BACKGROUND
one feature of in vitro produced mammalian embryos is the high frequency of early developmental failure brought on by sub-optimal culture environments  <cit> .
fewer than half of all in vitro fertilized  bovine embryos reach the blastocyst stage of development  <cit>  with many of these unable to sustain development following embryo transfer  <cit> .
the reasons for this high rate of in vitro embryo demise remains unclear, but it has been proposed as a protective mechanism for preventing further development of abnormal, poor quality embryos.
almost half of all arrested human embryos display chromosomal abnormalities  <cit> , and significantly more chromosomal aberrations are observed, alongside delayed development, for in vitro produced bovine embryos compared to their in vivo-derived counterparts  <cit> .
blastomeres with characteristic features of apoptosis, such as cytoplasmic, nuclear and dna fragmentation, have been detected in both in vitro and in vivo derived embryos, indicating that high levels of apoptosis might play a role in early embryo death  <cit> .
in situ tunel labelling has identified a greater incidence of apoptotic nuclei in cultured bovine blastocysts compared to those derived in vivo  <cit> .
interestingly, we  <cit>  and others  <cit>  have observed no morphological or biochemical signs of apoptosis during the early 2– <dig> cell cleavage stages of bovine embryogenesis.
it is at this early developmental stage that approximately 14% of all in vitro produced  bovine embryos permanently arrest in a senescence-like state   <cit> .
cellular or replicative senescence is the well known in vitro phenomenon whereby most proliferating somatic cell types irreversibly cease in their replicative capacity after a fixed number of population doublings and display distinct morphological features of "aged" cells  <cit> .
senescence is triggered by a p53-dependent signalling cascade associated with the shortening of telomeres, the tandem g-rich repetitive, non-coding dna sequences and specific binding proteins at the ends of linear chromosomes  <cit> .
we have previously shown that permanent cell growth arrest of cultured bovine somatic cells is associated with telomere shortening  <cit> , increased levels of serine 20-p <dig> phosphorylation, and elevated levels of oxidative damage  <cit> .
since permanent growth arrest/cellular senescence occurs prematurely under conditions of elevated oxidative stress  <cit>  and can be activated by disruption of the telomere structure itself, even at maximally long telomere lengths  <cit> , we hypothesize that arrested mammalian embryos permanently arrest in their development by a similar stress signalling pathway.
at the cellular level, an oxidative stress sensor that can provide a key understanding of cell senescence and permanent embryo arrest is p66shc, the recently identified protein belonging to the shc family of adaptors for signal transduction in mitogenic and apoptotic responses.
p66shc is involved in signal transduction pathways that regulate the cellular response to oxidative stress and life span and has been shown to act as a downstream target of p53-mediated elevation of intracellular oxidants, cytochrome c release and apoptosis  <cit> .
p66shc is important in the signalling response to ros and propagation of apoptotic signals in mouse fibroblasts  <cit> .
remarkably, p66shc mice exhibit increased resistance to oxidative stress and have a ~30% longer lifespan than wild-type control animals with no apparent side effects  <cit> .
we have measured elevated levels of p66shc mrna and protein in senescent somatic cells and in permanently arrested 2– <dig> cell bovine embryos  <cit> .
recently, we have demonstrated that p66shc is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ros  <cit> .
taken together, these data suggests that p66shc regulates an oxidative stress-induced senescence pathway that triggers permanent embryo arrest.
the objective of this study was to elucidate a functional role for p66shc in embryo arrest by selectively decreasing p66shc levels in bovine oocytes and embryos using the post-transcriptional gene silencing technique of rna interference .
RESULTS
quantification of p66shc, h2a and p <dig> mrna transcripts
determination of the number of p66shc, h2a and p <dig> mrna transcripts was carried out by real time pcr in arrested 2– <dig> cell embryos collected at day  <dig> post-insemination, in early cleaving embryos ) that show very low percentage of embryo arrest and in late cleaving embryos  that are more likely to arrest, in accordance to previous observations  <cit> .
these data are summarized in table  <dig> along with the amounts of these specific gene transcripts in day  <dig> blastocysts.
significantly greater quantities of p66shc mrna transcripts  were measured in both arrested  and late cleaving embryos  that are more likely to arrest compared to early cleaving  <dig> hpi embryos.
no differences in h2a or p <dig> mrna levels were detected among early and late cleaving embryos, arrested embryos, and blastocysts.
the numbers of p66shc mrna transcripts at the blastocyst stage were significantly  lowered compared to those measured in early cleaving and arrested embryos .
values represent mean ± sem
different superscripts differ significantly
determination of the number of p66shc shrnai molecules to microinject
in the attempt to down-regulate p66shc mrna expression, we wanted to microinject the amount of p66shc hairpin rnai molecules equivalent to the number of copies of p66shc transcripts measured in arrested 2– <dig> cell embryos.
the average amount of p66shc mrna transcripts in arrested 2– <dig> cell embryos, as detected by real time pcr, was  <dig>  ±  <dig>  × 10- <dig> pg/embryo .
using the size of the amplified pcr fragment  and the molecular weight of the nucleotides, the quantity  of p66shc mrna in arrested 2– <dig> cell embryos were transformed to equivalent numbers of p66shc mrna transcripts of approximately  <dig>  ±  <dig>  therefore, approximately  <dig>  p66shc short hairpin rnai molecules were microinjected in the subsequent experiments.
to further verify a dose-dependent silencing/reduction of p66shc protein levels we also injected twice  the amount of p66shc shrnai molecules.
