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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

Photomicrographs of frozen/thawed and vitrified/warmed mouse embryos at the morula and blastocyst stage of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. MitoTracker Orange, and DCDH FDA were used to label mitochondria and ROS, respectively. Nuclear chromatin was stained with Hoechst 33258. Representative photomicrographs showing mt distribution pattern and ROS intracellular localization in a control morula (row A) and a control blastocyst (row B) with P/P mt pattern, a SF morula with SA pattern (row C), a SF blastocyst with P/P pattern (row D), a VF morula with SA pattern (row E) and a VF blastocyst with P/P pattern (row F). In embryos at the blastocyst stage, a higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by cryopreservation. For each embryo, the corresponding epifluorescence images showing nuclear chromatin (line 1) and confocal images showing mt distribution pattern (line 2), ROS localization (line 3), mt/ROS merge (line 4) are shown. Scale bar represents 20 μm.
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Fig3: Photomicrographs of frozen/thawed and vitrified/warmed mouse embryos at the morula and blastocyst stage of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. MitoTracker Orange, and DCDH FDA were used to label mitochondria and ROS, respectively. Nuclear chromatin was stained with Hoechst 33258. Representative photomicrographs showing mt distribution pattern and ROS intracellular localization in a control morula (row A) and a control blastocyst (row B) with P/P mt pattern, a SF morula with SA pattern (row C), a SF blastocyst with P/P pattern (row D), a VF morula with SA pattern (row E) and a VF blastocyst with P/P pattern (row F). In embryos at the blastocyst stage, a higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by cryopreservation. For each embryo, the corresponding epifluorescence images showing nuclear chromatin (line 1) and confocal images showing mt distribution pattern (line 2), ROS localization (line 3), mt/ROS merge (line 4) are shown. Scale bar represents 20 μm.

Mentions: In Figure 3, representative photomicrographs of mouse morulae and blastocysts of control (rows A, B), slow freezing (rows C, D) and vitrification (rows E, F) groups and showing nuclear chromatin configuration (lane 1), and corresponding P/P or SA mt pattern (lane 2), intracellular ROS localization (lane 3) and mt/ROS merge (lane 4) are reported. In morulae and blastocysts with heterogeneous P/P mt pattern, in all blastomeres, there were detectable highly fluorescent signals of the mt-specific probe in the form of continuous rings around the nuclei and clusters of mitochondria at the cortex (Figure 3, A2, B2, D2, F2) which was reported in previous studies as indication of healthy embryos [51,52]. Due to reduced blastomere cytoplasmic size in embryos at the morula or blastocyst developmental stages, P/P mt clustering were almost overlapping. In morulae and blastocysts with homogeneous SA pattern, small mt aggregates diffused throughout the blastomere cytoplasm were observed (Figure 3, C2, E2). In addition, in embryos at the blastocyst stage, a higher number of red fluorescent spots was found on the trophoectoderm compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderm compared with ICM (Figure 3, B2, D2, F2). This feature was observed in all groups (22% [5/23], 23% [6/26] and 32% [15/47] for control, SF and VF embryos: not significant) thus it was not influenced by CP procedures. Intracellular ROS localization did not vary upon CP and intracellular ROS appeared diffused throughout the cytoplasm of embryonic blastomeres at any stage of development (Figure 3, lane 3) apart areas/sites of mitochondria/ROS overlapping (Figure 3, lane 4).Figure 3


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)

Photomicrographs of frozen/thawed and vitrified/warmed mouse embryos at the morula and blastocyst stage of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. MitoTracker Orange, and DCDH FDA were used to label mitochondria and ROS, respectively. Nuclear chromatin was stained with Hoechst 33258. Representative photomicrographs showing mt distribution pattern and ROS intracellular localization in a control morula (row A) and a control blastocyst (row B) with P/P mt pattern, a SF morula with SA pattern (row C), a SF blastocyst with P/P pattern (row D), a VF morula with SA pattern (row E) and a VF blastocyst with P/P pattern (row F). In embryos at the blastocyst stage, a higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by cryopreservation. For each embryo, the corresponding epifluorescence images showing nuclear chromatin (line 1) and confocal images showing mt distribution pattern (line 2), ROS localization (line 3), mt/ROS merge (line 4) are shown. Scale bar represents 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Photomicrographs of frozen/thawed and vitrified/warmed mouse embryos at the morula and blastocyst stage of development as assessed for their nuclear chromatin and bioenergy/oxidative potential. MitoTracker Orange, and DCDH FDA were used to label mitochondria and ROS, respectively. Nuclear chromatin was stained with Hoechst 33258. Representative photomicrographs showing mt distribution pattern and ROS intracellular localization in a control morula (row A) and a control blastocyst (row B) with P/P mt pattern, a SF morula with SA pattern (row C), a SF blastocyst with P/P pattern (row D), a VF morula with SA pattern (row E) and a VF blastocyst with P/P pattern (row F). In embryos at the blastocyst stage, a higher number of red fluorescent spots is evident on the trophoectoderma (white arrows) compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderma compared with ICM. This feature can be observed in embryos of both groups, thus it was not influenced by cryopreservation. For each embryo, the corresponding epifluorescence images showing nuclear chromatin (line 1) and confocal images showing mt distribution pattern (line 2), ROS localization (line 3), mt/ROS merge (line 4) are shown. Scale bar represents 20 μm.
Mentions: In Figure 3, representative photomicrographs of mouse morulae and blastocysts of control (rows A, B), slow freezing (rows C, D) and vitrification (rows E, F) groups and showing nuclear chromatin configuration (lane 1), and corresponding P/P or SA mt pattern (lane 2), intracellular ROS localization (lane 3) and mt/ROS merge (lane 4) are reported. In morulae and blastocysts with heterogeneous P/P mt pattern, in all blastomeres, there were detectable highly fluorescent signals of the mt-specific probe in the form of continuous rings around the nuclei and clusters of mitochondria at the cortex (Figure 3, A2, B2, D2, F2) which was reported in previous studies as indication of healthy embryos [51,52]. Due to reduced blastomere cytoplasmic size in embryos at the morula or blastocyst developmental stages, P/P mt clustering were almost overlapping. In morulae and blastocysts with homogeneous SA pattern, small mt aggregates diffused throughout the blastomere cytoplasm were observed (Figure 3, C2, E2). In addition, in embryos at the blastocyst stage, a higher number of red fluorescent spots was found on the trophoectoderm compared with the inner cell mass, indicating differences in mt number/cell between these two embryo lineages and higher mt/number and aggregate formation in the trophoectoderm compared with ICM (Figure 3, B2, D2, F2). This feature was observed in all groups (22% [5/23], 23% [6/26] and 32% [15/47] for control, SF and VF embryos: not significant) thus it was not influenced by CP procedures. Intracellular ROS localization did not vary upon CP and intracellular ROS appeared diffused throughout the cytoplasm of embryonic blastomeres at any stage of development (Figure 3, lane 3) apart areas/sites of mitochondria/ROS overlapping (Figure 3, lane 4).Figure 3

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