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Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

Somoskoi B, Martino NA, Cardone RA, Lacalandra GM, Dell'Aquila ME, Cseh S - Reprod. Biol. Endocrinol. (2015)

Bottom Line: After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts.However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

View Article: PubMed Central - PubMed

Affiliation: Department and Clinic of Obstetrics and Reproduction, Szent Istvan University, Budapest, Hungary. somoskoibence@gmail.com.

ABSTRACT

Background: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts.

Methods: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.

Results: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

Conclusions: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

No MeSH data available.


Related in: MedlinePlus

Effects of slow freezing and vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts, as related to mt pattern. In each group, energy status (Panel a) intracellular ROS levels (Panel b) and mt/ROS colocalization (Panel c) are expressed as in Figure 4 and Figure 5. Vitrified embryos with P/P pattern had significantly higher energy status than their SA counterparts and significantly higher ROS levels and mt/ROS colocalization than P/P controls (P < 0.05). Numbers of analyzed embryos per group are indicated on the bottom of each histogram. One-way ANOVA followed by Multiple Comparison Dunn’s or Dunnett’s met hods: comparisons among morula stage embryos: a,b P < 0.05; comparisons among blastocyst stage embryos: c,d P < 0.05.
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Fig6: Effects of slow freezing and vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts, as related to mt pattern. In each group, energy status (Panel a) intracellular ROS levels (Panel b) and mt/ROS colocalization (Panel c) are expressed as in Figure 4 and Figure 5. Vitrified embryos with P/P pattern had significantly higher energy status than their SA counterparts and significantly higher ROS levels and mt/ROS colocalization than P/P controls (P < 0.05). Numbers of analyzed embryos per group are indicated on the bottom of each histogram. One-way ANOVA followed by Multiple Comparison Dunn’s or Dunnett’s met hods: comparisons among morula stage embryos: a,b P < 0.05; comparisons among blastocyst stage embryos: c,d P < 0.05.

Mentions: Mitochondrial activity, intracellular ROS levels and mitochondria/ROS colocalization were evaluated at the equatorial plane of embryos which were cryopreserved at the morula or blastocyst stage, having a round shape and thus allowing the confocal quantification software set-up in areas describing continuous surfaces [39,52]. Energy status, expressing embryonic mt activity, was significantly reduced upon application of both cryopreservation procedures in embryos at the morula stage, whereas it did not change in embryos at the blastocyst stage (Figure 4, Panel a; P < 0.05). Intracellular ROS levels were significantly increased in VF embryos at the morula stage (Figure 4, Panel b; P < 0.05) compared with controls whereas they did not change in blastocyst stage embryos. Moreover, ROS levels were significantly increased in VF compared with SF embryos. Mitochondria/ROS colocalization significantly increased in VF embryos compared with controls in both developmental stages (Figure 4, Panel c; P < 0.05). In Figure 5, quantification bioenergy/redox data were separated according to embryo grade. Due to the absence of grade C embryos in control morulae and blastocysts and in vitrified blastocysts, the column representing grade C embryos is lacking in these groups. In addition, only one grade B morula, one grade B blastocyst and one grade C blastocyst were examined, thus data of these samples are represented as single values and were not statistically analyzed. Statistical analysis revealed that energy status of SF grade C morulae was significantly lower than that of control grade A morulae (Figure 5, Panel a; P < 0.05). No differences were found for this parameter between treatments (SF vs controls, VF vs controls and SF vs VF) among grade A and among grade B embryos. No differences were found for intracellular ROS levels and mt/ROS colocalization between treatment and for any embryo grade. Figure 6 shows quantification bioenergy/redox data separated according to mt pattern. Due to the absence of control blastocyst with SA pattern with regular round shape, the corresponding column is lacking. Statistical analysis revealed that, for energy status, no significant differences between SF and controls for embryos having either P/P or SA pattern were observed, indicating that both embryo types contributed equally to energy status reduction observed after SF. Instead, energy status was significantly higher in VF embryos with P/P mt pattern compared with those having SA mt pattern, both at the morula and the blastocyst stage, indicating that, mt activity reduction in vitrified embryos is associated with the appearance of the SA pattern (Figure 6, Panel a; P < 0.05); 2) for intracellular ROS levels, significantly higher values were observed in VF embryos with P/P pattern compared with fresh ones with P/P pattern, both in morulae and blastocysts (Figure 6, Panel b; P < 0.05); 3) the same significances were revealed for mt/ROS colocalization (Figure 6, Panel c; P < 0.05). In embryos showing SA mt pattern, no differences were observed for any bioenergy/redox parameter between both treatments and controls.Figure 4


Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification.