successful selective down-regulation of p66shc
to assess whether p66shc expression was selectively down-regulated by the injection of approximately  <dig>  and  <dig>  p66shc short hairpin rnai molecules, we quantified p66shc mrna and protein levels in  <dig> hpi embryos by real time pcr and by quantitative immunoflorescence, respectively.
the ivf control group contained  <dig>  ±  <dig>  × 10- <dig> pg of p66shc mrna, the vehicle alone microinjected group  <dig>  ±  <dig>  × 10- <dig> and the p66shc shrnai microinjected group  <dig>  ±  <dig>  × 10- <dig> pg of p66shc transcripts .
therefore, this result demonstrated that microinjection of oocytes at the gv stage with the p66shc shrnai molecule resulted in significantly lower levels of p66shc mrna  by 54% attained in comparison to the control and vehicle microinjected groups.
to confirm that the down-regulation was selective towards p66shc mrna, p66shc shrnai treatment did not significantly affect  the mrna levels of the housekeeping gene histone h2a in the three groups considered .
in addition, real time pcr evaluation of the p <dig> mrna levels following p66shc down-regulation was also performed obtaining no significant differences  in p <dig> mrna transcript abundance among the control, vehicle microinjected and p66shc shrnai molecule microinjected groups .
the p66shc protein levels and distribution patterns in non-injected and vehicle microinjected groups were not different, displaying relatively high immunofluorescence intensities and punctate staining patterns in the cytoplasm and nuclei of all embryos analyzed .
unexpectedly, a peri-nuclear ring of staining around the nuclei was readily apparent .
in contrast,  <dig> hpi embryos that had been injected with ~ <dig>   and ~ <dig>   p66shc shrnai molecules showed visible reductions in p66shc fluorescence staining intensities.
control embryos treated with the secondary fitc-conjugated antibody alone  exhibited virtually no staining .
quantitative analysis of the relative signal strength of p66shc fluorescence revealed a significant  reduction in pixel numbers for both low  and high  p <dig> shrnai microinjected groups compared to non-injected and vehicle injected controls .
the knockdown efficiency of p66shc protein was  <dig> % for  <dig>  shrnai molecules and  <dig> % for  <dig>  shrnai molecules compared to p66shc levels in vehicle-injected embryos.
effects of p66shc knockdown on in vitro embryo development
the percentage of embryo cleavage varied significantly among the three groups with  <dig>  ±  <dig> % of oocytes cleaved after fertilization in the control non-injected group,  <dig>  ±  <dig> % in the group microinjected with only the vehicle and  <dig>  ±  <dig> % when the p66shc hairpin rnai molecules were microinjected  .
the developmental frequencies to the blastocyst stage, calculated as the number of blastocysts over the total number of cleaved oocytes, was  <dig>  ±  <dig> %,  <dig>  ±  <dig> % and  <dig>  ±  <dig> % for the control, vehicle and p66shc rnai microinjected groups respectively  .
subsequent injections of scrambled short-interfering rna control molecules, non-specific for any endogenous transcripts, had no affect on embryo arrest frequencies and developmental potential of bovine embryos compared to vehicle-injected embryos .
interestingly, while there were no significant differences in the percentage of arrested 2– <dig> cell embryos between the control  and the group microinjected with the vehicle , there was a significant decrease  in the percentage of permanent embryo arrest in the group where p66shc was down-regulated , which showed only  <dig>  ±  <dig> % of cleaved embryos arresting.
assessment of spontaneous oocyte cleavage revealed  <dig>  ±  <dig> % oocytes spontaneously cleaved in the control group, a significant decrease  in spontaneous cleaved oocytes after microinjection with  <dig>  ±  <dig> % in the vehicle microinjected group, and no spontaneous oocyte cleavage in the p66shc microinjected group.
the differences in spontaneous oocyte cleavage between the vehicle and p66shc shrnai injected groups were not significant  .
no differences in p66shc transcript levels were detected in embryos that reached the blastocyst stage
quantification by real time pcr of p66shc mrna in blastocysts collected at day- <dig> post-insemination from the three groups, showed no significant differences in p66shc mrna levels  among the different groups , with  <dig>  ±  <dig>  × 10- <dig> pg of p66shc mrna in the control group,  <dig>  ±  <dig>  × 10- <dig> pg in the vehicle microinjected group and  <dig>  ±  <dig>  × 10- <dig> pg in the p66shc shrnai microinjected group.
the amount of p66shc transcripts in each group reflects the amount normally detected at the blastocyst stage .
quantification of the housekeeping gene, h2a, was used as a control and showed no significant differences  .
discussion
the results presented here demonstrate the successful application of rna interference  in selectively diminishing the oxidative stress adaptor protein p66shc during in vitro bovine embryo development.
based on our previous findings that showed significantly elevated levels of p66shc and reactive oxygen species in permanently arrested 2– <dig> cell bovine embryos  <cit> , the goal in this study was to decrease p66shc levels in developing bovine oocytes and embryos to elucidate a functional role for p66shc in early embryo arrest.
we significantly decreased the number of p66shc mrna transcripts and protein by approximately 54% by microinjecting ~ <dig>  short hairpin rnai molecules specific for the bovine p66shc mrna sequence into gv stage oocytes.
concomitant with this reduction in p66shc mrna and protein levels, we observed a significant reduction in permanently arrested 2– <dig> cell embryos of approximately 93%.
therefore, this study clearly demonstrates a direct functional association between high levels of p66shc with early embryo arrest.
however, down-regulation of p66shc also induced a significant reduction in the number of cleaved embryos and blastocysts signifying that it may play a vital role in facilitating early embryonic development as well.
