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.

Controlled stress improves oocyte performance--cell preconditioning in assisted reproduction.

A recently emerged concept utilizing a controlled environmental impact as a treatment for cells and tissues aims to improve neither the in vitro conditions nor the procedures, but the cell itself. Hydrostatic pressure stress emerged as the most controllable and most effective stressor, proving the principle that controlled stress improves cell performance in in vitro procedures, whereas further studies using different stressors (osmotic, oxidative or mechanic stresses) supported the principle. The present summary reviews studies of various stress treatments to treat oocytes of three species (murine, porcine, human) before vitrification, in vitro maturation, enucleation and somatic cell nuclear transfer. Eventually, cleavage and blastocyst rates and--in cases when hydrostatic pressure was used--blastocyst cell number and birth rates as well were significantly improved compared to untreated controls.

Stress preconditioning of boar spermatozoa: a new approach to enhance semen quality.

Semen preparation and cryopreservation require finely adjusted procedures. Gametes are sensitive to environmental stresses, so in vitro procedures aim to minimize the inevitable harmful conditions. Applying stress to precondition cells has only been investigated recently. Studies demonstrated that by utilizing a well defined and properly applied hydrostatic pressure (HP) stress treatment to spermatozoa before in vitro storage, cryopreservation or insemination, cell survival and fertility improved compared with untreated controls. The birth of healthy piglets from treated fresh or frozen-thawed semen demonstrates the in vivo safety of the procedure. Although the biological mechanism is still unclear, several processes incorporating cellular stress response might explain the observations. This paper summarizes results, background, aspects and considerations of HP treatment for porcine semen. The new principle, i.e. to improve the stress tolerance by a defined sublethal stress may outline a new strategy in assisted reproductive technologies with unique theoretical and practical consequences.

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.

High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification.

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4+/-2.4%) or 130 (13.1+/-3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 degrees C or 25 degrees C. Oocytes pressurized at 37 degrees C had a significantly higher blastocyst (14.1+/-1.4%) rate than those treated at 25 degrees C (5.3+/-1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.