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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

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.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Oocytes are the core cell of many biotechnical procedures. Matured oocytes are used as recipient cells after enucleation to accommodate DNA of a different cell to build a new organism. Oocytes are matured and fertilized in vitro in human- and animal-assisted reproductive procedures; resulting zygotes cleave and develop in culture, and, if transferred, develop to term, all of these miraculously resulting in a healthy offspring: a procedure that can function with 50% or greater. The contribution of the oocyte is vital for all of these processes. A good quality oocyte with proper nuclear and cytoplasmic maturation that was not corrupted during the in vitro manipulations would offer a significantly higher chance for a viable offspring compared with compromised counterparts. In other words, proper procedures and competent cells together confer a higher chance for success.

Procedures are being optimized to find the proper environment and manipulation techniques. Conversely, the quality of the oocytes depends on many factors. The ability of the oocyte to acquire developmental competence and to support early embryonic development is the result of the regulation of its transcriptional activity and the synergic actions of gonadotropins and growth factors (Menezo and Elder 2011). Regulation of transcriptional activity includes timely translation of stored maternal transcripts, post-translational modification of stored or newly synthesized proteins (this sets the exact timing for cellular events), and processes involved in degradation of proteins and mRNAs (Song and Wessel 2005; Stitzel and Seydoux 2007; Evsikov and Marin de 2009). All of these are influenced by hormones, genetic, nutritional, immunological factors and age, as well as environmental impacts (Grøndahl et al. 2010).

A recently emerged concept utilizing a controlled environmental impact (e.g. high hydrostatic pressure) as a treatment for cells and tissues aims to improve neither the in vitro conditions nor the procedures, but the cell itself. Stronger cells will perform better: as a further interpretation of William Harvey’s claim stating that ‘ex ovo omnia’, a more competent oocyte would confer a better success for all procedures originating from the oocyte.

Controlled stress improves cells’ performance (Pribenszky et al. 2010a,b). The emphasis is on the definition ‘controlled’. For example, although uncontrolled sheer stress during moving embryo culture dishes or vigorous pipetting may harm cells, controlled mechanic stress applied at the right time of oocyte development might precondition cells, enabling them to perform better (Xie et al. 2006; Mizobe et al. 2010).

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 various stressors (osmotic, oxidative or mechanic stresses) supported the principle.

Here, we summarize and compare defined stress treatment protocols for oocytes using hydrostatic pressure, osmotic, oxidative or mechanic impacts. First, oocytes’ stress tolerance is described for the given stressor – as the initial and most important step for building up a treatment protocol – then, the results of routine, state-of-the-art procedures [vitrification, in vitro maturation, in vitro culture, somatic cell nuclear transfer (SCNT)] are compared, with or without stress preconditioning of oocytes.

Stress Preconditioning of Oocytes

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Sublethal stress was originally used in food industry to apply a sequence of mild treatments to foodstuff to reduce microbial load, but at the same time preserve food quality. However, in sharp contrast with the expected effect, Wemekamp-Kamphuis et al. (2002) reported that the proliferation of Listeria monocytogenes was not decreased but significantly increased as a consequence of sequential treatment with cold shock and hydrostatic pressure (HHP). It appears that biological effects of the first sublethal treatment preconditioned the bacteria, protecting them from the detrimental effects of the second sublethal treatment.

This observation eventually motivated research to apply sublethal stress to gametes, embryos, stem cells to improve success rates of in vitro procedures such as cryopreservation, in vitro maturation, in vitro culture, SCNT, extended in vitro storage, or even artificial insemination.

Initially, HHP treatment was chosen for a sublethal stressor, because of its unique and outstanding features: (i) acts instantly and uniformly at every point of the cell, (ii) features zero penetration problems or gradient effects, (iii) can be applied with the highest precision, consistency, reliability and safety, (iv) functions with an extremely high safety margin and wide therapeutic range for the cells and (v) has no or minimal cell-to-cell variation. Each of these features clearly differentiates it from all other environmental stressors.

As for the first step, the HHP stress tolerance of the oocytes had to be defined together with proper handling procedures. As soon as treatment parameters were defined, it was used to precondition oocytes before enucleation, vitrification or in vitro maturation. Based on these experiments, further stress factors (osmotic, oxidative and mechanic stresses) were also evaluated and published by various groups.