since permanent embryo arrest occurs before the major activation of the embryonic genome  <cit> , microinjection of the p66shc hairpin rnai molecules was required at the germinal vesicle  stage to maximize the effects of rnai mediated reduction of endogenous p66shc transcripts.
microinjection was carried out on cumulus-intact gv oocytes, but this presented several challenges.
accordingly, care was taken to minimize the damage to cocs by the holding pipette, which readily stripped off the cumulus cells, and to reduce oocyte injury by the injection pipette since the ooplasm contents were not easily visible and piercing of the gv was possible .
denuded immature oocytes do exhibit a decreased ability to reach the second metaphase , with a greater incidence of abnormal fertilization, reduced pronuclei formation, and reduced cleavage and blastocyst development  <cit> .
when we microinjected embryos without the p66shc shrnai molecules , we found a significant decrease in these parameters with  <dig>  ±  <dig> % of embryos cleaved,  <dig>  ±  <dig> % of embryos that develop to the blastocyst stage and  <dig>  ±  <dig> % of embryos that arrested.
it appears that the micromanipulation procedure itself affects the developmental potential of ivp bovine embryos reaching the blastocyst stage in vitro.
a similar decrease in cleavage and blastocyst rates has been reported after pronuclear injection of bovine zygotes  <cit> .
to improve oocyte survival following microinjection of rnai molecules the use of cytochalasin b and cycloheximide treatment prior to puncture have improved oocyte survival rates to levels comparable to non-punctured control oocytes  <cit> .
only a limited number of studies have been carried out using rna interference in mammalian oocytes and embryos  <cit> .
svoboda et al.
were able to down-regulate the expression of specific target genes up to 80% in a dose-dependent manner.
they microinjected short hairpin rnai molecules in mouse oocytes, obtaining a viable down-regulation using a range between  <dig>  ×  <dig> and  <dig>  ×  <dig> molecules in a volume of  <dig> pl  <cit> .
wianny and zernicka-goetz  microinjected mouse oocytes with  <dig>  to  <dig>  μg/μl of hairpin rnai molecules in a larger volume, up to  <dig> pl, to down-regulate their genes of interest.
marked success was also achieved by microinjection of the short interfering sirna expression vector directly into the nuclei of 2-cell mouse embryos in a concentration of  <dig> ng/μl  <cit> .
in the present study, approximately  <dig>  p66shc hairpin rnai molecules were microinjected in gv-stage bovine oocytes, representing the number of p66shc mrna molecules previously quantified in arrested 2-cell embryos  <cit> .
the microinjection volume we utilized needed to be significantly smaller than what was used previously for mouse oocytes.
microinjection of a volume greater than  <dig>  pl resulted in mechanical lysis of the gv bovine oocytes.
this species-difference might be due to a greater fragility of bovine oocytes versus mouse oocytes or to different parameters, such as the optimal pressure utilized for the microinjection and the microinjection needle diameter.
early cleaving co-culture embryos known to display high developmental potential and a low incidence of embryo arrest  <cit> , exhibit an average p66shc mrna concentration of  <dig>  ±  <dig>  × 10- <dig> pg per embryo, while late cleaving embryos, which are significantly more likely to arrest, contain a p66shc mrna amount of over twice as much  per embryo .
the amount of p66shc mrna is even higher in arrested 2– <dig> cell embryos collected at day- <dig> post-insemination, at  <dig>  ±  <dig>  × 10- <dig> pg per embryo .
these results, along with the consensus that the major activation of embryonic transcription occurs at the 8– <dig> cell stage in bovine embryos  <cit> , suggest that the increased levels of p66shc mrna and protein in arrested embryos may be due to an increase in p66shc mrna stability and/or protein stability through post-transcriptional or post-translational modifications.
recently, the small gtpase rac <dig> has been shown to induce phosphorylation-dependent increase in the stability of p66shc in various somatic cell lines  <cit> .
future experiments will elucidate whether the increase in p66shc in arrested embryos is due to de novo transcription, increased transcript stability and/or protein stability.
there was no increase in spontaneously cleaved oocytes upon rnai microinjection detected in this study, in contrast to the results obtained in the mouse  <cit> .
spontaneous oocyte cleavage induced by the microinjection procedure itself, while occurring in some species such as mouse  <cit> , humans  <cit>  and hamsters  <cit> , does not readily occur in the bovine  <cit> .
in the absence of chemical activation, approximately 4% of injected bovine oocytes cleaved  <cit> , which is consistent with the results presented in this study where  <dig>  ±  <dig> % of injected oocytes spontaneously cleaved following microinjection.
most spontaneously cleaved bovine oocytes exhibit developmental delay/arrest  <cit> , suggesting that the reduction in spontaneous cleaved oocytes in the microinjected groups compared to non-injected controls  may account for the reduction in embryo arrest.
however, there was also significant reduction in spontaneous oocyte cleavage in the vehicle-microinjected group without a concomitant reduction in the proportion of embryos permanently arresting at the 2– <dig> cell stage.
this result further supports that the decrease in arrested embryos observed for the p66shc shrnai microinjected group was not due to a reduction in spontaneous oocyte cleavage but to the knockdown in p66shc levels.
p66shc knock-out mice are viable, but the fertility of p66shc null females has not been reported  <cit> .
in this study, rnai-mediated reduction in p66shc mrna and protein significantly reduced the proportion of embryos arrested at the 2– <dig> cell stage , but only  <dig>  ±  <dig> % developed to the blastocyst stage , suggesting that escape from arrest leads to embryo demise by other means at later developmental stages and that p66shc has a variety of functions during early embryo development.
