Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos

Fouladi-Nashta, Ali A.; Alberio, Ramiro; Kafi, M.; Nicholas, Ben; Campbell, Keith; Webb, Robert I.

doi:10.1016/s1472-6483(10)60827-9

Cited by 84 publications

(53 citation statements)

References 30 publications

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“…Since the frequency of apoptotic blastomeres was not affected by methionine status, it is likely that the reduction in glutathione concentrations among embryos receiving methionine was not sufficient to compromise cellular function. As reported elsewhere [20,31], the incidence of apoptosis was lower for trohectoderm than inner cell mass, indicating differences between these two cell types in either the amount of reactive species generation or antioxidant defenses.…”

Section: Discussionsupporting

confidence: 77%

“…Procedures for determination of numbers of inner cell mass (ICM) and trophectoderm (TE) cells in combination with TUNEL labeling of nuclei for determination of apoptosis was performed as described by Fouladi-Nashta et al [20]. Briefly, blastocysts at Day 8 after insemination were harvested and then incubated in a 4-well plate containing 10 mM KPO4, pH 7.4, 0.9% (w/v) NaCl and 1 mg/ ml PVP (PBS-PVP) with 50 μg/ml propidium iodide and 0.5% (v/ v) Triton X-100 for 30 sec at 37 C. Embryos were washed twice in PBS-PVP and incubated in a 50 μl microdrop of PBS-PVP with 4% (w/v) paraformaldehyde and 1 μg/ml Hoescht 33258 for 15 min, Embryos were washed twice in PBS-PVP, permeabilized in a 50 μl microdrop of PBS-PVP containing 0.5% (v/v) Triton X-100 for 5 min at room temperature, washed again twice in PBS-PVP, and incubated in a 50 μl microdrop of TUNEL reaction mixture (containing fluorescein isothiocyanate-conjugated dUTP and the enzyme terminal deoxynucleotidyltransferase) according to manufacturer's instructions for 1 h at 37 C in the dark.…”

Section: Cell Number and Apoptosismentioning

confidence: 99%

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Methionine Requirements for the Preimplantation Bovine Embryo

Bonilla

Luchini²,

Devillard³

et al. 2010

Journal of Reproduction and Development

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Abstract. The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium -bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 μmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 μmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 μmol/ l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 μmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine.In conclusion, the methionine requirement for preimplantation development is between 14 and 21 μmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of embryonic mortality.

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Section: Discussionsupporting

confidence: 77%

Section: Cell Number and Apoptosismentioning

confidence: 99%

Methionine Requirements for the Preimplantation Bovine Embryo

Bonilla

Luchini²,

Devillard³

et al. 2010

Journal of Reproduction and Development

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“…Day 7 sheep and day 8 cattle blastocysts were assessed using a differential staining technique combined with TUNEL labelling, which discriminates blastomeres from the ICM from those of the TE (Fouladi-Nashta et al 2005). Briefly, following permeabilisation, TE cells were stained with 30 mg/ml propidium iodide for 5 min, and fixed in 4% PFA with 10 mg/ml bisbenzimide (Hoechst 33342) for 20 min prior to incubation in small droplets of In situ Cell Death Detection Kit solution (Roche) for 45 min at 37 8C.…”

Section: Western Blotting and Immunostaining Of Bovine Oocytes And Emmentioning

confidence: 99%

Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre-implantation embryo

Kwong¹,

Adamiak²,

Gwynn³

et al. 2010

Reproduction

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Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expression for genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicle and pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1 and FOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase 1A were absent in all ovarian cells, and transcripts for FOLR2 were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosa cells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantation embryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovine culture media at either 1 or 10 mM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthine and thymidine (100 mM) increased the proportion of embryos that developed to blastocysts, but the cell number was reduced by 20%. The reduced uptake of [ 35 S] methionine into intra-cellular S-adenosylmethionine and S-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellular pools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status during the peri-conceptional period.Reproduction (2010) 139 705-715

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“…Day 8 blastocysts were stained using a differential staining combined with TUNEL labeling technique for counting the number of healthy and apoptotic cells in ICM and TE compartments as previously described (Fouladi-Nashta et al 2005). Briefly, TE cells were (Fig.…”

Section: Differential Staining Of Blastocysts and Detection Of Apoptomentioning

confidence: 99%

Oocyte quality in lactating dairy cows fed on high levels of n-3 and n-6 fatty acids

Fouladi-Nashta

Wonnacott²,

Gutiérrez³

et al. 2009

Reproduction

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Different fatty acid (FA) sources are known to influence reproductive hormones in cattle, yet there is little information on how dietary FAs affect oocyte quality. Effects of three dietary sources of FAs (supplying predominantly palmitic and oleic, linoleic (n-6) or linolenic (n-3) acids) on developmental potential of oocytes were studied in lactating dairy cows. A total of 12 Holstein cows received three diets containing rumen inert fat (RIF), soyabean or linseed as the main FA source for three periods of 25 days in a Latin-square design. Within each period, oocytes were collected in four ovum pick-up sessions at 3-4 day intervals. FA profiles in plasma and milk reflected profiles of dietary FA sources, but major FAs in granulosa cells were not affected. Dietary FA source did not affect plasma concentrations of leptin, insulin, IGF1, GH, or amino acids. RIF led to a higher proportion of cleaved embryos than soya or linseed, but blastocyst yield and embryo quality were not affected. It is concluded that the ovary buffers oocytes against the effects of fluctuations in plasma n-3 and n-6 FAs, resulting in only modest effects on their developmental potential.

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Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos

Cited by 84 publications

References 30 publications

Methionine Requirements for the Preimplantation Bovine Embryo

Methionine Requirements for the Preimplantation Bovine Embryo

Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre-implantation embryo

Oocyte quality in lactating dairy cows fed on high levels of n-3 and n-6 fatty acids

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