Sublethal Hydrostatic Pressure Research Articles

New data concerning oocyte maturationand embryo production in vitro

It is reviewed last science data concerning oocyte maturation and pig embryo production in vitro. The aim of the work is to present new data concerning oocyte maturation and embryo production in vitro. Efficiency of different assistant reproductive methods can be improved by treatment of gametes and embryos with sublethal hydrostatic pressure. COCs denudation is better to perform 44 hours from the initiation of maturation procedure. Last 11 hours of maturation is better to perform in medium with decreased quantity of NaCl. Lipid content in oocyte significantly correlates with number of granulose cells per follicle. Larger size pre- and post pubertal oocytes are characterized by greater ability to development. Supplementation of culture medium with glycine leads to increase number of blastocyst cells. Addition of fibroblast growth factor to COCs culture medium benefits its development and blastocyst formation. COCs maturation normalization decreases polyspermy. Bringing endothelial growth factor in COCs medium culture increases rate of blastocyst formation. Different cells processes require different quantity of calcium ion oscillations. Temperature of liquid of preovulatory follicle can be indicator for oocyte quality. 14-days culture of COCs, recovered from early antral follicles, on polyacrylamide gel, results in achievement of larger size oocytes. It is improved method for optical observation of fertilization. It is worked out morula - blastocyst transportation for long distances. Introduction of Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel. Conclusion: last literature data show that row of different means of improvement for oocyte maturation and obtaining embryo in vitro is worked out.

High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

Controlled hydrostatic pressure stress downregulates the expression of ribosomal genes in preimplantation embryos: a possible protection mechanism?

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression

Sublethal stress treatment has been reported to enhance gametes' performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at -80°C until gene expression analysis. Re-expansion and hatching rates after vitrification-warming were significantly (P<0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.

Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes' and embryos' performance

The principal approach in in vitro embryo culture and manipulation has been a defensive one: procedures aim to satisfy passively the supposed or real physiological needs of gametes and embryos. Similarly, during cryopreservation the aim is to cause minimal damage to cells whilst attempting to obtain the highest achievable cell survival. However, carefully chosen and precisely controlled sublethal stress treatment of cells has been described to improve embryos' and gametes' performance, and, as a consequence, subsequent morphological survival, fertilisation, in vitro development, pregnancy and farrowing rates improved considerably compared with untreated controls. This review summarises studies that open up a new approach: instead of - and besides - trying to passively reduce the harm to cells during in vitro manipulations and culture, procedures may also prepare the cells themselves to ward off or reduce the damage by turning up the cells' own, inner capacities.

Stress for stress tolerance: improving cell survival by sublethal stress treatment of eggs before vitrification – pilot study

OBJECTIVE: Sublethal environmental stress induces general adaptation of cells and makes them more resistant during subsequent interventions such as cryopreservation. In the present study sublethal hydrostatic pressure (HP) stress was used to treat mouse and human oocytes before vitrification in order to increase post-warming survival and developmental competence.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38 °C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P<0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P<0.05) and TE (P<0.01) cell number and a lower percentage of picnotic nuclei (P<0.05) compared with the control group. Significantly lower abundance for BAX (P<0.01) and OCT4 (P<0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Sublethal Hydrostatic Pressure Pre-Treatment: Effect in Bovine Embryos Gene Expression.

Sublethal Hydrostatic Pressure Pre-Treatment: Effect in Bovine Embryos Gene Expression.

High Hydrostatic Pressure Treatment of Porcine Oocytes before Handmade Cloning Improves Developmental Competence and Cryosurvival

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.

Transcriptional response reveals translation machinery as target for high pressure in Lactobacillus sanfranciscensis

The effect of sublethal hydrostatic pressure on the transcriptome of Lactobacillus sanfranciscensis was determined using a shot-gun-microarray. Among the 750 spots that passed quality analysis 42 genes were induced, while six were repressed when cells were incubated at 45 MPa for 30 min. The nature of genes and their differential expression clearly indicate cellular efforts to counteract a decrease in translational capacity. The majority of high pressure affected genes were found to encode either translation factors (EF-G, EF-TU), ribosomal proteins (S2, L6, L11), genes changing translational accuracy or molecular chaperones (GroEL, ClpL). These data agree with previously reported effects observed in in vitro studies as well as with physiological and proteomic data. This study provides in vivo evidence to identify ribosomes and impaired translation among primary targets for high pressure treatment. The observed induction of heat as well as cold shock genes (e.g. hsp60, gyrA) may be explained as a result of high pressure affected protein synthesis.

Physiological responses to sublethal hydrostatic pressure in Yarrowia lipolytica

In this study some physiological and biochemical modifications induced by sublethal hydrostatic pressures, ranging between 200 and 800 bar, in four Yarrowia lipolytica strains were investigated. In particular, the changes in the proteolytic and lipolytic activities, as well as the hydrophobicity and cellular fatty acid composition of four strains, were analysed in order to carry out a preliminary investigation on response or adaptation mechanisms. This paper shows the involvement of the membrane systems and of fatty acid desaturase enzymes in the active response to sublethal doses, in addition to the enhancement of extracellular proteases.