Hydrostatic Pressure Treatment of Oocytes

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Stress tolerance and protocol setup

The treatment protocol for porcine oocytes was set up by Pribenszky et al. (2008) and Du et al. (2008a,b,c) and was also applied to murine and human eggs, with minor modifications. In brief, oocytes (immature (GV stage) or in vitro matured (MII. stage) were aspirated into 0.5 ml straws (IMV, France) in media used to manipulate cells outside the incubator (Fig. 1). For porcine oocytes, TCM-199 medium supplemented with Hepes (TCMH) was used, whereas for mouse or human oocytes, G-Mops (Vitrolife, Go¨teborg, Sweden) was used.

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Figure 1.  Oocytes or cumulus–oocyte complexes loaded into artificial straws ready for HHP treatment. Straws were closed by iron balls or plastic plugs

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For stress tolerance studies, porcine cumulus–oocyte complexes were randomly distributed among 12 treatment groups and controls. Treatments were done simultaneously, testing a matrix of treatment parameters including pressures of 20, 40, 60 and 80 MPa, and treatment times of 30, 60 and 120 min, both at room and body temperatures. Presumptive zygotes were then cultured in vitro until 7 days after HHP treatment. Programmable hydrostatic pressure machines (HHP100, Cryo-Innovation Ltd., Hungary) were used to execute the treatments (Figs 2 and 3).

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Figure 2.  HHP100: programmable hydrostatic pressure generating device (Cryo-Innovation Ltd., Hungary). a. Pressure chamber with the lid closed. b. Touch screen that is used to run preset or designed programs uploaded from the PC; sets pressure, time and temperature parameters for a current program; shows actual pressure and temperature parameters in the pressure chamber and stores data of previous cycles. The HHP100 is able to produce controlled hydrostatic pressure in the range of 2–90 MPa

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Figure 3.  PC user interface of the HHP100. (a, b) Panel ‘a’ shows an example of designing a unique pressure profile with oscillating curve between 15 and 75 MPa; Panel ‘b’ shows a standard treatment curve running at room temperature with a set 20 MPa treatment level

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Oocytes treated in the range of 60–80 MPa did not even cleave, whereas those treated with 20 MPa/60 min resulted in a 46% blastocyst rate compared with the control group of 22% (p < 0.05) (Pribenszky et al. 2008).

In the subsequent step, the effect of recovery time was assessed. The HHP treatments were followed by different equilibration periods. Equilibration meant that after the treatment oocytes were removed from the HHP machine and placed back into the incubator in in vitro maturation medium for the corresponding time. When the equilibration time was complete, vitrification or enucleation followed. The optimal treatment that resulted the highest improvement in oocyte competence was treatment of cumulus-oocyte complexes (COCs) at 37°C with 20 MPa for 60 min, followed by 1 or 2 h of equilibration before vitrification or enucleation, respectively (Du et al. 2008a,b,c; Pribenszky et al. 2008).

Treatment media

The HHP treatment did not cause parthenogenetic activation in the studies when TCMH medium was used to hold oocytes during treatment. As various physical (mechanical, electronic, osmotic), chemical and other treatments (Machaty and Prather 1998; Machaty et al. 1999; Somfai et al. 2007; Tian et al. 2007) were reported to cause parthenogenetic activation, further experiments were conducted to investigate the effect of various holding media with different Ca2+ concentrations on possible-HHP induced parthenogenetic activation of the oocytes. The media included TCMH medium, mannitol-PVA fusion medium with (MPVA+Ca2+) or without Ca2+ and Mg2+ (MPVA). HHP did not induce parthenogenetic activation in TCMH, but only in MPVA+Ca2+ with low Ca2+ concentration and MPVA. The highest activation efficiency was achieved with 10 min HHP treatment using 10 or 20 MPa for oocytes in MPVA+Ca2+ or MPVA, respectively (Figure 4). In the light of these results, the possible source of Ca2+ during activation was investigated. Even after a total of 30 min wash with TL-HEPES-PVA buffer without Ca2+ before HHP treatment in MPVA, oocytes could still be activated, indicating the possibility of an intracellular Ca2+ source. It was concluded that parthenogenetic activation could be induced by HHP, but only in certain holding media with low or zero Ca2+ content.

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Figure 4.  Parthenogenetic activating effect of various HHP treatments in three treatment media

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Microscopic morphology of the oocytes after HHP treatment also revealed differences caused by the applied holding media; in that regard, the perivitelline space was considerably enlarged in all treated groups with MPVA+Ca2+ and MPVA; however, no such changes were observed in untreated groups, or treated groups in TCMH holding medium (Lin et al. 2010).