the reduction in blastocyst yield might suggest rnai toxicity to the oocytes/embryos, but this is not likely since histone h2a and p <dig> mrna levels were unchanged for both the 2-cell embryo and blastocyst stages after p66shc rna interference.
any insult/toxicity would greatly affect the expression of most genes  <cit> , rather than isolated individual loci.
the use of long  dsrna molecules have been shown to trigger a sequence non-specific interferon response  <cit> , however the use of short interfering and short hairpin rnai molecules in mammalian cells and embryos have not shown any toxicity or interferon response  <cit> .
the lack of alterations in p <dig> mrna levels also suggest that p <dig> is not affected by p66shc mrna down-regulation, consistent with our previous observations, which propose that embryo arrest and death are p53-independent events in early bovine embryos  <cit> .
the levels of p66shc transcripts after a 54% p66shc knockdown with ~ <dig>  shrnai molecules in  <dig> hpi, 2– <dig> cell embryos  is equivalent to the amount of p66shc mrna normally observed in a developing bovine blastocyst  , suggesting that we were working in a physiological range typically measured in blastocysts.
at the blastocyst stage no differences in p66shc mrna levels were observed between the control, vehicle microinjected and p66shc rnai microinjected groups .
this result indicates that the rnai molecules either induced only a temporary effect on endogenous p66shc levels, or that an optimal concentration of p66shc is necessary for continued development.
besides p66shc's role in normal cell management of stress and damage repair, p66shc functions in regulating mitochondrial metabolism  <cit> .
in the absence of p66shc, mouse embryonic fibroblasts and pc <dig> cells both exhibit a reduction in mitochondrial oxidative phosphorylation capacity, which was partially offset by increased levels of aerobic glycolysis and lactate production  <cit> .
this altered mitochondria metabolism in p66shc deficient cells is the most likely explanation for the reduced blastocyst frequencies observed for the p66shc knockdown embryo group.
quantitative assessment at timed stages of development coupled with a titrated silencing of p66shc under culture conditions with varying oxygen levels and energy sources will help clarify the metabolic role of this protein during early embryo development.
p66shc is an adaptor protein that is activated in response to oxidative stress and appears to play a crucial role in mediating cell death and cellular senescence  <cit> .
conversely, trinei et al.
showed that p66shc is an oxidative stress inducer, increasing reactive oxygen species  production  <cit> .
it is possible that the reduction in permanently arrested 2– <dig> cell embryos in the p66shc shrnai injected group is due to a decline in mitochondrial ros production and an increase in stress resistance.
recent studies have shown that oxidative stress-activated p66shc can translocate into the mitochondrial intermembrane space where it interacts with reduced cytochrome c to produce h2o <dig> which promotes the opening of permeability transition pores allowing for the release of ros and other constituents into the cytosol  <cit> .
the localization of p66shc protein throughout and as distinct foci within the cytoplasm of bovine embryos corroborates with this data.
ros are involved in the redox-dependent regulation of various cellular functions, including energy metabolism and the response to stress or growth signals  <cit> .
our previous research demonstrated that elevated oxygen tensions increase the levels of p66shc, intracellular ros and oxidative damage in bovine fibroblasts and embryos  <cit> , supporting other work that shows that oxidative stress mediates p66shc's function  <cit> .
interestingly, embryos injected with the high dose  of p66shc shrnai molecules appeared to cleave faster than those injected with the low does  of p66shc rnai hairpin molecules , supporting p66shc's other known role in antagonizing mitogenic signalling  <cit> .
embryo arrest and continued embryo development may therefore be regulated, in part, by the translocation of p66shc into the inter-mitochondrial space where it controls trans-membrane potential altering mitochondrial metabolism and/or generating and releasing ros into the cytosol causing cellular damage that triggers permanent cell cycle arrest at a developmental stage  that is in an anti-apoptosis state  <cit>  or apoptosis at other developmental stages.
p66shc has been shown to regulate redox-dependent inactivation of members from the forkhead transcription factor family triggering their translocation from the nucleus to the cytosol  <cit> .
forkhead transcription factors have been reported to regulate the expression of anti-apoptosis genes and several antioxidant enzymes, including superoxide dismutase  and catalase  <cit> .
the nuclear and peri-nuclear localization of p66shc observed in this study and elsewhere  <cit>  may account for this extra-mitochondrial role for p66shc.
rnai-mediated knockdown of p66shc in embryos already deemed arrested and in embryos at later developmental stages will reveal the developmental capacity of arrested embryos and may uncover the aetiology of the permanently arrest state and other p66shc function during later stages of early embryo development.
finally, there is now supporting evidence that telomere-driven senescence is triggered by mitochondrial production of ros  <cit> .
ros-mediated disruption of the telomere structure may explain the high rates of developmental arrest of in vitro produced embryos  <cit> .
preliminary evidence in our lab detecting phosphorylated histone γ-h2a.x foci in arrested 2– <dig> cell embryos but not in proliferating 2– <dig> cell embryos support this possibility since phosphorylation of histone h2a.x appears to facilitate the formation of dna damage foci around uncapped telomeres  <cit> .
based on the immunolocalization studies it is tempting to speculate that p66shc may be involved in a cycling cascade of events that triggers and maintains permanent embryo arrest through a ros-mediated and response pathway that permanently maintains a telomeric dna damage state.
