Hypoxic in vitro culture reduces histone lactylation and impairs pre-implantation embryonic development in mice

Abstract Background Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. Results We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. Conclusions We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.

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143 EFFECTS OF BOAR SEMINAL PLASMA IN IN VITRO CULTURE OF PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab143 ◽

2013 ◽

Vol 25 (1) ◽

pp. 219

Author(s):

J. H. Moon ◽

S. J. Kim ◽

J. T. Kang ◽

S. J. Park ◽

J. Y. Choi ◽

...

Keyword(s):

In Vitro Culture ◽

Seminal Plasma ◽

Harmful Effect ◽

Developmental Rate ◽

Blastocyst Formation ◽

Cleavage Rate ◽

Significant Difference ◽

Protective Medium ◽

La Jolla

Seminal plasma consisting of carbohydrates, proteins, and lipids not only serves as a nutritive and protective medium for sperm cells but also play a pivotal role in inducing the tolerance to pre-existing immune cells as well as improving the intra-uterine conditions for implantation of fertilized embryos (Guerin et al. 2009 Hum. Reprod. Update 15, 517–535). However, the effects of seminal plasma in in vitro culture of fertilized embryos are unknown. In the present study, the seminal plasma was separated from the second fraction of a normal farm boar (n = 1) by centrifugation and filtered seminal plasma was stored at –30°C until use. In a preliminary experiment, the optimal activity of seminal plasma was evaluated by incubating the embryos for different time intervals. To investigate the developmental rates, electrically (EA) (triplicates, n = 490) or chemically (CA) (quintuplicates, n = 599) activated 2-day-old porcine embryos were incubated for 3 h in PZM-5 medium (Funakoshi Co., Tokyo, Japan, Catalog no. IFP0410P) containing 0% (EA: n = 122 and CA: n = 152), 0.1% (EA: n = 123 and CA: n = 148), 0.5% (EA: n = 122 and CA: n = 150), or 1% (EA: n = 123 and CA: n = 149) seminal plasma. Similarly, the developmental rate of chemically activated 2-day-old somatic cell nuclear transferred porcine embryos (quadruplicates, n = 239) was studied after incubation with 0% (n = 119) or 0.1% (n = 120) seminal plasma for 3 h. A significant difference was noticed only in the rate of blastocyst formation in the chemically activated embryos treated with 0.1% seminal plasma (31.7 v. 24.8% in the 0% group, ANOVA; P < 0.05; Prism5, GraphPad Software Inc., La Jolla, CA, USA). None of the treatments showed a significant effect on the cleavage rate and cell numbers of blastocysts. In conclusion, the seminal plasma did not show any harmful effect on early embryos development. Furthermore, the seminal plasma (0.1%) improved the rate of blastocyst formation among the chemically activated nuclear transferred embryos. The results of this preliminary study suggest that the addition of seminal plasma during embryo transfer could increase the rate of pregnancy in pig. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

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35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab35 ◽

2011 ◽

Vol 23 (1) ◽

pp. 124

Author(s):

C. Feltrin ◽

M. Machado ◽

L. M. V. Queiroz ◽

M. A. S. Peixer ◽

P. F. Malard ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Zona Pellucida ◽

Blastocyst Stage ◽

Aggregation State ◽

Developmental Potential ◽

In Vitro Development ◽

Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

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Effects of Hypoxia on Differentiation of Mesenchymal Stem Cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190823144928 ◽

2020 ◽

Vol 15 (4) ◽

pp. 332-339

Author(s):

Wei Chen ◽

Yi Zhuo ◽

Da Duan ◽

Ming Lu

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Oxygen Concentration ◽

Atmospheric Oxygen ◽

Physiological State ◽

The Body ◽

Alveolar Epithelial Cells ◽

Differentiation Potential ◽

Alveolar Epithelial

Mesenchymal Stem Cells (MSCs) are distributed in many parts of the human body, including the bone marrow, placenta, umbilical cord, fat, and nasal mucosa. One of the unique features of MSCs is their multidirectional differentiation potential, including the ability to undergo osteogenesis, adipogenesis, and chondrogenesis, and to produce neurons, endothelial cells, Schwann cells, medullary nucleus cells, cardiomyocytes, and alveolar epithelial cells. MSCs have thus become a hot research topic in recent years. Numerous studies have investigated the differentiation of MSCs into various types of cells in vitro and their application to numerous fields. However, most studies have cultured MSCs under atmospheric oxygen tension with an oxygen concentration of 21%, which does not reflect a normal physiological state, given that the oxygen concentration generally used in vitro is four to ten times that to which MSCs would be exposed in the body. We therefore review the growing number of studies exploring the effect of hypoxic preconditioning on the differentiation of MSCs.