HHP treatment to improve in vitro maturation and in vitro embryo production

Nuclear and cytoplasmic maturation of the oocyte have been considered the most significant parameters affecting the success of in vitro fertilization (IVF) (Krisler 2004). The efficiency of oocyte maturation in human IVF procedures is still suboptimal; during ART cycles, only approximately 5% of fresh oocytes produce a baby (Patrizio and Sakkas 2009). In fact, cytoplasmic competence in oocytes, that is, the ability to produce embryos with high developmental potential, is poorly defined biochemically.

The transition from oocyte to fertilized egg (zygote) involves many changes, including protein synthesis, protein and RNA degradation, and organelle remodelling. These changes occur concurrently with the meiotic divisions that produce the haploid maternal genome. Accumulating evidence indicates that the cell-cycle regulators that control the meiotic divisions also regulate the numerous changes that accompany the oocyte-to-zygote transition. In studies described below, a sublethal HHP stress promoted competence of the maturing oocyte to produce a better quality embryo.

Pig

Germinal vesicle stage oocytes surrounded by cumulus cells were aspirated into 0.5 ml straws in TCMH medium. Straws were then treated as described above, at room temperature (24°C; HHP-treated group). Control groups included GV oocytes kept at 24°C for 60 min (Control group I) and unaffected oocytes kept in the incubator in IVM medium for the corresponding time (Control group II.) Following that, COCs were released from straws and incubated in the original IVM (in vitro maturation) medium until the start of the parthenogenetic activation. Cleavage rates, day 6 blastocyst rates and blastocysts’ cell number were compared between groups. Generalized linear model was used for statistical analysis (values with p < 0.05 were regarded as different). Results are presented in Table 1 (Pribenszky et al. 2008).

Table 1.   Blastocyst rate and cell number of HHP-treated GV oocytes followed by IVM, PA and IVC
Experimental groupBlastocyst rate (%)Cell number
  1. Values are mean percentage ± SEM.

  2. *Differs significantly from the control.

Untreated control47 ± 248 ± 3
Control, kept at room temperature39 ± 347 ± 4
HHP treated54 ± 2* (p < 0.05)60 ± 3* (p < 0.01)

The study was repeated independently by Kurome et al. in 2011 (unpublished results of Mayuko Kurome, Barbara Kessler and Eckhard Wolf; Molecular Animal Breeding and Biotechnology, LMU Munich, Germany) with a slight modification: HHP treatment of the COCs was performed at 38°C. In total, 608 COCs were used in seven replicates; they were distributed into treatment and control groups. Results are shown in Table 2.

Table 2.   Blastocyst rate and cell number of HHP-treated GV oocytes followed by IVM, PA and IVC
Experimental groupBlastocyst rate (%)Cell number
  1. Values are mean percentage ± SEM.

  2. *Differs significantly from the control.

Untreated control20 ± 258.7 ± 3
HHP treated24.3 ± 280.9 ± 4* (p < 0.01)

In the presented studies, oocytes treated with a single controlled hydrostatic pressure stress at the beginning of the maturation process developed to the blastocyst stage with a higher chance than controls. Whereas maturation and cleavage rates were not affected at either of the studies, treatment significantly increased the cell number of the blastocyst with p < 0.01 in both cases. HHP treatment of immature oocytes increased embryo quality during in vitro embryo production.

HHP treatment to improve cryotolerance of oocytes

Oocyte cryopreservation has become one of the most challenging approaches to restore fertility of chemo- and radiation therapy treated women with compromised ovarian function (Falcone and Bedaiwy 2005) and improve reproductive flexibility of in vitro assisted reproductive technologies in humans (Porcu and Venturoli 2006; Vajta and Kuwayama 2006; Vajta and Nagy 2006). In animals, endangered species and premium genetics from specific breeds could be rescued, whereas the expense and disease transmission during storage and transportation could be minimized (Vajta 2000). Although vitrification of human oocytes can result in pregnancy rates comparable to that of the fresh ones as the result of newly defined techniques (Cobo et al. 2008; Rienzi et al., 2010), improvements are still needed, especially in animals, including pigs (Somfai et al. 2012).