CONCLUSIONS
in conclusion, using the post-transcriptional gene silencing approach of rna interference by microinjecting short hairpin rnai molecules into bovine oocytes/embryos, we were able to selectively diminish the levels of p66shc and correlated this knockdown with a significant reduction in the incidence of 2– <dig> cell embryo arrest and with a significant decrease in embryo development to the blastocyst stage.
these apparent contradictory roles for p66shc in both permanent embryo arrest and developmental competence implies that p66shc functions must be tightly regulated.
escape from permanent cell cycle arrest may alleviate some cases of age-related infertility, or at least provide a less controversial source of embryonic stem cells from arrested embryos original deemed as non-viable, biological waste.

 one feature of in vitro produced mammalian embryos is the high frequency of early developmental failure brought on by sub-optimal culture environments. fewer than half of all in vitro fertilized bovine embryos reach the blastocyst stage of development with many of these unable to sustain development following embryo transfer. the reasons for this high rate of in vitro embryo demise remains unclear, but it has been proposed as a protective mechanism for preventing further development of abnormal, poor quality embryos. almost half of all arrested human embryos display chromosomal abnormalities, and significantly more chromosomal aberrations are observed, alongside delayed development, for in vitro produced bovine embryos compared to their in vivo-derived counterparts. blastomeres with characteristic features of apoptosis, such as cytoplasmic, nuclear and dna fragmentation, have been detected in both in vitro and in vivo derived embryos, indicating that high levels of apoptosis might play a role in early embryo death. in situ tunel labelling has identified a greater incidence of apoptotic nuclei in cultured bovine blastocysts compared to those derived in vivo. interestingly, we and others have observed no morphological or biochemical signs of apoptosis during the early 2– cell cleavage stages of bovine embryogenesis. it is at this early developmental stage that approximately 14% of all in vitro produced bovine embryos permanently arrest in a senescence-like state. cellular or replicative senescence is the well known in vitro phenomenon whereby most proliferating somatic cell types irreversibly cease in their replicative capacity after a fixed number of population doublings and display distinct morphological features of "aged" cells. senescence is triggered by a p53-dependent signalling cascade associated with the shortening of telomeres, the tandem g-rich repetitive, non-coding dna sequences and specific binding proteins at the ends of linear chromosomes. we have previously shown that permanent cell growth arrest of cultured bovine somatic cells is associated with telomere shortening, increased levels of serine 20-p phosphorylation, and elevated levels of oxidative damage. since permanent growth arrest/cellular senescence occurs prematurely under conditions of elevated oxidative stress and can be activated by disruption of the telomere structure itself, even at maximally long telomere lengths, we hypothesize that arrested mammalian embryos permanently arrest in their development by a similar stress signalling pathway. at the cellular level, an oxidative stress sensor that can provide a key understanding of cell senescence and permanent embryo arrest is p66shc, the recently identified protein belonging to the shc family of adaptors for signal transduction in mitogenic and apoptotic responses. p66shc is involved in signal transduction pathways that regulate the cellular response to oxidative stress and life span and has been shown to act as a downstream target of p53-mediated elevation of intracellular oxidants, cytochrome c release and apoptosis. p66shc is important in the signalling response to ros and propagation of apoptotic signals in mouse fibroblasts. remarkably, p66shc mice exhibit increased resistance to oxidative stress and have a ~30% longer lifespan than wild-type control animals with no apparent side effects. we have measured elevated levels of p66shc mrna and protein in senescent somatic cells and in permanently arrested 2– cell bovine embryos. recently, we have demonstrated that p66shc is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ros. taken together, these data suggests that p66shc regulates an oxidative stress-induced senescence pathway that triggers permanent embryo arrest. the objective of this study was to elucidate a functional role for p66shc in embryo arrest by selectively decreasing p66shc levels in bovine oocytes and embryos using the post-transcriptional gene silencing technique of rna interference.  quantification of p66shc, h2a and p mrna transcripts determination of the number of p66shc, h2a and p mrna transcripts was carried out by real time pcr in arrested 2– cell embryos collected at day post-insemination, in early cleaving embryos ) that show very low percentage of embryo arrest and in late cleaving embryos that are more likely to arrest, in accordance to previous observations. these data are summarized in table along with the amounts of these specific gene transcripts in day blastocysts. significantly greater quantities of p66shc mrna transcripts were measured in both arrested and late cleaving embryos that are more likely to arrest compared to early cleaving hpi embryos. no differences in h2a or p mrna levels were detected among early and late cleaving embryos, arrested embryos, and blastocysts. the numbers of p66shc mrna transcripts at the blastocyst stage were significantly lowered compared to those measured in early cleaving and arrested embryos. values represent mean ± sem different superscripts differ significantly determination of the number of p66shc shrnai molecules to microinject in the attempt to down-regulate p66shc mrna expression, we wanted to microinject the amount of p66shc hairpin rnai molecules equivalent to the number of copies of p66shc transcripts measured in arrested 2– cell embryos. the average amount of p66shc mrna transcripts in arrested 2– cell embryos, as detected by real time pcr, was ± × 10- pg/embryo. using the size of the amplified pcr fragment and the molecular weight of the nucleotides, the quantity of p66shc mrna in arrested 2– cell embryos were transformed to equivalent numbers of p66shc mrna transcripts of approximately ± therefore, approximately p66shc short hairpin rnai molecules were microinjected in the subsequent experiments. to further verify a dose-dependent silencing/reduction of p66shc protein levels we also injected twice the amount of p66shc shrnai molecules. successful selective down-regulation of p66shc to assess whether p66shc expression was selectively down-regulated by the injection of approximately and p66shc short hairpin rnai molecules, we quantified p66shc mrna