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56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab56 ◽

2008 ◽

Vol 20 (1) ◽

pp. 109 ◽

Cited By ~ 1

Author(s):

B. S. Song ◽

J. S. Kim ◽

X. L. Jin ◽

Y. Y. Lee ◽

Y. J. Cho ◽

...

Keyword(s):

Developmental Rate ◽

Cumulus Cells ◽

Complete Medium ◽

Blastocyst Formation ◽

Blastocyst Development ◽

Cleavage Rate ◽

Developmental Potential ◽

Frozen Semen ◽

Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL–1 hCG, and 10 ng mL–1 epidermal growth factor (EGF) at 38.5�C and 5% CO2 in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 � 106 sperm mL–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean � SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 � 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 � 2.7%, 38/45), and iSCNT (89.3 � 2.7%, 100/110, monkey; 89.3 � 3.3%, 45/49, pig; and 86.0 � 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 � 3.0%, 2/49, and 7.9 � 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 � 2.9%, 38/117, and 26.7 � 2.8%, 12/88, respectively; P < 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 � 2.7%, 78.1 � 2.1%, and 82.3 � 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.

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35 EFFECT OF CULTURE AT LOW OR ATMOSPHERIC OXYGEN TENSION IN SOMATIC DONOR CELLS FOR HORSE NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab35 ◽

2013 ◽

Vol 25 (1) ◽

pp. 165

Author(s):

A. Gambini ◽

J. Jarazo ◽

A. De Stefano ◽

F. Karlaninan ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Oxygen Tension ◽

Embryo Development ◽

Atmospheric Oxygen ◽

Culture Conditions ◽

Chi Square ◽

Reconstructed Embryos ◽

Donor Cells

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the well system as 3 reconstructed embryos per well. Cleavage and blastocyst formation (7–8 days) of the experimental groups were assessed. In vitro development, on a per-well and RE basis, was compared using the chi-square test. No statistical differences were observed in cleavage [(1): 48/84, 57%; (2): 54/87, 62%). No difference was observed in blastocyst rates on a per-well basis [(1): 5/28, 18%; (2): 4/29, 14%] or on a per-RE basis [(1): 5/84, 6%; (2): 4/87, 5%]. This work suggests that the oxygen tension during the in vitro culture of somatic donor cells does not affect the quantity of the cloned equine blastocyst produced. Further studies are required to determine if these conditions would affect in vivo embryo development.

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Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification

Reproduction Fertility and Development ◽

10.1071/rd14203 ◽

2016 ◽

Vol 28 (7) ◽

pp. 886 ◽

Cited By ~ 2

Author(s):

Roser Morató ◽

Míriam Castillo-Martín ◽

Marc Yeste ◽

Sergi Bonet

Keyword(s):

In Vitro Culture ◽

Embryo Quality ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Apoptotic Cells ◽

Total Cell Number ◽

Developmental Potential ◽

High Degree

The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24 h. Re-expansion rates were recorded at 3 and 24 h and total cell number and apoptotic cells were determined at 24 h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification–warming procedures and length of in vitro culture, as expanding and hatching–hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.

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Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms20174203 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4203 ◽

Cited By ~ 8

Author(s):

Marta Osrodek ◽

Mariusz Hartman ◽

Malgorzata Czyz

Keyword(s):

Cell Line ◽

Cell Lines ◽

Oxygen Concentration ◽

Melanoma Cell ◽

Atmospheric Oxygen ◽

Melanoma Cells ◽

In Vitro Studies ◽

Cell Phenotype ◽

Dependent Manner

Cancer cell phenotype largely depends on oxygen availability. The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue. Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2). A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines. Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner. Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells. Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner. In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.