Pig

A total of 1668 porcine IVM oocytes were used by Du et al. (2008a,b,c) in control and treatment groups, treated in TCMH as described above. 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 equilibration times between treatment and the start of the vitrification, when compared with the 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 post-HHP equilibration were performed at 37 or 25°C. Oocytes pressurized at 37°C had a significantly higher blastocyst (14.1 ± 1.4%) rate than those treated at 25°C (5.3 ± 1.1%; p < 0.01) or untreated control (1.3 ± 1.3%). Overall, HHP pretreatment considerably improved developmental competence of vitrified pig in vitro-matured (IVM) oocytes.

Mouse

In vivo matured mouse oocytes (COCs) were treated as described above, at 37°C. After a short incubation period, state-of-the-art cryopreservation followed as described before, using the Cryotop method (Kuwayama et al. 2005). Eggs were fertilized by ICSI using a Piezo-driven system (Kuretake et al. 1996) after warming, then cultured and transferred to recipient females. Post-warming survival rate was similar between the groups, but cleavage and blastocyst rates, blastocyst ICM cell number, and, most importantly, birth rates were significantly higher in the oocytes HHP stressed before vitrification (Table 3).

Table 3.   Success rates of oocyte vitrification with or without HHP treatment. Embryos were either transferred at the two-cell stage or cultured to the blastocyst stage for cell count
 Experimental groupSurvival rate (%)Cleavage rate (%)Birth rate (%)Blastocyst rate (%)Blastocyst ICM cell number
  1. Values are mean percentage ± SEM.

  2. *Differs significantly from the control.

Untreated control7142125017 ± 1
HHP treated7351*27*60*21 ± 2*
Human

Discarded IVM or not fertilized human oocytes were used for this pilot study. Oocytes (110) were randomly distributed to treatment or control groups. Treatment groups were treated as described above. Morphological survival and parthenogenetic activation were the tools to assess survival after cryopreservation. MediCult’s Oocyte Freezing kit was used for cryopreservation as described by the manufacturer (MediCult, Denmark, today Origio, Denmark). The parthenogenetic activation protocol included 10 μm calcimicin treatment for 5 min, followed by incubation with 5 mm DMAP for 3 h. None of the activated oocytes developed to the blastocyst stage, possibly due to their inappropriate cytoplasmic maturation; however, HHP treatment increased rates of both survival (62 vs 67%) and activation (30 vs 40%) rate for control and treated groups, respectively (Matyas et al. 2010; Pribenszky et al. 2010a,b).

HHP treatment to improve the efficacy of SCNT

Authors aimed to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by hand-made cloning (HMC) after HHP treatment of the recipient oocytes. In vitro-matured porcine oocytes were treated with 20 MPa as described above, then equilibrated for 2 h before enucleation. Two cell lines (from Day 40 fetuses of Yucatan and Danish Landrace breeds (LW1 – 2)) were used as donor cells for nuclear transfer. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method as referred above. The blastocyst rate was higher in the HHP-treated groups compared with the control groups at both of the cell lines (p < 0.01). Cell number of the blastocysts was similar; however, subsequent blastocyst vitrification resulted in significantly higher survival rate in the HHP group compared with the control group after thawing (Table 4.).

Table 4.   Blastocyst rate, cell number, blastocysts’ cryosurvival and birth rates achieved by the HHP treatment of recipient oocytes before SCNT, compared with untreated controls
Experimental groupBlastocyst rate (%) with different donor cell linesCell numberCryosurvival of vitrified/warmed blastocysts (%)Birth of piglets
Yucatan cell lineLW1-2 cell line
  1. Values are mean percentage ± SEM.

  2. *Differs significantly from the control.

Control28.9 ± 346.4 ± 449 ± 530 ± 4
HHP treated57.0 ± 2*68.2 ± 4*56 ± 462 ± 4*2

Representative embryo transfer of reconstructed embryos from the HHP group resulted in the birth of two healthy piglets by natural delivery on day 122 of gestation.

The authors concluded that sublethal HHP treatment of porcine oocytes before HMC improved in vitro developmental competence and cryotolerance and supported embryonic and foetal development, as well as pregnancy establishment and maintenance during pregnancy, up to the birth of healthy piglets (Du et al. 2008a,b).