and protein levels in hpi embryos by real time pcr and by quantitative immunoflorescence, respectively. the ivf control group contained ± × 10- pg of p66shc mrna, the vehicle alone microinjected group ± × 10- and the p66shc shrnai microinjected group ± × 10- pg of p66shc transcripts. therefore, this result demonstrated that microinjection of oocytes at the gv stage with the p66shc shrnai molecule resulted in significantly lower levels of p66shc mrna by 54% attained in comparison to the control and vehicle microinjected groups. to confirm that the down-regulation was selective towards p66shc mrna, p66shc shrnai treatment did not significantly affect the mrna levels of the housekeeping gene histone h2a in the three groups considered. in addition, real time pcr evaluation of the p mrna levels following p66shc down-regulation was also performed obtaining no significant differences in p mrna transcript abundance among the control, vehicle microinjected and p66shc shrnai molecule microinjected groups. the p66shc protein levels and distribution patterns in non-injected and vehicle microinjected groups were not different, displaying relatively high immunofluorescence intensities and punctate staining patterns in the cytoplasm and nuclei of all embryos analyzed. unexpectedly, a peri-nuclear ring of staining around the nuclei was readily apparent. in contrast, hpi embryos that had been injected with ~ and ~ p66shc shrnai molecules showed visible reductions in p66shc fluorescence staining intensities. control embryos treated with the secondary fitc-conjugated antibody alone exhibited virtually no staining. quantitative analysis of the relative signal strength of p66shc fluorescence revealed a significant reduction in pixel numbers for both low and high p shrnai microinjected groups compared to non-injected and vehicle injected controls. the knockdown efficiency of p66shc protein was % for shrnai molecules and % for shrnai molecules compared to p66shc levels in vehicle-injected embryos. effects of p66shc knockdown on in vitro embryo development the percentage of embryo cleavage varied significantly among the three groups with ± % of oocytes cleaved after fertilization in the control non-injected group, ± % in the group microinjected with only the vehicle and ± % when the p66shc hairpin rnai molecules were microinjected. the developmental frequencies to the blastocyst stage, calculated as the number of blastocysts over the total number of cleaved oocytes, was ± %, ± % and ± % for the control, vehicle and p66shc rnai microinjected groups respectively. subsequent injections of scrambled short-interfering rna control molecules, non-specific for any endogenous transcripts, had no affect on embryo arrest frequencies and developmental potential of bovine embryos compared to vehicle-injected embryos. interestingly, while there were no significant differences in the percentage of arrested 2– cell embryos between the control and the group microinjected with the vehicle, there was a significant decrease in the percentage of permanent embryo arrest in the group where p66shc was down-regulated, which showed only ± % of cleaved embryos arresting. assessment of spontaneous oocyte cleavage revealed ± % oocytes spontaneously cleaved in the control group, a significant decrease in spontaneous cleaved oocytes after microinjection with ± % in the vehicle microinjected group, and no spontaneous oocyte cleavage in the p66shc microinjected group. the differences in spontaneous oocyte cleavage between the vehicle and p66shc shrnai injected groups were not significant. no differences in p66shc transcript levels were detected in embryos that reached the blastocyst stage quantification by real time pcr of p66shc mrna in blastocysts collected at day- post-insemination from the three groups, showed no significant differences in p66shc mrna levels among the different groups, with ± × 10- pg of p66shc mrna in the control group, ± × 10- pg in the vehicle microinjected group and ± × 10- pg in the p66shc shrnai microinjected group. the amount of p66shc transcripts in each group reflects the amount normally detected at the blastocyst stage. quantification of the housekeeping gene, h2a, was used as a control and showed no significant differences. discussion the results presented here demonstrate the successful application of rna interference in selectively diminishing the oxidative stress adaptor protein p66shc during in vitro bovine embryo development. based on our previous findings that showed significantly elevated levels of p66shc and reactive oxygen species in permanently arrested 2– cell bovine embryos, the goal in this study was to decrease p66shc levels in developing bovine oocytes and embryos to elucidate a functional role for p66shc in early embryo arrest. we significantly decreased the number of p66shc mrna transcripts and protein by approximately 54% by microinjecting ~ short hairpin rnai molecules specific for the bovine p66shc mrna sequence into gv stage oocytes. concomitant with this reduction in p66shc mrna and protein levels, we observed a significant reduction in permanently arrested 2– cell embryos of approximately 93%. therefore, this study clearly demonstrates a direct functional association between high levels of p66shc with early embryo arrest. however, down-regulation of p66shc also induced a significant reduction in the number of cleaved embryos and blastocysts signifying that it may play a vital role in facilitating early embryonic development as well. since permanent embryo arrest occurs before the major activation of the embryonic genome, microinjection of the p66shc hairpin rnai molecules was required at the germinal vesicle stage to maximize the effects of rnai mediated reduction of endogenous p66shc transcripts. microinjection was carried out on cumulus-intact gv oocytes, but this presented several challenges. accordingly, care was taken to minimize the damage to cocs by the holding pipette, which readily stripped off the cumulus cells, and to reduce oocyte injury by the injection pipette since the ooplasm contents were not easily visible and piercing of the gv was possible. denuded immature oocytes do exhibit a decreased ability to reach the second metaphase, with a greater incidence of abnormal fertilization, reduced pronuclei formation, and reduced cleavage and blastocyst development. when we microinjected embryos without the p66shc shrnai molecules, we found a significant decrease in these parameters with ± % of embryos cleaved, ± % of embryos that develop to the blastocyst stage and ± % of embryos that arrested. it appears that the micromanipulation procedure itself affects the developmental potential of ivp bovine embryos reaching the blastocyst stage in vitro. a similar decrease in cleavage and blastocyst rates has been reported after pronuclear injection of bovine zygotes. to improve oocyte survival following microinjection of rnai molecules the use of cytochalasin b and