Pirredda ◽  
Sara Mira ◽  
Carmen Martín
Keyword(s):  
Dna Methylation ◽  
In Situ Conservation ◽  
Plant Conservation ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Regenerated Plants ◽  
Before And After ◽  
Methylation Patterns

Epigenetic variation, and particularly DNA methylation, is involved in plasticity and responses to changes in the environment. Conservation biology studies have focused on the measurement of this variation to establish demographic parameters, diversity levels and population structure to design the appropriate conservation strategies. However, in ex situ conservation approaches, the main objective is to guarantee the characteristics of the conserved material (phenotype and epi-genetic). We review the use of the Methylation Sensitive Amplified Polymorphism (MSAP) technique to detect changes in the DNA methylation patterns of plant material conserved by the main ex situ plant conservation methods: seed banks, in vitro slow growth and cryopreservation. Comparison of DNA methylation patterns before and after conservation is a useful tool to check the fidelity of the regenerated plants, and, at the same time, may be related with other genetic variations that might appear during the conservation process (i.e., somaclonal variation). Analyses of MSAP profiles can be useful in the management of ex situ plant conservation but differs in the approach used in the in situ conservation. Likewise, an easy-to-use methodology is necessary for a rapid interpretation of data, in order to be readily implemented by conservation managers.

scholarly journals Combined in situ Physical and ex-situ Biochemical Approaches to Investigate in vitro Deconstruction of Destarched Wheat Bran by Enzymes Cocktail Used in Animal Nutrition

2019 ◽  
Vol 7 ◽  
Author(s):  
Marine Deshors ◽  
Olivier Guais ◽  
Virginie Neugnot-Roux ◽  
Xavier Cameleyre ◽  
Luc Fillaudeau ◽  
...  
Keyword(s):  
Wheat Bran ◽  
Ex Situ ◽  
Animal Nutrition

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Long-term conservation of potato genetic resources: Methods and status of conservation

2019 ◽  
pp. 717-725
Author(s):  
Jane Muthoni ◽  
Hussein Shimelis ◽  
Rob Melis
Keyword(s):  
Genetic Resources ◽  
Slow Growth ◽  
Plant Genetic Resources ◽  
Ex Situ ◽  
Growth Media ◽  
Medium Term ◽  
Natural Habitats

Plant genetic resources (PGRs) play an important role in agriculture, environment protection, cultural property and trade; they need to be conserved. There are two fundamental approaches for the conservation of PGRs: in situ and ex situ. In situ conservation is the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings. Ex situ preservation is the storage of seeds or plant materials under artificial conditions to maintain their long term viability and availability for use. Genebanks employ seed storage, field collections of living plants and in vitro storage (tissue culture or cryopreservation) for ex situ preservation of PGR. Storage of orthodox seeds, which are tolerant to low moisture content and low temperatures at appropriate temperature and humidity, is the most convenient ex situ conservation method. Plants that produce recalcitrant seeds or non-viable seeds are conserved in field genebanks as well as in-vitro in slow growth media for short-to-medium term and cryopreservation in liquid nitrogen at -1960C for long-term periods. Cryopreservation is very expensive and needs trained personnel; this could explain why this method is rarely used for conservation of plant genetic resources in most developing countries. Potato tubers are bulky and highly perishable; the crop is generally conserved as clones either in field genebanks (with annual replanting), in-vitro conservation in slow growth media for short-to-medium term and cryopreservation for long term. Field genebanks are expensive to maintain and the crop is exposed to many dangers; hence, cryopreservation is the only feasible method for long term conservation. However, given the high cost of cryopreservation, long-term conservation of potato genetic resources is poorly developed in most resource-poor countries leading to high rates of genetic erosion. This paper looks into the various methods that that can be applied to conserve potato genetic resources and the status of conservation of potatoes in major genebanks and some countries.

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells

2017 ◽  
Vol 117 (01) ◽  
pp. 105-115 ◽  
Author(s):  
Yvonne Schaletzki ◽  
Marie-Luise Kromrey ◽  
Susanne Bröderdorf ◽  
Elke Hammer ◽  
Markus Grube ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Apical Plasma Membrane ◽  
Dense Granules ◽  
Haematopoietic Cells ◽  
And Function

SummaryThe multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knockdown of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leuk aemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.Supplementary Material to this article is available at www.thrombosis-online.com.

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