Osmotic Stress Treatment of Oocytes

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Stress tolerance and protocol setup

Temporary increase of NaCl concentration on cryotolerance and developmental competence of porcine oocytes was tested by Lin et al. (2009a,b). Survival rates were compared after 1 h exposure to seven elevated NaCl concentrations (from 0.25 to 4%, equivalent to 361–to 1306 mOsmol). Survival rate after treatment was only reduced at 1.5 and 2% NaCl concentrations (710 and 850 mOsmol). In subsequent experiments, oocytes were exposed to 593 mOsmol NaCl, equilibrated for 1 or 2 h, vitrified, then subjected to parthenogenetic activation or used as recipients for SCNT. Blastocyst rates increased in both cases after NaCl treatment compared with untreated controls. (Lin et al. 2009a). In a forthcoming experiment, further chemicals were tested to provide increased osmotic pressure of the treatment medium, including NaCl, sucrose or trehalose applied at the same osmotic level (588 mOsmol). Subsequently, COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period, cumulus cells were removed and the oocytes were subjected to further treatments, as described below (Lin et al. 2009b). All treatments improved blastocyst rate; however, NaCl had no influence, whereas trehalose and sucrose reduced the cell number of the blastocysts.

Hyperosmotic treatment to improve cryotolerance of oocytes

Porcine in vitro-matured cumulus–oocyte complexes were exposed to 588 mOsmol NaCl, sucrose or trehalose solutions for 1 h, allowed to recover for a further 1 h, vitrified (using the Cryotop method as described above), warmed and subjected to parthenogenetic activation (as described above). Both Day 2 (Day 0 = day of activation) cleavage and Day 7 blastocyst rates were increased after NaCl, sucrose and trehalose osmotic treatments compared with untreated controls (Table 5.).

Table 5.   Continued in vitro development of oocytes treated with different osmotic agents of 588 mOsmol for 1 h, followed by cryopreservation/warming, parthenogenetic activation and IVC
Experimental groupBlastocyst rate (%)
  1. Values are mean percentage ± SEM.

  2. Different superscripts in a column differ significantly from each other.

Untreated control3 ± 1a
NaCl treated9 ± 2b
Sucrose treated8 ± 2ab
Trehalose treated9 ± 3b

Hyperosmotic treatment to improve the efficacy of SCNT

The COCs were treated with 588 mOsmol NaCl, sucrose or trehalose, then used as recipients for SCNT (Day 0). Cleavage rates on Day 1 did not differ between the NaCl-, sucrose-, trehalose-treated and the untreated control groups, but blastocyst rates on Day 6 were higher in all treated groups compared with control. Cell numbers were significantly lower in the sucrose- and trehalose-treated groups, whereas the NaCl-treated group did not affect cell number compared with the control group (Table 6). In conclusion, treatment of porcine oocytes with osmotic stress improved blastocyst rate, but not the cell number after enucleation and SCNT (Lin et al. 2009a,b).

Table 6.   Blastocyst rate and cell number of blastocysts originating from oocytes treated with different osmotic agents of 588 mOsmol for 1 h before SCNT
Experimental groupBlastocyst rate (%)Mean cell number
  1. Values are mean percentage ± SEM.

  2. Different superscripts in a column differ significantly from each other.

Control47 ± 4a63 ± 4a
NaCl64 ± 2b62 ± 6a
Sucrose69 ± 5b48 ± 3b
Trehalose65 ± 3b48 ± 4b

Oxidative Stress Treatment of Oocytes

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Stress tolerance and protocol setup

The effect of controlled oxidative stress to IVM bovine oocytes was investigated based on previous studies about sublethal HHP stress treatments. For all experiments, immediately before fertilization, COCs were incubated in various concentrations (low, 0.01 or 0.1 μm; medium, 1 or 10 μm; high, 100 μm; or very high, 1 mm) of H2O2 for 1 h before fertilization. Because pyruvate can neutralize the effect of peroxide (Morales et al. 1999), the different concentrations were diluted in maturation medium without pyruvate. Meanwhile, two control groups were kept in maturation medium without pyruvate and normal maturation medium, respectively. After incubation, COCs were washed in HEPES-TALP. After H2O2 exposure, mature oocytes were fertilized. At 24 h post-insemination, presumed zygotes were denuded and cultured in modified SOF medium. Embryos were evaluated for blastocyst development, total cell number and apoptotic cell ratio. Fertilization and penetration rates were comparable between the groups; only very high concentrations of H2O2 (1 mm) resulted in significantly lower fertilization (22.1 ± 5.25%) and penetration rates (34.4 ± 5.22%) in comparison with all other groups (Vandaele et al. 2010).

