Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

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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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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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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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135 EFFECT OF L-ERGOTHIONEINE SUPPLEMENTATION DURING CULTURE ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO (BUBALUS BUBALIS)

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab135 ◽

2015 ◽

Vol 27 (1) ◽

pp. 160

Author(s):

G. Zullo ◽

A. Salzano ◽

G. Bifulco ◽

V. Longobardi ◽

G. Albero ◽

...

Keyword(s):

Oxidative Stress ◽

In Vitro Culture ◽

Embryo Development ◽

Production Efficiency ◽

Cell Function ◽

Embryo Viability ◽

Cleavage Rate ◽

Mammalian Embryo ◽

Morphological Criteria

It is known that in vitro mammalian embryo development is negatively affected by the increased oxidative stress occurring under culture conditions. The oxidative damage of cell components via reactive oxygen species interferes with proper cell function. Buffalo embryos are particularly sensitive to oxidative stress because of their high lipid content (Boni et al. 1992 Acta Med. Vet. 38, 153–161). l-Ergothioneine (LE) is a powerful scavenger of hydroxyl radicals (OH) and an inhibitor of iron or copper ion-dependent generation of OH from hydrogen peroxide (H2O2). The aim of this study was to evaluate whether enriching the in vitro-culture medium with LE improves in vitro embryo production efficiency in buffalo. Abattoir-derived buffalo oocytes (n = 854, over 6 replicates) were in vitro matured and fertilized according to standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). Twenty hours after IVF presumptive zygotes were cultured in SOFaa supplemented by 8 mg mL–1 BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2, 88% N2, in humidified air, at 38.5°C with 0 (control; n = 214), 0.05 mM LE (n = 217), 0.1 mM LE (n = 204), and 1 mM LE (n = 219). Cleavage rate was assessed at the time of change of culture (Day 5) and the cleaved elements were cultured for a further 2 days. The embryos obtained by the end of culture, i.e. on Day 7 post-IVF, were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson, Nelson 2010 Manual of the International Embryo Transfer Society 86–105). The percentages of total transferable embryos and Grade 1 and 2 blastocysts in relation to cleaved oocytes were recorded. Because the chronology of development is known to be one of the most reliable parameters for assessing quality, the percentage of fast-developing embryos, i.e. hatched and expanded blastocysts, was also recorded. Data were analysed by Chi-squared test. Cleavage rate was not affected by the treatment (71.4, 66.8, 68.7, and 63.0%, respectively, with 0, 0.05, 0.1, and 1 mM LE). The total embryo output increased in groups supplemented with 0.05 and 0.1 mM LE (31.3, 42.2, 43.8, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). However, the enrichment of in vitro culture with 0.1 mM LE also increased the percentage of Grade 1 and 2 blastocysts compared with the control and to 1 mM LE (21.6, 30.9, 33.9, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). Likewise, 0.1 mM LE gave higher percentages of fast developing embryos than the control and 1 mM LE groups. In conclusion, these results demonstrated a beneficial effect of LE during culture on buffalo in vitro embryo development. The dose response trial indicated that the optimal concentration is 0.1 mM that also influenced the chronology of development and hence embryo viability.

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Developmental potential of day 13 porcine embryonic disk under in vitro culture conditions

In Vitro Cellular & Developmental Biology ◽

10.1007/bf02624185 ◽

1988 ◽

Vol 24 (12) ◽

pp. 1165-1172 ◽

Cited By ~ 3

Author(s):

Roy W. Silcox ◽

Bryan H. Johnson

Keyword(s):

In Vitro Culture ◽

Culture Conditions ◽

Developmental Potential ◽

Embryonic Disk

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Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

Zygote ◽

10.1017/s0967199414000677 ◽

2014 ◽

Vol 24 (1) ◽

pp. 18-30 ◽

Cited By ~ 10

Author(s):

Alejandra E. Velasquez ◽

Fidel O. Castro ◽

Daniel Veraguas ◽

Jose F. Cox ◽

Evelyn Lara ◽

...

Keyword(s):

Gene Expression ◽

Survival Rate ◽

In Vitro Culture ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryo Morphology ◽

Developmental Potential ◽

Bovine Blastocyst

SummaryEmbryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

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

Epigenetics & Chromatin ◽

10.1186/s13072-021-00431-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Wanting Yang ◽

Peijun Wang ◽

Pengbo Cao ◽

Shuang Wang ◽

Yuxiao Yang ◽

...

Keyword(s):

In Vitro Culture ◽

Oxygen Concentration ◽

Epigenetic Modification ◽

Atmospheric Oxygen ◽

Hypoxic Condition ◽

Developmental Rate ◽

Nuclear Accumulation ◽

Nuclear Staining ◽

Developmental Potential

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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