cycloheximide treatment prior to puncture have improved oocyte survival rates to levels comparable to non-punctured control oocytes. only a limited number of studies have been carried out using rna interference in mammalian oocytes and embryos. svoboda et al. were able to down-regulate the expression of specific target genes up to 80% in a dose-dependent manner. they microinjected short hairpin rnai molecules in mouse oocytes, obtaining a viable down-regulation using a range between × and × molecules in a volume of pl. wianny and zernicka-goetz microinjected mouse oocytes with to μg/μl of hairpin rnai molecules in a larger volume, up to pl, to down-regulate their genes of interest. marked success was also achieved by microinjection of the short interfering sirna expression vector directly into the nuclei of 2-cell mouse embryos in a concentration of ng/μl. in the present study, approximately p66shc hairpin rnai molecules were microinjected in gv-stage bovine oocytes, representing the number of p66shc mrna molecules previously quantified in arrested 2-cell embryos. the microinjection volume we utilized needed to be significantly smaller than what was used previously for mouse oocytes. microinjection of a volume greater than pl resulted in mechanical lysis of the gv bovine oocytes. this species-difference might be due to a greater fragility of bovine oocytes versus mouse oocytes or to different parameters, such as the optimal pressure utilized for the microinjection and the microinjection needle diameter. early cleaving co-culture embryos known to display high developmental potential and a low incidence of embryo arrest, exhibit an average p66shc mrna concentration of ± × 10- pg per embryo, while late cleaving embryos, which are significantly more likely to arrest, contain a p66shc mrna amount of over twice as much per embryo. the amount of p66shc mrna is even higher in arrested 2– cell embryos collected at day- post-insemination, at ± × 10- pg per embryo. these results, along with the consensus that the major activation of embryonic transcription occurs at the 8– cell stage in bovine embryos, suggest that the increased levels of p66shc mrna and protein in arrested embryos may be due to an increase in p66shc mrna stability and/or protein stability through post-transcriptional or post-translational modifications. recently, the small gtpase rac has been shown to induce phosphorylation-dependent increase in the stability of p66shc in various somatic cell lines. future experiments will elucidate whether the increase in p66shc in arrested embryos is due to de novo transcription, increased transcript stability and/or protein stability. there was no increase in spontaneously cleaved oocytes upon rnai microinjection detected in this study, in contrast to the results obtained in the mouse. spontaneous oocyte cleavage induced by the microinjection procedure itself, while occurring in some species such as mouse, humans and hamsters, does not readily occur in the bovine. in the absence of chemical activation, approximately 4% of injected bovine oocytes cleaved, which is consistent with the results presented in this study where ± % of injected oocytes spontaneously cleaved following microinjection. most spontaneously cleaved bovine oocytes exhibit developmental delay/arrest, suggesting that the reduction in spontaneous cleaved oocytes in the microinjected groups compared to non-injected controls may account for the reduction in embryo arrest. however, there was also significant reduction in spontaneous oocyte cleavage in the vehicle-microinjected group without a concomitant reduction in the proportion of embryos permanently arresting at the 2– cell stage. this result further supports that the decrease in arrested embryos observed for the p66shc shrnai microinjected group was not due to a reduction in spontaneous oocyte cleavage but to the knockdown in p66shc levels. p66shc knock-out mice are viable, but the fertility of p66shc null females has not been reported. in this study, rnai-mediated reduction in p66shc mrna and protein significantly reduced the proportion of embryos arrested at the 2– cell stage, but only ± % developed to the blastocyst stage, suggesting that escape from arrest leads to embryo demise by other means at later developmental stages and that p66shc has a variety of functions during early embryo development. the reduction in blastocyst yield might suggest rnai toxicity to the oocytes/embryos, but this is not likely since histone h2a and p mrna levels were unchanged for both the 2-cell embryo and blastocyst stages after p66shc rna interference. any insult/toxicity would greatly affect the expression of most genes, rather than isolated individual loci. the use of long dsrna molecules have been shown to trigger a sequence non-specific interferon response, however the use of short interfering and short hairpin rnai molecules in mammalian cells and embryos have not shown any toxicity or interferon response. the lack of alterations in p mrna levels also suggest that p is not affected by p66shc mrna down-regulation, consistent with our previous observations, which propose that embryo arrest and death are p53-independent events in early bovine embryos. the levels of p66shc transcripts after a 54% p66shc knockdown with ~ shrnai molecules in hpi, 2– cell embryos is equivalent to the amount of p66shc mrna normally observed in a developing bovine blastocyst, suggesting that we were working in a physiological range typically measured in blastocysts. at the blastocyst stage no differences in p66shc mrna levels were observed between the control, vehicle microinjected and p66shc rnai microinjected groups. this result indicates that the rnai molecules either induced only a temporary effect on endogenous p66shc levels, or that an optimal concentration of p66shc is necessary for continued development. besides p66shc's role in normal cell management of stress and damage repair, p66shc functions in regulating mitochondrial metabolism. in the absence of p66shc, mouse embryonic fibroblasts and pc cells both exhibit a reduction in mitochondrial oxidative phosphorylation capacity, which was partially offset by increased levels of aerobic glycolysis and lactate production. this altered mitochondria metabolism in p66shc deficient cells is the most likely explanation for the reduced blastocyst frequencies observed for the p66shc knockdown embryo group. quantitative assessment at timed stages of development coupled with a titrated silencing of p66shc under culture conditions with varying oxygen levels and energy sources will help clarify the metabolic role of this protein during early embryo development. p66shc is an adaptor protein that is activated in response to oxidative stress and appears to play a crucial role in mediating cell death and cellular senescence. conversely, trinei et al. showed that p66shc is an oxidative stress inducer, increasing reactive oxygen species production. it