Oxidative stress treatment to improve in vitro maturation and in vitro embryo production

Exposure of oocytes to high H2O2 concentration (50–100 mm) produced more blastocysts in comparison with the control group (47.3 and 31.8%, respectively). The mean number of blastomeres at the blastocyst stage (7 days post-insemination) varied between 100 and 120 and was not different between groups. Interestingly, the mean apoptotic cell ratio of the high H2O2 group did not differ from the control, but was significantly lower in comparison with low or medium H2O2.

Short-term exposure of mature COCs to high concentrations of H2O2 induced stress tolerance during further development, manifested as enhanced embryo development, but it did not affect apoptosis in blastocysts. In contrast, low to medium H2O2 concentrations significantly increased apoptosis in Day 7 blastocysts. Neither the higher GSH levels in mature oocytes nor the enhanced penetration or fertilization rate was the cause of the positive effect of H2O2 on oocyte competence. The beneficial effect of H2O2 on embryo development should therefore be related to other (uncharacterized) effects of H2O2, presumably on the oocyte.

The positive effect of H2O2 at the end of the maturation period is not mediated by increased GSH content or improved fertilization, but is an effect on the long term up to the morula or blastocyst stage more than 4 days later (Vandaele et al. 2010).

Mechanic Stress Treatment of Oocytes

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Stress tolerance and protocol setup

Mizobe et al. (2010) tried to mimic the ciliary beating of oviductal epithelial cells and contraction of oviductal smooth muscle by applying mechanic stimuli to oocytes. Authors hypothesized that mechanical vibration of dishes containing oocytes and embryos during in vitro maturation and in vitro culture might improve production efficiency of blastocysts. Mechanical vibrations at a frequency of 20 Hz and accelerations of ±0.33 G and ±0.11 G in the x-axis and y-axis directions, respectively, were applied. The COCs were cultured with mechanical vibration for 5, 10 or 60 s at intervals of 10, 30, 60 or 90 min. Control COCs were cultured without mechanical vibration. After culture, some oocytes were examined for in vitro maturation. The control group was activated and cultured without mechanical vibration. The effects of intervals of mechanical vibration during in vitro maturation on the nuclear maturation and parthenogenetic development of oocytes were examined. In a forthcoming experiment, oocytes matured without mechanical vibration were activated and cultured with mechanical vibration for 5 s at intervals of 10, 30, 60, 180, 360 or 720 min. Control oocytes were cultured without mechanical vibration. The effects of mechanical vibration during in vitro maturation of recipient oocytes and/or in vitro culture after reconstruction on the development of SCNT embryos were also examined. Oocytes matured with or without mechanical vibration for 5 s at intervals of 60 min were enucleated and fused with donor cells. Fused embryos were cultured with (for 5 s at intervals of 60 min) or without mechanical vibration. Mechanical vibration during in vitro culture after activation did not affect the blastocyst formation of oocytes (Table 7).

Table 7.   Blastocyst formation rate of SCNT embryos matured or cultured with or without mechanic stimuli
Experimental groupsBlastocyst rate (%)Mean cell number of the blastocysts
Mechanic stimuli during IVMMechanic stimuli during IVC
7.4 ± 0.934.8 ± 1
+9.4 ± 0.935.7 ± 1.5
+13 ± 0.336.8 ± 1
++17.6 ± 2.538 ± 1.4

Treatment durations of 5 and 10 s per 60 min increased blastocyst rates, whereas other treatments caused no changes. Blastocyst cell number was the same in all groups (Mizobe et al. 2010).

A different group has investigated a further mechanic stimulus, sheer stress, by applying a non-static, namely tilting culture system. To set tilting parameters, uniform radial velocity, the maximum tilt angle and the holding time at the maximum tilt angle were defined. First, the plate is tilted to the positive maximum tilt angle. Second, the tilting plate is held static, next, the plate is tilted to the negative maximum tilt angle. Last, the tilting plate is held with no motion.

