Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts

Park, Sae Young; Kim, Eun Young; Cui, Xiang‐Shun; Tae, Jin; Lee, Won Don; Kim, Nam Hyung; Park, Se Pill; Lim, Jin Ho

doi:10.1017/s0967199406003649

Cited by 100 publications

(66 citation statements)

References 29 publications

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“…In this study, there was a 2.4-fold increase (P < 0.0001) in the apoptosis rate of vitrified (49.4 ± 1.9) in relation to fresh embryos (20.8 ± 1.1). Similar apoptosis profiles were observed in other studies, which demonstrated increases of 3.7-fold (Park et al, 2006) and 1.7-fold (Márquez-Alvarado et al, 2004) in the apoptosis rate of cryopreserved embryos compared with fresh embryos.…”

Section: Cryopreservation Of Bovine Embryossupporting

confidence: 87%

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

Sanches¹,

Zangirólamo²,

Silva³

et al. 2017

Anim Reprod

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In the last several years, the high demand for embryo production has resulted in the need to study new methods to make the cryopreservation of bovine embryos produced in vitro more efficient. Despite the advantages offered by in vitro embryo production (IVEP), the major challenge to its greater dissemination is to improve embryonic survival after cryopreservation. Embryos that are produced in vitro are less resistant to cryopreservation compared to those produced in vivo, which is due to the higher accumulation of lipids in their cells, among other factors. In this context, changes in the culture conditions such as the addition of lipolytic chemical substances and the adjustment of fetal calf serum in the medium have been proposed to decrease the lipid amount within the embryos. Several years ago, vitrification allowed good results for in vitro produced (IVP) embryos because of its simplicity, speed and low cost. More recently, another technique applied to simplify the embryo post-thawing rehydration step in vivo, direct transfer (DT), is a strategy that has proven to be of interest in helping to overcome limitations to the cryopreservation of in vitro produced embryos. DT has been performed by commercial laboratories, ensuring good embryo viability after thawing. However, commercial and operational limitations prevent the large-scale use of these techniques. Thus, this review aims to discuss the use of strategies to improve the postcryopreservation survival capacity and the aspects to be overcome so that the cryopreservation of IVP embryos becomes a well-established and commercially applicable technique in addition to presenting new guidelines for embryo transfer (ET) programs from a better selection of recipients.

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Section: Cryopreservation Of Bovine Embryossupporting

confidence: 87%

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

Sanches¹,

Zangirólamo²,

Silva³

et al. 2017

Anim Reprod

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“…The expression pattern of development and/or apoptosis related genes reflects embryo quality [2,24,25]. BIRC5 (survivin) is important for optimal embryo development and it suppresses apoptosis in preimplantation embryos [25].…”

Section: Discussionmentioning

confidence: 99%

“…BIRC5 (survivin) is important for optimal embryo development and it suppresses apoptosis in preimplantation embryos [25]. Although HSPA1A is a constitutive gene in embryos, its expression level is linked to the response to cellular stress [26].…”

Section: Discussionmentioning

confidence: 99%

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Development and quality of bovine embryos produced in vitro using growth factor supplemented serum-free system

Dhali¹,

Anchamparuthy²,

Butler³

et al. 2011

OJAS

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The influence of a growth factor supplemented serum-free system on the development, gene expression, and cryotolerance of in vitro produced bovine embryos was investigated. To assess the embryo development and gene expression in blastocysts, abattoir-derived oocytes (obtained from 3 -10 or <3 mm follicles) were matured and fertilized in serum-free media and cultured in synthetic oviductal fluid supplemented with fetal bovine serum (FBS, 4%), epidermal growth factor (EGF, 10 ng/mL), insulin like growth factor-1 (IGF-1, 100 ng/mL), stem cell factor (SCF, 50 ng/mL) or combinations of the growth factors. Expressions of selected gene transcripts were relatively quantified in the d 8 blastocysts. To assess the cryotolerance, d 4 morulae (derived from 3 -10 mm follicles and cultured with the supplementation of FBS or combinations of the growth factors) were vitrified, thawed and cultured (with respective supplementations). Total cell number and DNA fragmentation in blastocysts derived from the vitrified morulae were assessed through TUNEL assay. The rate (%) of cleavage, blastocyst and expanded/hatched blastocyst did not differ among the culture medium supplementations within the follicle size of 3 -10 mm (range 65.1 ± 4.3 -75.4 ± 3.9; 22.4 ± 3.9 -36.4 ± 3.6; and 11.2 ± 2.9 -23.3 ± 3.2, respectively) or <3 mm (range 59.3 ± 4.2 -74.5 ± 3.7; 15.0 ± 3.5 -28.7 ± 4.5; and 9.3 ± 2.8 -17.3 ± 2.7, respectively). Nevertheless, significantly lower (P < 0.05) cleavage and blastocyst rates with FBS and lower blastocyst rate with SCF supplementations were observed for the oocytes derived from <3 compared to 3 -10 mm follicles. The expression patterns of BCL-2, BAX, HSP1A1, GJA1 and BIRC5 transcripts varied significantly (P < 0.05) in all cases, except for BIRC5 in the blastocysts derived from 3 -10 mm follicles. Following thaw and culture, the development (%) of vitrified morulae into expanded/hatched blastocysts was lower (P < 0.01) with the supplementation of growth factors compared to FBS. In contrast, total number of cells and DNA fragmentation index in blastocysts were not different among the treatments. In conclusion, the growth factor supplemented serum-free system was satisfactory for in vitro bovine embryo production. Nevertheless, the system was not efficient when embryos were derived from <3 mm follicles and cultured with SCF. Additionally, gene expression patterns and cryotolerance of the embryos were affected with the treatments of growth factors compared to serum.