Somoskoi B, Martino NA, Cardone RA, Lacalandra GM, Dell'Aquila ME, Cseh S - Reprod. Biol. Endocrinol. (2015)

Effects of slow freezing and vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts, as related to mt pattern. In each group, energy status (Panel a) intracellular ROS levels (Panel b) and mt/ROS colocalization (Panel c) are expressed as in Figure 4 and Figure 5. Vitrified embryos with P/P pattern had significantly higher energy status than their SA counterparts and significantly higher ROS levels and mt/ROS colocalization than P/P controls (P < 0.05). Numbers of analyzed embryos per group are indicated on the bottom of each histogram. One-way ANOVA followed by Multiple Comparison Dunn’s or Dunnett’s met hods: comparisons among morula stage embryos: a,b P < 0.05; comparisons among blastocyst stage embryos: c,d P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419566&req=5

Fig6: Effects of slow freezing and vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts, as related to mt pattern. In each group, energy status (Panel a) intracellular ROS levels (Panel b) and mt/ROS colocalization (Panel c) are expressed as in Figure 4 and Figure 5. Vitrified embryos with P/P pattern had significantly higher energy status than their SA counterparts and significantly higher ROS levels and mt/ROS colocalization than P/P controls (P < 0.05). Numbers of analyzed embryos per group are indicated on the bottom of each histogram. One-way ANOVA followed by Multiple Comparison Dunn’s or Dunnett’s met hods: comparisons among morula stage embryos: a,b P < 0.05; comparisons among blastocyst stage embryos: c,d P < 0.05.
Mentions: Mitochondrial activity, intracellular ROS levels and mitochondria/ROS colocalization were evaluated at the equatorial plane of embryos which were cryopreserved at the morula or blastocyst stage, having a round shape and thus allowing the confocal quantification software set-up in areas describing continuous surfaces [39,52]. Energy status, expressing embryonic mt activity, was significantly reduced upon application of both cryopreservation procedures in embryos at the morula stage, whereas it did not change in embryos at the blastocyst stage (Figure 4, Panel a; P < 0.05). Intracellular ROS levels were significantly increased in VF embryos at the morula stage (Figure 4, Panel b; P < 0.05) compared with controls whereas they did not change in blastocyst stage embryos. Moreover, ROS levels were significantly increased in VF compared with SF embryos. Mitochondria/ROS colocalization significantly increased in VF embryos compared with controls in both developmental stages (Figure 4, Panel c; P < 0.05). In Figure 5, quantification bioenergy/redox data were separated according to embryo grade. Due to the absence of grade C embryos in control morulae and blastocysts and in vitrified blastocysts, the column representing grade C embryos is lacking in these groups. In addition, only one grade B morula, one grade B blastocyst and one grade C blastocyst were examined, thus data of these samples are represented as single values and were not statistically analyzed. Statistical analysis revealed that energy status of SF grade C morulae was significantly lower than that of control grade A morulae (Figure 5, Panel a; P < 0.05). No differences were found for this parameter between treatments (SF vs controls, VF vs controls and SF vs VF) among grade A and among grade B embryos. No differences were found for intracellular ROS levels and mt/ROS colocalization between treatment and for any embryo grade. Figure 6 shows quantification bioenergy/redox data separated according to mt pattern. Due to the absence of control blastocyst with SA pattern with regular round shape, the corresponding column is lacking. Statistical analysis revealed that, for energy status, no significant differences between SF and controls for embryos having either P/P or SA pattern were observed, indicating that both embryo types contributed equally to energy status reduction observed after SF. Instead, energy status was significantly higher in VF embryos with P/P mt pattern compared with those having SA mt pattern, both at the morula and the blastocyst stage, indicating that, mt activity reduction in vitrified embryos is associated with the appearance of the SA pattern (Figure 6, Panel a; P < 0.05); 2) for intracellular ROS levels, significantly higher values were observed in VF embryos with P/P pattern compared with fresh ones with P/P pattern, both in morulae and blastocysts (Figure 6, Panel b; P < 0.05); 3) the same significances were revealed for mt/ROS colocalization (Figure 6, Panel c; P < 0.05). In embryos showing SA mt pattern, no differences were observed for any bioenergy/redox parameter between both treatments and controls.Figure 4

Bottom Line: After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts.However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

View Article: PubMed Central - PubMed

Affiliation: Department and Clinic of Obstetrics and Reproduction, Szent Istvan University, Budapest, Hungary. somoskoibence@gmail.com.

ABSTRACT

Background: The ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts.

Methods: Mouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.

Results: Cryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.

Conclusions: This study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

No MeSH data available.


Related in: MedlinePlus