The maximum tilt angle that caused mineral oil to spill out was defined and set the limit of the maximum tilt angle to approximately 20°. Furthermore, excess uniform radial velocity also induces spill out of the mineral oil. The minimum radial velocity at which the mineral oil spilled from the 35-mm dish was 240°/s when the maximum tilt angle was 20°. The oil did not spill out when the tilt angle was 10°. It is necessary to increase the maximum tilt angle and radial velocity to move embryos in the microdrop. However, this study set the maximum tilt angle and radial velocity so that they did not result in the spilling out of the mineral oil, but still allowed the observation of embryo motion in the video rate recording. Thus, the maximum tilt angle was 10–20° and the radial velocity was approximately 1°/s. Frozen 2-cell-stage embryos were cultured at a maximum tilt angle of 20° with a holding time of 1 min. The plate was rotated at 1°/s to reach a total tilt of 20°. Frozen-thawed human embryos were also used to test the system. As the consequence, the culture system used did not affect blastocyst rate, but increased cell number of the embryos by 16–26% (Matsuura et al. 2010).

Mechanic stress treatment to improve in vitro maturation and in vitro embryo production

Mechanical vibration during in vitro culture (ICV) after activation did not affect blastocyst formation (11.6 ± 5.2–16.5 ± 3.0%) of in vitro activated porcine oocytes. However, blastocyst formation rates after activation of oocytes matured with mechanical vibration for 5 s at intervals of 30–60 min or for 10 s at intervals of 60 min were higher than those of oocytes matured without mechanical vibration (25.7 ± 2.0–28.1 ± 2.7% vs 12.3 ± 1.4% and 25.8 ± 1.8% vs 15.7 ± 1.9%, respectively). Therefore, mechanical vibration enhanced the cytoplasmic maturation of in vitro-matured pig oocytes, resulting in improvement of their parthenogenetic development (Mizobe et al. 2010).

In another study, the effects of continuous tilting of the culture dish during IVM of porcine COCs and/or IVC following IVF of oocytes on early development to the blastocyst stage and the cell number of blastocysts were investigated. The tilting culture during IVM has improved the diameter of COCs after IVM, whereas maturation itself was unaffected. Although the results and their interpretation is conflicting (blastocyst rate and average cell number in the control group: 28.3% and 30.4; group tilted during IVM: 29.1% and 30.1, group tilted during IVC: 24.9% and 34.1), the authors concluded that tilting culture was beneficial for embryo development (Koike et al. 2010).

Mechanic stress treatment to improve the efficacy of SCNT

Mechanical vibration during IVM and/or IVC with different intervals and frequencies, as described before, was used. Mechanical vibration for 5 s at intervals of 60 min during in vitro maturation of oocytes did not affect fusion with miniature pig somatic cells after enucleation. However, the blastocyst formation rate of SCNT embryos was improved (p < 0.05) by mechanically vibrating recipient oocytes for 5 s at intervals of 60 min during in vitro maturation, regardless of the presence or absence of the same treatment during in vitro culture. The treatment did not affect cell number of the blastocysts.

Conclusions

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Controlled stress treatment of oocytes helped to improve their survival and continued in vitro development during in vitro maturation, cryopreservation, enucleation and SCNT. However, the level and way of improvement and treatment consistency differed among treatments. The effect of the stress treatment was more evident as the in vitro (and in vivo) development progressed, showing that it is the function of the cell that is preserved, maintained and enhanced primarily. For example, hydrostatic pressure stress treatment of mouse oocytes before vitrification resulted in similar post-warming survival, 20–25% increase in blastocyst rate and blastocyst cell number, and more than doubled birth rates, compared with untreated controls.

Four types of stressors used to precondition oocytes are described in this review. Hydrostatic pressure, osmotic and oxidative stresses all followed the same principle based on the same claim. First, stress treatment protocols were defined by stress tolerance studies aiming to define a sublethal stress level that does not cause irreversible damage to the cells, but improve their survival rates after different in vitro manipulations. Second, protocols were refined and were compared with untreated controls to define improvement levels and consistency. All of these studies were based on the premise that the sublethal stress treatment triggered cellular stress reaction, which actually improved the cells’ resistance and capacity, at the post-transcriptional level.

Authors claimed at experiments using mechanic stimuli that they mimic the in vivo environment by either shaking or tilting the embryo culture dishes during IVM. As oocyte maturation in vivo takes place in the ovaries, it seems unlikely that mechanic vibration is a physiological promoter of nuclear and cytoplasmic maturation in vivo. Rather, it is a cellular stress reaction as a response to controlled sublethal mechanic stimuli that may support improved developmental competence of the treated cells, reported in the studies. Coincidentally, bovine oocytes experiencing heat stress during the first 12 h of meiotic maturation had equivalent or higher development after parthenogenetic activation compared with unstressed controls (Rispoli et al. 2011).