is possible that the reduction in permanently arrested 2– cell embryos in the p66shc shrnai injected group is due to a decline in mitochondrial ros production and an increase in stress resistance. recent studies have shown that oxidative stress-activated p66shc can translocate into the mitochondrial intermembrane space where it interacts with reduced cytochrome c to produce h2o which promotes the opening of permeability transition pores allowing for the release of ros and other constituents into the cytosol. the localization of p66shc protein throughout and as distinct foci within the cytoplasm of bovine embryos corroborates with this data. ros are involved in the redox-dependent regulation of various cellular functions, including energy metabolism and the response to stress or growth signals. our previous research demonstrated that elevated oxygen tensions increase the levels of p66shc, intracellular ros and oxidative damage in bovine fibroblasts and embryos, supporting other work that shows that oxidative stress mediates p66shc's function. interestingly, embryos injected with the high dose of p66shc shrnai molecules appeared to cleave faster than those injected with the low does of p66shc rnai hairpin molecules, supporting p66shc's other known role in antagonizing mitogenic signalling. embryo arrest and continued embryo development may therefore be regulated, in part, by the translocation of p66shc into the inter-mitochondrial space where it controls trans-membrane potential altering mitochondrial metabolism and/or generating and releasing ros into the cytosol causing cellular damage that triggers permanent cell cycle arrest at a developmental stage that is in an anti-apoptosis state or apoptosis at other developmental stages. p66shc has been shown to regulate redox-dependent inactivation of members from the forkhead transcription factor family triggering their translocation from the nucleus to the cytosol. forkhead transcription factors have been reported to regulate the expression of anti-apoptosis genes and several antioxidant enzymes, including superoxide dismutase and catalase. the nuclear and peri-nuclear localization of p66shc observed in this study and elsewhere may account for this extra-mitochondrial role for p66shc. rnai-mediated knockdown of p66shc in embryos already deemed arrested and in embryos at later developmental stages will reveal the developmental capacity of arrested embryos and may uncover the aetiology of the permanently arrest state and other p66shc function during later stages of early embryo development. finally, there is now supporting evidence that telomere-driven senescence is triggered by mitochondrial production of ros. ros-mediated disruption of the telomere structure may explain the high rates of developmental arrest of in vitro produced embryos. preliminary evidence in our lab detecting phosphorylated histone γ-h2a.x foci in arrested 2– cell embryos but not in proliferating 2– cell embryos support this possibility since phosphorylation of histone h2a.x appears to facilitate the formation of dna damage foci around uncapped telomeres. based on the immunolocalization studies it is tempting to speculate that p66shc may be involved in a cycling cascade of events that triggers and maintains permanent embryo arrest through a ros-mediated and response pathway that permanently maintains a telomeric dna damage state.  in conclusion, using the post-transcriptional gene silencing approach of rna interference by microinjecting short hairpin rnai molecules into bovine oocytes/embryos, we were able to selectively diminish the levels of p66shc and correlated this knockdown with a significant reduction in the incidence of 2– cell embryo arrest and with a significant decrease in embryo development to the blastocyst stage. these apparent contradictory roles for p66shc in both permanent embryo arrest and developmental competence implies that p66shc functions must be tightly regulated. escape from permanent cell cycle arrest may alleviate some cases of age-related infertility, or at least provide a less controversial source of embryonic stem cells from arrested embryos original deemed as non-viable, biological waste. 
experiments were comprised of a control group undergoing ivf alone and two groups microinjected with and without p66shc shrnai molecules prior to ivf. no significant differences in p66shc mrna levels were observed among the three groups at the blastocyst stage. the relative signal strength of p66shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66shc shrnai microinjected groups compared to controls. these results show that p66shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos. excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. the aim of this study was to selectively diminish p66shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by rna-mediated interference to study the effects of p66shc knockdown on in vitro fertilized bovine embryos. the amount of p66shc mrna quantified by real time pcr was significantly lowered upon p66shc shrnai microinjection. a significant decrease in the incidence of arrested embryos upon p66shc shrnai microinjection was detected compared to ivf and microinjected controls along with significant reductions in both cleavage divisions and blastocyst development. this reduction was selective for p66shc mrna, as both histone h2a and p mrna levels were not altered. approximately – short hairpin rnai molecules specific for p66shc were microinjected into bovine germinal vesicle stage oocytes or zygotes.
 excessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. we hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66shc. the aim of this study was to selectively diminish p66shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by rna-mediated interference to study the effects of p66shc knockdown on in vitro fertilized bovine embryos.  approximately – short hairpin rnai molecules specific for p66shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. experiments were comprised of a control group undergoing ivf alone and two groups microinjected with and without p66shc shrnai molecules prior to ivf. the amount of p66shc mrna quantified by real time pcr was significantly lowered upon p66shc shrnai microinjection. this reduction was selective for p66shc mrna, as both histone h2a and p mrna levels were not altered. the relative signal strength of p66shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66shc shrnai microinjected groups compared to controls. a significant decrease in the incidence of arrested embryos upon p66shc shrnai microinjection was detected compared to ivf and microinjected controls along with significant reductions in both cleavage divisions and blastocyst development. no significant differences in p66shc mrna levels were observed among the three groups at the blastocyst stage.  these results show that p66shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos. 