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Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus

Zygote ◽

10.1017/s0967199414000161 ◽

2014 ◽

Vol 23 (4) ◽

pp. 525-536 ◽

Cited By ~ 21

Author(s):

Xue-Ming Zhao ◽

Jiang-Tao Min ◽

Wei-Hua Du ◽

Hai-Sheng Hao ◽

Yan Liu ◽

...

Keyword(s):

Formation Rate ◽

Developmental Rate ◽

Mrna Levels ◽

Developmental Potential ◽

Cumulus Oophorus ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Bovine Oocytes

SummaryThis study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10−9 M melatonin (DOs + MT), cumulus–oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

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125 SWINE EMBRYOS WITH HIGH POTENTIAL TO DEVELOP IN VITRO HAVE LESS γH2A.X AND MORE NBS1 PROTEINS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab125 ◽

2010 ◽

Vol 22 (1) ◽

pp. 221

Author(s):

A. R. S. Coutinho ◽

V. Bordignon

Keyword(s):

Dna Damage ◽

In Vitro Culture ◽

Western Blotting ◽

Protein Detection ◽

Developmental Competence ◽

Developmental Potential ◽

In Vitro Development ◽

Artificial Environment ◽

Vitro Development

The developmental potential of embryos produced in vitro is lower than those produced in vivo. The artificial environment and the stressful conditions of culture may affect embryo development through various mechanisms including DNA damage and, consequently, cell death. We hypothesized that the developmental competence of in vitro-cultured embryos is influenced by mechanisms signalling DNA damage and repair processes. Therefore, the aim of the study was to assess these processes by systematic quantification of phosphorylated histone H2A.X (γH2A.X) and p95 or nibrin protein (NSB1) in early- and late-cleaved swine embryos cultured in vitro. Studies from several groups including ours have demonstrated superior in vitro development for early-cleaved (within 24 h of culture) compared with late-cleaved (between 24 and 48 h) embryos. The presence of γH2A.X is associated with the DNA double-strand breaks, and NBS1 is involved in the process of DNA damage repair. These proteins were detected by both immunofluorescence and western blotting. Swine embryos were produced by parthenogenetic activation using in vitro-matured oocytes. Oocyte maturation, activation, and embryo culture were conducted as previously described (Che L et al. 2007 Theriogenology 67 1297-1304). At 24 and 48 h after activation, embryos were categorized as early- and late-cleaved, and were collected for protein detection on D2-3, D4-5, or D6-7 of culture. A minimum of 3 replicates were performed per treatment. The amount of protein in relation to the β-actin at D2-3, D4-5, and D6-7 as revealed by western blotting was 76.4% ± 1.3, 63.3% ± 10.5, and 43.2% ± 11.2 for γH2A.X and 60.2% ± 4.2, 67.3% ± 13.2, and 61.3% ± 6.2 for NBS1, respectively. Comparisons between early and late-cleaved groups were then performed by immunoflorescence detection of both proteins. Differences between groups were verified using Student’s t-test. The average proportion of cells that were positively stained for γH2AX at D2-3, D4-5, and D6-7 of culture was 64.4% ± 2.6 (n = 178) v. 65.92% ± 3.7 (n = 114; P = 0.7), 55.7% ± 2.4 (n = 121) v. 59.8% ± 4.7 (n = 62; P = 0.4) and 29.1% ± 2.1 (n = 137) v. 43.5% ± 3.4 (n = 41; P = 0.001), for early v. late-cleaved embryos. The values for NSB1 staining were 13.9% ± 3.8 (75) v. 3.9% ± 3.0 (34; P = 0.09), 50.5% ± 4.2 (66) v. 35.8% ± 6.0 (33; P = 0.05), and 51.0% ± 4.5 (n = 54) v. 38.2% ± 5.5 (n = 24; P = 0.1). These findings confirm the presence of γH2A.X and NBS1 proteins in swine embryos during all stages of in vitro culture. We further show that early cleaved embryos have a lower proportion of γH2A.X and a higher proportion of NSB1-positive cells compared with late-cleaved embryos. Together, these findings suggest that early cleaved embryos that have a superior capacity for in vitro development are better prepared to repair DNA damage during in vitro culture. Supported by NSERC.

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