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“…However, the period of post fertilization culture environment can have a significant impact on mRNA abundance (Lonergan et al, 2003b). Moreover, the survival of an embryo in a specific culture system can be done by monitoring the levels of mRNA and this could an indirect way of quality assessment in the embryo & Park et al,2006. This further generate information on expression patterns used as genetic markers and gives an indication of abnormal embryo during their preimplantation stages.…”

Section: Cx43glut1 G6pdmentioning

confidence: 99%

145 the Role of Cell Apoptosis on in Vitro-Produced Beef Cattle Embryos

Nkadimeng

2017

Reprod. Fertil. Dev.

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Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. Exposure of pre-implantation embryos to a variety of cellular stresses can induce apoptosis in all or a fraction of blastomeres. Among the conditions that can induce apoptosis in cattle embryo production in vitro are heat shock, media composition, oxygen levels, and tumour necrosis factor (TNF)-α. The aim of the study was therefore to evaluate the in vitro culture environment of embryos in terms of cell apoptosis through caspase and DNA fragmentation evaluation. A total of 1200 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The cumulus-oocyte complexes were randomly allocated into 2 maturation temperatures, 39°C and 41°C (200/temperature /replicated 6 times), and cultured in M199 + FSH-LH-oestradiol medium under oil at 100% humidity and 5% CO2 for 24 h. Postmaturation, oocytes were subjected to normal subsequent embryo conditions (Rynkowska et al. 2011 Biotechnol. Comput. Biol. Bionanotechnol. 92, 45–53). All matured oocytes were fertilized for 6 h with frozen-thawed Nguni bull semen from 10 bulls replicated 5 times at a concentration of 265 × 106 sperm cells/mL in Bracket and Oliphant medium under oil. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate post IVF. Produced embryos were divided into 3 groups: 2–4 cell, ≥8 cell embryos, and blastocyst. Two- to four-cell embryos were removed at Day 2, ≥8 cell at Day 5 (mainly 8-cell, morula, and cavitated blastocyst), and expanded blastocyst at Day 7 of embryo culture to determine caspase activity and DNA fragmentation for evidence of apoptosis. Caspase was performed using colourimetric assay on a 96-well microplate reader and monitored at 450 nm reference filter. The DNA fragmentation was examined using the TUNEL assay method and imaging was done using the Alexa Fluor® 488 emission at 519 nm. Data were analysed using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA), and Shapiro-Wilks test was used on the standardized residuals to test for deviations from normality, means of significant effects were compared using Student’s t-l.s.d. at the 95% confidence interval. There was no significant difference on the cleavage rates [39°C (72.0 ± 22.70) and 41°C (67.2 ± 18.9)] and blastocysts [39°C (11.4 ± 2.6) and 41°C (11.2 ± 6.3)] from both maturation temperatures. Caspase activity showed no significant difference on 2–4 cell [39°C (0.015 ± 0.001) and 41°C (0.016 ± 0.002)] and ≥8 cell embryos at both temperatures [39°C (0.022 ± 0.007a) and 41°C (0.032 ± 0.013)]. However, blastocyst differed significantly at both temperatures [39°C (0.037 ± 0.012) and 41°C (0.053 ± 0.005)]. A higher (P < 0.05) DNA fragmentation was observed at 2–4 cell (6.5 ± 2.9), ≥8 cell (13.3 ± 3.1), and blastocyst (48.0 ± 8.2) for embryos produced from 41°C matured oocytes compared to the 39°C maturation group [2–4 cell (2.2 ± 1.2), ≥8 cell (4.5 ± 1.9), and blastocyst (9.7 ± 6.7)]. It is therefore concluded that embryos produced from 41°C matured oocytes can have the same developmental capacity as the 39°C maturation group. Moreover, both temperatures can show signs of apoptosis, however, with more apoptosis evidence in the 41°C than the 39°C maturation group.

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Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts

Cited by 100 publications

References 29 publications

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

Cryopreservation of in vitro-produced embryos: challenges for commercial implementation

Development and quality of bovine embryos produced in vitro using growth factor supplemented serum-free system

145 the Role of Cell Apoptosis on in Vitro-Produced Beef Cattle Embryos

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