Stress treatments of oocytes were performed in human (HHP), mouse (HHP) and porcine (HHP, oxidative, osmotic and mechanic stresses) species in processes like vitrification (HHP, osmotic stresses), in vitro maturation (HHP, oxidative and mechanic stresses) and SCNT (HHP, osmotic and mechanic stresses). Hydrostatic pressure stress treatment proved to be the most effective as of increasing blastocyst rate and cell number of the blastocysts originating from treated oocytes. Offspring was born from HHP-treated eggs both following cryopreservation, ICSI and IVC, and following enucleation, SCNT and IVC (Tables 8–10).

Table 8.   Effect of stress treatment of different stressors on the cryotolerance of oocytes of different species
Stress typeSpeciesCryosurvivalCleavageBlastocystBlastocyst cell no.Birth
  1. nd, no data; +, improved (p < 0.05); +++, highly improved (p < 0.01); 0, no difference.

HHPMouse0++++++
Human++ndndnd
Pig+++++ndnd
OsmoticPignd++ndnd
Table 9.   Effect of stress treatment of the recipient porcine oocytes with various stressors on the efficacy of SCNT
Stress typeCleavageBlastocystBlastocyst cell no.Blastocyst cryotoleranceBirth
  1. SCNT, somatic cell nuclear transfer.

  2. nd: no data; +: improved (p < 0.05); +++: highly improved (p < 0.01); 0: no difference; −: reduced.

HHPnd++++++
Osmotic++−/0ndnd
Mechanic0+0ndnd
Table 10.   Effect of stress treatments of immature porcine oocytes during IVM followed by parthenogenetic activation and IVC
Stress typeMaturationCleavageBlastocystBlastocyst cell no.
  1. +, improved (p < 0.05); 0, no difference.

HHP00++
Oxidative00+0
Mechanic00+0

Other studies examined the HHP stress effect on ovine and bovine blastocysts and also reported increased cell number of blastocysts. The HHP treatment of ovine blastocyst increased cell number and decreased the apoptotic cell ratio in routine IVP studies and also in experiments incorporating vitrification/warming (Ledda et al. 2010; Bogliolo et al. 2011), whereas cell number of HH- treated bovine blastocysts increased significantly 2 days after treatment (C. Diez, unpublished data, 2011).

Vandaele et al. (2010) found that oxidative stress did not increase fertilization or penetration rates in porcine oocytes; neither higher GSH levels in mature oocytes nor the enhanced fertilization rate was the cause of the positive effect of H2O2 on oocyte competence.

Further studies described that HHP stress did not affect gene expression level at the oocyte stage, whereas it was significantly affected after the activation of the embryonic genome (Pribenszky et al. 2010a,b). The affected pathways among the 676 significantly changed genes were characterized; the most outstanding categories that were clustered together were related to ribosome or translation. The majority of the altered genes had significantly downregulated expression in response to HHP treatment. The authors concluded that ribosomal processes may play a central role in the HHP-treated and fertilized oocytes during pre-implantation development. Stress effects in the transcriptional processes were visible after the embryonic genome activation showing downregulation in the most energy consuming processes. Reduced energy consumption may give a developmental advantage to the embryo, in accordance with previous findings for the improved developmental competence of HHP-treated gametes and embryos (Bock et al. 2010; Pribenszky et al. 2010a).

Although several studies reported negative effects of environmental stress, authors of the cited experiments fine-tuned various stress effects to precondition/treat cells. As a conclusion, controlled, sublethal stress treatment of oocytes improved oocyte performance in these studies, suggesting a rather general approach of the principle. However, stress types differ significantly if consistency and effect size is regarded.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References

Studies with human and mouse oocytes were supported by NKTH (Hungary) OM-00069/2008, EGG_Care; travel expenses of A. Dinnyes were supported by TÁMOP 4.2.2/B-10/1-2010-011.

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  2. Contents
  3. Introduction
  4. Stress Preconditioning of Oocytes
  5. Hydrostatic Pressure Treatment of Oocytes
  6. Osmotic Stress Treatment of Oocytes
  7. Oxidative Stress Treatment of Oocytes
  8. Mechanic Stress Treatment of Oocytes
  9. Conclusions
  10. Acknowledgements
  11. Conflicts of interest
  12. References
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